Far-Sighted Vision US Astronomers’ Latest List of Priorities Holds Valuable Lessons for Other Scientific Disciplines

Home , Marshall Rosenbluth

www.nature.com/nature Vol 466 | Issue no. 7309 | 19 August 2010 Far-sighted vision US astronomers’ latest list of priorities holds valuable lessons for other scientific disciplines.

n 1964, a flood of post-Sputnik funding led US astronomers to draw On the ground, a second wide-field telescope will image the entire up a list of large projects that could benefit the field. The result was visible sky every three nights, generating petabytes of data that will Ithe first decadal survey, a ranked wish list of new telescopes to build be accessible to astronomers all over the world. over the next ten years. The survey was an instant hit with politicians The survey also attempts to bring more credibility to cost esti- and funders, and similar priority lists are today drawn up in a range mates by providing independent assessments of each project’s budget. of disciplines. This approach is a hard lesson learned from the 2001 decadal review, But astronomy’s decadal reviews have run into trouble of late, with whose top priority, the James Webb Space Telescope, has more than overambitious lists, and projects busting their budgets and their quadrupled in price. schedules. The latest survey, published last week (see page 910), The new strategy is certainly open to criticism. For one thing, there seeks to correct some of the fundamental problems with the previ- is a limit to how much astronomy’s many subdisciplines can share — ous reviews — and scientists in other fields drawing up their own an X-ray telescope is very different from an infrared telescope, for wish lists would be well advised to take note. example. It is also unclear how deep the commitment to cooperation The past few decadal surveys made the mistake of trying to runs: the wide-field space telescope is a rebranding of a previous appease all of astronomy’s subdisciplines. Recent lists have, for dark-energy mission, and those outside the field are suspicious that example, included purpose-built instruments for fields such as X-ray their work will be a lower priority than the original purpose. And the and infrared astronomy, without assessing whether everything can scrupulous budgeting means that some daring projects now receive actually be built in the decade at hand. At the same time, individual lower priority than more conservative proposals. projects have grown in size and complexity, causing delays and spi- The panellists hope to address these potential flaws with a more flex- ralling costs. These problems have slowed each decade’s agenda and ible decadal process. Rather than treating their document as carved have damaged the survey’s credibility with the funding bodies it is in stone until 2020, the researchers call for a standing committee to meant to entice. carry out periodic reassessments of projects, along with a review of This year’s survey aims to address these flaws while facing up to the progress in various priority areas, such as planet hunting. Both can the realities of the ongoing financial crisis. It seeks to satisfy several change depending on how current and prospective projects perform. subfields at once by endorsing large, flexible telescopes. In space, The latest survey has clearly rescued the decadal process from tor- the panel’s top priority is a wide-field infrared survey telescope that pidity. The list is relevant and affordable. Other disciplines planning will hunt for extrasolar planets, study galaxy formation and measure their own reviews should follow its lead, as it promises to be a steady dark energy — the mysterious force accelerating cosmic expansion. guide for a bumpy decade ahead. ■

The WHO finally released the names of the committee members After the pandemic on 11 August. And, as critics alleged, several of them have links to industry. But it would be shocking if they did not. Pharmaceutical Despite some mistakes, the World Health companies play a key role in the response to pandemics, and they Organization handled the flu outbreak well. control a wealth of research knowledge on drugs and vaccines. But for the WHO to withhold their names until now caused unnecessary arlier this month, Margaret Chan, director-general of the World self-inflicted damage. Health Organization (WHO) in Geneva, confirmed two things that This should not distract from the post-pandemic verdict that the Emany scientists already knew to be true: the H1N1 influenza pan- emergency committee, the WHO and its allied national health agen- demic is over, and the world was lucky. A disease that could have mutated cies around the world deserve praise — and our thanks. Throughout into a highly lethal strain turned out to be comparatively mild. the crisis, they generally walked the difficult line between hype and Chan also reiterated that the WHO needs to review its handling dangerous understatement with aplomb. Although vaccines proved of the pandemic. It should certainly revisit its decision to grant ano- hard to make and their distribution could have been improved, the nymity to the experts who sat on its emergency committee, and who level of coordination between various bodies and researchers is wielded huge control over the decision to announce a global public- heartening. health emergency. The WHO insists that anonymity was necessary Of course, mistakes were made. But those who handled the world’s to prevent committee members from being influenced. But it helped response to H1N1 deserve better than false accusations of industry to fuel accusations that members from industry were just trying to self-interest. Governments, industry and academics worked well sell more vaccine. In retrospect, openness would have tempered that together when faced with a potentially disastrous threat — and will criticism. hopefully do so when called upon again. ■

NANOBIOTECHNOLOGY Tiny cell transistor

Science 329, 830–834 (2010) Science/AAAS A nanometre-sized transistor disguised as part of a biological membrane has infiltrated a living cell (pictured) and measured its electrical activity. Charles Lieber and his colleagues at Harvard University in Cambridge, Massachusetts, created their hairpin-shaped device out of a silicon nanowire with a tiny transistor on the elbow of the bent pin and an electrical contact on each of the pin’s two arms. They coated the elbow’s tip with phospholipids — the main constituent of cell membranes — tricking the membrane into accepting the tip and pulling it inwards. The authors made a device less than 50 nanometres wide — smaller than many virus particles. The team poked the probe into a single cultured embryonic chicken heart cell and used it to record a series of voltage peaks corresponding to the beating of the cell. For a longer story on this research, see go.nature.com/t4z6hK

AGEING fluorinated several small molecules, including Telescope to show that Saturn’s dazzling a cholesterol drug, attaining high yields under aurorae change intensity in time with the Proteins clump with age milder reaction conditions. radio emissions. This suggests that the two are PLoS Biol. 8, e1000450 (2010) Although the new method requires an physically linked. Further studies of Saturn’s Ageing worms accumulate protein clumps additional synthetic step, it should be suitable magnetosphere may uncover the reason for similar to those observed in humans with for late-stage fluorination of complex small the radio period’s odd pace, the authors say. Alzheimer’s and Huntington’s disease. molecules such as pharmaceuticals, the Cynthia Kenyon and her colleagues at authors say. MATERIALS SCIENCE the University of California, San Francisco, searched for proteins made by the nematode ASTRONOMY Decorating graphene Caenorhabditis elegans that would not dissolve Nano Lett. doi:10.1021/nl1024744 (2010) in detergents — a sign that the proteins Saturn’s beat In order for single-atom-thick sheets of would aggregate into insoluble clumps. The Geophys. Res. Lett. doi:10.1029/2010GL044057 (2010) carbon, or graphene, to be used in sensors, researchers found 461 proteins that become The ringed planet has rhythm. Researchers transistors and other devices, reactive more insoluble as the worms age. Several of the previously observed a regular pulsation in the molecules must be attached to them so that proteins were similar to those that are found ultraviolet glow of Saturn’s aurorae, and now the sheets can be chemically bonded to other clumped and tangled in the brains of patients show that this beats in time with the planet’s materials. Mingdi Yan at Portland State with Alzheimer’s disease. radio emissions. University in Oregon and her colleagues Furthermore, mutations that slow In the 1980s, when the Voyager spacecraft report a simple way to do this. ageing in C. elegans by interfering with an detected an electromagnetic pulse oscillating They created three variants of insulin-signalling pathway also delayed the in a recurring, 11-hour period, this was perfluorophenylazide, a compound that accumulation of insoluble proteins. The thought to be tied to the planet’s rotation. But reacts with graphene’s carbon–carbon bonds results suggest that disease is not the only over the years the beat has sped up and slowed when heated or illuminated with a mercury factor to blame for protein aggregation, with down. Jonathan Nichols at the University of lamp. Each variant carried a different ageing playing a part as well. Leicester, UK, and his colleagues analysed molecular group. By mixing each variant images (pictured) from the Hubble Space individually with a solution of graphene ORGANIC CHEMISTRY flakes, the team attached a specific molecule ter to the graphene, preparing the carbon sheet S

Fantastic fluorination eice for further chemical bonding. l J. Am. Chem. Soc. doi:10.1021/ja105834t (2010) Previous methods were less controlled, so , Univ. Univ. , Attaching fluorine to small organic molecules attached a variety of molecules to graphene at S can improve their pharmacological varying densities. ichol properties. But the process often relies on n

IMMUNOLOGY SA/J.

palladium catalysts and harsh reaction e conditions. ASA/ Tobias Ritter and his colleagues at Harvard Killer cells help n University in Cambridge, Massachusetts, J. Exp. Med. doi:10.1084/jem.20092749 (2010) have developed an alternative approach The primary job of natural killer (NK) based on a silver catalyst. Using the relatively cells, a type of immune cell, is to destroy inexpensive silver oxide, they successfully host tissue infected by pathogens. The cells

are also thought to boost autoimmunity several small, mostly non-overlapping, among other candidates. The latest hot under certain circumstances by acting on differences. material, graphene — single-atom-thick other immune cells in the lymph nodes. When participants were compared sheets of carbon — seems to home in on Fu-Dong Shi at St Joseph’s Hospital and individually with data from the groups, SVM tumours and, with the help of a laser, can heat Medical Center in Phoenix, Arizona, and analysis identified ASD in as many as 90% of up and kill them from within. his colleagues now report that NK cells have cases, which is comparable to the accuracy of Zhuang Liu at Soochow University in a stronger effect on autoimmunity in the behavioural diagnosis. Suzhou, China, and his colleagues coated central nervous system. nanometre-scale graphene sheets with The researchers studied a mouse model GEOSCIENCE polyethylene glycol to increase their of multiple sclerosis, a disease in which solubility and stability in the body. They then the immune system attacks myelin, the Ocean acid control injected the material into tumour-bearing protective sheath surrounding nerve fibres Geophys. Res. Lett. doi:10.1029/2010GL043181 (2010) mice (pictured left) and found high levels in the brain and spinal cord. They found that As atmospheric carbon dioxide levels of graphene accumulation in their tumours NK cells limit inflammation and the immune rise, some of that gas dissolves in ocean after 24 hours (right). response against myelin antigens. Increasing waters, lowering the surface pH the number of NK cells in the central nervous and potentially harming marine system protected the animals from disease, ecosystems. Quick and aggressive whereas limiting the cells’ numbers made emissions reductions are key to symptoms worse. The researchers speculate minimizing this acidification, that drugs used to treat multiple sclerosis may say Dan Bernie at the Met Office exert their positive effect by increasing NK Hadley Centre in Exeter, UK, and his cell numbers. co-workers. By coupling a climate model to NEUROBIOLOGY ocean and terrestrial carbon models, the researchers simulated the Autism detector effect of more than 100 emissions J. Neurosci. 30, 10612–10623 (2010) scenarios on ocean pH. The model Brain scans may be sufficient to identify outputs indicate that, without any people with autism spectrum disorder mitigation strategy in place, global (ASD), thanks to a new application of a type mean surface pH would drop from of data analysis. current levels of 7.9–8.3 to between Christine Ecker and her colleagues at 7.67 and 7.81 by 2100. But in an . the Institute of Psychiatry at King’s College aggressive mitigation scenario, in which The team administered the graphene to c London scanned the brains of 20 adults emissions peak in 2016 and then decrease by another set of 10 mice with breast tumours hem. So hem. diagnosed with ASD and 20 other volunteers 5% each year, pH would end up at around 8. and shone lasers at the growths. The tumours c

using magnetic resonance imaging. The disappeared the following day and did not Am. researchers’ support vector machine CANCER THERAPEUTICS regrow during the 40-day experiment. (SVM) analysis — which is also used in face Tumours in control mice that did not receive recognition — searched the data for subtle Nano tumour killer either the graphene or the laser treatment differences in cortex morphology between Nano Lett. doi:10.1021/nl100996u (2010) grew rapidly, killing the mice in about 16 days. the two groups, using several parameters Potential weapons against cancer are not Although a small toxicity study did not previously linked to ASD, such as cortical limited to small-molecule drugs, with reveal any obvious side effects, the authors say thickness and cortical folding. This identified nanomaterials such as carbon nanotubes that more safety studies are needed.

JOURNAL CLUB is common in tumours, also rate in the cell’s energy-producing component in mediating a novel contributes to a cellular shift from a organelles, the mitochondria, function of p53: the regulation of Gerry Melino metabolic pathway called oxidative resulting in increased generation energy metabolism. Medical Research Council, phosphorylation to a less efficient of the cell’s fuel source, ATP This is an attractive and University of Leicester, UK one known as glycolysis. This (W. Hu et al. Proc. Natl Acad. Sci. provocative hypothesis. There A cancer biologist weighs up shift, called the Warburg effect, is USA 107, 7455–7460; 2010). are some understandable p53, metabolism and cancer. characteristic of tumour cells. Meanwhile, Carol Prives at discrepancies in the data, Two papers shed light on this Columbia University in New York which suggests that additional The classic tumour-suppressor possibility. Both show that GLS2, and her co-workers find that mechanisms may be contributing gene, p53, plays a pivotal part in an enzyme involved in oxidative GLS2 expression is lost, or greatly to the metabolic changes. halting the cell cycle and inducing phosphorylation, is regulated decreased, in liver cancers, and Nevertheless, these two papers programmed cell death in response by p53 under stressed and non- that overexpression of GLS2 provide a potential mechanism to DNA damage. However, recent stressed conditions. Arnold Levine reduces the number of tumour linking the metabolic and genetic data suggest that it also has a at the Institute for Advanced cell colonies formed (S. Suzuki characteristics of tumours. role in cellular metabolism. I have Study in Princeton, New Jersey, et al. Proc. Natl Acad. Sci. USA 107, become intrigued by the possibility and his colleagues also show that 7461–7466; 2010). The results View the archive at http://blogs. that the inactivation of p53, which GLS2 increases the respiration reveal that GLS2 is an important nature.com/nature/journalclub

● policy y/AP AR

Sugar-beet ban: Farmers in d

the United States have been HAU blocked from planting genetically modified sugar beets after a C M. K. US federal judge revoked the government’s 2005 approval of the crop. The decision on 13 August means that the beets — which at present provide around half of the US sugar supply — cannot be grown until the US Department of Agriculture (USDA) completes an environmental-impact statement, which could take until the end of April 2012. Environmental organizations including the Sierra Club and the Center for Food Safety, headquartered in California and DiSEASE FolloWS DElUGE iN pAKiSTAN Washington DC, respectively, sued the USDA in 2008 for As nearly three weeks of floods in Pakistan displaced tens of millions of people and killed around 1,500, the approving the sugar beet without United Nations this week warned of the spread of acute diarrhoea and water-borne diseases such as dysentery adequately assessing the effects and cholera. In a report released on 16 August, the UN Office for the Coordination of Humanitarian Affairs added that it could have on weeds and that the floods would have a catastrophic effect on agricultural livelihoods, with extensive damage to standing nearby conventional crops. crops — such as maize (corn), cotton and rice — and stored planting seeds, including the major staple food crop wheat. The summer monsoon was exacerbated this year by an unusual jet-stream pattern in the upper atmosphere; the same weather phenomenon has been linked to the Russian heatwave and resulting peat fires. Berry bank threatened: Europe’s largest repository of rare berries and fruit faces and for exoplanets should be top providing incoming students closure after a Moscow court priority for large space activities with personal analyses of three ruled on 11 August against an (projects exceeding US$1 billion). common genetic variants. The appeal to preserve it. Pavlovsk See page 910 for more. educational programme had Experimental Station, an drawn criticism for its handling 84-year-old gene bank outside Pandemic over: The World of ethical and legal issues since St Petersburg, houses more Health Organization (WHO) its announcement in May (see than 5,000 crop varieties, but a announced on 10 August Nature 465, 845–846; 2010). government development agency that the world is no longer California’s health department wants to bulldoze the site. The experiencing an H1N1 influenza ruled on 11 August that such Vavilov Institute of Plant Industry virus pandemic. An emergency analyses constitute medical in St Petersburg, which runs the committee, which convened information, so must be station, has a month to appeal that day, said that countries conducted under a doctor’s the decision. The Twitter account were generally not reporting order and at a clinically certified of Russia’s president, Dmitri out-of-season outbreaks of the lab. The university will now not Medvedev (@KremlinRussia_E), flu strain, and that H1N1 would release individual results, but will stated on 13 August that the issue probably take on the behaviour NUMBER still analyse some 700 student would be “scrutinised”, giving of a seasonal flu virus. Margaret cRUNcH samples received, and discuss campaigners hope. See go.nature. Chan, director-general of the them in bulk. com/YJpZmb for more. WHO, said the pandemic had turned out better than feared 12 m ha Harvard first: Harvard US astronomy survey: The US because the virus hadn’t mutated The amount of land has once again topped the National Academy of Science into a more lethal form and drug (an area the size of influential university rankings has released its decadal survey, resistance hadn’t developed. “We Benin) lost every year list produced by the Institute of a much-anticipated report have been aided by pure good to desertification, Higher Education at Shanghai recommending the astronomy luck,” she said. according to the United Jiao Tong University in China. and astrophysics research projects Nations, which on Harvard, based in Cambridge, that US agencies should fund over ● 16 August launched Massachusetts, has been ranked the next ten years. The report, RESEARcH a ten-year campaign number one ever since the table published on 13 August, says Student gene-testing dropped: to halt the advance of was first produced in 2003. that a space telescope that could The University of California, deserts. Released last week, the survey search for clues to dark energy Berkeley, has been barred from uses six indicators, including

Nobel prizes won, publications

MAGES in Nature and Science, and highly SoUND y I y cited researchers in various ETT BiTES THE WEEK

/G subject categories. In individual disciplines, Princeton University “He’s been in slow- in New Jersey took top spot for motion fall for the AHEAD OOMBERG mathematics and the University 19–27 AUGUSt /Bl last three years, but of California, Berkeley, led in it hasn’t slowed him the International Congress chemistry. of Mathematicians — the down one bit.” discipline’s largest gathering,

ISHWANATHAN Weapons lab lawsuit: A held once every four years — Michael Tomasello, a P. V P. takes place in Hyderabad, India. non-profit watchdog filed a psychologist at the Max Planck lawsuit on 16 August to stop the Institute for Evolutionary based in Tokyo. Atul Sobti the prestigious Fields Medal construction of a US$4-billion Anthropology in Leipzig, (pictured) had led Ranbaxy, will be awarded at its opening weapons facility at Los Alamos Germany, comments on the a major player in the generic ceremony. National Laboratory in New misconduct investigation into drugs market and headquartered ➧ www.icm2010.org.in prominent Harvard University Mexico. The Los Alamos Study psychologist Marc Hauser. See in Gurgaon, for little more Group says that the nuclear page 908 for more. than a year. He took the helm 22–26 AUGUSt facility, the core of the Chemistry after the US Food and Drug the American Chemical Society and Metallurgy Research Source: New York Times, 13 August Administration accused the holds its autumn meeting in Replacement (CMRR) project, company of falsifying safety Boston, Massachusetts, with has violated federal law by failing data. Ranbaxy announced net the theme of chemistry for to produce an environmental- profits of 3.3 billion rupees combating disease. impact statement. An extensive (US$71 million) for the year’s ➧ go.nature.com/Ad8G6E redesign of the CMRR project second quarter, down 50% on the means that an impact statement previous year but above analysts’ 22–28 AUGUSt for an earlier version of the expectations. the 28th International facility in 2003 no longer applies, Ornithological Congress the group says. Pharma deal: Generic drugs discusses all things bird-related maker Aspen Pharmacare, based in Campos do Jordão, São Paulo, in Durban, South Africa, will Brazil. ● BUSiNESS buy the drugs manufacturing ➧ www.acquaviva.com.br/ioc2010/ Battery start-up: A123 Systems, unit of Australian company a rechargeable-battery company Sigma Pharmaceuticals for spun out of the Massachusetts A$900 million (US$806 million). public offering. In a US Securities Institute of Technology (MIT) New Jersey, and MIT, based in Aspen, which is 19% owned and Exchange Commission and based in Watertown, Cambridge. A123, meanwhile, by pharmaceutical giant filing dated 16 August, the Massachusetts, has spawned an says that it has ended a deal with GlaxoSmithKline, headquartered company says that it would use energy-storage company, 24M Chrysler to provide batteries for in London, and is Africa’s largest the proceeds for further research Technologies. The venture will the firm’s electric cars, although drugs manufacturer, wanted and development relating to its develop rechargeable power it promised a new deal with an to expand its position in the sequencing technology, which units that combine elements unnamed car manufacturer. Australian generics market. It reads off DNA sequences at from fuel cells and liquid-based announced the deal on 16 August. single-molecule resolution in real batteries. 24M has US$10 million Ranbaxy resignation: The time (see Nature 465, 145; 2010). in venture-capital funding, and chief executive of India’s biggest Gene-sequencing IPO: Pacific It would also use the money shares a $6-million grant from the drug maker, Ranbaxy, resigned Biosciences, a gene-sequencing to boost marketing, sales and US Advanced Research Projects unexpectedly last week, citing technology company in Menlo manufacturing of its sequencers, Agency-Energy with Rutgers differences of opinion with Park, California, aims to raise planned for commercial launch University in New Brunswick, parent company Daiichi Sankyo, US$200 million in an initial later this year.

BUSINESS WATCH PHARMA’S NEXT BLOCKBUSTER? STEMS

A new generation of hepatitis C drugs speciﬁcally Sy A generation of drugs with the potential to cure Vertex’s telaprevir both block HCV’s protease targets the virus. hepatitis C is set to flood the market. This month, enzyme and — combined with the standard

Phase I ESEARCH

Vertex Pharmaceuticals, based in Cambridge, treatment — cured 66% and 72% of patients NS5A R Phase II or IIB Massachusetts, and drug behemoth Merck, respectively in phase III trials. inhibitors Phase III headquartered in Whitehouse Station, New If the drugs are approved by mid-2011, as their Polymerase AGIENT Jersey, both released promising results from manufacturers hope, they will take the early inhibitors (non- late-stage clinical trials of their leading drugs lead in an HCV-drug field that could grow to nucleoside) against the hepatitis C virus (HCV). be worth US$15 billion by 2017, according to Polymerase S SOURCE: inhibitors The virus, which infects liver cells and can Irena Melnikova, a life-sciences analyst at TVM (nucleoside)

cause cirrhosis and liver cancer, affects about Capital in Boston, Massachusetts. Competitors Mechanism of action Protease 3% of the world’s population. Currently, half of in a crowded field (see chart) are developing inhibitors the patients with HCV are cured by a course other protease inhibitors, as well as drugs that of an immune-boosting protein and a general target HCV’s polymerase enzyme, and its NS5A 0 2 4 6 8 10 12 14 16 antiviral, although the treatment can have protein, which is involved in replication and viral Number of drugs in clinical trials against hepatitis C virus serious side effects. Merck’s drug boceprevir and assembly. See go.nature.com/rkncZt for more.

907 © 2010 Macmillan Publishers Limited. All rights reserved Vol 466|19 August 2010 NEWS Harvard probe kept under wraps Researchers call for the release of findings of the Marc Hauser misconduct investigation.

When news broke last week that famed Harvard University evolutionary psychologist Marc Hauser had been investigated for scientific misconduct, it was no surprise to many in the field. Rumours had been flying for three years, ever since university officials arrived to snatch computers from Hauser’s laboratory at the start of the inquiry. By the time Harvard completed its investigation in January, the gossip had become standard cocktail-hour fare at conferences. Now, after a Boston Globe story threw a sudden spotlight on the investigation, some researchers are voicing frustration with Harvard’s refusal to release the details of its findings. Hauser studies the evolution of key human characteristics, such as morality, language and mathematical ability, by tracing the origins of these traits in non-human primates. A popular professor and mentor, his research output has been diverse and prodigious, generating about one peer-reviewed paper per month for the past four years and forming the basis for popular articles, books and media appearances. Now that allegations of misconduct have surfaced, those working in related areas are adamant that a full account of any problems with Hauser’s published work is needed. “Sci- entists working in these areas, some of whom would like to build on Marc’s results, need to Evolutionary psychologist Marc Hauser’s research on primate cognition has been criticized. know exactly which may have been the results 2 of misconduct,” says Robert Seyfarth, an evolu- agencies that provide funding for Hauser’s of the Royal Society , which demonstrates that bis R tionary psychologist at the University of Penn- work. Both the National Science Foundation rhesus monkeys living on an island off Puerto sylvania in Philadelphia, and one of Hauser’s and the Office of Research Integrity at the Rico can correctly read specific human ges- 3

graduate-school advisers. “Keeping things National Institutes of Health, have declined tures, has been corrected. In an addendum to iedman/Co R

secret simply fuels rumours.” to comment on the matter, but the practice the paper, Hauser and his co-authors write that F R. Some even worry that without additional in such cases is that findings are made public field notes and video records from the study details, the field as a whole could become only if government officials conclude that a were found to be “incomplete”, leading two of tainted. “It is disastrous,” says primatologist researcher has acted improperly. the authors, Hauser and Justin Wood, now at Frans de Waal of Emory University in Atlanta, Harvard has also acknowledged that three the University of Southern California in Los Georgia. “This is a very small field — if one of Hauser’s publications have been singled out Angeles, to return to the island to repeat the prominent person is under suspicion, then for correction. None is among his most influ- experiments. The new data match the previ- everyone comes a little bit under suspicion.” ential, judging from citation data. One, a 2002 ously reported results, they write. Researchers close to those involved say paper in Cognition1, is being retracted, says the The status of the third paper4, published that the Harvard investigation, journal’s editor, Gerry Altmann in Science in 2007, is still up in the air, says launched after three of Hauser’s “Keeping things of the University of York, UK. In Ginger Pinholster, a spokeswoman for the graduate students became trou- secret simply fuels the retraction letter, Hauser takes journal’s publisher, the American Association bled by how he interpreted his rumours.” responsibility for the error, but for the Advancement of Science in Washington data and reported their concerns fails to describe precisely what DC. On 27 June, Wood wrote to the journal to to the university, has discovered eight instances was wrong with the paper, which reports that report that data for this paper were also miss- of misconduct. Hauser, who has taken a one- cotton-top tamarins — diminutive New World ing. Wood and Hauser have submitted new year period of leave from Harvard, has not monkeys with punk-rock hair — can learn to data that are now under review, but the edi- responded to requests for comment. Harvard distinguish between different patterns of vow- torial team is uncomfortable about making a will not discuss its investigation, but says the els and consonants, just as human infants do. decision without knowing the full results of the results have been reported to the two federal Meanwhile, a 2007 paper in the Proceedings Harvard investigation, Pinholster says.

Nickel allergy tracked to receptor Why humans are allergic to Yookji the metal but mice are not. go.nature.com/Fb88v7

Hauser had his share of critics even before the investigation began. In 1995, a Fda challenges stem-cell clinic paper from Hauser’s group in the Proceed- ings of the National Academy of Sciences5 How should clinics that treat patients Medicine Society (ICMS), based in Salem, caught the eye of evolutionary psycholo- with injections of their own stem cells be Oregon, an association of 1,100 physicians gist Gordon Gallup of the State University regulated? That question is about to test and patients that he co-founded and of of New York at Albany. The paper asserted the jurisdiction of the US Food and Drug which he is medical director. that tamarins can recognize their reflec- Administration (FDA) in a landmark legal Centeno and his supporters say that the tion in a mirror rather than assuming that battle — and is fuelling a war of words FDA’s request for an injunction is another the reflection is another monkey. Gallup between doctors marketing such therapies blow for stem-cell clinics in their David- was intrigued — his earlier work6 had indi- and academics who urge caution. and-Goliath struggle with an industry-led cated that although chimpanzees could The FDA asserted its authority on alliance that wants to put a stranglehold on recognize themselves in a mirror, monkeys 6 August, when it requested a federal stem-cell therapies and restrict individuals’ could not. He asked to see video footage of injunction from the US District Court of the use of their own cells. In an open letter on the experiment. District of Columbia to prevent stem-cell 30 July, ICMS executive director David But when Gallup reviewed the tapes, he clinic Regenerative Sciences in Broomfield, Audley accused the International Society

says he found no evidence of self-recogni- Colorado, from preparing for Stem Cell Research (ISSCR), C tion. He published his concerns7 in Animal its treatments. The company based in Deerfield, Illinois, and Behaviour in 1997. Hauser published a isolates, cultures and processes including some 3,500 stem-cell 8 rebuttal in the same issue , but four years adult stem cells from a patient’s researchers, of setting out to Clini hultz later, in a paper in the American Journal of bone marrow or synovial fluid. close their clinics. Motivated by sC Primatology9, reported that he had been Doctors then inject the cells to the interests of a pharmaceutical

unable to reproduce the results of the treat fractures, torn tendons industry unlikely to profit from Centeno– earlier paper. and other ailments. The clinic the treatments, Audley says, That does not necessarily mean the origi- charges patients US$7,000– the society wants to “change the nal claim was wrong, says Mark Liberman, 9,000, carries out about laws in all civilized countries to a linguist at the University of Pennsylvania. 20 procedures each month, outlaw these therapies”. When Subtle variations between experiments can and says it will fight the FDA’s Christopher Centeno, questioned by Nature, however, lead to contradictory results without clearly injunction. Unlike conventional medical director of Audley admitted he had no hard indicating that one result is wrong. But Gal- bone-marrow transplants Regenerative Sciences. evidence for these assertions. lup thinks that the paper should have been of blood-forming stem cells, ISSCR president Elaine Fuchs withdrawn or corrected, especially given Regenerative Sciences’ procedure relies on of the Rockefeller University in New York his experience with the raw data. “Unfor- mesenchymal stem cells that can potentially denies the claims. Although the society gets tunately, I think most people are unaware transform into bone, cartilage or fat. 12% of its funding from industry, its aim of the published failure to replicate,” he says, In July 2008, the FDA told Regenerative is to “motivate basic science” and not to noting that the original 1995 paper has been Sciences that its treatments are drugs support industrial interests, she says. cited 40 times, whereas the 2001 paper has according to the Federal Food, Drug and But the ISSCR is worried about unproven been cited only 10 times. Cosmetic Act, and biological products stem-cell treatments. In June, it established De Waal worries that the field will face under the Public Health Service Act. But the a service that, on request, will judge whether more problems as pressure builds for company did not apply for FDA approval and a treatment or clinic is safe and effective (see young professors to publish in high-profile continued to offer the treatment. Now the Nature 466, 7–8; 2010). Douglas Sipp of the journals. “Now scientists facing tenure are agency says that the company is not following RIKEN Centre for Developmental Biology asked to produce something new and excit- good manufacturing practice, and that the in Kobe, Japan, and a member of the ing that can be summed up in three pages,” treatment’s safety and efficacy is unproven. ISSCR’s Task Force on Unproven Stem Cell he says. “It’s craziness, because actually the But Christopher Centeno, Regenerative Treatments, says that “the consequences study of animal behaviour is painstak- Sciences’ medical director, argues that as the could be severe” if Regenerative Sciences ing, slow, laborious, and rarely leads to treatment uses a patient’s own stem cells, wins the US District Court case, likely unambiguous results.” ■ it is a medical procedure akin to in vitro to begin next summer. “Companies Heidi Ledford fertilization, and therefore none of the FDA’s would likely feel empowered to ignore business. He adds that his treatment has a requirements for demonstrable safety and 1. hauser, m. d. et al. Cognition 86, b15–b22 (2002). 2. hauser, m. d., Glynn, d. & Wood, j. Proc. R. Soc. B 274, much better safety record than conventional efficacy of autologous medicinal products, 1913–1918 (2007). surgery (C. J. Centeno et al. Curr. Stem Cell creating an ‘anything goes’ atmosphere,” he 3. hauser, m. d. & Wood, j. n. Proc. R. Soc. B doi:10.1098/ Res. Ther. 5, 81–93; 2010) and that animal says. “It would be, as they say, a bad thing.” rspb.2010.1441 (2010). (see go.nature.com/PiFFyf) and imaging But Centeno senses a landmark victory. 4. Wood, j. n. et al. Science 317, 1402–1405 (2007). 5. hauser, m. d. et al. Proc. Natl Acad. Sci. USA 92, (C. J. Centeno et al. Pain Physician 11, 343– “If we win, the entire regulatory structure for 10811–10814 (1995). 353; 2008) studies have proved it effective. autologous cell processing, with or without 6. Gallup, G. G. Science 167, 86–87 (1970). The FDA’s demand for scientific evidence culture, will be rewritten such that any 7. anderson, j. R. & Gallup, G. G. Anim. Behav. 54, from clinical trials “is a valid position. But physician using good practices and treating 1563–1567 (1997). 8. hauser, m. d. & kralik, j. Anim. Behav. 54, 1568–1571 it is not the only position,” Centeno told patients responsibly can use stem cells as part (1997). Nature. He says that it is sufficient to follow of his or her medical practice,” he says. ■ 9. hauser, m. d. et al. Am. J. Primatol. 53, 131–137 (2001). the guidelines of the International Cellular david Cyranoski

US survey sets cosmic priorities Dark energy rises to the top in decadal report ranking future astronomy and astrophysics projects.

Recently, a colleague of astronomer Claire Max factors can be derived from a three-dimen- Such data would contain subtle clues — in the jokingly told her that, come 13 August, half her sional survey of the surrounding Universe that distance–brightness relationships of super- friends would love her and half would never the LSST is well suited to provide. novae, the bending of light (micro lensing) from want to speak to her again. “Increasingly, we are able to ask new ques- background galaxies and the three-dimensional That is because Max, of the University of tions by querying huge databases,” says Tyson. clustering of matter in space — that can be used California, Santa Cruz, has for the past two “The key is to populate those databases with to independently measure dark energy. years been helping to craft US astronomy’s calibrated and trusted data.” WFIRST is effectively a rebranding of the latest decadal survey, an influential report The LSST is expected to help US astrono- Joint Dark Energy Mission, a NASA–DOE prepared for the National Research Council mers regain some momentum in ground-based collaboration. The new name, says one survey

that recommends which astronomy reviewer, signals that the $1.6-bil- . P and astrophysics projects NASA, the lion telescope is not a one-trick or c T

National Science Foundation (NSF) pony, but a way of serving other ss and the Department of Energy astronomical needs as well. The nc./L (DOE) should fund over the next survey committee stresses, for i ten years. example, that WFIRST could spot ions T Using input from 9 appointed micro lensing events caused when panels, 17 town-hall meetings and exoplanets — planets outside our 324 white papers from individual Solar System — pass briefly in research groups, the survey’s aim is to front of background stars in the assess opportunities and set priorities Milky Way. Although the method for US astronomy and astrophysics, is unsuitable for studying individ- while balancing scientific goals with ual solar systems in detail, it prom- Produc Mason Mason, T. fiscal realities. The report is now ises, through its sheer number of out, and although Max isn’t aware discoveries, to provide an unbiased of losing any friends, there are some sample of the kinds of planetary in the community who have more systems prevalent in the Galaxy. reason to thank her than others. This may not be enough to sat- Topping the list of the pleased isfy those who study exoplanets. “I and grateful is Anthony Tyson, an The Large Synoptic Survey Telescope will capture short-lived cosmic events. don’t think the report reflects that astrophysicist at the University of activity and the amount of young California, Davis, and director of the proposed astronomy at a time when European facilities people going into exoplanets,” says Sara Seager, Large Synoptic Survey Telescope (LSST), which have begun to dominate the field. To that end, an astrophysicist at the Massachusetts Institute ranks highest among the ground-based facili- the survey stresses the need for a swift decision of Technology in Cambridge. Seager notes that ties considered by the survey committee. When on which of two competing mega-telescopes the survey abandons support for the Space completed in 2015, the 8.4-metre telescope will should receive federal funding. Interferometry Mission, a project that would regularly sweep the entire visible sky in three The proposed Thirty Meter Telescope, on have detected planets slightly larger than Earth nights with a 3.2-gigapixel camera, capturing Mauna Kea in Hawaii, and the Giant Magellan through their gravitational effect on the stars short-lived phenomena ranging Telescope, envisioned for Las they orbit. However, Seager applauds another from fast-moving near-Earth “Increasingly, we Campanas in Chile, are both recommendation: a $100-million to $200-mil- asteroids to the flashes of super- are able to ask new supported by significant pri- lion allocation for the technical development novae in distant galaxies. vate money, and would have of a future exoplanet mission. Over ten years, the US$465- questions by querying many times the light-gathering Exoplanet researchers were stung when the million observatory will also huge databases.” power and resolution of today’s Terrestrial Planet Finder, a mission endorsed by build up an unprecedented largest telescopes. Realistically, the previous decadal survey was later cancelled 100-petabyte database for astronomers trying only one project will receive federal funds, owing to cost overruns. So they welcome the to discern the nature of two mysterious factors which the survey recommends should be chance to continue pursuing other approaches that shape the Universe. One is dark matter, between $257 million and $350 million. Given to observing Earth-like planets. thought to be an unknown particle or family of that Europe has also prioritized a 42-metre “Last week, I was worried,” says Webster particles beyond the standard model of physics. telescope, the European Extremely Large Tel- Cash, who is working on an exoplanet-mission Hidden in vast quantities among the galaxies, escope, a choice needs to be made now to avoid concept at the University of Colorado, Boulder. dark matter generates a gravitational pull that a counterproductive stalemate. “Now, I’m feeling like my career is going to be a has shaped the evolution of the Universe. The In space, the decadal survey proposes lot of fun for the next ten years.” ■ other factor is dark energy, the pervasive but the Wide Field Infrared Survey Telescope Adam Mann mysterious phenomenon that is causing cosmic (WFIRST), a 1.5-metre instrument that will See Editorial, page 903. For a full list of ranked expansion to accelerate. Crucial data on both map the whole sky at near-infrared wavelengths. projects, see go.nature.com/BrYViu.

booSt YoUR citatioNS T Analysis suggests that it o could pay to include more PH references. ock sT go.nature.com/SpQfPh i science panel gives hope in river-pollution dispute

BuEnoS AirES, ArgEntinA smarting from years of contention. orbis

A panel of scientists could help At the international court, research c resolve a festering environmental from each side was presented, dispute between Argentina and and subsequently trashed by its ua Press/ ua

Uruguay. The controversy, over opponents. H a paper-mill complex on the Gabriel Yorda, DINAMA’s head Uruguayan side of a shared river, of water quality, says that Uruguay has sparked years of protests. has monitored the river intensively

It prompted Argentina to take and seen no signs of pollution. Inés an/XinHua/Xin LL e

Uruguay to the International O’Farrell, a specialist in inland T an s

Court of Justice in The Hague, water studies from the University of . the Netherlands, accusing the Buenos Aires, counters that Uruguay r neighbouring country of ignoring sampled fewer sites than Argentina. environmental concerns. And it She adds that Argentinian scientists has seen scientists on each side took samples in summer — the accused of twisting data to suit their river’s most vulnerable time, when government’s agendas. the waters ebb, allowing ocean Now the two countries have tides to reverse the river’s flow and agreed to jointly monitor the potentially trap pollutants instead of Uruguay River, which forms their discharging them into the sea. common border, for pollution and O’Farrell and her colleagues say algal blooms. But poisoned relations Thousands of Argentinians protested against a Uruguayan paper mill. that their model of the river’s flow, between the two countries’ scientists mainly developed by scientists at could hamper cooperation. together, for the first time since 2005. An the Argentinian National University of The controversy began more than five advisory committee of four scientists — two La Plata, predicted a pollution-trapping years ago, after Uruguay granted two from Uruguay and two from Argentina — event matching the extent and distribution companies — ENCE, based in Madrid, and will also visit the mill up to a dozen times of a record algal bloom in February Botnia, headquartered in Espoo, Finland — a year and check that it is not polluting the 2009. Other data, she says, “indicate the permission to build paper-pulp mills in the river. Argentina’s foreign minister, Héctor presence of Eucalyptus globulus wood town of Fray Bentos near the river, not far Timerman, said on 4 August that if pollution fibres in the bloom, as well as nonylphenol from the Argentinian city of Gualeguaychú. is found, the country will again lobby to close contaminants” from detergent breakdown. In the first protest, in 2005, more than the mill. But DINAMA’s data for February 10,000 people blocked the Libertador The details of the deal will be decided 2009 show that levels of algae-fertilizing General San Martín Bridge, which connects this month. But more than two weeks into phosphates at sites supposedly affected the two countries. Protestors said that discussions, “we are still waiting to find by the factory were lower than at sites waste water from the mills flowing into out what the process of the monitoring will elsewhere. And nonylphenol is not among the river would carry pollutants such as be exactly”, says Jorge Rucks, the director the mill’s likely pollutants, says Yorda. sulphates and reactive chlorine compounds. of DINAMA, Uruguay’s environment When DINAMA dispatched Uruguayan Argentina has also claimed that Uruguay ministry. “At the moment we don’t know scientists to take samples directly from the violated a treaty by not seeking its who the scientists will be.” effluent pipes of the plant, Yorda says that permission for the mills. Experts from both countries are still they found none of the expected pollutants. Just months after the row reached the With so much ill will between the r e international court in 2006, ENCE pulled v scientists involved, the soon-to-be-formed i BRAZIL R out of its project. Botnia pressed ahead, y committee will have a tough job building a

u however, and the wrangling escalated after g trust. But Eugenio Lorenzo, the current u

r its mill — costing US$1.2 billion to set up, U head of the Uruguayan delegation to the making it Uruguay’s largest-ever foreign ARGENTINA bilateral administrative commission of URUGUA Y investment — started processing eucalyptus the Uruguay River, which carried out wood in 2007. Gualeguaychú monitoring from 1990 to 2005, is optimistic But a fresh round of diplomacy followed that renewed efforts will be successful. Fray Bentos Mercedes the appointment this March of José Mujica as “With a good system, we might be able Uruguay’s president, as well as a ruling by the to identify other sources of pollution on international court in April in which 11 out the river,” he says. Better environmental of 14 judges sided with Uruguay. On 28 July, Buenos Aires Montevideo monitoring, of course, may yet uncover Mujica and Argentina’s president Cristina more polluters — and political headaches — ■ Fernández de Kirchner announced that the 100 km Atlantic Ocean on the Uruguay River. countries would monitor the Uruguay River Anna Petherick

Jet reveals atmosphere’s secrets Marathon flights test models with first pole-to-pole snapshot of trace gases.

If only they could cash in the air miles. By flying nearly 50,000 kilometres between the Arctic Ocean and the Antarctic coast and repeatedly sampling the air at a broad range HIPPO/NCAR of altitudes, scientists are building the most detailed profile of the atmosphere yet. In spring this year, during its third of five planned missions, a specially outfitted Gulf- stream V jet, owned by the US National Sci- ence Foundation and operated by the National Center for Atmospheric Research in Boulder, Colorado, journeyed northwards, nearly reaching the Pole before turning south towards the Antarctic. The plane made occasional refuelling stops along the way, and then largely retraced its eastern Pacific route before returning to its home base (see map). As it flew, the plane (formerly Researchers have been using instruments on this adapted Gulfstream V jet to profile Earth’s atmosphere. known as HIAPER — the High-Performance Instrumented Airborne Platform for Environ- at Harvard University in Cambridge, Massa- taper off much more quickly than expected over mental Research) repeatedly climbed as high as chusetts, and the principal investigator for the the Pacific. Wofsy and his colleagues report that 13.7 kilometres and dipped down to a nail-biting US$4-million HIAPER Pole-to-Pole Observa- the first HIPPO flight in January 2009 found 150 metres above the ocean waves, all the while tions (HIPPO) project. “That’s like studying the levels of black carbon that average about five sampling more than 100 atmospheric constitu- ocean by studying what is on the surface of the times lower than predicted by an ensemble of ents, including greenhouse gases, aerosols and a ocean,” he says. In contrast, HIPPO can help 14 global aerosol models (J. P. Schwarz et al. suite of natural and industrial chemicals. modellers to test their ability to reproduce the Geophys. Res. Lett. doi:10.1029/2010GL044372, Now, early results from that flight and two atmosphere in three dimensions. in the press). The models underestimated how previous ones with the same aircraft, presented Among the surprises to come out of HIPPO much black carbon is being scrubbed out of on 9 August at a joint assembly of the American data are nitrous oxide concentrations that the air by precipitation, says lead author Joshua Geophysical Union and several Latin Ameri- consistently seem to increase with altitude. Schwarz, an atmospheric scientist with the can societies in Iguaçu Falls, Brazil, are yielding “Yet the models all show concentrations National Oceanic and Atmospheric Adminis- surprises in the distribution of trace gases and decreasing with altitude,” says Wofsy. The tration (NOAA) in Boulder. airborne pollution. implication, he adds, is that models are either Prabir Patra, a climate modeller at the Japa- Scientists generally have to rely on ground not properly accounting for the transport of nese Agency for Marine-Earth Science and measurements and then use mathematical nitrous oxide or they are missing a source of Technology in Yokohama who is already work- models to extrapolate upwards when they need the greenhouse gas. ing with Wofsy, says that the HIPPO data will to create a picture of the global atmosphere, The HIPPO team also found that black car- serve as a valuable new resource. By tracing says Steve Wofsy, an atmospheric researcher bon particles originating in Asia and beyond various constituents in the database back to their likely sources, he says, modellers should INCREDIBLE JOURNEY be able to glean information about circulation On its third of ﬁve planned ﬂights, the HIPPO team sampled the atmosphere from the Arctic to Antarctica. trends and then improve the way their models represent the atmosphere. Germany is currently developing a similar aircraft called the High Altitude and Long Range Research Aircraft, or HALO, which is expected to fly a parallel mission over the Atlantic Ocean in the coming years. For Wofsy, who gave up his seat to make room for NOAA’s laser-based black-carbon counter, the flights are bittersweet. Nevertheless, he has been up in the air for a few flights and has experienced the thrill of skimming over the waves at 150 metres, watching seals on ice floes as the jet whooshes past. “We want to see penguins,” he says, “but we haven’t seen any yet.” ■ Jeff Tollefson

E-mails spark ethics row The rapper Earl ‘DMX’ Simmons is not Berkeley’s ethical standards. In a letter to the known for his conciliatory lyrics. So Tyrone university, the company acknowledges an Hayes, a biologist and faculty member at the “ongoing difference of opinion” with Hayes University of California, Berkeley, drew from over atrazine, but states that the researcher Simmons’s lexicon earlier this year while has not responded to its attempts to interact writing a series of ‘trash-talking’ e-mails with him “on a scientific level”. to representatives of Syngenta, the world’s Hayes, who grew up in a tough South largest producer of the herbicide atrazine. Carolina community, says he reacted to Those e-mails, which Hayes says were intimidation from Syngenta in a language prompted by a heated encounter with a and style he felt appropriate. “Where I came Syngenta scientist, are included in an ethics from, people did a lot more,” he says. complaint that Syngenta filed with top This is not the first time Hayes has sent University of California officials on 19 July. offensive e-mails to employees of Syngenta. The Syngenta scientist concerned denies Earlier missives sparked a complaint in provoking Hayes. The action marks the latest February 2009. Berkeley responded with a in a series of bitter exchanges between the letter to the company saying that Hayes had Switzerland-based company agreed to refrain from using /AP and Hayes, whose work has offensive language in his ONE

J. M J. linked atrazine to adverse communications. effects on the environment Hayes, who says he was and on human health. not aware of the university’s Atrazine works by response, told Nature that inhibiting photosynthesis his latest e-mails were a in plants, but can disrupt reaction to a confrontation endocrine pathways in with a Syngenta scientist animals. Although banned in Springfield, Illinois. by the European Union, it Hayes was there to provide remains one of the most testimony on atrazine to the widely used herbicides in the state’s Environmental Health world, with tens of millions of Committee, and words were kilograms applied annually exchanged between the two to US farms and lawns. Biologist Tyrone Hayes. men shortly before Hayes In 2002, Hayes and his spoke. Illinois representative colleagues showed that developing male Karen Yarbrough (Democrat), a committee frogs exhibited female characteristics after member who was present, confirmed that exposure to atrazine1,2. In more recent an encounter occurred but did not know work3, he reports the same effect in an adult what was said. male frog of one species. The effects are Hayes’s research has figured prominently observed at exposure levels deemed safe by in legal actions against Syngenta and other the US Environmental Protection Agency manufacturers of atrazine. The chemical is (EPA). Hayes has also been a co-author on currently the subject of a class action lawsuit work that names atrazine as a potential launched against Syngenta by 15 water cause of reproductive cancers in humans4. providers in Illinois who are seeking at least Syngenta casts doubt on all these claims. The $350 million for facilities to purify drinking company’s website quotes EPA findings that water of atrazine, which they say poses a cite methodological problems with Hayes’s threat to human health. Syngenta says such research and suggest that there is insufficient claims are false. evidence to support his conclusions. A similar suit involving 17 water providers Mark Schlissel, dean of biological sciences across six Midwestern states is currently in at the University of California, Berkeley, progress in a federal court in East St Louis, says that the “decade long” dispute between Illinois. On its website, Syngenta says the Hayes and Syngenta has “moved increasingly cases are based on “questionable studies”. ■ into the personal arena”. Hayes met with Rex Dalton university officials on 6 August to discuss the 1. Hayes, T. B. et al. Proc. Natl Acad. Sci. USA 99, 5476–5480 latest incident, although Schlissel says that (2002). 2. Hayes, T. et al. Nature 419, 895–896 (2002). Hayes is not under investigation. 3. Hayes, T. B. et al. Proc. Natl Acad. Sci. USA 107, 4612–4617 Syngenta asserts that Hayes’s taunting (2010). and racy e-mails put him in violation of 4. Fan, W. et al. Environ. Health Perspect. 115, 720–727 (2007).

The bones of contention The skeleton may provide more than just structural support. Alla Katsnelson investigates the rise of bone as a metabolic regulator.

t first, Patricia Ducy was not particu- implications. “Gerard has really been larly fond of the mice she found her- one of the most creative thinkers in the self working with in 1994. One of the bone field for many years,” says Sun- Afirst strains genetically engineered to deep Khosla, an endocrinologist at the lack a bone-related protein — osteocalcin — Mayo Clinic in Rochester, Minnesota. Still, their cages reeked of urine. And when Ducy Karsenty is not without detractors, who say he dissected the mice, their bellies overflowed has overstated the case for a bone–metabolism with fat. “They are disgusting,” Ducy remem- connection. And because almost no one in the bers telling her supervisor. “They pee so much; field has worked directly on the idea, “the ques- I hate them!” tion people have is validation in Ducy was a postdoc at the University another lab”, says Khosla. of Texas MD Anderson Cancer Center in That is beginning to change. In Houston. Working with junior professor a study published last month1, bone Gerard Karsenty, she was using the mice to researchers at Johns Hopkins Uni- test the idea that osteocalcin helped regulate versity in Baltimore, Maryland, largely the way calcium phosphate is deposited onto stumbled on findings that affirm bone’s bone cells. Although the fat, smelly mice grew role in metabolism. It has taken a long time, slightly stronger and thicker bones than ordi- however, to put the pieces together. nary mice, osteocalcin didn’t seem to have any effect on mineralization. Its function remained The missing link a mystery. However, for Karsenty, who had Karsenty put his osteocalcin- originally trained in Paris as an endocrinolo- deficient mice aside for a time in gist, there was something familiar about the the late 1990s and plunged into unfortunate rodents’ symptoms: they seemed his new hypothesis using a protein to be diabetic. with a well-established metabolic Maybe, he thought, the osteocalcin- role: leptin. The gene for leptin — deficient mice indicated a direct link between which is secreted by fat cells and sup- the skeleton and energy metabolism, which goes presses appetite — had been cloned, PSAILA/SPL P. askew in diabetes. The connection made sense and by then, a mouse in which the leptin to Karsenty. The body’s maintenance of bone is gene was deleted (a knockout) was commer- Developing skeleton of a mouse embryo. Red a highly dynamic process orchestrated by two cially available. More intriguing for Karsenty, denotes bone and blue denotes cartilage. types of cell. Osteoblasts form and shape bone leptin had been found only in animals with tissue, and osteoclasts break it down. Karsenty bony skeletons, suggesting that it evolved with signalling directly to bone cells. The research- reasoned that this energy-intensive back-and- bone. Perhaps, he thought, the hormone also ers had to infuse it into the brain to return the forth process of remodelling might be coupled serves as a ‘molecular link’ connecting energy knockouts’ bones to normal2. by necessity to metabolism. Adult humans who metabolism to bone’s growth and decay. Karsenty’s lab spent the next decade fill- don’t eat — such as those with anorexia — lose When the leptin-knockout mice arrived in ing in details of the signalling pathway that bone mass, and obese people have beefy bones. late 1998, it was clear by a quick glance at an connected leptin, osteocalcin and bone (see Obesity even seems to protect X-ray that their bones were denser graphic), piecing the puzzle together largely some women from osteoporosis. “The best way than those of wild-type mice. By by knocking out different genes. Most homeo- The idea was radical. Most sci- to address then, Karsenty and his group static processes are regulated by the brain, and entists who study bone believed had moved to Baylor College of this one was no exception. Leptin exerts its the role of the skeleton was limited scrutiny is if Medicine, also in Houston, and in brake-pedal effect on bone growth by inhibit- to support and mineral exchange, your neighbour 1999 Karsenty brought in another ing synthesis of the neurotransmitter serotonin and the possibility that it could gets the same postdoc, endocrinologist Shu in the brainstem. This in turn dials down bone interact with other organ systems Takeda. Initially, Takeda says, he building — which is regulated by the sympa- was unheard of. But over the past results as you.” didn’t believe the idea would pan thetic nervous system, a neuronal pathway that 15 years, Karsenty and Ducy, who out, but the findings supported controls many unconscious bodily functions3. are now married and run collaborating labs at Karsenty’s vision. The knockout mice had a But the endocrine system works by feedback Columbia University in New York, have gradu- 40–50% increase in bone mass compared with loops. If a hormone released from fat cells ally shifted the bulk of their work towards test- wild-type mice, suggesting that leptin blocks can affect bone metabolism, the scientists ing the hypothesis, which could have clinical bone growth. But leptin didn’t seem to be reasoned, bone must in turn regulate energy

metabolism. And when they went back to their comes with the extra price of scrutiny, and the at Harvard School of Dental Medicine in Bos- osteocalcin knockouts they confirmed that the best way to address scrutiny is if your neigh- ton, Massachusetts. “Part of the problem is mice were glucose intolerant, insulin resistant, bour gets the same results as you.” that [Karsenty] has a tremendous tendency to and had fewer insulin-producing β-cells than The link between the skeleton and glucose overstate his findings. And that makes a lot of normal mice — they were diabetic. Karsenty metabolism has more than just theoretical people uncomfortable.” Karsenty says he isn’t concluded that an activated form of osteocalcin implications. The model has “great clinical surprised that his ideas garner criticism. “I will acts as an energy-regulating hormone4. relevance”, says Rosen. The most tantalizing say nothing to them — I will go back to the The idea still hadn’t gained much traction possibility in the short term, he lab and do more experiments to when Thomas Clemens, a bone biologist at says, is that osteocalcin could be “This has to be weaken the resistance.” Johns Hopkins University, came to study it. used as a way of promoting insulin Rosen says that the work is In 2006, one of his graduate students, Keertik secretion to treat diabetes. With proved right or pushing at the edges of knowledge Fulzele, became interested in the conflicting that in mind, Karsenty spun out a wrong quickly.” in valuable ways. “Even if he is medical literature on whether or not diabetic company called Escoublac in late 50% right, he has done a tremen- patients, who have poor insulin-mediated 2007. Head-quartered in Cambridge, Massa- dous service to the field.” Yet the other thorn control of glucose, are prone to osteoporosis. chusetts, it aims to capitalize on the connections in Karsenty’s theory, he says, is that women Fulzele suggested knocking out the insulin between osteocalcin and bone growth, insulin who take medication for osteoporosis to pre- receptor specifically in osteoblasts. “I did every- sensitivity and weight gain. vent bone resorption — bisphosphonates, for thing I possibly could to discourage him,” says example, or the newer antibody-based drug Clemens, who was convinced that they’d see an Thrown a bone Prolia (denosumab) that was approved in uninformative effect, but Fulzele insisted. Despite the therapeutic potential, several Europe and the United States in June — don’t Like the osteocalcin and leptin knockouts, pieces of the puzzle are still missing. “I guess seem to have significantly altered glucose lev- the insulin-receptor-knockout mice were fat if I wasn’t in it, I’d be sceptical too,” says Clem- els. This could mean that any control bone has and insulin-resistant, but unlike the other two ens. The Karsenty lab has yet to close the leptin on insulin and glucose is minor in humans, mutants, they had low bone mass. It was puz- loop and identify exactly how bone regulates says Rosen. Karsenty argues that such drugs zling, Fulzele recalls. “We spent about six or leptin levels. And so far, neither group has decrease osteocalcin levels and insulin secre- eight months arguing about it and doing all identified an osteocalcin receptor. Both are tion resulting in higher glucose levels, but that sorts of controls.” Just then, the Karsenty lab’s now examining how osteocalcin might prompt the change is subtle because they are not potent 2007 paper4 was published, and for both Cle- β-cells to stimulate insulin production. enough to whack the metabolism far out of bal- mens and Fulzele, the light dawned. Bone and The main outstanding issue, however, is how ance. Also, he notes, studies in the past few years metabolism were connected. closely these studies in mice reflect the physi- have reported a link between blood osteocalcin Coincidentally, Karsenty and his team had ology of humans. “My personal conviction levels and insulin sensitivity in humans6. created precisely the same knockout, and is that he is right overall, but that it may not Although supportive of a link, says Khosla, emerged with a similar story. Eating promotes be a key component of the human regulatory those observations have largely been made in insulin release, which activates bone remod- pathway,” says Roland Baron, a bone biologist overweight or obese individuals, who tend to be elling. Bone formation by osteoblasts insulin resistant and have low bone turnover produces more osteocalcin, and bone CLOSE TO THE BONE to begin with. “So is it cause and effect, or resorption (or break down) by osteoclasts Bone remodelling includes both building of bone (by cells just two things that happen together but are activates the hormone, releasing it from called osteoblasts) and destruction of bone (by osteoclasts). not causally related?” bone into the bloodstream. Osteocalcin This dynamic, energy-intensive process seems to interact Clinical data exist from studies on the in turn acts on pancreatic β-cells to boost with metabolism in multiple ways. osteoporosis drugs, but without a pro- insulin production and hence glucose spective study examining the relationship 1,5 Sympathetic uptake in the body. The two studies were nervous system between osteocalcin and obesity or diabetes published together in Cell this July. in humans, it will be impossible to know for Karsenty and Clemens suggest that Leptin inhibits sure. Karsenty is starting to collaborate with osteoblast formation Osteoblasts this bone pathway may facilitate glucose and osteocalcin increase levels clinical researchers to gather such data. absorption on a different time frame production of inactive “This has to be proved right or wrong from the well-established insulin pathway, osteocalcin quickly,” says Clemens. “There are lots of by which a jump in glucose levels spurs the Insulin promotes observations in the basic literature that are quick release of insulin. That direct path- bone remodelling not repeated because they are not going way would provide a rapid-response sys- to be changing clinical practice.” But the tem for glucose uptake, whereas the bone Bone destruction implications for current patients make pathway might oversee the longer-term activates osteocalcin this important, he says. “This won’t be left Leptin Bone equilibrium. Findings from the osteob- behind.” ■ last insulin-receptor knockouts1,5, “sort of Alla Katsnelson writes for Nature from complete the loop that insulin is really one New York. of the feedbacks on the bone-remodelling 1. Fulzele, K. et al. Cell 142, 309–319 (2010). system”, says Clifford Rosen, an osteoporo- Fat cells Insulin 2. Ducy, P. et al. Cell 100, 197–207 (2000). sis researcher at the Maine Medical Center Osteocalcin stimulates 3. Yadav, V. K. et al. Cell 138, 976–989 (2009). 4. Lee, N. K. et al. Cell 130, 456–469 (2007). Research Institute in Scarborough. β-cells to produce insulin 5. Ferron, M. et al. Cell 142, 296–308 (2010). “I think it was very important that Tom Pancreas 6. Strapazzon, G., De Toni, L. & Foresta, C. Osteoporos. came along,” says Karsenty. “Novelty Int. doi:10.1007/s00198-010-1322-2 (2010).

CROSSING The baRRIeR Researchers have rallied round a promising molecule for rescuing dying nerves. But getting it into the brain remains a daunting challenge, finds Brian Vastag. e ine blocks from the beach in Santa Mon- a smattering of evidence from initial trials in the feedback he has heard from prospective R ica, California, a small biotechnology humans show that GDNF can halt the damage investors. Pardridge — like other researchers a/Natu

company occupies one bay of a single- that follows stroke, interrupt drug addiction, — soldiers on, nevertheless, knowing that the R Nstorey commercial block, about the and slow or even reverse the neuronal death pay-off could be huge. “The therapeutic poten- width of a three-car garage. Founded by William march that incapacitates patients with Parkin- tial of GDNF is just enormous,” he says. R. Macu R. Pardridge, an endocrinologist at the University son’s or Huntington’s disease1. of California, Los Angeles (UCLA), ArmaGen “The excitement behind growth factors such A hit-or-miss history Technologies employs just five people: call it as GDNF is that not only could they be protec- GDNF was a star from the start. Discovered in garage-band biotech. From a small refrigera- tive, but there’s the possibility for regenerating or 1991 by researchers at Synergen, a biotechnology tor in the cramped space, Pardridge pulls out a rejuvenating some of the sick cells in the brain,” company based in Boulder, Colorado, GDNF black plastic tray. Within are four dozen small says Todd Sherer, vice-president of research pro- dramatically revived dishes of dying neurons. bottles labelled ‘AGT-190’, a drug that Pardridge grammes at the Michael J. Fox Foundation for When Synergen tested GDNF in monkeys with hopes will revolutionize treatment for several Parkinson’s Research, based in New York, which an induced form of Parkinson’s disease, the debilita ting brain diseases. has awarded about US$20 million in grants for treated monkeys trembled and spasmed much Starting with a radical idea for sneaking growth-factor research. less than their untreated cage mates. therapeutic proteins into the brain, Pardridge But a maddening hurdle remains: delivery. A year after announcing their results, in launched ArmaGen in 2004. This year, the com- Early surgical trials in which GDNF was deliv- 1994, Synergen was snapped up by a larger pany is close to achieving a milestone: a green ered through catheters to the brains of patients biotechnology firm, Amgen, for about $240 mil- light from the US Food and Drug Administra- with Parkinson’s disease failed to spread the lion. Kevin Sharer, Amgen’s chief operating tion (FDA) to carry out human safety tests of its protein to a sufficient proportion of damaged officer at the time, said the company made the first product. regions to do much good. Advancing on these decision to buy Synergen after seeing the before- AGT-190, says Pardridge, acts as a Trojan techniques, companies are moving forward with and-after footage of treated monkeys. As Sharer Horse. It sneaks across the barrier that sepa- more trials looking to circumvent the blood– told The New York Times: “We looked at that rates blood and brain tissue and delivers its brain barrier, but Pardridge is convinced that movie and said, ‘Buy this company’. Literally.” contents — a growth factor that can protect and his technology can reliably deliver GDNF to the Amgen quickly moved GDNF into human repair neurons. A long line of researchers has entire brain without the need for surgery. Still, trials. Between 1996 and 1999, 38 patients with heard the siren song of this naturally occurring his company is running out of funds, and there advanced Parkinson’s disease underwent sur- brain protein, called glial-cell-derived neuro- is no hint of investment forthcoming. “Nobody gery to place catheters in their brains. Hooked trophic factor (GDNF) — and for good reason. has succeeded at crossing the blood–brain bar- to a pump implanted in the abdomen, the cath- A thick stack of reports on animal studies and rier before, so why should we?” he asks, echoing eter delivered GDNF into the fluid-filled spaces

improvements in standard scores of movement unacknowledged — show-stopper in drug and motor skills. It was a “very successful trial”, development. Almost none of the hundreds says Erich Mohr, chief executive of MedGenesis of potential drugs for treating brain disorders Therapeutix, based in Victoria, Canada, which can penetrate the tight mesh of endothelial earlier this year acquired the rights to GDNF cells lining the blood vessels in the brain. This from Amgen. barrier protects the brain and keeps most large After that trial, Amgen quickly launched a molecules out of the cerebrospinal fluid that larger trial to test GDNF against a placebo. But bathes neurons. Still, the tantalizing hints of in this case, patients given GDNF fared no bet- effectiveness seen in some trials convinced ter than patients who received the researchers that GDNF was worth surgery without GDNF. “That trial “GDNF has pursuing. Although Amgen had little was basically designed to fail,” says interest in GDNF between 2004 and Clive Svendsen, a neuroscientist this lovely 2010, researchers continued to seek who was a consultant to Amgen on regenerative other strategies for getting the protein the study. Amgen chose a catheter capacity and into the brain. that was thicker than Gill’s, drip- Now, several biotechnology com- ping GDNF into the brain rather rejuvenating panies are launching a new round of than delivering it under pressure. ability.” surgical trials. One project will test Consequently, the GDNF solution next-generation catheters — similar to simply refluxed up the outside of the catheter, Gill’s design — for delivering GDNF protein. A says Svendsen, now director of the Regenera- second will, as early as next year, implant neural tive Medicine Institute at Cedars-Sinai Medical stem cells that have been programmed to pro- Center in Los Angeles. Amgen declined to com- duce GDNF into the spinal cords of patients with ment about the trial for this story. In 2004, amid the degenerative disorder amyotrophic lateral ArmaGen’s AGT-190 — a a huge spate of negative publicity, it mothballed sclerosis. And one study, already under way, is therapeutic protein fused to its GDNF project. delivering viruses that carry the gene encoding an antibody — might cross But shortly after, Gill and his colleagues made a growth factor that is closely related to GDNF directly from the blood into a tantalizing discovery. One of the patients in through a brain-implanted catheter. brain tissue in humans. Gill’s trial had died of a heart attack, and when Ceregene in San Diego, California, is the com- the team carefully sliced the man’s brain the pany taking this gene-therapy approach. Jeffrey researchers saw something amazing. Neuro- Ostrove co-founded the biotechnology com- between the main lobes of the brain in the hope nal fibres had sprouted in the patient’s puta- pany in 2001 and chose a growth factor called that GDNF would migrate deeper into the brain, men on the right side, where the catheter had neurturin, which acts much like GDNF, as a to the structures most affected by Parkinson’s been placed3. The man had described a huge potential treatment for Parkinson’s disease. The disease. This wishful strategy failed. Instead improvement in his quality of life over the 43 team at Ceregene packages the gene encoding of improvement, patients experienced nau- months for which he received the drug. To those neurturin into a gutted virus, and then infuses sea, delusions and chest pains, the exact cause in the field, the message was clear: his brain had the virus into the patient’s putamen under pres- of which has still not been uncovered. Amgen been healing. sure. There, the virus delivers the gene to brain halted the trial early. cells, which in turn should pump out the growth After shelving further trials, the com- New surgical trials factor, perhaps indefinitely, says Ostrove. pany made the protein available to interested GDNF isn’t the only drug that has run up But results released in 2008, from the compa- researchers. Steven Gill, a neurosurgeon at against delivery troubles. The blood–brain ny’s trial in 58 patients with advanced Parkinson’s Frenchay Hospital in Bristol, UK, partnered barrier has been an enormous — often disease, were disappointing. A year after surgery, with researchers at the Univer- patients who had received the altered virus fared e R sity of Kentucky in Lexington no better than patients who received sham sur-

and designed a new catheter to gery and no virus. Both groups improved about a/Natu push the drug directly into brain equally on standardized symptom scores. (For R regions affected by the disease. unknown reasons, sham surgery for Parkin- Delivered under pressure, the son’s disease produces a strong placebo effect.) Macu R. GDNF solution continuously dif- Despite raising some $70 million, Ceregene laid fused from the tip of the catheter off 30 of its 50 staff, and Ostrove pondered shut- into the putamen, a thumb-sized tering the programme. structure at the base of the fore- Then, donated brains from two trial patients brain that degenerates in individ- who had died suggested a path forwards. In uals with Parkinson’s disease. the trial, surgeons had infused the virus in In 2001, Gill inserted his eight locations across each patient’s putamen. catheters into the brains of five Yet Ostrove says that just 15% of the putamen patients. Although designed as expressed the neurturin gene — the delivery a safety trial, the changes were problem again. Persuaded that neurturin might swift and positive2. Over 12 Ruben Boado of ArmaGen collects a sample from a bioreactor still work if its delivery could be improved, months, all five patients showed used to grow therapeutic proteins. this June the Michael J. Fox Foundation gave

Ceregene $2.5 million to support another trial, catheter-based delivery. The zone immediately (NIH) grant funded the drug’s development, in which surgeons will infuse virus into both the surrounding the catheter is blasted with the and the company has spent a total of $4 mil- putamen and the substantia nigra, a structure drug, whereas tissue more than seven or eight lion on the project, including $1.5 million on in the midbrain. The company is also increas- millimetres away receives almost none. “Unless the monkey study. Large biotechnology coming the viral dose fourfold. Twenty-six patients the region of the brain you’re trying to reach panies, big drug makers and venture capitalists will undergo the revised procedure over the next is the size of a pinhead, a transcranial delivery alike have all rebuffed Pardridge’s entreaties for few months, and the same number will receive system isn’t going to work,” he says. investment. He has a stack of grant applications sham surgery. The scientists planning the catheter-based that he has submitted to the NIH; it is nearly GDNF trials disagree. Ostrove thinks that Cere- half a metre high. Most have been rejected. Back-door protein gene’s new surgical protocol will deliver GDNF Venture capitalists, too, “are very risk averse, ArmaGen is taking a different approach to to 25–30% of the putamen — and he predicts right now”, says Casey Lynch, a biotechnology

breaching the blood–brain analyst and president of the e barrier. Rather than going Neurotechnology Develop- R around it, with all the risks ment Foundation, based in San a/Natu that brain surgery entails, Francisco, California. It is just R Pardridge wants to enter one of several impediments through a biological back for Pardridge, including the Macu R. door. Pardridge has been expense of producing biologi- studying the barrier since cal drugs, as opposed to small 1970. After about a decade, he molecule drugs, which can be discovered a potential way in, synthesized chemically. an insulin receptor. Pardridge So Pardridge is stuck. He showed that insulin receptors has developed a new approach in the capillaries that feed the to delivering a promising brain brain are transporters — grab- drug. He has compiled thou- bing molecules of insulin and sands of pages of data required pulling them into the brain by the FDA. And by the end tissue. of this year, he may know Over the next 15 years, whether AGT-190 is safe to while at UCLA, Pardridge Garage-band biotech: ArmaGen’s commercial space in Santa Monica is close quarters. use in humans. But that might developed a monoclonal be the end of the line. A larger antibody that latches onto part of the brain’s that this will be sufficient coverage to reverse study of the drug’s efficacy would cost at least insulin receptor without interfering with the progression of Parkinson’s disease. Mohr $15 million, and Pardridge has no idea where insulin binding. The receptor pulls both the is confident that MedGenesis Therapeutix’s to get this money. insulin and the antibody through the blood– next-generation catheter will push GDNF into In many ways, GDNF is stuck, too. It is brain barrier, says Pardridge. He published this a larger proportion of the structure than earlier unclear whether surgical procedures, even if ‘Trojan Horse’ antibody design4 in 1995, and catheters did. effective, will be practical for a condition such then set to work engineering it as a vehicle for When asked about ArmaGen’s strategy, as Parkinson’s disease, which affects millions therapeutic proteins. Ostrove, Mohr and other scientists involved in of people worldwide, not to mention the many Pardridge and Ruben Boado, a molecular the surgical trials argue that the Trojan Horse other indications for which people have been biologist also at UCLA and ArmaGen, stitched will be felled ultimately by the very trait that eyeing it. The costs and dangers associated with the gene encoding the antibody together with Pardridge touts most: it hits the entire brain. putting a hole in someone’s head or spine make the gene encoding GDNF and, after several “You don’t want GDNF to go all over,” says it prohibitive for all but the most severe cases, painstaking years, worked out how to scale up Svendsen, pointing out that high doses of those unresponsive to other treatments. production of the hybrid protein, AGT-190. GDNF can cause neurons to make connections And yet, the development of GDNF as a treat- Animal studies funded by ArmaGen show that they shouldn’t. It could result in the same ment will probably continue, because its brain- that the Trojan Horse approach works: it gets side effects as seen in Amgen’s first human trial, healing properties are too tantalizing to pass GDNF into the brain5. A large meeting poster which essentially bathed the brain in GDNF. up. “It has this lovely regenerative capacity and hanging in Pardridge’s office displays the results: Pardridge argues that the doses of GDNF rejuvenating ability,” says Svendsen. “But how sections of a rhesus monkey brain stained a lurid delivered by AGT-190 will be much lower than it’s going to work across these different diseases, blue. (Rhesus monkeys, unlike other monkeys or those in the Amgen trials. But whether the Tro- which all have different mechanisms, I don’t mice, have a blood–brain barrier very similar to jan Horse approach is safe remains an open ques- think we’ll find out until after clinical trials.” ■ that of humans.) When the researchers injected tion, because no one has received the drug. Brian Vastag is a freelance reporter in the antibody into the animals’ veins and then That could change as early as this October, Washington DC. looked for it in the brain, they found it every- when 12 healthy volunteers in Kansas are slated 1. airaksinen, M. S. & Saarma, M. Nature Rev. Neurosci. 3, where. About 2% of the AGT-190 injected into to receive three doses of AGT-190. Pardridge 383–394 (2002). veins arrives in the brain. That’s about the same doesn’t know what will happen after that. Even 2. Gill, S. S. et al. Nature Med. 9, 589–595 (2003). as for antidepressants and other traditional, if all goes well, his company is almost out of 3. Love, S. et al. Nature Med. 11, 703–704 (2005). small-molecule, brain drugs that can cross the money. 4. Pardridge, W. M., Kang, Y. S., Buciak, J. L. & Yang, J. Pharm. Res. 12, 807–816 (1995). blood–brain barrier unassisted. ArmaGen is $1 million in the red on AGT- 5. Pardridge, W. M. & Boado, R. J. Pharm. Res. 26, Pardridge says there is a fatal flaw in surgical, 190. A $3-million National Institutes of Health 2227–2236(2009).

reviewed by the European Strategy Forum on Research Infrastructures (ESFRI). It has Leaders wanted endorsed 44 such projects, ranging from a €13-million (US$17-million) project called There’s room at the top for more old-fashioned charisma, IAGOS, which would use commercial jets says Colin Macilwain. to gather atmospheric data, to the €1.3-billion European Spallation Source. Some of the Burt Richter, the Nobel laureate and physicist projects involve large, single-site facilities; oth- who used to run the Stanford Linear Accelera- ers would be distributed across several coun- tor Center in California, once had to entertain tries. All involve big sums of money, and will Dana Rohrabacher, a prickly Republican con- require considerable management acumen to gressman inexplicably charged with oversee- bring to fruition. ing his lab. Richter took Rohrabacher home Conscious of the need to identify and train to show off his extensive pistol collection. future leaders, the European Commission is “I wanted him to know that not all physicists backing the Realizing and Managing Inter- were wimps,” Richter later told me. national Research Infrastructures (RAMIRI) Richter was of the generation that learned programme. RAMIRI organizes intensive work- directly from the unfettered clique that built the outside world. That’s on top of their routine shops at which veteran managers, senior civil the atomic bomb. The veterans of the Manhat- tasks of setting budgets, choosing senior staff servants and aspirant leaders exchange knowl- tan Project had taken physics out of the univer- and setting out strategy. edge on project management. sity lab and into the big world of politics and There is a shortage of men or women who can The first set of RAMIRI meetings took quid pro quos. They bequeathed the culture combine the charisma of ‘old-school’ scientific place in London, Hamburg and Grenoble last that scientists could do it all for themselves. leaders with the bureaucratic skills demanded summer, under the tutelage of John Wood, an An axiom of this culture is that major today. Developing such individuals is a tall engineer at Imperial College London and an projects should be led by top scientists, with order; but efforts to do so must be encouraged. experienced research administrator. Another little input from engineers or, heaven-forbid, Unless these efforts succeed, it is hard to see set of workshops is planned for next year, led by managers from business or industry. This is how science will build future facilities that are Carlo Rizzuto, president of the ELETTRA syn- perhaps unsurprising, given the low esteem in truly remarkable in scope and ambition. chrotron at Trieste, Italy, and chair of ESFRI. which most scientists hold non-scientific train- The University of Ljubljana is also starting a ing. It is nonetheless an aberration from what Bureaucratic behemoths master’s course next year in research adminis- happens in other spheres of human activity, The leadership question has been highlighted tration. “We used to select scientists as leaders, from construction to health care. this year by the shambles at ITER, the inter- on their ability to do science, and then leave It would be daft to suggest that there was a national fusion project. ITER last month them to make their own mistakes,” says Wil- ‘golden age’ of scientific leadership in which replaced its director, former diplomat and liam Barletta, director of the US Particle Accel- characters such as Richter sprang forth to occupy nuclear engineer Kaname Ikeda, just half- erator School, at Fermilab in Batavia, Illinois, every major job. And science is still producing way through his five-year term in office (see who will help to teach the Ljubljana course. exceptional leaders who work their way into Nature 465, 143; 2010). “People realize that projects are more compli- powerful positions: Steven Chu, the US energy ITER represents a particularly daunting lead- cated now, and that they need real training.” secretary, and Leszek Borysiewicz, who moves ership challenge. Partly as a result of its longevity A key challenge for such efforts is to train on next month from the UK Medical Research (ITER was conceived in 1984, and a brick hasn’t talented scientists in the mundane aspects of Council to run the University been laid yet), the visionaries project management — such as employment of Cambridge, spring to mind. “Major facilities and who might have got it built — law — without scaring them off or ironing out But today laboratory and laboratories need truly such as Marshall Rosenbluth, the personality traits that make great leaders. facility heads are often selected inspirational leaders.” a plasma physicist and protégé It is perhaps no longer possible to drive a less for their intellectual bril- of hydrogen-bomb developer great scientific project chiefly by force of per- liance than for being ‘good committee men or Edward Teller — are no longer with us. Placat- sonality. There is too much political account- women’ who can cope with the bureaucracy now ing its multiple international partners presents ability, too many rules, regulations, committees inherent to the task. The result is often mediocre special difficulties, as does operating within the and milestones. Old bears such as Richter and management by individuals who can get by, but straightjacket lovingly provided by the CEA, the Carlo Rubbia, the formidable but temperamen- can’t inspire. French atomic energy commission, which owns tal director of CERN from 1989 to 1993, might The problem is most acute at major sci- the ITER site at Cadarache. have struggled to cope with the constraints of entific facilities and at the agencies that run But these are merely acute manifestations today’s management environment. them, such as NASA and the US Department of the political, technical and administrative So it is good to see steps being taken to of Energy. Grant-giving agencies — even great challenges that face the leader of any major encourage individuals with the right stuff to ones, such as the US National Science Foun- scientific infrastructure project. These days, rise up into management positions, rather than dation — can roll along with leadership that almost every discipline of science includes at just sticking to their science, and recoiling from is merely competent. But major facilities and least one such behemoth — commonly, a data the hassle that leadership entails. ■ laboratories — especially new ones, most management project to enable researchers to Colin Macilwain is based in the United especially in a recession — need truly inspi- share the mountains of digital information that Kingdom. rational leaders to enthuse staff, charm civil they now generate. e-mail: [email protected] servants and politicians, and provide a face to In Europe, plans for such projects have been See go.nature.com/ILx8PC for more columns.

Nature’s special issue ‘Can Science Feed the World?’ (29 July 2010; www.nature.com/food) prompted a considerable response from readers.

Mitigate food loss to production — but have the lowest Reducing soil disturbance collecting information will be food security. is crucial to sustainable crop required to build a global system. feed more people Trevor Nicholls CABI, Wallingford, production in most environments. There is a growing disconnect right now Oxfordshire, OX10 8DE, UK The principles of no-tillage, between ambitious international e-mail: [email protected] permanent soil cover and agendas set by Western scientists The proposals for agricultural rotation need less energy and and the realities on the ground in monitoring systems by Jeffrey pesticide input than tillage- developing countries. To achieve Sachs and colleagues (Nature based alternatives, and are the policy and governance targets that 466, 558–560; 2010) will take Culturing practices foundations of conservation are associated with sustainable five years to mature and deliver can make roots more agriculture, which inhibits wind agriculture, conservation and value. We can make an immediate and water erosion. human welfare, monitoring should difference to agricultural advisory robust too Large grain harvesters flatten be participatory and decentralized services and farmers by providing The growth and performance of the soil with an axle load of 18–20 to take advantage of the wealth access to pragmatic advice about root systems can be enhanced by tonnes each — about twice that of experience and information strategies that are appropriate to altered management practices, allowed for heavy trucks. Driving embodied in traditional farming their local environment. not just by plant breeding (Nature these loads over soil destroys knowledge and practices and in At present, for example, there 466, 552–553; 2010). porosity and productivity, so decentralized governance regimes. are no reliable means of tracking Researchers in China, India, tillage seems the only option. Kamaljit S. Bawa University plant pests and diseases globally. Thailand and Japan have shown Yet compaction can be avoided of Massachusetts, Boston, So we lose 40% of what we grow how the structure and function by no-till cropping of permanent Massachusetts 02125, USA; to pest and disease damage to of rice roots, for instance, can be beds in controlled-traffic farming Ashoka Trust for Research in Ecology crops in the field, in transit and improved by modifying factors systems, in which the wheels of and the Environment, Bangalore, India during storage. This threat is such as seedling age, plant heavy machinery are restricted to e-mail: [email protected] set to increase as trade flows spacing, water management, hard permanent traffic lanes (see and climate change accelerate active soil aeration and sources of www.ctfsolutions.com.au). the movement of plant pests fertilization. Plants with better root Jeff Tullberg University of Queensland, and pathogens. By losing less, systems become more efficient Queensland 4069, Australia Track social and we can feed more people right at using water and fixing carbon e-mail: [email protected] economic impacts of now — without extra land, water, dioxide and are more protected Competing financial interests: food production energy or chemicals, or creating against drought and storm damage declared (see http://dx.doi. new crop varieties. Using data (see, for example, A. K. Thakur et al. org/10.1038/466920c for details). Food security depends on efficient and information that already Exp. Agr. 46, 77–98; 2010). distribution and affordability as exist, a knowledge bank to reduce Changing crop physiology well as on sustainable production losses in all major food and cash and morphology through altered (Nature 466, 558–560; 2010). crops could be up and running culturing practices can help Monitoring systems We are already seeing the within three years. The UK-based agriculture to meet the challenges outdated and acquisition of African farmland CABI (Centre for Agricultural of population growth and climate protectionist by Asian countries to feed their Bioscience International) already change in the decades ahead. own populations, and we can has a prototype that could start Norman Uphoff Cornell International None of Jeffrey Sachs and expect more dislocation globally delivering useful data on a few key Institute for Food, Agriculture and colleagues’ cited monitoring between sites of food production crops within a year. Development, New York 14853, USA systems (Nature 466, and consumption, exacerbated More funding for agricultural e-mail: [email protected] 558–560; 2010) meets the by growing urbanization. This will research is essential, but the innovation requirements for the aggravate distribution problems greatest challenge is to transfer comprehensive monitoring of and adversely affect affordability the knowledge we already have, agro-ecological zones worldwide. by increasing financial barriers applying known remedies and Reduce soil damage Information technologies now (transport costs) and economic simple technologies that farmers for more sustainable offer an array of approaches impediments (market distortions). can afford and readily adapt. crop production and tools for rapid collation, As well as ecological indicators Much of the hand-wringing by organization and dissemination of sustainable food production, developed countries about food Your series on solving world of interactive data, but most we need metrics to track socio- production skirts around this hunger in the future (Nature 466, monitoring systems are outdated economic factors affecting central problem: that however 531 and 546–561; 2010) focuses and protectionist. They are affordability and distribution gaps. much we know, we still do mainly on biological measures. often developed and maintained Don Gunasekera, John Finnigan Centre not get the information to the Better management of soil and in isolation, which severely for Complex Systems Science, CSIRO smallholders and communities cropping systems could improve reduces the utility of the data Marine and Atmospheric Research, in developing countries that productivity right away, with and information gathered. New Canberra ACT 2601, Australia are the backbone of local food minimal environmental threat. platforms and different ways of e-mail: [email protected]

920 © 2010 Macmillan Publishers Limited. All rights reserved Vol 466|19 August 2010 BOOKS & ARTS Long view of the Human Genome Project A bold attempt to tell the complicated story behind the human DNA sequence highlights that social change is needed before personalized medicine can take off, finds Jan Witkowski.

Drawing the Map of Life: Inside the Human tty E Genome Project by Victor K. McElheny Basic Books: 2010. 384 pp. $28, £16.99 S. JAFFE/AFP/G S. In 1985, Robert Sinsheimer, then chancellor of the University of California, Santa Cruz, convened a workshop to discuss sequencing the human genome. It was an audacious proposal: the longest genome that had been sequenced at the time was that of the Epstein- Barr virus, at 172,282 base pairs compared with 3 billion in human DNA. Sinsheimer’s initiative failed. Yet the idea gained momentum when, in 1988, James Watson was appointed associate director of the Office of Genome Research, part of the US National Institutes of Health (NIH). Watson declared 1990 the official start of the publicly funded NIH Human Genome Project (HGP). In 1998, Craig Venter and his company Celera Genomics, then in Rockville, Maryland, joined the race. Ten years ago in June, both projects announced a finish-line draw from President Bill Clinton’s White House. Febru- ary 2011 will mark a decade since the draft sequences were published. Genome-project pioneers: (left to right) Eric Lander, Robert Waterston, James Watson and Francis Collins. In Drawing the Map of Life, science jour- nalist and author Victor McElheny relates McElheny traces the various stages of the In 2000, HGP and Celera jointly announced the story of the HGP, from its methods to the HGP and the power struggles it engendered. the draft human genome sequences to people involved. He describes the project’s The project had two phases under different great fanfare. But acrimony and infighting tortuous path to success, and asks whether directors. Watson led the NIH effort from continued over data release until the its medical impacts live up to expectations. 1988 until his resignation in 1992, after ques- sequences were published in 2001. Celera Weaving together so many threads is a formi- tions were raised about his holding of stock in published in Science but sought special con- dable task. McElheny offers an entertaining biotechnology companies. McElheny describes ditions of access to its data for commercial narrative, but his book stops short of being a the resignation, but not the finding by the scientists. The HGP opposed any such restric- comprehensive history. US Department of Health and tions and instead published its sequence in He opens in the 1980s, when “We are only at Human Services that Watson had Nature, depositing its data in the open-access many of the technologies cen- the beginning of done nothing unethical. Francis GenBank database. tral to gene sequencing were Collins then took over direc- The completion of the HGP in 2003 was a developed: the polymerase chain interpreting the torship of the HGP, and saw it great triumph. With draft sequences finished reaction that multiplies DNA sequence.” through to the completion of the two years earlier than planned, it is transform- fragments for analysis; the use sequence in 2003. ing research. The pace of sequencing has since of restriction enzymes to sever DNA strands The book also describes the fierce competi- rocketed thanks to techniques that the project at particular sites; and the use of restriction tion between various commercial and academic hastened. Many complex organisms have now fragment length polymorphisms as mark- laboratories to isolate and sequence medically been sequenced, and their genomes will be ers in early searches for the genes involved in relevant genes. The most famous rival to the mined for years to come. inherited disorders. The mapping of the genes NIH project is Venter, a pioneer of large-scale Yet the era of genomic medicine has not yet underlying Huntington’s disease, Duchenne sequencing who left the NIH to set up The come to pass. McElheny is right to ask when muscular dystrophy and cystic fibrosis in the Institute for Genome Research to sequence we will see public-health returns on the huge late 1980s had a huge impact on clinical genet- small genomes. In 1998 Venter announced the investment in the HGP. He argues that social ics: suddenly the arrangement of the gene itself, formation of a new company, Celera Genom- change will be needed before genomic infor- rather than secondary markers, could be used ics, that would target the human genome. The mation can be integrated into current medical to reveal mutations. academics rallied to meet Celera’s challenge. practice and interpreted by the public. He asks,

for example, how physicians will use genetic what variants mean for the individual. James Shreeve has written a detailed study of data for diagnosis and treatment, and whether Drawing the Map of Life is one of many Venter’s contributions. individuals will welcome or fear knowledge of books that have been written about the HGP. All of these books are valuable; what is what their genomes hold for the future. The volume does not add much to earlier now needed is a scholarly history of the HGP. Such social change will follow, I believe, descriptions of the project’s genesis, such as Drawing the Map of Life is not that book, but it when useful applications of genomic infor- Genome by Jerry Bishop and Michael Wald- offers an enjoyable account of the project from mation become available. They might tell holz (Simon and Schuster, 1990) and The Gene origin to conclusion and beyond. ■ us how to alter our lifestyles to improve our Wars by Robert Cook-Deegan (W. W. Norton, Jan Witkowski is executive director of the health, or distinguish which drugs will be of 1994). In Cracking the Genome (Free Press, Banbury Center and a professor in the Watson benefit or have serious side effects, or may 2001), Kevin Davies brought us up to the com- School of Biological Sciences, Cold Spring Harbor guide the development of new drugs. But this pletion of the draft sequences. More recently, Laboratory, New York 11724, USA. He is co-author will take time. We are only at the beginning of protagonists John Sulston and Venter have of Recombinant DNA: Genes and Genomes. interpreting the sequence and understanding told their contrasting personal stories, while e-mail: [email protected] In Retrospect: Science — The Endless Frontier Vannevar Bush’s pivotal report that marked the beginning of modern science policy catapulted the phrase ‘basic research’ into popular usage, explains Roger Pielke Jr.

Science — The Endless Frontier. tty E

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Science — The Endless Frontier was published W H. 65 years ago last month. Commissioned by President Franklin D. Roosevelt and prepared by electrical engineer Vannevar Bush, who directed US government research during the Second World War, the document distilled the lessons of wartime into proposals for subsequent federal support of science. Although its bold recommendations were only partly implemented, the document is ripe for reappraisal today: it marked the beginning of modern science policy. Bush’s report called for a centralized approach to government-sponsored science, Engineer Vannevar Bush’s proposals led to the creation of the National Science Foundation in 1950. largely shielded from political accountability. The creation of the National Science Foun- of the atomic bomb, radar and penicillin meant along similar lines were made to no avail in dation in 1950, a small agency with a limited that Bush’s declaration that “scientific progress 1924 by the UK National Union of Scientific mandate, was far from the sweeping reform is essential” to public welfare found a recep- Workers (NUSW) and in 1929 by US agricul- set out in the 30-page report and its appen- tive audience. Bush also adopted innovative ture secretary Arthur Hyde. The poor response dices. However, its publication ushered in a language that capitalized on this new-found might have been due to the confused messages new era in which science was viewed as vital government credulity. offered to protect the integrity of pure research. for progress towards national goals in health, In particular, he broadened the meaning of In a 1921 essay, for example, the NUSW presi- defence and the economy. Government fund- the phrase ‘basic research’. In using it to refer dent declared that scientific research has “no ing for research and development consequently simultaneously to the demands of policy- industrial bearing at all” but later stated that it increased by more than a factor of ten from the makers for practical innovation and to the inter- is “the foundation of progress in industry”. Not 1940s to the 1960s. ests of scientists in curiosity-driven enquiry, he surprisingly, most policy-makers shrugged. The influence of Science —The Endless Fron- satisfied both sectors. Some political leaders did champion govern- tier stems largely from its timing, coming at Before the report, pleas by scientists to ment support for basic research before 1945. the tail end of a war in which science-based expand government support for research had Prior to Hyde’s appointment, US agriculture technology had been crucial. The development met with only limited success. Prominent calls secretary Henry C. Wallace had argued in

922 © 2010 Macmillan Publishers Limited. All rights reserved SOuRcE: R. PIELkE JR NATURE ence, had basic been to the production of a but hardly of real impact on a practical exist that had been regarded by many as interesting the phrase made it easy to convey that “work pleasing the politicians. Bush later recalled how scientists — and could meet national needs, be carried out for curiosity’s sake — satisfying scientists. By contrast, basic research could ofview science in terms of benefits only to mental’ research had long presented a narrow politicians. The concepts of ‘pure’ and ‘funda pragmatic compromise between scientists and ‘basic’ research descriptor helped to secure a language of science policy changed too. from the agriculturists to the physicists, and the completed policy its switchshipscience in When Wallace’s political fortunes fell, leader detonated — it was well positioned to influence. weeks before the Hiroshima atomic bomb was when the report was released — less than two the science and policy camps. This meant that credibility and good connections within both Scientific Research and Development, Bush had The Endless Frontier bour Vice-President Wallace to draft liaison between Roosevelt and Bush. During the war, the younger Wallace served as then as Roosevelt’s vice-president (1941–45). ture secretary under Roosevelt (1933–40) and Wallace, picked up the baton, first as agricul to see his vision realized, but his son, Henry A. catch up in the longer term. Wallace did not live reasoned presciently that consumption would prices and caused hardship for farmers. But he asurfeitefficient; of production depressed US agriculture was suffering from being too for investment was counter-intuitive because tural productivity. At the time, Wallace’s call research”more “basic agricul enhanceto of phrase) the that agency should the fund the early 1920s (one of the first narrow uses The fluid meaning of “basic research” galvanized science-policy discussions in the mid-twentieth century. Frontier bomb that had ended a war.” The publication of inscrutability,inherentWithits Bush’s Bush was selected by his friend and neigh Average usage* 0.0 2.0 0.5 3.0 2.5 3.5 USAGE OFTHEPHRASE“BASICRESEARCH” 1.0 1.5 entrenched the concept of government 1920 | Vol 466 | Nature Science The New York Times 19 August 2010 1930 . As director of the Office of Science — The Endless 1940 Report 1950 Science — 1960 ------*Running 5-year meanasaproportion of1920–2009 annualaverage.

© has made an ironic return. And science policy up to fill the gap; even ‘fundamental research’ such as ‘transformative research’, have sprung of the phrase “basic research”’). Other terms, mentions in declined since the early 1990s, as indicated by funds. Consequently, use of the phrase has today’s competitive environment for public inareinsufficient benefit descriptionsof research no longer seems to fit — nebulous efits to society. Theconcept fuzzy of basic its focus towards conferring measurable ben research”’). tions in 1957 (see ‘Usage of the phrase “basic 4 YorkNew Times is demonstrated by usage of the phrase in science and society discussions were reframed Benoît Godin in 2000. The speed with which basic research,” wrote science-policy scholar are what gave stability to the fuzzy concept of reports cemented it. “Institutions and statistics Science Foundation and countless other policy National the of up setting The discourse. policy in research scientific patronageof e-mail: [email protected] Colorado, Boulder, Colorado 80309, USA. and Technology Policy Research, University of Roger Pielke Jr up. catch to needs both transformed. Our framework for discussing was written, research and havepolicy been decades since unsettled nature of science policy. In the six relevant today both reflects and reinforces the The so-far futile search for a language that is demand relevance; scientists desire freedom. pursuingin ticians research: governments the different interests of scientists and poli known only to that community. and other monikers that have meanings largely robust science, use-inspired basic research studies: as collaborative assurance, socially itself has been renamed by scholars of science

0 mentions in 1944 to a peak of 159 men 159 of peak a tomentions 1944 in Words alone cannot bridge the gap between In recent decades, science policy has shifted 10 1970

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and probability appealing. even complex ideas such as calculus fairground rides, Ouellette makes examples, such as petrol mileage and basis of modern life. numbers and how maths forms the she overcame her own phobia of enticing facts. She describes how using a mix of humour, anecdote and Stephen Hawking. together with modern figures such as Johannes such as Nicolaus underpin the work of great scientists has spread across the ages, to unravels how Pythagoras’s influence how his convictions developed. She interest in mathematics arose and and his followers. She asks how his of the ancient Greek philosopher Ferguson pieces together the life story such as journeys to the ocean floor or looks to modern frontiers of discovery, programme in cold war politics, and origins of the planetary exploration (Viking, 2010). He examines the arc of human exploration in sets these missions within the wider Environmental historian Stephen Pyne System and witness interstellar space. Saturn, they will soon exit the Solar beneath Antarctica’s ice sheets. k epler and Isaac Newton, c opernicus, maths palatable Ouellette makes writer Jennifer 2010), science Diaries The Calculus with fear. In fills some people m writer 2010), science of Jupiter and neighbourhoods to visit the spacecraft the earliest explorers. Among probes are true the twin Voyager Launched in 1977, Pythagoras to all things. In order and unity that there is is rational, and that the Pythagoras held u sing everyday athematics k (Penguin, Voyager itty u niverse (Icon, OPINION

923 BOOKS iN BRieF Vol 466|19 August 2010 NEWS & VIEWS nasa ingqiu mao ingqiu J

Forest fires at northern latitudes (left) contribute to atmospheric pollutants that cause the Arctic haze (right).

Atmospheric chemistry A missing sink for radicals Jos Lelieveld Air pollution can cause a widespread haze in the Arctic. A study of the lower atmosphere there suggests that haze particles might take up free radicals, and so extend the lifetime of air pollutants in the region.

The lowest part of the atmosphere, the tropo- this haze are promoted by low removal rates ratio — the ratio of the OH volume to that of sphere, has a self-cleaning mechanism for deal- in the otherwise pristine Arctic environment. air — within the sampled air parcels was about ing with pollution. This mechanism is largely The aerosol particles in the haze are composed 0.02 parts per trillion (p.p.t.) up to altitudes of regulated by hydrogen oxides (the radicals OH of sulphates, nitrates, black carbon, heavy met- 7 kilometres, increasing to about 0.08 p.p.t. and HO2, collectively abbreviated to HOx), als, dust and organic matter, the last of which at altitudes of 10 km. When they compared because the reaction of OH with pollutant originates largely from forest fires3–5. There is these data with the predictions of a widely used gases is the first step in the pollutants’ removal. growing concern that pollutant emissions in atmospheric-chemistry model, they found that The OH can then be recycled in chains of the region will rise sharply if the Arctic Sea ice the model tends to underestimate OH concen- radical reactions in which HO2 is an inter- continues to recede, allowing shipping activity trations in the boundary layer (the lowermost mediate. What’s more, hydrogen peroxide to increase. region of the atmosphere that is in contact with 1 (H2O2, the reaction product of HO2) is a sink Mao et al. took measurements of a suite of Earth’s surface), but overestimates OH concen- for HOx because it is deposited on Earth’s atmospheric compounds as part of the Arctic trations in the upper troposphere. The under- surface, but it is also the main source of HOx in Research of the Composition of the Tropo- estimate in the boundary layer might be related the upper troposphere, both in the Arctic1 and sphere by Aircraft and Satellites (ARCTAS) to the model’s neglect of bromine chemistry 2 6 at lower latitudes . Reporting in Atmospheric campaign . The measurements were taken in — near the surface, HOx radicals can interact Chemistry and Physics, Mao et al.1 describe April 2008 by aircraft over Alaska, northern with reactive bromine (Br and BrO), which their study of the Arctic troposphere, from Canada, Greenland and the North Pole, and catalyses radical cycles, and can even lead to which they conclude that haze particles can targeted compounds that are appropriate for ozone destruction at polar sunrise7,8. interrupt the tropospheric cleaning mecha- constraining models of atmospheric chem- Mao et al. found that the observed mixing 1 nism by taking up HO2. Surprisingly, this istry, including HOx radicals and peroxides. ratios of nitrogen oxides , which are expected process can reduce the concentrations of OH, The conditions during the study were typical to increase OH concentrations, are well repro- HO2 and H2O2 by about one-third compared for the Arctic, with low sun angles at polar duced by the model. Their further analysis of with concentrations generated in conditions sunrise, intense cold (of about –17 to –47 °C) these data indicated that a large fraction (more without aerosol particles. and sunlight reflecting off the ice surface. than 40%) of the nitrogen oxides detected is Arctic haze has been reported since the Because the aircraft sampled the atmosphere anthropogenic. But the mixing ratios that they 1950s, and is prevalent in winter and spring. It almost exclusively in clear skies, chemistry observed for OH, HO2 (about 3 p.p.t.) and H2O2 is mainly attributed to the long-distance trans- within clouds did not play a part in deter- (about 300 p.p.t.) were much lower than the port of air pollution from Eurasian industrial mining the concentrations of the measured values predicted by the model. This is consist- sources, in particular coal burning in northern compounds. ent with the results of an earlier study9, which Russia3. The high pollutant concentrations in The authors observed that the OH mixing also showed that high-latitude measurements

1 of H2O2 were substantially lower than predicted clearly inconsistent with the authors’ data — a need for comprehensive investigations of concentrations. so if Mao and colleagues’ theory is correct, multiphase aerosol photochemistry, both in ■ The discrepancies between the predictions where is the putative H2O2 generated by aerosol the field and in the laboratory. and Mao and colleagues’ results cannot be uptake going? Jos Lelieveld is in the Atmospheric Chemistry attributed to errors in the model with regard Measurements10 taken using mass spectro- Department, Max Planck Institute for Chemistry, to the chemical sources of HOx, because a simi- metric techniques show that atmospheric Mainz 55020, Germany, and The Cyprus lar disagreement is found in other simulations aerosols are largely organic, consisting of Institute, 1645 Nicosia, Cyprus. that factor in these sources, at concentrations compounds that are in an intermediate oxida- e-mail: [email protected] recorded in the same data set as that used by tion state. The compounds originate from the the authors. Rather, the discrepancies point chemically reduced hydrocarbons emitted by 1. mao, J. et al. Atmos. Chem. Phys. 10, 5823–5838 (2010). 2. Colomb, a. et al. Environ. Chem. 3, 244–259 (2006). to chemistry that is completely absent from vegetation, from biomass burning, and can be 3. shaw, g. E. Bull. Am. Meteorol. Soc. 76, 2403–2413 (1995). the model — a missing sink of HOx radicals. processed in the atmosphere to highly oxidized, 4. quinn, P. K. et al. Tellus B 59, 99–114 (2007). The authors propose that multiphase reac- volatile reaction products. It is not known 5. Warneke, C. et al. Geophys. Res. Lett. 36, L02813 doi:10.1029/2008gL036194 (2009). tions (those that involve more than one phase which of the reaction products volatilize and 6. Jacob, D. J. et al. Atmos. Chem. Phys. 10, 5191–5212 (2010). of matter) on aerosol particles could be the to what extent these compounds take part in 7. Barrie, L. a. et al. Nature 334, 138–141 (1988). 8. neuman, J. a. et al. Atmos. Chem. Phys. 10, 6503–6514 missing sink. solution photochemistry with HOx radicals. To The aerosol scheme of the model used by answer these questions, and to confirm Mao (2010). 1 9. Cantrell, C. a. et al. J. Geophys. Res. 108, 8361 Mao et al. explicitly accounts for the uptake and colleagues’ hypothesis concerning the doi:10.1029/2002JD002198 (2003). and subsequent oxidation of sulphur dioxide role of aerosols in HO2 uptake, there is clearly 10. Jimenez , J. L. et al. Science 326, 1525–1529 (2009). by H2O2 in solution, but represents multiphase processes involving nitrogen oxides and HO2 by using parameters known as reactive uptake coefficients (γ). Mao et al.1 compiled values of GeNe eXpressioN γ(HO2) — the fraction of HO2 collisions with a given type of aerosol particle that results in loss of the radicals in reactions — for differ- how plants avoid incest ent aerosol types, as quantified from previ- Daphne goring and Emily indriolo ously published laboratory investigations. They found that aqueous aerosols (droplets of Different versions of the same gene can be either dominant or recessive. aqueous solutions) become more efficient at A small non-coding RNA mediates such differences in dominance as part reactively removing HO2 from the gas phase with decreasing temperature. This is because of a system that prevents inbreeding in plants. the sticking coefficient (the rate of absorption of a gas by aerosol particles) and the Henry’s Many flowering plants have elaborate systems diploid nature of plants: they carry two alleles law coefficient (the gas solubility in water) for to distinguish between pollens that are of each gene, and so two different S-haplotypes. HO2 are relatively high at low temperatures. acceptable for fertilization and those that are Normally, either S-haplotype can cause pollen 1,2 Furthermore, γ(HO2) is higher in the presence unsuitable . For plants of the Brassica family, rejection if it is shared by the anther and the of certain transition metals, especially copper. one such system is self-incompatibility — pistil (Fig. 1b). However, there are exceptions From their analysis, Mao et al. conclude that, rejection of genetically similar gametes. On to this rule: in the anther, one S-haplotype can to significantly affect the tropospheric concen- page 983 of this issue, Tarutani et al.3 describe be dominant to the other, masking the recessive 5 tration of HOx, the value of γ(HO2) must be at how small non-coding RNAs might intro- S-haplotype . For example, Tarutani et al. show least 0.1 — that is, the probability that HO2 will duce another level of mate selection through that, if the anther genome carries the pollen- react with a given type of aerosol particle must the epigenetic regulation of genes mediating dominant S9-haplotype and the pollen-reces- be greater than 10%. What’s more, the effect of self-incompatibility. sive S60-haplotype, pollen rejection occurs only HO2 uptake is largest in the cold upper tropo- Three genes regulate self-incompatibility if the pistil has the S9-haplotype. A pistil with 1,2 sphere, even though aerosol concentrations in Brassica: SP11/SCR, SRK and SLG . These the S60-haplotype will accept pollen grains from are relatively low in this region. If the authors’ genes are inherited together as a unit, and the S9 S60 anther, because the pollen-recessive hypothesis is correct, it implies that HO2 uptake each comes in many versions, or alleles, such S60-haplotype is suppressed (Fig. 1c). on aerosols may also be an important factor for as SRK1, SRK2 and SRK3; each allele produces How does the dominant–recessive relation- the cold upper troposphere at lower latitudes, a different version of the same protein. Dif- ship between these S-haplotypes emerge? The and not just in Arctic regions. ferent allele combinations of these genes are authors3 reveal that a fourth gene, SP11 meth- When Mao et al. incorporated the aerosol called S-haplotypes (Fig. 1a); the S1-haplotype, ylation inducer (SMI), which is tightly linked to uptake of HO2 into their model, they found for example, represents SP11/SCR1, SRK1 and the gene trio mediating self-incompatibility, is that the revised model successfully predicted SLG1. When the S-haplotype of the male responsible for this altered trait. the experimentally observed HOx concentra- parent (the pollen-producing anther) matches The same team of researchers has previously tions at various altitudes. This bodes well for the S-haplo type of the female parent (the pistil) discovered6 that, when a plant carries a pollen- their theory, but several major unknown fac- the pollen is rejected (Fig. 1b). The key deter- dominant and a pollen-recessive S-haplotype, tors remain to be investigated. For example, in minants of this system are the pollen-specific the SP11/SCR gene from the pollen-recessive – + water, HO2 can dissociate into O2 and H ions, SP11/SCR protein and its receptor, the pistil- S-haplotype is turned off. The group also and take part in ion chemistry and chains of specific SRK protein. Interaction between found7 that suppression of the pollen-recessive radical reactions. Unfortunately, only a few of these proteins activates the pollen-rejection SP11/SCR gene correlates with methylation of these reactions have been studied, and so their response in the pistil1,2,4. Only proteins encoded the SP11/SCR promoter sequence — a process influence on atmospheric chemistry is poorly by matching alleles from each S-haplotype can known to cause gene silencing. The key out- known. What’s more, the self-reaction of HO2 interact (for instance, SP11/SCR1 can bind only standing question was how this very selective in solution forms H2O2, as does the reaction of to SRK1), thus avoiding unnecessary pollen silencing of SP11/SCR occurs. The authors now – 3 HO2 with O2 . It has been assumed that such rejection. show that SMI is the missing link. H2O2 is released to the gas phase, but this is Another level of complexity is the inherent The SMI gene is part of both the genomic 926 © 2010 Macmillan Publishers Limited. All rights reserved accepts the S type type — when both — from the dominant to the recessive plant are accepted by the pistil, and so the pollen grains from the tein is not produced to activate SRK gene. Accordingly, the SP11/SCR silencing of — the pollen-recessive in methylation of the promoter of — and so from the pollen-dominant et anther. Take Figure (sRNA) that produced is specifically in the (Fig. and that carrying the recessive regioncontaining dominant lands on the recessive ( instance rejection by the pistil occurs when the pollen-producing anther shares the same and Brassica well-known genes, Figure 1 NATURE an evolutionarily conserved change. When the that the pollen-recessive SMI 60

al. But why does this occur in only one direction -haplotype in the anther (through the action of the S 3 gene? Tarutani and co-workers discovered

60 showed that production of an c b a 1a). It encodes a small non-coding RNA Anther pollen-recessive. plants. In this example, two different | Dominant Co-dominance |

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break-through such as occurred perhaps combined with a tidal century slight land sinkage, Somewhere about the fourteenth peat digging in the Middle Ages. that the broads resulted from now points to the conclusion it would seem that everything outcome of these investigations philological, botanical, etc. As the of view—geological, historical, attacked from various points in recent years, and it has been has been devoted to this problem were formed?... Much research themselves how these large lakes holiday makers have ever asked the “Broads”... But how many Thousands of people know 50 From situation. telegraphy may alter the whole long the development of wireless generation ago. Very probably ere so strong as it may have been a case for a long trial does not seem to be a serious matter. Thus the of a single chronometer is unlikely length of time, and the breakdown seldom isolated for any great the increase of speed, a ship is done?... In the present day, with the place where it should be national Observatory exactly for the Navy, but is our great work is doubtless most valuable has enormously increased. The work borne by the Observatory 1880, so that the burden of this we learn, more than trebled since chronometers rated daily has, 596 ... The average number of being rated (at Greenwich) was chronometers and watches the average daily number of In the year ending 1910, May 100 From his present enjoyment is due. his forerunners, to whose labours remember the old peat workers, let him spare a moment to Barton or proletarian Wroxham, aristocratic Ranworth or stately the holiday maker is sailing over relative to the sea... So whether the new lower level of the land could not drain away owing to workings, and this flood water a few years ago, flooded the peat ye ye Nature Nature A A rs rs 18 August 1910. 20 August 1960. AG AG o o NEWS &VIEWS

10,

927 50 & 100 YEARS AGO NEWS & VIEWS NATURE|Vol 466|19 August 2010

response — seems unnecessary, it is a bio- of gene silencing between select combina- 1. Franklin-Tong, V. E. Self-Incompatibility in Flowering Plants logically relevant mechanism. Recently, two tions of pollen-recessive S-haplotypes, which (springer, 2008). 8,9 2. Chapman, L. a. & goring, D. R. J. Exp. Bot. 61, 1987–1999 studies examined the role of dominance indicates that other unknown factors might (2010). in this system using theoretical modelling also be involved. Regardless of this, Taru- 3. Tarutani, Y. et al. Nature 466, 983–986 (2010). and found that modifiers leading to pollen- tani and colleagues’ paper is an intriguing 4. ivanov, R., Fobis-Loisy, i. & gaude, T. Trends Plant Sci. 15, dominant S-haplotypes (such as SMI sRNA) example of sRNA-mediated regulation of 387–394 (2010). 5. Thompson, K. F. & Taylor, J. P. Heredity 21, 345–362 (1966). would be favoured, because these plants could dominant–recessive patterns of Mendelian 6. shiba, H. et al. Plant Cell 14, 491–504 (2002). 8 mate with more plants in the population . The inheritance. ■ 7. shiba, H. et al. Nature Genet. 38, 297–299 (2006). pressures of inbreeding depression also seem Daphne Goring and Emily Indriolo are in the 8. schoen, D. J. & Busch, J. W. Evolution 63, 2099–2113 to favour the evolution of dominance among Department of Cell and Systems Biology, (2009). 9 9. Llaurens, V., Billiard, s., Castric, V. & Vekemans, X. Evolution S-haplotypes . University of Toronto, Toronto M5S 3B2, Canada. 63, 2427–2437 (2009). A remaining puzzle is the observation10 e-mail: [email protected] 10. Kakizaki, T. et al. Plant Cell Physiol. 44, 70–75 (2003).

Astrophysics the mass of our Sun, shine millions of times more brightly and explode as supernovae within a few million years of their birth. The Waves on orion’s shores least massive stars that eventually explode are John Bally those born with about 8 times the Sun’s mass, and that live for about 40 million years. Con- Waves have been discovered in the molecular cloud surrounding the Orion trast this with the life expectancy of our Sun: nebula, generated by shearing flows in the cloud. This finding provides clues roughly 10 billion years. Since the formation of our Galaxy and the to the way filamentary substructures form in the interstellar medium. birth of our Solar System 4.5 billion years ago, many generations of massive stars have come Massive stars have a profound effect on evo- converted into long-lived, low-mass stars. But and gone. Each generation converted much of lution in the cosmos. Their brilliant light can just a few per cent of the mass of these gases its hydrogen and helium into heavier elements outshine all other stars in star-forming gal- formed short-lived massive stars. The most and, on exploding, recycled this heavier mater- axies, and their cores are the source of most massive were born with more than 100 times ial to enrich the ISM. Today, between 1 and 2% elements other than hydrogen and of the mass of the ISM consists of ele- sa helium. Their intense radiation, ments forged by the thermonuclear E stellar winds and supernova explo- fires in the cores of massive stars. sions sculpt surrounding interstellar Orion B North During their brilliant lives, mas- gas clouds and determine the molecular sive stars ionize their parent clouds, clouds’ state. The Orion nebula1,2, cloud driving stellar winds that create located about 400 parsecs from the expanding bubbles of plasma. Over Sun, is the nearest site of ongoing the past 12 million years, the Orion massive-star formation. It is one Barnard’s region’s giant molecular clouds of the best-known environments loop (Fig. 1) have given birth to tens of in which interactions between thousands of stars, including dozens massive stars and the surrounding Orion of massive ones. The Orion nebula, interstellar medium (ISM) can be nebula which has spawned more than 1,000 studied in detail. Orion continues low-mass stars and at least half a to provide new insights into the dozen massive ones, is the youngest physics of the ISM, particularly into region of massive-star birth in the Orion A the inter actions between massive molecular Orion constellation. stars and the surrounding clouds cloud Within the past million years, the that gave birth to them. Reporting nebula’s luminous, hot, massive stars in this issue (page 947), Berné et al.3 have started to dissociate the back- provide evidence for a new class of ground molecular cloud, creating a such interactions — waves at the ‘blister’ of plasma that has a temper- interface between the nebula and the ature of 10,000 kelvin. In this proc- background molecular cloud. Figure 1 | the constellation of orion. This image was taken by the ess, ultraviolet radiation cleaves the The dense, molecular phase of the Planck satellite using high-frequency radio emissions, and covers bonds of molecules, heats and ion- ISM is the raw material for star and a region of 13°×13°. The turquoise structures consist of interstellar izes atoms, and accelerates the result- planet formation. Massive stars drive dust and are mostly associated with molecular clouds in which stars ing plasma to form a wind blowing a ‘galactic ecology’, recycling matter are forming. The two largest clouds, Orion A and Orion B, have a towards Earth and over parts of the from interstellar clouds into stars combined mass of about 100,000 times that of the Sun. The red regions adjacent dense cloud. Doppler shifts and back again into the ISM. Our trace hydrogen gas that has been ionized by dozens of massive stars of the plasma’s spectrum have shown Milky Way galaxy contains between over the past 13 million years. The supernova explosions of some of that the plasma flows with speeds of 10 billion and 100 billion stars, and these stars have created a bubble that is squeezing and compressing tens of kilometres per second, form- the clouds. The large red arc, known as Barnard’s loop, marks the there is sufficient material in its ISM ing a shearing layer over parts of the outer edge of this bubble. The Orion nebula, located in front of the 4 to make billions more. Over the past compressed northern end of the Orion A cloud, and in the interior background cloud . This situation 13 billion years, most of the Milky of the hot, supernova-heated bubble, marks the most recent site of might be familiar to anyone who Way’s hydrogen and helium that massive-star birth in Orion. Berné et al.3 have discovered waves has spent time on a beach, watch- originated in the Big Bang has been in the molecular cloud of Orion. ing waves breaking on the shore.

Distant winds blowing over the water form and Space Astronomy, University of Colorado at 3. Berné, o., marcelino, n. & Cernicharo, J. Nature 466, a shearing layer and excite the formation of Boulder, Boulder, Colorado 80309-0389, USA. 947–949 (2010). 3 4. o’Dell, C. R. Annu. Rev. Astron. Astrophys. 39, 99–136 (2001). waves. Berné et al. now report that shearing e-mail: [email protected] 5. Bally, J., Langer, W. D., stark, a. a. & Wilson, R. W. flows between the plasma in the nebula and Astrophys. J. 312, L45–L49 (1987). the background molecular cloud form a hydro- 1. Bally, J. in Handbook of Star Forming Regions Vol. 1 6. Heitsch, F., slyz, a. D., Devriendt, J. E. g., Hartmann, L. W. (ed. Reipurth, B.) 459–483 (asP monogr. Publs, 2008). & Burkert, a. Astrophys. J. 648, 1052–1065 (2006). dynamical effect known as a Kelvin–Helmholtz 2. o’Dell, C. R., muench, a., smith, n. & Zapata, L. in 7. Vázquez-semadeni, E., Ryu, D., Passot, T., gonzález, R. F. & instability (KHI), which generates waves on Handbook of Star Forming Regions Vol. 1 (ed. Reipurth, B.) gazol, a. Astrophys. J. 643, 245–259 (2006). the surface of the cloud — the ‘shores’ of the 544–590 (asP monogr. Publs, 2008). 8. von Kármán, T. Aerodynamics (Dover, 1994). Orion nebula. But why should we care about waves in Orion? There are several reasons. The waves discovered by Berné and colleagues are an DNA repAir example of how energy and momentum are injected into the ISM by massive stars, and provide clues about the self-regulation of star for- Blocking ubiquitin transfer mation. For example, instabilities such as KHIs april Rose and Christian schlieker can develop in a complex manner, growing in amplitude until the wave energy degrades into The protein OTUB1 inhibits DNA repair without using its enzymatic activity. turbulence — analogous to the chaos that fol- Instead, it sequesters a protein that is required for the assembly of certain lows the breaking of ocean waves on a shore. Berné and colleagues’ results also indicate forms of ubiquitin chain, which function as key signals during repair. new directions for the numerical modelling of interstellar plasmas. Modification of proteins with ubiquitin — extend ubiquitin chains that have been added Unlike ocean waves, however, the shear flows a small, evolutionarily highly conserved by RNF8, which operates downstream of the in Orion are mediated by ultraviolet radiation. polypeptide — is crucial for the activity of enzyme ATM kinase. Recent studies4,5 show This radiation penetrates the shearing layer of cellular DNA-repair machinery1. Ubiquityla- that mutations in RNF168 underlie RIDDLE the KHI, creating an insulating sheet of plasma tion is a reversible process involving specific syndrome, a disorder that is characterized by that separates the dense cloud from the nebula’s proteases called de ubiquitylating enzymes immuno deficiency and sensitivity to ioniz- out-flowing wind, which should prevent the (DUBs), which detach ubiquitin tags from ing radiation. Little is known about negative KHI from developing. How, then, did the waves protein substrates. On page 941 of this issue, regulators of this signalling pathway. develop in the presence of ultraviolet radiation? Nakada et al.2 describe OTUB1, an atypi- Nakada et al.2 set out to search for such nega- Berné and colleagues’ answers3 provide clues cal DUB that prevents ubiquitin attachment, tive regulators by looking for DUBs whose about the history of the Orion nebula.They rather than detaching bound ubiquitin, and in depletion results in an increase in ubiqui- propose that the waves formed before the birth this way inhibits DNA repair. tin chains linked through the lysine residue of the nebula’s most massive star. At this time, Ubiquitin is usually attached to the side at position 63 (K63) of ubiquitin. This type there was less ultra violet radiation flooding the chains of the lysine amino-acid residues in its of ubiquitin linkage is commonly found in environment, and so the layer separating the substrate. This attachment is a carefully con- the signalling components involved in DNA shearing plasma flow from the cloud would trolled pro cess that involves the coordinated repair. OTUB1 was the only protein that, when have been thinner than today. action of an E1 ubiquitin-activating enzyme, depleted, caused this type of response. This Radiation-mediated shear flows may be a an E2 ubiquitin-conjugating enzyme and an E3 result is surprising because, in vitro, OTUB1 universal feature of interactions in the ISM. ubiquitin–protein ligase enzyme. Several ubiq- has strict specificity for K48-linked ubiquitin The origin of star-forming molecular clouds, uitin molecules can be attached to a substrate, chains, which target proteins to the proteasome and of their filamentary substructure5, remains forming ubiquitin chains, and the ubiquitin for degradation6. a hotly debated topic among astronomers. mol ecules within these chains can be linked The authors also show that increasing the One popular theory6,7 posits that dense clouds through different lysine residues in the ubiq- expression of OTUB1, or, more surprisingly, form behind shock waves, where lower-den- uitin itself. The prevailing view is that DUBs a catalytically inactive derivative of it, greatly sity ISM flows converge head-on as a result of operate by proteolytically cleaving either the reduces the abundance of the ubiquitylated expanding bubbles, stellar winds, supernova amide (isopeptide) bond that links ubiquitin to proteins at cellular sites containing DNA- explosions or spiral density waves (regions its substrate or the bond that links one ubiquitin repair proteins. What’s more, when the expres- of higher density associated with the spiral molecule in the chain to the next3. Nakada and sion of either protein was increased together arms of galaxies). Head-on collisions of clouds, colleagues’ observations challenge this view. with RNF168, the ubiquitylation of the his- however, are much less likely than collisions When a double-strand break occurs in DNA, tone H2AX and other chromatin-associated with shear flows. The study and modelling this triggers a signalling cascade that leads to proteins was reduced markedly. These data2 of ultraviolet-mediated shear flows, such as extensive modification of the chromatin (com- point to an inhibitory mechanism that involves those in Orion, may thus provide new insights plexes of DNA and proteins such as histones) the binding activity of OTUB1 rather than the into the formation of filamentary molecular surrounding the break; such modifications detachment of ubiquitin by it. clouds. On Earth’s shores and in its atmos phere, include phosphorylation and ubiquitylation. Nakada and co-workers further show that the convergence of flows with shear sometimes Depending on their precise architecture, ubiq- OTUB1 binds to UBC13 — the E2 enzyme gives rise to long cylinders of vortical motion uitin chains contribute to the recruitment of that interacts with the E3 ligases RNF8 and (so-called von Kármán vortex streets8). In factors that are involved in DNA repair and RNF168 — as well as to other E2 enzymes of the ISM, perhaps ultraviolet-mediated con- cell-cycle control1. the same subfamily. Moreover, OTUB1 and verging and shearing flows, combined with The E3 ligases that are involved in repair- its catalytically inactive variant both blocked entrained magnetic fields and self-gravity, ing DNA double-strand breaks are RNF8 and UBC13-mediated formation of short ubiqui- provide an explanation for the origins of RNF168, which cooperate with the E2 enzyme tin chains. The enhanced polyubiquitylation filamentary clouds. ■ UBC13 in the attachment of ubiquitin to spe- normally seen in the additional presence of John Bally is in the Department of Astrophysical cific histones and potentially to other chro- RNF168 was also inhibited. and Planetary Sciences, Center for Astrophysics matin-bound proteins. RNF168 is thought to This work provides strong evidence that

elegantly show that OTUB1 binds prefer- DNA repair could have therapeutic relevance. Inhibition of entially to the E2 enzyme that is primed for Nakada et al. found that reducing the level Ub-chain formation ubiquitin transfer, a finding that favours the of OTUB1 expression restores the pro cess of last of the three possibilities mentioned above homologous recombination in cells in which Ub (Fig. 1). As the authors suggest, in a physiologi- ATM kinase is inhibited. Thus, OTUB1 deple- UBC13 DNA cal context OTUB1 may set the threshold for tion can, in principle, mitigate DNA-repair (E2) OTUB1 initiating the signalling that is induced by DNA defects. This observation makes the inter action double-strand breaks. between OTUB1 and UBC13 an attractive Together with a growing body of evidence target for therapeutic intervention, with OTUB1 7 Ionizing dissociation that DUBs have non-canonical activity , this particular relevance for disorders affecting radiation paper2 presents an appealing model for how DNA repair and for use in combination with several DUBs that lack the signature cata- radiation therapy. ■ lytic triad (a specific sequence of amino acids April Rose and Christian Schlieker are in the involved in catalysis) might nevertheless fulfil Department of Molecular Biophysics and essential regulatory functions. Several differ- Biochemistry, Yale University, New Haven, Ub Ub ent E2 enzymes interact with OTUB1 (refs 2, 8) Connecticut 06520-8114, USA. and other DUBs8, so these unusual mecha- e-mail: [email protected] Substrate protein nisms might also control the activity of other E2 enzymes. 1. messick, T. E. & greenberg, R. a. J. Cell Biol. 187, 319–326 RNF168 Double-strand (2009). break A key question is how the inhibitory effect of 2. nakada, s. et al. Nature 466, 941–946 (2010). OTUB1 is relieved when acute DNA damage 3. Komander, D., Clague, m. J. & urbé, s. Nature Rev. Mol. Cell occurs. OTUB1 has multiple phosphorylation Biol. 10, 550–563 (2009). 9 4. Doil, C. et al. Cell 136, 435–446 (2009). sites , which might provide an additional layer 5. stewart, g. s. et al. Cell 136, 420–434 (2009). Ub of control by allowing its affinity for UBC13 6. Wang, T. et al. J. Mol. Biol. 386, 1011–1023 (2009). Ub to be modulated. Whether OTUB1 regu- 7. Hanna, J. et al. Cell 127, 99–111 (2006). K63-linked Ub-chain 8. sowa, m. E., Bennett, E. J., gygi, s. P. & Harper, J. W. Cell formation Ub lates other pathways that rely on UBC13 also Ub 138, 389–403 (2009). remains to be established. 9. Edelmann, m. J., Kramer, H. B., altun, m. & Kessler, B. m. DNA-damage response DNA-damage The demonstration that OTUB1 inhibits FEBS J. 277, 2515–2530 (2010).

eVoLUtioNAry BioLoGy Recruitment of DNA-repair factors oh sibling, who art thou?

Figure 1 | otUB1 does it differently. Nakada andrew Cockburn and co-workers’ data2 suggest that the deubiquitylating enzyme OTUB1 sequesters the Help from earlier offspring in rearing a subsequent brood should evolve E2 enzyme UBC13 in its ubiquitin (Ub)-loaded more easily when the mother is strictly monogamous. A comparative study form, preventing the formation of ubiquitin of birds provides evidence in support of this view. chains. (Usually, deubiquitylating enzymes detach ubiquitin from their substrate proteins.) If the cell’s DNA is damaged by ionizing Cooperative breeding, in which more than two The authors’ approach involved use of the radiation, OTUB1 dissociates from UBC13~Ub, individuals combine to rear a single brood of phylogenetic comparative method, which allowing the formation of K63-linked ubiquitin young, has evolved repeatedly in animals, and relates changes in the trait of interest on dif- chains on substrate proteins in the presence of most commonly in insects and birds. This situ- ferent branches of the tree of life to changes in the E3 enzyme RNF168. Such ubiquitylation of ation poses an evolutionary paradox: because potential explanatory factors. This approach is chromatin-bound proteins near sites of DNA individuals have only two parents, some of the well known and frequently used, but Cornwal- double-strand breaks is a signal that recruits carers in these cooperative societies are helping lis et al. estimate the correlation by applying DNA-repair factors and cell-cycle regulators. to raise young that are not their own. The evo- a novel and very general Bayesian statistical lutionary biologist W. D. Hamilton famously technique4. The response variable they initially interaction between OTUB1 and UBC13 solved part of the puzzle by pointing out that investigated was whether birds bred coopera- has a role in inhibiting DNA repair. Several helping to rear siblings can be completely anal- tively, an activity that they conclude declines mechanisms, which are not necessarily mutu- ogous to rearing your own offspring, as both strongly with an index of female infidelity — ally exclusive, may be at work. These include, actions facilitate the propagation of your own the proportion of broods in which some young first, direct competition for UBC13 between genes in subsequent generations. are sired by extra-group (or extra-pair) males. OTUB1 and the E3 ligase; and second, masking However, many females are so promiscuous The effect works in both directions: transition of the lysine side chains of ubiquitin, prevent- that young will be uncertain whether they share to cooperative breeding occurs more coming chain extension. Third, E2~ubiquitin — paternal genes with other young produced by monly in monogamous species, and transitions the thioester-linked ubiquitin, which then their mother. This argument has been used to from cooperation to non-cooperation increase transfers ubiquitin to a target lysine — might suggest that the evolutionary transition to and with infidelity. be masked, preventing the processing of the from cooperative care will depend on the extent The evolution of mechanisms to detect next ubiquitin molecule. to which the mother mates monogamously1,2. relatedness would allow potential helpers to In all cases, one would expect a stoichio- On page 969 of this issue, Cornwallis and col- overcome the uncertainty that flows from infi- metric, rather than a catalytic, effect, which leagues3 exploit the great variation in the mat- delity. However, the strength of evolutionary Nakada et al.2 indeed observe in vitro. Using ing systems of cooperatively breeding birds to selection for sophisticated means of recogniz- a molecular mimic of E2~ubiquitin, they provide support for this hypothesis (Fig. 1). ing kinship will vary. It is sufficient to have a

BB the incidence of cooperative breeding. a We are most likely to learn about the forces

FFREY D FFREY that lead to cooperation by studying pairs of o E g species sitting close to the point of evolutionary transition. To take a different case, the study of primitively social wasps might tell us more about the conditions that produce sociality in insects than does research on army ants in which the single queen could be attended by a million workers. Likewise, investigations of closely related birds that either lack or exhibit a low level of cooperative breeding might allow the most illuminating direct tests of the monogamy hypothesis. Although such primitively social birds lack the charisma of the most highly developed avian cooperative societies, there is new impetus for adding them to the already impressive array of birds in which molecular tools have been used to dissect patterns of parentage. ■ Andrew Cockburn is in the Division of Evolution, Ecology and Genetics, Research School of Biology, Australian National University, Canberra Figure 1 | monogamy and cooperation. About 10% of birds, including these grey-crowned babblers ACT 0200, Australia. (Pomatostomus temporalis), breed cooperatively. The research of Cornwallis et al.3 indicates that e-mail: [email protected] cooperation is more likely to evolve if the breeding female is monogamous. 1. Boomsma, J. J. Curr. Biol. 17, R673–R683 (2007). 2. Boomsma, J. J. Phil. Trans. R. Soc. B 364, 3191–3207 rule-of-thumb that presumes kinship if the cooperative behaviour can drive the incidence (2009). female is always faithful; assuming limited of fidelity rather than vice versa, and that the 3. Cornwallis, C. K., West, s. a., Davis, K. E. & griffin, a. s. relatedness also works if the female is highly coevolution of fidelity and cooperation could Nature 466, 969–972 (2010). 4. Hadfield, J. D. J. Stat. Software 33(2), 1–22 (2010). promiscuous. Somewhere in the middle of be much more complex than is assumed in 5. Cockburn, a. Annu. Rev. Ecol. Syst. 29, 141–177 (1998). these extremes, it would pay to know whether Cornwallis and colleagues’ analysis. None- 6. griffith, s. C., owens, i. P. F. & Thuman, K. a. Mol. Ecol. 11, you are related or not and to adjust your care theless, their study provides a welcome and 2195–2212 (2002). 7. mulder, R. a., Dunn, P. o., Cockburn, a., Lazenby-Cohen, accordingly. Consistent with this view, Corn- exciting direction for empiricists, who have K. a. & Howell, m. J. Proc. R. Soc. Lond. B 255, 223–229 wallis et al. found that the ability of birds to failed miserably to produce models that predict (1994). vary their investment in offspring according to kinship was most pronounced at intermediate levels of infidelity. The phylogenetic comparative method can eArthQUAKes be extremely powerful, but has some limita- tions. First, particularly with transitions to complex behavioural states, defining whether Double trouble at tonga the transition has occurred is not always Kenji satake straightforward. Cooperative breeding in birds can be confined to family groups, can A puzzling case is presented by the occurrence of two large but dissimilar occur among completely unrelated individu- 5 earthquakes at almost the same time and place. One must have acted as als, or can be a bewildering mixture of the two . Cornwallis et al. use a restrictive definition, the trigger, but which one and how did it do so? and define cooperative breeding as applying only to family groups, which is reasonable, One earthquake can set off others. Most Australia plate (specifically its Tonga block), as their index of infidelity is defined as mat- triggered earthquakes are aftershocks that result in an area where these two plates are moving ing outside such groups. However, using this from adjustments on and near the plane of the towards one another at about 20 centimetres definition excludes a lot of the complexity that fault that produced a larger mainshock. In this per year — faster than at any other plate con- makes cooperative breeding most interesting, issue, Beavan et al.1 (page 959) and Lay et al.2 vergence worldwide. Despite this, the Tonga and potentially underemphasizes direct ben- (page 964) take up the intriguing example of two trench had somehow failed to produce a sin- efits to the provider of help relative to those earthquakes that overlapped in time and were gle great earthquake of magnitude 8 or larger derived indirectly through kinship. adjoined in location, but differed drastically in since 1917. That each of the September 2009 The second universal problem of using cor- mechanism. The authors reach opposing con- earthquakes attained magnitude 8 thus came as relations in evolutionary biology is that the clusions about which earthquake began first, something of a surprise to geophysicists. direction of causation is often uncertain. For and how one earthquake triggered the other. The main earthquake visible in seismic example, in Australian fairy wrens (Malurus The earthquakes in question occurred on records was not a typical subduction-zone species), which, despite ubiquitous coopera- 29 September 2009 in the southwest Pacific, earthquake. Great earthquakes and associated tive breeding, are the least faithful of all birds6, near the Tonga trench. An associated tsu- tsunamis are caused by sudden slip occurring females exploit the availability of helpers as nami claimed close to 200 lives across Samoa, on the plate interface, releasing accumulated an alternative source of care for their young, American Samoa and Tonga1,2. The earth- compressional strain between the two plates. allowing them to increase the extent to which quakes ultimately resulted from the descent, A recent example of such a plate-boundary they cuckold their mates7. This indicates that or subduction, of the Pacific plate beneath the earthquake is the magnitude-8.8 earthquake

Administration (NOAA). These waveforms are sensitive to the parent earthquake because a Slip rate B2 plate-boundary earthquakes and outer-rise 1 B1 Beavan et al. earthquakes produce opposite sea-surface dis- B1 is slow and not detected Time placement above the earthquake fault. NOAA by seismic data modellers implicitly assumed a plate-boundary Ground displacement earthquake model in their successful real-time Observed seismograms data assimilation to forecast the far-field tsu- nami5. Beavan et al. likewise show that the tsunami waveforms recorded at the DART sta- L2 buried in seismic data tions are better explained by a plate-interface Slip rate L1 L2 earthquake, and they obtained the best match Lay et al.2 by postulating the occurrence of a slow plate- Time boundary earthquake before the outer-rise earthquake. They point out that this sequence b Tonga Samoa can be explained by static stress change, as in Displacement detected by GPS the Kuril example. When two earthquakes occur nearly simul- Tsunami sources taneously, the signal from the later event may be buried in the seismic waves from the first. 2 Trench Outer rise Lay et al. carried out non-routine, detailed and Australian plate L2 comprehensive analyses of the available seis- (Tonga block) B1 B2 mic data, and succeeded in detecting signals L1 from earthquakes after the outer-rise earthquake. Their model indicates that the main Paciﬁc plate outer-rise earthquake triggered the rupture of the plate boundary by shaking it. Such dynamic Pull force triggering is plausible: it has been documented on faults hundreds of kilometres from the initiating earthquake6. But it is still difficult to tell whether the plate- interface earthquake really happened later. If Figure 1 | interpretations of the two tonga-trench earthquakes of 29 september 2009. a, Beavan 1 2 that event was generated slowly in compari- et al. (B) and Lay et al. (L) come to different conclusions about the order in which the earthquakes son to seismic-wave periods, it would not have happened. B1 and L1 denote which, respectively, the authors consider to have occurred first. But been detected in ordinary seismic records1. seismologically, neither interpretation is clear-cut: if an earthquake is slow and the slip rate is Analysis of ultra-long-period seismograms2 small (B1), the signal may be undetected in regular seismic records. Alternatively, if the second earthquake (L2) happens soon after the first one, the signal can be buried in seismic records. can indicate the existence of such slow earth- b, Depiction of the Tonga trench, where the Australian and Pacific plates meet. The two groups1,2 quakes, but it is difficult to achieve an accurate agree that B1/L2 was an interplate earthquake, due to compressional stress, at the boundary estimate of timing from such ultra-long-period between the plates; and that the main, visible, earthquake (B2/L1) was an intraplate event that records. Lay et al.2 locate the plate-boundary occurred at the outer rise due to extensional stress. Beavan et al.1 drew their conclusions from earthquake (as a pair of subevents) close to the GPS measurements and models of tsunami waveforms. Lay and colleagues’ interpretation rests on trench. It has been shown that the shallower analyses of the available seismic data. on the plate interface and closer to the trench axis slip occurs, the slower it is7. Therefore, that rattled central Chile and set off a tsunami the load on some neighbouring faults and the plate-boundary earthquake rupture might on 27 February 2010. By contrast, the main subtracts from the load on others. These stress have been slow. Tonga event resulted from extensional fault- changes may hasten or retard earthquakes, Taken together, the two papers1,2 leave ing that occurred in an area known as the outer respectively4. uncertainty as to which of the two earth- rise, where the descending plate begins to bend In the Tonga case, both Beavan et al.1 and Lay quakes happened first. And, until we learn into the trench. et al.2 found that a plate-boundary earthquake which of them was the cause and which the Still, it is not a geophysical surprise to find a was associated with the outer-rise earthquake effect, it will be difficult to know whether the great outer-rise earthquake. Several have been (Fig. 1). The strongest evidence for this find- trigger was the release of static stress on an recorded during the past 100 years, and they ing comes from satellite geo desy. By comparing extensional fault, or of dynamic stress on a are easily explained by downward pull by the pre- and post-earthquake measurements from compressional one. ■ descending plate. This pull force can be trans- northern Tonga, made by the Global Position- Kenji Satake is at the Earthquake Research mitted towards the outer rise if the two plates ing System (GPS), Beavan et al.1 estimate that Institute, University of Tokyo, Bunkyo-ku, do not accumulate strain on the plate-bound- 35 cm of horizontal movement occurred in Tokyo 113-0032, Japan. ary fault, and it can also increase suddenly if the a direction opposite to that expected for an e-mail: [email protected] plate boundary breaks in a great earthquake. A outer-rise earthquake. Continuous GPS meas- 1. Beavan, J. et al. Nature 466, 959–963 (2010). November 2006 plate-boundary earthquake of urements, such as those made during the 2010 2. Lay, T. et al. Nature 466, 964–968 (2010). magnitude 8.3 along the Kuril trench set off Chilean earthquake, were not available, how- 3. ammon, C. J., Kanamori, H. & Lay, T. Nature 451, 561–565 an extensional outer-rise earthquake of magni- ever; such measurements could have pinpointed (2008). tude 8.1 just two months later3, by causing the which earthquake happened first. 4. stein, R. s., King, g. C. P. & Lin, J. Science 265, 1432–1435 subducting plate to pull away from the outer Clues to the earthquakes’ sequence can also (1994). 5. http://nctr.pmel.noaa.gov/samoa20090929 rise. In a generic case, such triggering results be found in tsunami waveforms recorded on 6. gomberg, J., Reasenberg, P. a., Bodin, P. & Harris, R. a. from a change in static stress. Sudden displace- bottom-pressure (DART) sensors operated Nature 411, 462–466 (2001). ment on a fault during an earthquake adds to by the US National Oceanic and Atmospheric 7. Bilek, s. L. & Lay, T. Nature 400, 443–446 (1999).

932 © 2010 Macmillan Publishers Limited. All rights reserved NATURE|Vol 466|19 August 2010 NEWS & VIEWS oBitUAry stephen henry schneider (1945–2010) a voice of reason in climate-change science and policy. CE Anyone who ever met Stephen lymphoma, a disease he overcame i

Schneider won’t forget the occasion. in 2002. ERV I first encountered him when I was an Schneider was deeply committed s s EW undergraduate at Stanford University to public outreach and to training n RD o

in 1997. Minutes after I stepped into the next generation of scientists. F an his office, curious about his work After joining the faculty of Stanford T

but unsure what to say to a famous in 1992, he championed graduate o/s CER climatologist, my nervousness was and undergraduate environmental i C

swept away by a deluge of information. programmes and science literacy for a. . He rapidly delivered a synthesis of all students. He fought tirelessly against L climate-change science and the need misinformation about climate change for action, an amusing deconstruction and what he called “mediarology”, the of the arguments of climate-change tendency of the media to portray a topic deniers and an invitation to take his as a two-sided debate, with each side course, ‘Climate modeling and theory’. carrying equal weight. Later, while making notes to try to He always separated scientific facts capture all I’d learned, I realized I had A postdoc at NASA’s Goddard Institute for and personal opinions, emphasizing what met a brilliant scientist who possessed a Space Studies in New York City in 1971 gave is well established and where uncertainties unique talent for both explaining complex Schneider his start. A year later, he moved remain — and why those uncertainties do not issues and encouraging others to care to the National Center for Atmospheric lessen the urgency of societal action. In 1992, about them. Research in Boulder, Colorado, where he he was awarded a MacArthur fellowship Schneider, who died on 19 July, was a remained on the staff until 1996. for his ability to integrate and convey the pioneer in climate-change research. He was In 1971, Schneider drew attention and results of climate-change research through one of the first to develop and use numerical criticism for a paper he published in Science public and classroom lectures, congressional models of the climate system to address with S. Ichtiaque Rasool. It suggested that the testimonies, media appearances and his own issues such as the effects of greenhouse cooling effects of aerosols could dominate the research. He served as a scientific adviser gases, aerosols and clouds on Earth’s warming effects of greenhouse gases. In this to all eight US presidential administrations radiation balance. He also made an art nascent field, findings quickly emerged that from Nixon to Obama. of communicating the science; for four showed the effects of aerosols to be regional Among his many honours, Steve was decades, he strove to inform the public rather than global, and that warming effects elected to the National Academy of Sciences and policy-makers about the growing and would dominate. Schneider himself was in 2002 and was a leading contributor to now overwhelming evidence supporting proud that he got the “wrong answer for the the Intergovernmental Panel on Climate human-caused climate change. right reasons”. His conclusions were based on Change (IPCC). A co-author of all four IPCC Schneider grew up on Long Island, the evidence and modelling tools available Assessment Reports, he was preparing to New York. Early on, his favourite pastimes at the time. On re-examining the issue in the serve as a coordinating lead author of the included roaming the American Museum light of new evidence using new tools, he was chapter on ‘Detection and attribution of of Natural History in New York City and the first to correct his earlier calculations in observed impacts’ for the forthcoming gazing at the rings of Saturn through his the literature in 1975. fifth report. homemade telescope. He received a Bachelor Much of Schneider’s interdisciplinary Coming of age within a traditional of Science degree in mechanical engineering research focused on how to model and better scientific paradigm in which objectivity in 1966 and a PhD in plasma physics in 1971, understand Earth’s interconnected systems, was considered to be compromised by both from Columbia University. While at including human society. He also strove to outreach, Steve demonstrated that scientists Columbia, several experiences pushed him quantify the risks posed by climate change can also speak as citizens. His own outreach towards the study of climate change. using probabilistic methods. He founded the efforts made him an extensive world traveller, In 1968, the university was the stage for interdisciplinary journal Climatic Change and despite a packed schedule, he always violent protests against the institution’s in 1975, which focuses on research relevant found time to fit in birdwatching forays to affiliation with defence research to policy, and served as editor-in-chief add to his ‘life list’ or to sample wine from organizations that supported the Vietnam until his death. He strongly believed that a nearby vineyard with his wife and frequent War. Previously uninvolved in university scientists should provide policy-makers with scientific collaborator Terry Root. The politics, Schneider became a mediator information about the relative likelihood of importance of living life with purpose between students and trustees. He learned possible outcomes and their consequences. and passion is a legacy he leaves to all to consider differing views and to search for In his view, policy-makers could then use who knew him. common values on which solutions could this knowledge to make informed decisions michael D. mastrandrea be built. Then in 1970, the first Earth Day about which risks are acceptable — value Michael D. Mastrandrea is the deputy director, (originally created by US senator Gaylord judgements that extend beyond the answers science, for the Intergovernmental Panel on Nelson to inspire awareness and appreciation scientific research can provide. As he Climate Change Working Group II, and is also of the environment) impressed on him described in his 2005 book, The Patient at the Woods Institute for the Environment, the importance of climate change as an From Hell, Schneider applied this kind Stanford University, Stanford, California 94305, environmental and societal problem, as of ‘risk assessment’ to his own treatment USA. well as an emerging field of research. options when diagnosed with mantle cell e-mail: [email protected]

Type IIA topoisomerase inhibition by a new class of antibacterial agents

Benjamin D. Bax1, Pan F. Chan2, Drake S. Eggleston3{, Andrew Fosberry3, Daniel R. Gentry2, Fabrice Gorrec3{, Ilaria Giordano4, Michael M. Hann1, Alan Hennessy4, Martin Hibbs3, Jianzhong Huang2, Emma Jones3, Jo Jones3, Kristin Koretke Brown5, Ceri J. Lewis3, Earl W. May2{, Martin R. Saunders1, Onkar Singh1, Claus E. Spitzfaden1, Carol Shen2, Anthony Shillings3, Andrew J. Theobald3, Alexandre Wohlkonig4{, Neil D. Pearson2 & Michael N. Gwynn2

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A˚ crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor ‘bridges’ the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Bacterial type IIA topoisomerases (DNA gyrase and topoisomerase region of GyrB and the amino-terminal region of GyrA) contains the IV (or topo IV)) are the targets of the quinolone antibiotics that have regions required to bind and cleave DNA13,14, and residues from B9 been in use since 19621. The emergence of multidrug-resistant strains TOPRIM and A9 domains of different subunits come together to form of bacteria—such as methicillin-resistant S. aureus (MRSA)—and the catalytic site(s)12,15,16. However, structural insights into the mech- the lack of new classes of antibacterial agents in advanced clinical anism of cleavage (and religation) of the G segment have remained development is a growing threat2–4. Bacterial type IIA topoisomerases elusive5, as all available structures have DNA bound in the cleaved state are A2B2 tetramers that cleave and reseal DNA to transform its topo- and cleavage/religation-competent configurations have not been logy, including, in the case of DNA gyrase, the introduction of nega- reported8,12. tive supercoils5,6. Covalent bond formation between a tyrosine from We created a new class of type IIA topoisomerase inhibitor17, novel the GyrA subunit7 of DNA gyrase and a 59 phosphate generates a bacterial topoisomerase inhibitors (NBTIs), and other researchers 4-base-pair (bp) staggered break in the DNA, allowing the passage of have recently described further NBTI class compounds18–20. Unlike another DNA duplex through this break5. Quinolones stabilize the fluoroquinolones, inhibition by this class is generally not associated cleaved form of the DNA, and a recent 4 A˚ crystal structure with with stabilization of double-strandedly cleaved DNA complexes19,20. Streptococcus pneumoniae topo IV showed two quinolone molecules We determined the 2.1 A˚ crystal structure of an NBTI in a pre-cleavage binding in the cleaved DNA, preventing the free 39 hydroxyls from complex with S. aureus DNA gyrase and a 20-bp DNA duplex, as well attacking the phosphotyrosines to religate the DNA8. No direct inter- as a 3.35 A˚ structure complex with a fluoroquinolone (ciprofloxacin), action between quinolone and protein was evident. However, at 4 A˚ giving detailed structural insight into how DNA is cleaved by type IIA this is the only liganded structure, antibacterial or anticancer, topoisomerases, including details of the role of the metal-binding reported for this drug target class in complex with DNA. The flexibility TOPRIM domains in catalysis21. and complexity of the enzymes have been a considerable challenge for structural studies of drug action. Target inhibition and antibacterial activity of GSK299423 Structural studies of type IIA topoisomerases have revealed three GSK299423 (Fig. 1), an NBTI that is structurally and mechanistically protein ‘gates’ (Supplementary Fig. 1) that can open and close to allow distinct from fluoroquinolones, is derived from a chemical series the passage of the transport- (T) segment9–11 DNA duplex, and have originating from an unbiased antibacterial screen17. GSK299423 shown how the gate (G) segment is bent by the enzyme12. In yeast Topo shows potent inhibition of supercoiling by DNA gyrase from S. aureus II, the central B9A9 region (corresponding to the carboxy-terminal (half-maximum inhibitory concentration (IC50)of146 5 nM;

1Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK. 2Antibacterial Discovery Performance Unit, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline, 1250 Collegeville Road, Collegeville, Pennsylvania 19426, USA. 3Molecular Discovery Research, GlaxoSmithKline, Third Ave, Harlow, Essex, CM19 5AW, UK. 4Antibacterial Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. 5Molecular Discovery Research, GlaxoSmithKline, 1250 Collegeville Rd., Collegeville, Pennsylvania 19426, USA. {Present addresses: Innovalyst, 1000 Centre Green Way, Suite 200, Cary, North Carolina 27513, USA (D.S.E.); MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK (F.G.); OSI Pharmaceuticals, 1 Bioscience Park Drive, Farmingdale, New York 11735, USA (E.W.M.); Vrije Universiteit Brussel, VIB Department of Molecular and Cellular Interactions, Pleinlaan 2, 1050 Brussels, Belgium (A.W.). 935 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010

Optimization and crystallization of a protein–DNA–NBTI complex O O O F To understand the inhibitory function of GSK299423, we determined OH a2.1A˚ crystal structure in complex with DNA and S. aureus DNA GSK299423 N N N gyrase. Briefly, efforts to generate a particle suitable for crystallization HN N O went through several iterations. First, the translational fusion of the N N C-terminal region of GyrB to the N-terminal region of GyrA (GyrB27– Ciprofloxacin S A56). An apo crystal structure of this construct was determined at 3 A˚ N (Fig. 2c). Second, mutation of the catalytic Tyr 123 to a phenylalanine (GyrB27–A56(Tyr123Phe)) and the substitution of Mn21 for Mg21; Figure 1 | The chemical structures of GSK299423 and ciprofloxacin. each increased markedly the stability of the ternary complex in our gel filtration assay (Supplementary Fig. 6). GyrB27–A56(Tyr123Phe) in Supplementary Fig. 2) and Escherichia coli (IC , 100 nM; Sup- 50 complex with a 20-bp DNA homoduplex23 with optimized sequence plementary Fig. 3); the IC was not affected by a GyrA(Ser83Leu) 50 and GSK299423 provided a structure at 3.5 A˚ showing the binding site mutation in E. coli DNA gyrase. Fluoroquinolone resistance in diverse for the compound (see Supplementary Table 2 and Supplementary pathogens is most commonly associated with mutations in this residue Fig. 7 for crystallographic details). Although we do not rule out the in DNA gyrase and topo IV, and the ciprofloxacin control showed a possibility that a crystal structure of GSK299423 liganding the cataly- 40-fold increase in IC50 for the mutant E. coli enzyme. In DNA-cleavage tically active Tyr-123 construct is achievable, we have not been suc- experiments, ciprofloxacin stabilized characteristic double-strand cessful so far, possibly owing to the heterogeneous states resulting from breaks with S. aureus DNA gyrase, whereas GSK299423 did not, but the single-strand cleavage equilibrium that exist in the inhibition com- markedly differed in generating single-strand breaks (Supplemen- plex. Use of the Tyr123Phe construct enabled us to stabilize a homo- tary Fig. 4). The single-strand breaks seem to be associated with cova- genous pre-cleavage conformation trapped by the inhibitor, with a lent phosphotyrosyl-protein–DNA complex formation as they were construct that was otherwise demonstrated to be cleavage competent. 22 reversible by salt and EDTA buffer treatment (Supplementary Fig. Last, the small, flexible Greek key (or tail) domain (residues 544–579) 5). We observed the induction of single-strand DNA breaks to varying in the GyrB subunit, which did not make contact with the DNA in the degrees for other inhibitor analogues in this structural class, with both 3.5 A˚ structure, was deleted (GKdel) and replaced with two amino S. aureus and E. coli DNA gyrases. acids to give a GyrB27–A56(GKdel/Tyr123Phe) construct that signifi- GSK299423 was approximately 70 times more potent against cantly improved resolution to 2.1 A˚ (the Greek key domain may be S. aureus DNA gyrase than reported for NXL101, an NBTI class com- involved in T-segment navigation24). The fused protein with the Greek pound that progressed to human trials20. It was also over 2,000 times key deleted retained DNA cleavage activity comparable to wild-type S. more potent than ciprofloxacin (IC50,316 10 mM) for inhibition of aureus DNA gyrase when the active site Tyr 123 was intact (Sup- S. aureus DNA gyrase supercoiling. In S. aureus, fluoroquinolones are plementary Fig. 4), and bound short pieces of duplex DNA in the usually more active against topo IV than DNA gyrase, and highly lethal presence of NBTI (and not in the absence of NBTI) (C.E.S., S. double-stranded DNA breaks are generated by quinolone inhibition; Brooks and A. West, unpublished results). nevertheless, the antibacterial activity of GSK299423 against S. aureus and MRSA, against which marketed fluoroquinolones have limited Structure of S. aureus DNA gyrase–DNA–NBTI complex utility owing to widespread resistance, is particularly notable. The course of the DNA in the S. aureus complex (Fig. 2) is broadly Furthermore, GSK299423 has potent antibacterial activity against a similar to the yeast Topo II complex with a double-nicked 34-bp broad spectrum of Gram-positive and Gram-negative bacterial patho- duplex of DNA12. The GyrA Ile 175 residue sits in between base-pairs gens, including clinical isolates with fluoroquinolone resistance C16 and C17, bending the DNA in a similar way to that seen in the mediated by DNA gyrase and topo IV mutations (Supplementary yeast Topo-II–DNA complex12. We observed more interactions with Table 1). Mutations conferring high-level resistance to fluoroquino- the DNA (Fig. 3a) in the S. aureus complex than those seen in the yeast lones in an isogenic S. aureus strain also did not affect GSK299423 complex, and the CAP (or winged helix, WHD) domains are in susceptibility. Laboratory-selected DNA gyrase mutations conferring slightly different (,4A˚ ) relative positions, making different interac- resistance to NBTIs20 are distinct from, but proximal to, mutations tions with the DNA (Supplementary Fig. 8). Comparison of these two conferring fluoroquinolone resistance, reflecting the closeness of the structures indicates that for DNA cleavage, the correct positioning of respective inhibitor binding sites as revealed here. The lack of cross- the catalytic tyrosine on the CAP domain with respect to the TOPRIM resistance with fluoroquinolones and activity against multidrug- domain seems to be achieved by a rigid body-domain movement. resistant hospital and community pathogens, including MRSA, is a The GyrA dimer interface in the apo S. aureus gyrase structure is key attribute of the NBTI class. similar to E. coli and Mycobacterium tuberculosis GyrA59 apo

abc

90º

Figure 2 | The GyrB27–A56 fusion protein. a, b, Orthogonal views of 2.1 A˚ the complex the a3 helices slide approximately 6 A˚ past each other (indicated complex with GSK299423 (yellow, solid) and DNA (green). One fused by red arrows) to align the red stripes (Ala 68 and Gly 72) underneath the GyrB27–A56(GKdel/Tyr123Phe) subunit is shown in grey, the other in red compound (in b). Molecular figures generated with Pymol40. (GyrB) and blue (GyrA). c,3A˚ apo GyrB27–A56 structure. On formation of 936 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 ARTICLES

a D508 G82 Y123 E435 H46 R238 G178 R K N463 R471 R122 2+ H79 K43 H S438 Mn H81 R92 R272 K460 R458 3′ 5′ 19 18 12 9 8 321 CCGG AATT CCGGG GCC GA

A G C CCGGGT AAG CCC TTGG 21 3 8 9 18 19 20

5′ 3′ R458 K R92 Mn2+ S438 H R272 H81 N463 K G178 K43 H79 Y123 R122 R471 R R238 S330 H46 G82 D508E435 b c

S84 Mn2+ D83 Mn2+ M121 M121 Y123F M121

M121 M75 M75 M75 A68 V71 V71 G72

Figure 3 | The 2.1 A˚ GyrB27–A56 complex with GSK299423 and DNA. is shown in stick representation with GSK299423 (yellow) half-way between a, Schematic diagram showing interactions (,3.2 A˚ )ofS. aureus DNA the two Mn21 ions (purple) at the active sites. The 3.35 A˚ ciprofloxacin gyrase with DNA (dotted lines, side chains; solid lines, main chain). structure (black lines) is shown superposed with ciprofloxacins in GSK299423 is indicated by a yellow rectangle. Interactions from one spacefilling (grey carbons) representation, and Mn21 ions are shown in covalently fused subunit are coloured by domain (as in Supplementary Figs black. c, The oxathiolopyridine ring of GSK299423 sits in a largely 7 and 8), whereas those from the other subunit are in black. The arrows hydrophobic pocket at the dimer interface. indicate where Tyr 123 would cleave DNA. b, The 2.1 A˚ GSK299423 complex dimers11,25,26, but on binding DNA the GyrA subunits slide some 6 A˚ of Ala 68, indicating that the CH2 group is acting as an unusual C–H past one another (Fig. 2c), decreasing the distance between the two hydrogen-bond donor27. This interaction may explain the structure- catalytic tyrosines from 29.3 A˚ in the apo structure to 23.2 A˚ in the activity relationship (SAR) showing that compounds with electron- GSK299423 complex (Ca–Ca distances). Of importance for the withdrawing groups adjacent to the methylene are more potent19.At inhibition mechanism the DNA is stretched and slightly untwisted the entrance to the protein pocket a basic nitrogen from GSK299423 between the two active sites, the scissile phosphates being some 25.9 A˚ makes an interaction with Asp 83, and mutations of Asp 83 and apart versus 15–21 A˚ apart in A or B DNA (Fig. 3 and Supplementary Met 121 have been reported to affect the activity of NBTI class com- Table 3), and the relative translation of the GyrA subunits opens up pounds20, consistent with data we have generated for GSK299423 the non-catalytic pocket used by the inhibitor on the two-fold axis at itself. The novel binding mode provides a structural basis for the GyrA dimer interface (Figs 2 and 3c). why NBTIs are able to overcome target-mediated fluoroquinolone resistance28. The binding mode of NBTIs GSK299423 binds midway between the two active sites, with the Comparison of the NBTI- and quinolone-binding sites quinoline-carbonitrile group sitting in between the two central base A 3.35 A˚ crystal structure (twinned) of the S. aureus GyrB27– pairs of the stretched DNA (Fig. 3) and the oxathiolo-pyridine group A56(GKdel/Tyr123Phe) construct with a 20-bp double-nicked occupying the non-catalytic pocket that opens up between the two DNA and ciprofloxacin (Supplementary Fig. 7) has electron density GyrA subunits (Fig. 3c). The structure indicates that the compound next to Ser 84, into which we can model ciprofloxacin in a similar does not directly inhibit the DNA cleavage–religation reaction as it is position to that reported for moxifloxacin (ref. 8) but in a different not bound in the active sites, and we observed some single-strand orientation (Supplementary Fig. 14), consistent with our further cleavage (Supplementary Fig. 4). The compound stabilizes a pre- work to be reported (A.W. et al., submitted). Two quinolones sit in cleavage enzyme–DNA complex (Supplementary Fig. 6) and inhibits the DNA between bases 8 and 9 (numbered as in Fig. 3a). The binding strand separation. site for the NBTI is close to, but does not overlap with, the two The pocket between the two GyrA subunits occupied by the quinolone binding sites (Fig. 3b). Comparison of the 3.35 A˚ cipro- oxathiolo-pyridine group (Fig. 3c) is largely hydrophobic, with resi- floxacin crystal structure with the GSK299423 structures shows a dues Ala 68, Gly 72, Met 75 and Met 121 making van der Waals inter- rigid body rotation of some 9u at the GyrA dimer interface, slightly actions with the compound. Sequence analysis shows that this pocket increasing the separation between the two halves of the double- is highly conserved in GyrA and ParC (topo IV) from Gram-negative strandedly cleaved DNA in the ciprofloxacin structure (Supplemen- and Gram-positive bacteria but not in eukaryotes (Supplementary tary Fig. 14); this is consistent with NBTIs stabilizing a conformation Fig. 9b). The methylene group at the ‘bottom’ of the compound preceding generation of double-stranded DNA breaks (ref. 20 and (Fig. 3c) is some 3.2 A˚ away from the main-chain carbonyl oxygen Supplementary Fig. 4). 937 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010

˚ Two metal-binding positions 2.8 A apart at each active site DNA DNA a b O O The 2.1 A˚ S. aureus crystal structure liganded with GSK299423 shows Glu B435 Glu B435 the first view of this class of enzyme in a cleavage-competent config- – 2.1 O O O uration in complex with uncleaved DNA—giving detailed insights 2.7 GyrB 2.4 O + NH2 2.3 P H2N into the cleavage–religation mechanism of type IIA topoisomerases. 2.1 2+ – 2.0 H O – In the complex, the catalytic tyrosine (Tyr 123) has been mutated to a 2 Mn O O O O HN phenylalanine so that cleavage cannot occur and this residue is point- O O Asp B508 H O Arg A′122 ing directly at the scissile phosphate (Fig. 4a and Supplementary 2 1 Asp B510 O– 2 GyrA′ Fig. 10). The single Mn ion we observe is at a position not previ- Asp B510 Tyr A′123 ously demonstrated structurally, 2.7 A˚ from the bridging 39 oxygen, and is positioned to stabilize the developing negative charge as the 39 Figure 5 | Metal-binding site associated with cleavage of DNA by type IIA 1 2 topoisomerases. a, Simulated annealing omit map (Fo 2 Fc) in which waters oxygen becomes the leaving group (Fig. 5a, b). The other five Mn 21 21 coordinating oxygens (from Glu B435, Asp B508, a non-bridging and residues coordinating the Mn ion were omitted but the Mn was included (contoured at 5s; 5,000K simulated annealing refinement). oxygen from the scissile phosphate and two waters) are between b ˚ ˚ 21 ˚ , Diagrammatic depiction of the pre-cleavage complex as in the 2.1 A crystal 2.3 and 2.0 A from the Mn ion (close to the expected ,2.15 A structure, with the Tyr oxygen modelled onto the mutant Phe (Tyr A9123).

a A7 21 G8 G9 G10 (ref. 29)). The interaction between the Mn ion and the 39 oxygen K460 has been predicted from biochemical experiments and ‘‘greatly accelerates rates of enzyme-mediated DNA cleavage’’30. We name this ˚ E435 newly observed metal binding site in the 2.1 A pre-cleavage structure R33 D512 the ‘39 site’. In our ciprofloxacin structure with cleaved DNA we also observe a R122 single Mn21 ion at each active site, but its position is approximately 2.8 A˚ from that seen in the GSK229423 complex (Fig. 3b and D510 D508 Supplementary Fig. 11). We observe different orientations for the Y123F side chain of Asp B508 depending on which position it is coordinat- b ing the metal ion in (Supplementary Fig. 12). The metal position in our ciprofloxacin structure corresponds to that observed in the yeast complex with DNA12 (Fig. 4b), and we name this the ‘Y site’, as it is close to the catalytic tyrosine in the yeast complex (Supplementary E449 Fig. 12). In both of these structures the DNA is double-strandedly R690 D530 nicked and the scissile phosphate is absent from the DNA. These structures indicate that when the phosphotyrosine reapproaches Y782 the active site before religation, a single metal ion will be present at the Y site (the two halves of the DNA that separate to allow passage of D528 D526 the T segment are differently shaded in Fig. 3a). 30–32 c Biochemical experiments have led to the paradigm that type IIA topoisomerases use two metal ions at the active site and that both must be present for cleavage or religation of the DNA backbone30. We have demonstrated with two distinct, trapped conformations of the same enzyme construct—cleaved and uncleaved DNA, respectively—that there are indeed two metal-binding positions at the cleavage–ligation active site, but our structural studies have not yet shown both sites occupied at the same time. An intriguing possibility is a mechanism involving the movement of a single metal33 between the two sites, ,2.8 A˚ from the 39 site to the Y site, as the scissile phosphate inverts and cleavage proceeds (Supplementary Discussion). d Type 1A topoisomerases cleave a single DNA strand to modify DNA topology, and have a TOPRIM domain and a catalytic tyrosine on a CAP domain34 at the active site of a protein with an otherwise unrelated structure to type IIA topoisomerases. The similarity of the R330 E7 active sites between type IA (Fig. 4d) and type IIA (Fig. 4a, b) topoisomerases, together with their ability to form 59-phosphotyrosine K8 linkages and a requirement for metal ions for full activity, ‘‘indicates that type IA and type IIA topoisomerases evolved from a common ancestor and probably utilize similar mechanisms’’12. Several confor- D105 D103 Y328F mations of type IA topoisomerases have been observed with bound DNA35,36 but, surprisingly, metal binding sites have not yet been Figure 4 | Comparison of topoisomerase active sites. a, The active site of identified. The remarkable similarity between the configurations of the 2.1 A˚ S. aureus complex. The Mn21 ion is the purple sphere. b, The active site in the 3 A˚ yeast Topo II complex12 (PDB accession 2RGR). The Mg21 ion the three evolutionarily conserved acidic side chains on the TOPRIM is the green sphere. c, The structures in a and b are superposed based on the domains in the pre-cleavage conformation of S. aureus gyrase and the TOPRIM domains, the metal ions are 2.7 A˚ apart. d, The active site of a pre- type IA topoisomerase (compare Fig. 4a and d) indicates that type 1A cleavage complex of type IA topoisomerase35 (PDB accession 1I7D). No topoisomerases probably use catalytic metals in a similar manner to metals were observed adjacent to the TOPRIM domain. type IIA topoisomerases. 938 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 ARTICLES

Is the Asp B512–Arg A33 ion pair a conformational switch? METHODS SUMMARY The three acidic residues (Glu B435, Asp B508 and Asp B510) that we GSK299423 synthesis is described in Supplementary Methods. Antibacterial observed coordinating the metal ion directly (or through waters) are activity (minimum inhibitory concentrations) was determined as described in conserved in all TOPRIM domains21. However, the TOPRIM Supplementary Methods. domains of type IIA topoisomerases have a fourth conserved acidic S. aureus GyrB–A fusion construct production and activity. An initial gene fusion residue, Asp B512 in S. aureus GyrB (Supplementary Table 4), which construct encoding GyrB27–A56 was made from a full-length gyrB–gyrA fusion by 31 deleting the N terminus of GyrB and the C terminus of GyrA using polymerase is implicated in cleavage–religation and metal-ion coordination . chain reaction (PCR). Three further constructs were derived by mutating this initial But in the S. aureus complex, Asp B512 points away from the catalytic gyrB27–A56 fusion construct to express GyrB27–A56(Tyr123Phe), GyrB27– site and makes an inter-subunit ion pair with Arg A33 (Fig. 4a), and a A56(GKdel), or GyrB27–A56(GKdel/Tyr123Phe) (constructs in which the catalytic similar interaction is seen in the yeast Topo II structure with DNA12,15 Tyr was mutated to Phe, and/or the 37-residue Greek key domain was deleted and (Asp 530–Arg 690; Fig. 4b). In our 2.1 A˚ ternary complex structure, replaced with two amino acids). The four (untagged) constructs were expressed in the peptide plane between residues A32 and A33 has been rotated so E. coli and proteins were purified as in Supplementary Methods. S. aureus GyrB27– that Arg A33 has Q-y angles (2165u, 269u) outside the normal A56 fusion protein constructs were assayed in gel shift assays as described in allowed regions of the Ramachandran plot. This conformational Supplementary Methods. change alters the angle that the a1 helix makes with the CAP domain, Complex analysis, optimization and purification. Complex formation with protein, GSK299423 and DNA duplex (12–37 bp) was assayed, in a variety of a and may contribute to the observed ordering of the 1 helix and the buffer conditions, by analytical size-exclusion chromatography (measuring rest of the linker region (B611–A30) in the ternary complex. absorption at 280 nm and 254 nm). Stable complexes of GSK299423, a 20-bp In our S. aureus DNA gyrase apo structure, this Arg A33–Asp B512 DNA homoduplex (palindrome) and either GyrB27–A56(Tyr123Phe), or ion pair is not observed. Arg A33, at the end of the first helix a1of GyrB27–A56(GKdel/Tyr123Phe) were purified on a gel filtration column in a GyrA, has normal main-chain Q-y angles, and the linker region buffer containing 5 mM MnCl2. (B611–B644 and A2–A26) and several loop regions pointing into Crystallization, data collection and structure determination. Diffraction data the DNA binding canyon are disordered (Supplementary Fig. 8). If were collected on single frozen crystals at the European Synchrotron Radiation the side chain of Asp B512 rotates at the end of the catalytic cycle to Facility (ESRF). Initially the apo-GyrB27–A56 structure was solved by molecular point towards the metal-binding centre (Fig. 4a), this would break replacement and refined. Complexes of GyrB27–A56(Tyr123Phe) and GyrB27– the Asp B512–Arg A33 ion pair and could contribute to the disorder- A56(GKdel/Tyr123Phe) with DNA and GSK299423 (Supplementary Figs 13 and 15) were determined by molecular replacement using domains from the apo- ing of the S. aureus DNA gyrase linker region (B611–B644, A2–A28) GyrB27–A56 structure and refined. The complex with ciprofloxacin was deter- and release of the religated DNA at the end of the catalytic cycle. mined by molecular replacement using domains from the 2.1 A˚ GSK299423 Thus, the Asp B512–Arg A33 ion pair may act as a conformational structure and refined (see Supplementary Table 2 for details). switch to help communicate information about the state of cleavage of the DNA from the active sites to the rest of the protein37. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Conclusions Received 11 October 2009; accepted 20 May 2010. The 2.1 A˚ NBTI complex provides new insights at unprecedented Published online 4 August 2010; corrected 19 August 2010 (see full-text HTML resolution into the complex catalytic cycle of a highly flexible molecu- version for details). lar machine, a type IIA topoisomerase. The conformation revealed 1. Mitscher, L. A. Bacterial topoisomerase inhibitors: quinolone and pyridone includes structural demonstration of a second metal-binding site antibacterial agents. Chem. Rev. 105, 559–592 (2005). involved in the TOPRIM domain nucleotidyl phosphotransferase. 2. Talbot, G. H. et al. Bad bugs need drugs: an update on the development pipeline The structure is consistent with our biochemical demonstration that, from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clin. Infect. Dis. 42, 657–668 (2006). unlike the fluoroquinolones, the stabilized equilibrium state has 3. Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! An update from the uncleaved and single-strandedly cleaved DNA. In our NBTI complex, Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009). the uncleaved DNA is stretched and untwisted between the two active 4. Payne, D. J., Gwynn, M. N., Holmes, D. J. & Pompliano, D. L. Drugs for bad bugs: sites. In the absence of inhibitor the DNA is likely to be similarly confronting the challenges of antibacterial discovery. Nature Rev. Drug Discov. 6, 29–40 (2007). stretched just before double-strand cleavage is completed, but the 5. Schoeffler, A. J. & Berger, J. M. DNA topoisomerases: harnessing and constraining NBTI inhibits both the double-strand DNA cleavage and the sub- energy to govern chromosome topology. Q. Rev. Biophys. 41, 41–101 (2008). sequent large conformational change. The NBTI GyrA binding site 6. No¨llmann, M. et al. Multiple modes of Escherichia coli DNA gyrase activity is well conserved in bacteria and is distinct from the fluoroquinolone revealed by force and torque. Nature Struct. Mol. Biol. 14, 264–271 (2007). binding sites, consistent with the observed potent antibacterial activity 7. Horowitz, D. S. & Wang, J. C. Mapping the active site tyrosine of Escherichia coli DNA gyrase. J. Biol. 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Crystal structure of the breakage-reunion domain of development for serious infections is a cause for growing medical DNA gyrase. Nature 388, 903–906 (1997). concern; more people now die of MRSA infection in US hospitals 12. Dong, K. C. & Berger, J. M. Structural basis for gate-DNA recognition and bending by type IIA topoisomerases. Nature 450, 1201–1205 (2007). than of human immunodeficiency virus (HIV)/AIDS and tuber- 13. Gellert, M., Fisher, L. M. & O’Dea, M. H. DNA gyrase: purification and catalytic culosis combined3, and we are actively exploring NBTIs for treatment properties of a fragment of gyrase B protein. Proc. Natl Acad. Sci. USA 76, of Gram-positive and Gram-negative infections. Anti-type-IIA- 6289–6293 (1979). topoisomerase therapeutics have important roles as medicines for 14. Reece, R. J. & Maxwell, A. Probing the limits of the DNA breakage-reunion domain 38 of the Escherichia coli DNA gyrase A protein. J. Biol. Chem. 266, 3540–3546 treating infections and for cancer chemotherapy , and determina- (1991). tion of the adjacent binding sites for ciprofloxacin and NBTIs opens 15. Liu, Q. & Wang, J. C. Identification of active site residues in the ‘‘GyrA’’ half of up a structural basis for new strategies to develop drugs that target the yeast DNA topoisomerase II. J. Biol. Chem. 273, 20252–20260 (1998). central DNA-cleaving gate. 16. Liu, Q. & Wang, J. C. Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases. Proc. Natl Acad. Sci. USA 96, 881–886 Note added in proof: While in proof a structure of S. cerevisiae Topo II (1999). with DNA was published showing two Zn ions in comparable posi- 17. Coates, W. J. et al. Preparation of Piperidinylalkylquinolines as Antibacterials. tions to those we demonstrate with Mn ions39. European patent 1051413 (1999). 939 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010

18. Gomez, L. et al. Novel pyrazole derivatives as potent inhibitors of type II 37. Mueller-Planitz, F. & Herschlag, D. Coupling between ATP binding and DNA topoisomerases. Part 1: synthesis and preliminary SAR analysis. Bioorg. Med. cleavage by topoisomerase II: a unifying kinetic and structural mechanism. J. Biol. Chem. Lett. 17, 2723–2727 (2007). Chem. 283, 17463–17476 (2008). 19. Wiener, J. J. M. et al. Tetrahydroindazole inhibitors of bacterial type II 38. Nitiss, J. L. Targeting DNA topoisomerase II in cancer chemotherapy. Nature Rev. topoisomerases. Part 2: SAR development and potency against multidrug- Cancer 9, 338–350 (2009). resistant strains. Bioorg. Med. Chem. Lett. 17, 2718–2722 (2007). 39. Schmidt, B. H., Burgin, A. B., Deweese, J. E., Osheroff, O. & Berger, J. M. A novel 20. Black, M. T. et al. Mechanism of action of the antibiotic NXL101, a novel and unified two-metal mechanism for DNA cleavage by type II and 1A nonfluoroquinolone inhibitor of bacterial type II topoisomerases. Antimicrob. topoisomerases. Nature 465, 641–645 (2010). Agents Chemother. 52, 3339–3349 (2008). 40. DeLano, W. L. The PyMOL Molecular Graphics System. Æhttp://www.pymol.orgæ 21. Aravind, L., Leipe, D. D. & Koonin, E. V. Toprim—a conserved catalytic domain in (2008). type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and Supplementary Information is linked to the online version of the paper at RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998). www.nature.com/nature. 22. Pan, X.-S., Gould, K. A. & Fisher, L. M. Probing the differential interactions of quinazolinedione PD 0305970 and quinolones with gyrase and topoisomerase Acknowledgements We thank H. Hiasa, A. Maxwell, J. C. Wang and D. B. Wigley IV. Antimicrob. Agents Chemother. 53, 3822–3831 (2009). for discussions. We thank S. Erskine, A. West and S. Brooks for experimental work 23. Gmunder, H., Kuratli, K. & Keck, W. In the presence of subunit A inhibitors DNA that helped initiate this project. We thank P. Rowland for help with data collection gyrase cleaves DNA fragments as short as 20 bp at specific sites. Nucleic Acids and processing, the antimicrobial profiling group at GlaxoSmithKline (GSK) for Res. 25, 604–611 (1997). antibacterial data, and K. Smith and D. Payne and members of the project team for 24. Fu, G. et al. Crystal structure of DNA gyrase B9 domain sheds lights on the discussions. A.W. was supported by the Wellcome Trust Seeding Drug Discovery mechanism for T-segment navigation. Nucleic Acids Res. 37, 5908–5916 (2009). Initiative and contract HDTRA1-07-9-0002 with the US Department of Defense 25. Edwards, M. J. et al. A crystal structure of the bifunctional antibiotic (DoD) Joint Science and Technology Office for Chemical and Biological Defense simocyclinone D8, bound to DNA gyrase. Science 326, 1415–1418 (2009). (JSTO-CBD), and the Defense Threat Reduction Agency (DTRA) Transformational 26. Tretter, E. M., Schoeffler, A. J., Weisfield, S. R. & Berger, J. M. Crystal structure of Medical Technologies (TMT). The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the DoD or the DNA gyrase GyrA N-terminal domain from Mycobacterium tuberculosis. the US Government. Proteins 78, 492–495 (2010). 27. Jeffrey, G. A. & Saenger, W. Hydrogen Bonding in Biological Structures. (Springer, Author Contributions M.N.G. and D.R.G. defined the relevant domains for 1991). crystallography by mutational analysis. K.K.B. performed gene sequence analysis 28. Tanaka, M., Wang, T., Onodera, Y., Uchida, Y. & Sato, K. Mechanism of quinolone and designed B27–A56 translation fusion. J.H. and E.W.M. made the initial fusion resistance in Staphylococcus aureus. J. Infect. Chemother. 6, 131–139 (2000). constructs, and tested them for activity. J.H., C.S. and P.F.C. performed studies with 29. Bock, C. W., Katz, A. K., Markham, G. D. & Glusker, J. P. Manganese as a GSK299423 on target potency and the inhibition mechanism and demonstrated replacement for magnesium and zinc: functional comparison of the divalent ions. the lack of cross-resistance with quinolones. B.D.B. and M.R.S. designed the Greek J. Am. Chem. Soc. 121, 7360–7372 (1999). key deletion. A.F. made the Greek-key-deletion and catalytic Tyr-to-Phe 30. Deweese, J. E., Burgin, A. B. & Osheroff, N. Human topoisomerase IIa uses a two- constructs. J.J. optimized expression in fermentors and grew cells for purification. metal-ion mechanism for DNA cleavage. Nucleic Acids Res. 36, 4883–4893 M.H., E.J., A.S. and A.F.T. purified apo protein and complexes of various constructs (2008). for crystallography and assay. I.G. and A.H. designed and synthesized the 31. Noble, C. G. & Maxwell, A. The role of GyrB in the DNA cleavage-religation compound. C.E.S. performed analytical size exclusion chromatography reaction of DNA gyrase: a proposed two metal-ion mechanism. J. Mol. Biol. 318, experiments that were used to find a stable particle that could be crystallized. F.G. 361–371 (2002). crystallized apo and GSK299423 complex in fluidigm chips. O.S. and A.W. grew 32. Sissi, C. & Palumbo, M. Effects of magnesium and related divalent metal ions in quinolone crystals by microbatch. B.D.B. grew apo and GSK299423 complex topoisomerase structure and function. Nucleic Acids Res. 37, 702–711 (2009). crystals by vapour diffusion, solved and refined structures. C.J.L. lead biochemistry. 33. Oezguen, N. et al. A ‘‘Moving Metal Mechanism’’ for substrate cleavage by the N.D.P. led chemistry and provided compound. D.S.E., M.N.G. and N.D.P. initiated DNA repair endonuclease APE-1. Proteins 68, 313–323 (2007). and led the project. B.D.B. and M.N.G. wrote the manuscript with the assistance of 34. Berger, J. M., Fass, D., Wang, J. C. & Harrison, S. C. Structural similarities between M.M.H. and the other authors. topoisomerases that cleave one or both DNA strands. Proc. Natl Acad. Sci. USA 95, Author Information Atomic coordinates and structure factors for the reported 7876–7881 (1998). structures have been deposited in the Protein Data Bank (PDB) under accession 35. Changela, A., DiGate, R. J. & Mondragon, A. Crystal structure of a complex of a codes 2XCO, 2XCQ, 2XCR, 2XCS and 2XCT. Reprints and permissions information type IA DNA topoisomerase with a single-stranded DNA molecule. Nature 411, is available at www.nature.com/reprints. The authors declare no competing 1077–1081 (2001). financial interests. Readers are welcome to comment on the online version of this 36. Changela, A., DiGate, R. J. & Mondragon, A. Structural studies of E. coli article at www.nature.com/nature. Correspondence and requests for materials topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA should be addressed to B.D.B. ([email protected]) or M.N.G. conformational intermediate. J. Mol. Biol. 368, 105–118 (2007). ([email protected]).

43,44 METHODS when expressed as a fusion of the two subunits . A full-length gyrase B–A fusion construct was made minus the N-terminal Met of GyrA. The subsequent Synthesis of GSK299423. GSK299423, or 6-(methyloxy)-4-(2-{4-[([1,3]oxathiolo S. aureus truncated gyrB27–A56 fusion was PCR amplified using the full-length [5,4-c]pyridin-6-ylmethyl)amino]-1-piperidinyl}ethyl)-3-quinolinecarbonitrile gyrB–A-fusion construct as template and the following oligonucleotide primers: dihydrochloride, was made in three stages. The first was the synthesis of 1,1- forward primer, 59-CGTAAATCAGCcatgGATGTAGCAAGTCTTCCAGG-39; dimethylethyl (1-{2-[3-cyano-6-(methyloxy)-4-quinolinyl]ethyl}-4-piperidinyl) reverse primer, 59-TCTTCTAAATCgaattcctaACCTAATTGGATTTCAGTA carbamate. CG-39. (Gene sequences are denoted by capital letters, and restriction sites of A mixture of 4-ethenyl-6-(methyloxy)-3-quinolinecarbonitrile (20 g, 95.2 mmol, EcoRI/NcoI are underlined.) for synthesis see ref. 41) and 1,1-dimethylethyl 4-piperidinylcarbamate (21.8 g, PCR amplification was performed using Pwo DNA polymerase and the fol- 109 mmol) in dimethylformamide (80 ml) under argon was treated with tetra- lowing conditions: denaturation at 94 uC for 1 min, annealing at 60 uC for 1 min, methylguanidine (0.4 ml) and everything was stirred at ambient temperature extension at 72 uC for 10 min, repeated for 30 cycles. The resulting gyrB27–A56 (,23 uC). After 3 h the reaction was evaporated, treated with toluene (60 ml) and PCR products were purified after agarose gel electrophoresis using a Qiagen gel then evaporated to dryness. The crude was purified by silica chromatography using a extraction kit, digested with EcoRI/NcoI and ligated into pET28a1 vector that 0–10% MeOH/dichloromethane (DCM) gradient to afford compound 1 (37 g, had been digested similarly and calf-intestinal-alkaline-phosphatase- (CIAP) 95%). treated. After transformation into E. coli TOP10 cells, positive clones were iden- Melting point (m.p.): 129–131 uC; high resolution mass spectrometry tified by DNA sequencing to verify the integrity of the insert. (HRMS) (m/z): [M1H]1 calculated for C H N O 411.2396; found 23 31 4 3 A second construct, deleting the Greek key domain of GyrB residues 618–644 411.2376; 1H-NMR (600 MHz, DMSO-d ), d 8.90 (singlet (s), 1H), 8.01 (doublet 6 and replacing the deleted region with Thr-Gly was made using overlap extension (d), coupling constant (J) 5 9.00 Hz, 1H), 7.58 (double doublet (dd), J 5 2.50, PCR to generate PCR fragments either side of the intended region of deletion. 9.00 Hz, 1H), 7.49 (d, J 5 2.50 Hz, 1H), 6.70 (broad d, 1H), 3.97 (s, 3H), 3.45– N-terminal fragment forward primer (1), 59-GcatatgGATGGATGTAGCAAG 3.49 (multiplet (m), 2H), 3.15–3.35 (m, 1H), 2.85–2.96 (m, 2H), 2.61–2.72 (m, 13 TCTTCCAGGTAAATTAGCC-39; N-terminal fragment reverse primer (2), 2H), 2.05–2.15 (m, 2H), 1.61–1.70 (m, 2H), 1.30 (s, 11H); C-NMR (151 MHz, 59-ACCGGTCGGTGGCTGTGCAATATACACATAGCC-39; C-terminal frag- d DMSO-d6): 158.4, 154.8, 151.7, 147.5, 144.2, 131.4, 126.5, 124.6, 117.1, 107.0, ment forward primer (3), 59-GTGTATATTGCACAGCCACCGACCGGTTA 102.9, 77.3, 57.5, 55.8, 52.0, 47.4, 31.8, 28.6, 28.2. CAAAGGTCTTGGAGAAATG-39; C-terminal fragment reverse primer (4), 4-[2-(4-Amino-1-piperidinyl)ethyl]-6-(methyloxy)-3-quinolinecarbonitrile. 1,1- 59-GctcgagCTAACCTAATTGGATTTCAGTACGACG-39. Dimethylethyl(1-{2-[3-cyano-6-(methyloxy)-4-quinolinyl]ethyl}-4-piperidinyl)- PCR was carried out using the above primers, PfuTurbo high-fidelity DNA carbamate (37 g, 90.2 mmol) was dissolved in chloroform (100 ml) and a 4 M polymerase (Invitrogen) and the following conditions: denaturation at 95 uC for solutionof HCl in dioxane was added (150 ml). The reaction was stirred atambient 1 min, annealing at 60 uC for 1 min, extension at 72 uC for 5 min, repeated for 30 temperature (,23 uC) for 2 h and then methanol and toluene were added and the cycles. The resulting PCR products were purified as above, combined in ratios of reaction was evaporated. The residue was dissolved in 10% MeOH/DCM, basified 1:1, 1:3 and 3:1 and used as template to generate the final deletion construct to neutral pH with a saturated solution of sodium bicarbonate and extracted three encoding GyrB27–A56(GKdel) using primers 1 and 4 (see earlier) under the times with 10% MeOH/DCM. Combined organics were dried, filtered and eva- same PCR conditions. The resulting deletion PCR product was purified as porated to afford compound 2 (27 g, 97%). 1 described earlier and subcloned into TOPOTA 2.1 (Invitrogen). Site-directed m.p.: 116–120 uC; HRMS (m/z): [M1H] calculated for C18H23N4O 1 mutagenesis was carried out using the Quickchange SDM kit (Stratagene) and 311.1872; found 311.1862; H-NMR (600 MHz, DMSO-d6): d 8.91 (s, 1H), the following primers were used to introduce a silent change to disable an 8.03 (d, J 5 9.00 Hz, 1H), 7.55 (dd, J 5 2.50, 9.00 Hz, 1H), 7.47 (d, J 5 2.50 internal NdeI restriction site: forward primer (5), 59-GGTATTAGACG Hz, 1H), 3.95 (s, 3H), 3.35–3.45 (m, 2H), 2.60–2.90 (m, 5H), 2.05–2.15 (m, TGCGTATGAAACAGGTCG-39; reverse primer (6), 59-CGACCTGTTTCATAC 13 2H), 1.75–1.85 (m, 2H), 1.35–1.45 (m, 2H); C-NMR (151 MHz, DMSO-d6): d GCACGTCTAATACC-39. 158.8, 152.0, 147.7, 144.4, 131.7, 126.8, 125.0, 117.4, 107.3, 103.1, 57.4, 56.1 51.5, The resulting clones were DNA sequenced to identify clones with the disabled 47.9, 31.7, 28.9. NdeI site, plasmid-DNA isolated (Maxiprep, Qiagen) and digested with the 6-(Methyloxy)-4-(2-{4-[([1,3]oxathiolo[5,4-c]pyridin-6-ylmethyl)amino]-1- restriction enzymes NdeI/XhoI to release the fragment encoding GyrB27– piperidinyl}ethyl)-3-quinolinecarbonitrile dihydrochloride (GSK299423). A56(GKdel). The resulting fragment was ligated into the expression vector 4-[2-(4-Amino-1-piperidinyl)ethyl]-6-(methyloxy)-3-quinolinecarbonitrile pET43b that had been CIAP-treated and NdeI/XhoI-digested. After transforma- (651 mg, 2.1 mmol) and [1,3]oxathiolo[5,4-c]pyridine-6-carbaldehyde (350 mg, tion into E. coli TOP10 cells, pET43b1/ GyrB27–A56(GKdel) constructs were 2.1 mmol, for synthesis see ref. 41) in chloroform (15 ml), methanol (15 ml) and verified as described earlier. o molecular sieves were heated at 80 C for 24 h under argon. The reaction mixture Two further constructs were made by mutating the initial GyrB27–A56 and was cooled down to ambient temperature (,23 uC) and sodium triacetoxybor- GyrB27–A56(GKdel) fusion constructs to change the active site Tyr to Phe using ohydride (900 mg, 4.2 mmol) was added. The reaction was stirred at room tem- site-directed mutagenesis and the following primers: forward primer (7), perature overnight. The solvents were removed and the residue was purified by 59-CAGCAATGCGTTTTACTGAAGCGCG-39; reverse primer (8), 59-CGCG silica chromatography using a 0–20% MeOH/DCM gradient to afford compound CTTCAGTAAAACGCATTGCTG-39. The resulting positive clones encoding 3 (660 mg, 68%). The free base was converted to dihydrochloride salt by treatment GyrB27–A56(Tyr123Phe) or GyrB27–A56(GKdel/Tyr123Phe) were confirmed with excess 1 M HCl in diethyl ether. by double-strand DNA sequencing. 1 m.p.: 165 uC (decomposition point); HRMS (m/z): [M1H] calculated for The optimized fermentation conditions for all the fusion constructs in E. coli 1 C25H28N5O2S 462.1964; found 462.1968; H-NMR (600 MHz, DMSO-d6 1 BL21DE3* were as follows: –80 uC preserved culture (Microbank bead) was inocu- 21 D2O): d 8.84 (s, 1H), 8.04 (s, 1H), 8.00 (d, J 5 9.00 Hz, 1H), 7.60 (dd, lated into a 500-ml seed flask containing Luria broth (LB) with 100 mgml ampi- J 5 2.50, 9.00 Hz, 1H), 7.43 (s, 1H), 7.42 (d, J 5 2.50 Hz, 1H), 5.80 (s, 2H), cillin, 1% glucose and incubated for 14 h at 30 uC at 240 r.p.m. (Multitron shaker 4.21 (s, 2H), 3.95 (s, 3H), 3.77–3.85 (m, 2H), 3.67–3.74 (m, 2H), 3.44–3.51 (Infors), 5 cm throw). A 2% inoculum was transferred to a 20-l Biolafitte fermenter (m, 1H), 3.30–3.36 (m, 2H), 3.11–3.22 (m, 2H), 2.34–2.40 (m, 2H), 1.97–2.05 containing 15-l LB medium plus 100 mgml21 ampicillin and 1% glucose. The 13 21 (m, 2H); C-NMR (151 MHz, DMSO-d6 1 D2O): d 160.2, 154.9, 148.3, 148.2, culture was incubated at 37 uC, 340 r.p.m. and 12 l air min until cell density 145.7, 144.8, 141.1, 132.1, 130.0, 127.4, 126.6, 118.2, 117.4, 108.4, 103.5, 77.5, (A600 nm 5 3.0) was reached, and then induced with 0.5 mM isopropyl-b-D- 57.2, 54.7, 52.4, 51.1, 48.3, 26.5, 26.4. thiogalactosidase (IPTG; BioSynth). The temperature was reduced to 18 uC for a Minimum inhibitory concentrations. The bacterial isolates used in the anti- further 20 h before harvest. bacterial activity assays were obtained from the culture collection of GSK, repre- Purification of GyrB27–A56. A five-column purification protocol (described senting commonly occurring bacterial pathogens. Ciprofloxacin was obtained below), which included protease inhibitors in buffers throughout, was developed. from US Pharmacopeia. Minimum inhibitory concentrations of the tested anti- Lysis was carried out by incubating 183 g of E. coli cell paste with 550 ml of lysis bacterial agents were determined from two independent experiments by using buffer (20 mM Tris, 50 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol (DTT), broth microdilution methods according to Clinical and Laboratory Standards 1mgml21 lysozyme, BLAP ( a protease inhibitor cocktail consisting of 2 mgml21 Institute guidelines42. The minimum inhibitory concentration was the lowest each of bestatin, leupeptin, aprotinin and pepstatin), 1 mM benzamidine, 0.2 mM concentration of an antibacterial that showed no visible growth after incubation phenylmethylsulphonyl fluoride (PMSF), pH 8.0), sonication on ice (15 min) and at 37 uC for 18–24 h, with a starting inoculum of ,5.5 3 105 colony forming centrifugation at 47,900g for 60 min. Seven-hundred millilitres of clear super- units (CFU) per ml. natant were recovered (containing some 13.6 g of protein). The protein was Generation and expression of S. aureus GyrB–GyrA fusion constructs. loaded onto a 200-ml Q sepharose fast flow column, washed to baseline with Bacterial type IIA topoisomerases normally exist as separate subunits which, buffer 1A (20 mM Tris, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, BLAP, 1 mM when combined, have functional activity; they have been shown to be functional benzamidine, 0.2 mM PMSF, pH 8.0) and eluted in a segmented gradient over

three column volumes with buffer 1B (the same as buffer 1A but with 1 M NaCl in size-exclusion chromatography buffer (20 mM HEPES, 5 mM MnSO4, pH 7.1 instead of 50 mM NaCl). The protein (1.0 g) was then incubated with 300 units of and 100 mM Na2SO4) or the same buffer without MnSO4 for 12 h at 4 uC. Size- benzonase for 30 min, and loaded onto a 200-ml heparin sepharose HP column, exclusion chromatography was performed by injecting 5 ml of ternary complex washed to baseline with buffer 1A and then eluted with a segmented gradient of solution on a 4.6 3 30 mm SW3000 column (TOSOH) at 0.2 ml min21. Elution buffer 1B. The purified protein (120 mg) was then dialysed against 10 litres of times were converted to apparent molecular weight (MWapp) using cytochrome buffer A. A third column, a 20-ml Source 15Q, was also eluted with a segmented C (12.4 kDa), carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase gradient of buffer 1B. Size-exclusion chromatography on a Superdex 200PG in (150 kDa), b-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin buffer 4 (10 mM Tris, 50 mM NaCl, 1 mM EDTA, 2.5 mM DTT, BLAP, pH 8.0), (669 kDa) (all from Sigma) as calibration standards. was followed by a final anion exchange column (20 ml MonoQ), again loading in The sequence of one strand for each of the DNA duplexes shown in buffer 1A and eluting with a segmented gradient to buffer 1B. The final purified Supplementary Fig. 6b is shown below; duplexes were made by annealing com- fusion truncate (10 mg) was .95% pure. plementary oligonucleotide strands. Thirty-seven-base oligonucleotide, Purification of GyrB27–A56(Tyr123Phe), GyrB27–A56(GKdel) and GyrB27– 59-CGCGCGATCTGTGCGGGTGATCCTATGGCTGATGGCC; 33-base oligo- A56(GKdel/Tyr123Phe). The purification protocol was followed as described nucleotide, 59- CGATCTGTGCGGGTGATCCTATGGCTGATGGCC; 29-base above for S. aureus GyrB27–A56 except that the final MonoQ column was oligonucleotide, 59- GATCTGTGCGGGTGATCCTATGGCTGATG; 25-base omitted for GyrB27–A56(Tyr123Phe), the Q sepharose fast flow and heparin oligonucleotide, 59- TCTGTGCGGGTGATCCTATGGCTGA; 21-base oligonu- sepharose HP columns were scaled such that the protein load per ml of matrix cleotide, 59- TGTGCGGGTGATCCTATGGCT. remained constant, and the dialysis step before the Source 15Q column was For crystallography, a series of self-complementary 20-bp oligonucleotides replaced by dilution to a salt concentration of 200 mM NaCl using a low- derived from the 21-base oligonucleotide above were investigated by analytical conductivity buffer 5 (10 mM Tris, 0.5 mM EDTA, 0.5 mM DTT, pH 8.0). To size-exclusion chromatography. The effect of mutating each base on complex maximize protein recovery, the sample was re-applied to this column after stability was investigated. dialysis into buffer 1A containing 100 mM NaCl. Multiple runs on the size- All oligonucleotides were obtained from Eurogentec (http://www.eurogentec. exclusion columns were run such that the percentage load (ml loaded per ml com), and were fast protein liquid chromatography (FPLC) or gel purified. column) remained constant. DNA sequences used for crystallography. The DNA used for purification and Purification of GyrB27–A56(Tyr123Phe), 20-base duplex DNA and crystallization of the two complexes with GSK299423 was a 20-bp homoduplex, GSK299423 ternary complex. To form the ternary complex, 6 mg of purified which we call 20-20 (both strands have the same sequence): 20-20, 59- S. aureus GyrB27–A56(Tyr123Phe) protein was incubated with a twofold molar AGCCGTAGGGCCCTACGGCT-39,39- TCGGCATCCCGGGATGCCGA-59. excess of 20-20 DNA (over the protein dimer; see later for the sequence of the The DNA used for complex with ciprofloxacin consisted of four strands, two 20-base homoduplex) and a fourfold molar excess of compound (over the 12-base oligonucleotides and two 8-base oligonucleotides. The 8-base and 12- protein monomer) for 4 h at 4 uC. The complex was then purified using a base polymers were annealed to give two duplexes with a complementary 4-bp G3000SW size-exclusion column (TOSOH) performed in buffer 6 (20 mM overhang: 8A 59-TGTGCGGTGTACCTACGGCT-39 12B; 12A 39-ACAC HEPES, 100 mM Na2SO4, 5 mM MnCl2, pH 7.0). Eluted fractions corresponding GCCACATGGATGCCGA-59 8B. Other DNA sequences were successfully crys- to the dimeric complex were then concentrated using a 30 kDa molecular mass tallized with quinolones, but these invariably led to almost perfect merohedrally cut-off centrifuge unit, yielding 2 mg of purified ternary complex. twinned crystals. Purification of GyrB27–A56(GKdel/Tyr123Phe), 20-base duplex DNA and Crystallization and structure determination of apo GyrB27–A56. Two apo GSK299423 ternary complex. A similar protocol was followed as described crystal structures were determined from the S. aureus GyrB27–A56 fusion protein. earlier to generate the purified ternary complex. Twelve milligrams of purified First, the structure of apo calcium was determined at 3.1 A˚ . The S. aureus GyrB27– S. aureus GyrB27–A56(GKdel/Tyr123Phe) protein was incubated with the A56 fusion protein (10 mg ml21 in 10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM 20-20 DNA and a sixfold molar excess of compound overnight at 4 uC. The EDTA, 2.5 mM DTT) was crystallized by the hanging-drop vapour diffusion process yielded 5 mg of purified ternary complex. method using 6% PEG 1000, 0.1 M imidazole, pH 8.0, 150 mM calcium acetate Purification of S. aureus GyrA and GyrB. Full-length S. aureus GyrA (relative as the precipitating agent. After serial transfer to 20% PEG 1000, the crystal was molecular mass (Mr), 99.5 kDa) was expressed in E. coli and purified by strep- dipped in paratone and frozen in liquid nitrogen. Data were collected on beamline tomycin sulphate precipitation, ion exchange chromatography and size exclu- ID14-4 at the ESRF. Data were processed with Mosflm45 and merged with pro- sion chromatography to give 5.4 mg of protein. Full-length S. aureus GyrB (Mr, gams from the CCP4 suite46. The structure was determined by molecular replace- 72.5 kDa) was expressed in E. coli and purified by heparin sepharose and hydro- ment with AMORE46 using the E. coli GyrA5911 subunit as a search model. The 459 phobic interaction chromatography to give 7.7 mg of protein. amino acid residues in the search model have a sequence identity of 55.6% with DNA supercoiling assays. The supercoiling activity of S. aureus DNA gyrase was the corresponding GyrA residues in the S. aureus GyrB27–A56 fusion protein. The measured in a 20 ml reaction containing 2 nM S. aureus gyrase, 250 ng relaxed B9 subunit (B27) was built using the Saccharomyces cerevisiae structures for the B9 pBR322 plasmid DNA, 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 700 mM pot- region (PDB accession codes 1BGW and 1BJT) as a template to aid in tracing (the assium glutamate, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 6.5% (w/v) B9 regions of S. aureus and S. cerevisiae have a sequence identity of some 26%). Ten glycerol, 0.1 mg ml21 bovine serum albumin (BSA) and 1 mM ATP. For E. coli rounds of refinement of the apo calcium structure with Refmac47 and CNX48 gave gyrase supercoiling assays, 1.7–4.2 nM of wild-type or GyrA(Ser83Leu) quino- a model containing some 171 amino acids in the B9 domain, 459 in the A9 domain lone-resistant E. coli DNA gyrases (purchased from Inspiralis) were incubated and a single calcium ion with an R factor (R free) of 23.3 (30.2). A final eleventh with 500 ng relaxed pBR322 plasmid DNA in a 30 ml reaction containing the round of refinement was carried out after the apo magnesium structure had been same buffer mixture but without the potassium glutamate. All reactions were refined (Supplementary Table 2). Second, we defined the apo magnesium struc- incubated at 37 uC for 1 h and stopped by the addition of BlueJuice gel loading ture at 2.98 A˚ . Protein (10 mg ml21 in 10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM buffer (Invitrogen) containing 1 mM EDTA or 1% SDS. Reactions were analysed EDTA, 2.5 mM DTT) was crystallized by free interface diffusion in a Fluidigm by electrophoresis in 1% w/v agarose gels followed by ethidium bromide stain- chip49 using 12.75% PEG 3350, 85 mM magnesium formate, 15% glycerol as the ing. IC50 values were determined by quantifying the amount of supercoiled DNA precipitating agent. The crystal was extracted from the chip into the precipitating formed by densitometry and fitting the data by nonlinear regression with GraFit agent and frozen in liquid nitrogen, before data collection on beamline ID14-4 at software (Erythacus). Compound IC50 values reported are the averages of three the ESRF. Data were processed as above. The R-free reflections were transferred determinations. (and extended to higher resolution) from the apo calcium data set and the apo DNA cleavage assay. Two-hundred-and-fifty nanograms of supercoiled pBR322 magnesium structure was refined for five rounds (see Supplementary Table 2). plasmid DNA was incubated with either 200 nM full-length S. aureus gyrase or The two structures are essentially the same except for a single calcium ion seen on 200 nM S. aureus GyrB27–A56(GKdel) in 16 ml reactions containing 35 mM the two-fold axis in the apo calcium structure. Several loop regions pointing into 21 Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 5 mM DTT, 360 mgml BSA, the DNA binding groove are disordered in the apo structures. 6.5% (w/v) glycerol and 1.5 mM ATP, in the presence or absence of various Crystallization and structure determination of GSK299423 ternary com- concentrations of ciprofloxacin or GSK299423 at 37 uC for 60 min. DNA was plexes. Two GSK299423 complex crystal structures were determined: the first released by further incubation with 0.2% (w/v) SDS and 0.1 mg ml21 proteinase with GyrB27–A56(Tyr123Phe) and 20-20 DNA, the second with GyrB27– Kat37uC for 60 min. Samples were analysed by electrophoresis in 1% agarose A56(GKdel/Tyr123Phe) and 20-20 DNA. First, the GSK299423 complex was gels containing 0.5 mgml21 ethidium bromide followed by photography under determined at 3.5 A˚ . The gel-purified ternary complex of GSK299423, 20-20 ultraviolet illumination. DNA and GyrB27–A56(Tyr123Phe) (in 100 mM Na2SO4, 20 mM HEPES, Analytical size-exclusion chromatography. Ternary complexes were formed by 5 mM MnCl2, pH 7.0) was crystallized by free interface diffusion in a Fluidigm incubation of S. aureus GyrB27–A56 or GyrB27–A56(Tyr123Phe) mutant (final X-ray chip using 12% PEG 4000, 100 mM NaCl, 100 mM sodium citrate as a concentration 5 mM), double-stranded DNA (10 mM) and compound (50 mM) precipitating agent. The crystal was extracted from the chip into precipitating

agent with 15% glycerol and frozen into liquid nitrogen before data collection on structure was refined with phenix.refine using twin law 5 h 2 k 2 l, refined twin beamline ID23-1 at the ESRF. The 3.5 A˚ GSK299423 complex data set was pro- fraction 0.308. The compound was built into electron density adjacent to GyrA cessed with Mosflm45 and programs from the CCP4 suite46. The structure was Ser 84, but the quality of the electron density maps for the central four bases and determined by molecular replacement using domains from the apo magnesium the compounds was not very good. The structure contains two dimers in the structure, and refined with Refmac47 and PHENIX50; the refinement of the 3.5 A˚ asymmetric unit and in all four subunits the Mn21 ion at the TOPRIM active site structure was completed after the 2.1 A˚ structure had been determined. Second, is clearly defined and is at the same position (within experimental error). This the GSK299423(GKdel) complex was determined at 2.1 A˚ . The gel-purified metal position is very similar (within ,0.4 A˚ in superposed structures) to that 21 12 53 GyrB27–A56(GKdel/Tyr123Phe) complex (9 mg ml in 33 mM Na2SO4, seen previously . Structures were built with Coot and generally have good 13 mM HEPES, 13 mM NaCl, 2 mM MnCl2, pH 7.0) was crystallized by the geometry. hanging-drop vapour diffusion method using 18% PEG 5000 MME, 0.1 M BisTris, pH 6.5 as a precipitating agent. The crystal was transferred into 15% 41. Miller, W. H. & Seefeld, M. A. Preparation of Naphthalenes, Quinolines, Quinoxalines glycerol with precipitating agent before freezing in liquid nitrogen for data and Naphthyridines as Antibacterial Agents. United States patent 7,691,850 B2 (2010). collection on beamline ID23-1 at the ESRF. The data processing, structure deter- 42. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial mination and refinement were as described earlier. The R-free reflections were Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard 8th edn selected in thin resolution shells to avoid bias due to non-crystallographic sym- (Clinical and Laboratory Standards Institute, 2008). metry51. The DNA was initially built into good density by omitting the ribose 43. Lavasani, L. S. & Hiasa, H. A ParE–ParC fusion protein is a functional sugars so that the phosphates and bases could be refined as rigid groups before topoisomerase. Biochemistry 40, 8438–8443 (2001). adding the ribose sugars in subsequent refinement rounds. The compound sits 44. Trigueros, S. & Roca, J. A GyrB–GyrA fusion protein expressed in yeast cells is able on a non-crystallographic two-fold axis and the observed electron density for the to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II. compound represents two equivalent two-fold related orientations. The quality Genes Cells 7, 249–257 (2002). of the electron density maps was good, the positions of the sulphur atom in the 45. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and compound were readily identifiable from difference maps, and the position of image plate data. Joint CCP4 1 ESF-EAMCB Newsl. Protein Crystallogr. 26 (1992). 46. Collaborative Computational Project, Number 4. The CCP4 suite: programmes the compound was clear in the density map (Supplementary Fig. 14). for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994). Crystallization and structure determination of ciprofloxacin ternary complex 47. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular ˚ (3.35 A). DNA cleavage is not required for the binding of quinolone drugs to the structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 DNA-gyrase– DNA complex52. The complex was made by mixing 25 mlof (1997). 2 GyrB27–A56(GKdel/Tyr123Phe) (8.3 mg ml 1 in 20 mM HEPES, 100 mM 48. Bru¨nger, A. T. et al. Crystallography and NMR system: a new software suite for Na2SO4, 5 mM MnCl2, pH 7.0) with 2 ml of 8A–12A/B8–12B DNA (2 mM in macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998). water) and 2.5 ml ciprofloxacin (10 mM in methanol). Crystals were grown by a 49. Hansen, C. L., Skordalakes, E., Berger, J. M. & Quake, S. R. A robust and scalable microbatch method (under silicon oil), by mixing 1 ml of DNA complex with 1 ml microfluidic metering method that allows protein crystal growth by free interface of precipitating solution (11% PEG 3350, 150 mM BisTris, pH 6.2). Crystals were diffusion. Proc. Natl Acad. Sci. USA 99, 16531–16536 (2002). briefly transferred to a 16% glycerol cryo buffer and frozen in liquid nitrogen 50. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002). before data collection. Data were collected on beamline ID14-2 at the ESRF. Data 45 46 51. Kleywegt, G. J. & Jones, T. A. Where freedom is given liberties are taken. Structure were processed with Mosflm and merged programs from the CCP4 suite . 3, 535–540 (1995). Data were processed in an orthorhombic and three monoclinic cells before the 52. Critchlow,S.E.&Maxwell,A.DNAcleavageisnotrequiredforthebindingofquinolone structure was solved by molecular replacement using domains from the 2.1 A˚ drugs to the DNA gyrase–DNA complex. Biochemistry 35, 7387–7393 (1996). GSK299423(GKdel) structure. After initial rigid body refinement, R-free reflec- 53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta tions were selected in thin resolution shells to avoid bias due to twinning and the Crystallogr. D 60, 2126–2132 (2004).

Non-canonical inhibition of DNA damage- dependent ubiquitination by OTUB1

Shinichiro Nakada1,2, Ikue Tai1,2, Stephanie Panier3,4, Abdallah Al-Hakim3, Shun-ichiro Iemura5, Yu-Chi Juang3, Lara O’Donnell3, Ayako Kumakubo2, Meagan Munro3, Frank Sicheri3,4, Anne-Claude Gingras3,4, Tohru Natsume5, Toshio Suda2 & Daniel Durocher3,4

DNA double-strand breaks (DSBs) pose a potent threat to genome integrity. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DSBs elicit a signalling cascade that modifies the chromatin surrounding the break, first by ATM-dependent phosphorylation and then by RNF8-, RNF168- and BRCA1-dependent regulatory ubiquitination. Here we report that OTUB1, a deubiquitinating enzyme, is an inhibitor of DSB-induced chromatin ubiquitination. Surprisingly, we found that OTUB1 suppresses RNF168-dependent poly-ubiquitination independently of its catalytic activity. OTUB1 does so by binding to and inhibiting UBC13 (also known as UBE2N), the cognate E2 enzyme for RNF168. This unusual mode of regulation is unlikely to be limited to UBC13 because analysis of OTUB1-associated proteins revealed that OTUB1 binds to E2s of the UBE2D and UBE2E subfamilies. Finally, OTUB1 depletion mitigates the DSB repair defect associated with defective ATM signalling, indicating that pharmacological targeting of the OTUB1–UBC13 interaction might enhance the DNA damage response.

Ubiquitination is a post-translational modification where ubiquitin include histones H2A and H2AX3,7. This regulatory ubiquitination (Ub), a small polypeptide, is attached to an acceptor amino group, event promotes the independent recruitment to damaged chromatin usually on the lysine residue of a substrate1. Ubiquitination is initiated of the RAP80 (also known as UIMC1)–BRCA1 complex8–11 and 53BP1 by the E1 enzyme-dependent activation of ubiquitin followed by its (also known as TP53BP1, refs 3–7), which are critical for DNA repair transfer onto an E2 ubiquitin-conjugating enzyme. The resulting and the G2/M checkpoint. Although the identity of the E3 ubiquitin thioester-linked E2,Ub intermediate acts in concert with a ubiquitin ligases that participate in the DSB response is emerging, much less ligase (E3) to conjugate ubiquitin on substrates. In the case of RING- is known about the proteins that negatively regulate ubiquitin- type E3s, ubiquitin is directly transferred from the E2 to the sub- dependent DSB signalling and repair. Here we sought to identify and strate without an E3,Ub intermediate. This cycle can be repeated characterize negative regulators of RNF168. to form ubiquitin chains. The type of ubiquitin chain linkage is key for determining the outcome of ubiquitination. For example, Lys 48 OTUB1 inhibits the RNF168 pathway (K48)-linked Ub chains usually mark proteins for proteasomal degra- By virtue of their activity as ubiquitin isopeptidases, deubiquitinating dation. In contrast, Lys 63 (K63)-linked Ub (UbK63) chains are non- enzymes (DUBs)12 are prime candidates for negative regulators of degradative and often orchestrate protein complex assembly in DSB-induced ubiquitination. USP3 (refs 7, 13) and BRCC36 (also signalling pathways. Selection of the substrate is dependent on the known as BRCC3, ref. 14) are DUBs that are negative regulators of this E3 whereas the E2 can specify the type of ubiquitin chain formed. pathway, but neither of these two proteins affects RNF168 activity per UBC13, for example, is an E2 dedicated for the catalysis of UbK63 se. Therefore, we carried out a small interfering RNA (siRNA) screen in chains when it acts in complex with UEV1a or MMS2 (also known as U2OS cells to uncover DUBs whose depletion resulted in persistent UBE2V1 and UBE2V2, respectively). DNA damage-dependent ubiquitination after exposure to ionizing The importance of ubiquitination in the DSB response was recently radiation. Ubiquitin conjugation at the DSB site was detected by highlighted by the discovery that RIDDLE syndrome, an immuno- immunofluorescence using the FK2 anti-ubiquitin conjugate antibody deficiency and radiosensitivity disorder, is caused by mutations in (Fig. 1a). We first focused on a subfamily of 14 DUBs characterized by the gene coding for RNF168 (refs 2, 3) a RING-type ubiquitin ligase. an ovarian tumour (OTU) domain15, because members of this family RNF168 participates in a chromatin-based pathway of the DSB res- target UbK63 chains15–17. Of all the DUBs tested (Supplementary Table ponse, downstream of ATM, phosphorylated histone H2AX (c-H2AX; 1), the knockdown of OTUB1 resulted in a highly reproducible per- H2AX is also known as H2AFX), MDC1 and RNF8. The DSB-induced sistence of conjugated ubiquitin foci (Fig. 1b–d and Supplementary ubiquitination cascade is initiated by the RNF8-dependent conjuga- Fig. 2a, b). OTUB1 depletion also resulted in persistent 53BP1 foci in tion of ubiquitin on histones H2A (also known as HIST2H2AC) and U2OS cells (Fig. 1e and Supplementary Fig. 2c) and, importantly, this H2AX4–6. Histone ubiquitination promotes the accumulation of phenotype was rescued by the stable expression of an siRNA-resistant RNF168 at DSB sites where it cooperates with UBC13 to catalyse the OTUB1 transgene (Supplementary Fig. 2d–f). These results indicated formation of UbK63 chains on chromatin-bound substrates that likely that the molecular events downstream of RNF8/RNF168-dependent

1Center of Integrated Medical Research, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo, 160-8582, Japan. 2Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo, 160-8582, Japan. 3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, M5G 1X5, Ontario, Canada. 4Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, M5S 1A8, Ontario, Canada. 5Biological Systems Control Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan. 941 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010

a Fix: methanol b a 3 Gy IR OTUB2 OTU domain 234 24 h siRNA

RNAi Immuno- Control OTUB1 OTUB1 OTU domain 271 b 4 h fluorescence: IB: OTUB1 47D88 C91 H265 Flag–OTUB1 Anti- C91S S 53BP1 Flag–OTUB1 53BP1/DAPI 48 h 1 h conjugated IB: RNF168 AS A No IR ASA U2OS ubiquitin (FK2) ΔN IB: Histone H3 OTUB1 c c OTUB1 No IR 1 h 4 h 24 h C91S 2 Gy ΔN 2 Gy ASA OTUB2 Empty OTUB1 No IR Empty siControl C91S 0 50 100 Flag-positive cells with >10 53BP1 foci (%) 2 Gy Flag–OTUB1 d UbK63 Flag–OTUB1 UbK63/DAPI

ΔN siOTUB1 OTUB1 2 Gy d e Control Control OTUB1 OTUB1 C91S 100 RNF8 100 RNF168 ASA

80 80 2 Gy 60 60 ΔN 40 40 OTUB2 20 20

0 0 2 Gy Cells with >10 FK2 foci (%) 4 8 12 16 20 24 4 8 12 16 20 24 ASA

No IR Cells with > 10 53BP1 foci (%) No IR Time after IR (h) Time after IR (h) f Flag–OTUB1 e FK2 Flag–OTUB1 FK2/DAPI OTUB1 C91S OTUB1 ΔN ASA No IR 050100 3 Gy Flag-positive cells with > 3 UbK63 foci (%) Figure 2 | OTUB1 inhibits the DSB response downstream of RNF168 focal accumulation. a, Domain architecture of OTUB1 and OTUB2. Amino acid residues mutated in this study are depicted. b, HeLa cells transfected with the indicated Flag–OTUB1 or Flag–OTUB2 expression vectors were irradiated No IR with 2 Gy and processed for 53BP1 and Flag immunofluorescence 1 h post- ionizing radiation. Scale bar, 23 mm. c, Quantification of the data shown in b (mean 6 s.d., N 5 3). d, U2OS cells transfected as in b with the exception that cells were irradiated with 3 Gy and processed for UbK63 and Flag Figure 1 | OTUB1 modulates ubiquitination of chromatin after DNA immunofluorescence. Scale bar, 10 mm. e, Quantification of the data shown damage. a, Schematic of the siRNA screen. IR, ionizing radiation. b–e, U2OS in d (mean 6 s.d., N 5 3). cells transfected with the indicated siRNAs were irradiated with 3 (c, d)or2 (e) Gy and processed for conjugated ubiquitin (FK2; c, d) or 53BP1 (e) immunofluorescence at the indicated time points. IB, immunoblot. through an N-terminal extension of 46 amino acid residues that is b c absent in OTUB2 (ref. 20; Fig. 2a). Cells expressing a mutant form , Validation of OTUB1 depletion. , Representative micrographs. Scale bar, DN 16 mm. d, e, Quantification of cells with the indicated protein foci of OTUB1 lacking its N-terminal extension (OTUB1 ) formed (mean 6 s.d., N 5 3). f, U2OS cells transfected with Flag–OTUB1 were fixed 53BP1 foci, indicating that OTUB1 requires its N terminus to suppress 1 h after 3 Gy ionizing radiation, and processed for Flag and FK2 DSB signalling (Fig. 2b, c). Interestingly, the N-terminal region of immunofluorescence. Scale bar, 23 mm. OTUB1 contains a Ub-binding site19, suggesting that Ub-binding might be involved in the mechanism of inhibition. Finally, we made ubiquitination were upregulated following OTUB1 knockdown. the serendipitous observation that mutation of all three residues of Moreover, when we transiently overexpressed OTUB1, we observed the OTUB1 catalytic triad20, the D88A C91S H265A mutation a drastic suppression of DNA damage-induced ubiquitin conjugation (OTUB1ASA, Fig. 2a), resulted in an OTUB1 protein unable to inhibit on chromatin (Fig. 1f) and of 53BP1 focus formation (Fig. 2b, top the accumulation of 53BP1 at DSB sites (Fig. 2b, c). These data indi- panel). OTUB1 was therefore a prime candidate for a DUB that inhibits cated that the function of OTUB1 in dampening the DSB response was RNF168-dependent ubiquitination. independent of its catalytic activity but dependent on the integrity of OTUB1 is a DUB specific for K48-linked ubiquitin chains18,19,an the OTU domain. observation that we found at odds with its potential role as a negative Next, we sought to pinpoint the step during DNA damage signal- regulator of UbK63 chain formation at DSB sites. We therefore tested ling that was sensitive to OTUB1. We found that the formation of whether the inhibition of 53BP1 focus formation by OTUB1 was c-H2AX, MDC1, RNF8 and RNF168 foci was normal in cells over- dependent on its catalytic activity. We generated the C91S mutation expressing OTUB1 (Supplementary Fig. 4). Because RNF168 accu- in OTUB1, which abolishes its deubiquitinase activity20 (Fig. 2a and mulates at DSB sites by recognizing the products of RNF8-dependent Supplementary Fig. 3). To our surprise, the OTUB1(C91S) mutant ubiquitination3,7, we concluded that OTUB1 inhibited the DSB res- inhibited 53BP1 focus formation as efficiently as wild type (Fig. 2b, c). ponse downstream of RNF8 activity, at the level of RNF168. This phenotype was specific to OTUB1 since overexpression of Importantly, the formation of UbK63 chains at DNA damage sites, OTUB2, a highly related DUB20, did not suppress accumulation of the products of RNF168 activity, was suppressed by the overexpres- 53BP1 at DSBs (Fig. 2b, c). OTUB1 differs from OTUB2 primarily sion of OTUB1 and OTUB1(C91S), but not by that of OTUB1ASA or 942 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 ARTICLES

OTUB1DN (Fig. 2d, e). Similar results were obtained when cells were other at the sequence level (Supplementary Fig. 9), indicating that stained with conjugated ubiquitin antibodies (Supplementary Fig. 5). OTUB1 targets a subfamily of E2 enzymes. These results indicated that OTUB1 does not impact the ability of Because UBC13 is the only known E2 that cooperates with RNF168 RNF168 to accumulate at sites of DNA damage but rather that it during the DNA damage response, we first focused on UBC13 as a inhibits its activity. potential target of OTUB1. Co-immunoprecipitation analyses in To test this possibility further, we carried out in vivo ubiquitination U2OS cells stably expressing Flag–OTUB1 confirmed the interaction assays7 where myc-tagged ubiquitin was co-expressed with epitope- between OTUB1 and UBC13 (Supplementary Fig. 10). Importantly, tagged RNF168 and histone H2AX. Co-expression of these proteins OTUB1(C91S), but not OTUB2, OTUB1DN or OTUB1ASA interacted resulted in H2AX and chromatin ubiquitination (Fig. 3a, b). However, with UBC13 (Fig. 3d). Therefore, the ability of OTUB1 to inhibit when OTUB1 was co-expressed, we observed a marked reduction of RNF168-dependent ubiquitination correlates with its capacity to H2AX or chromatin ubiquitination in a manner that was dependent bind to UBC13. on the OTUB1 N-terminal region and the OTU domain, but inde- Using recombinant proteins, we observed that OTUB1 binds pendent of its catalytic activity (Fig. 3a, b). In addition, we observed directly to UBC13 whether or not UEV1a, a UBC13 cofactor, was that RNF168 auto-ubiquitination was also greatly reduced in cells present (Fig. 3e). OTUB1(C91S) also robustly bound to UBC13 but overexpressing OTUB1 (Supplementary Fig. 6). OTUB1 and its OTUB1ASA and OTUB1DN did not efficiently interact with UBC13 mutants were similarly expressed both in terms of levels and sub- (Fig. 3f). Whereas this result was consistent with our immuno- cellular distribution (Supplementary Fig. 7). OTUB2 overexpression precipitation studies, the DN mutant (and OTUB2) did show residual also failed to inhibit RNF168-stimulated chromatin ubiquitination UBC13 binding, and this was especially evident under different bind- (Supplementary Fig. 8a). Lastly, under the same conditions, over- ing conditions (Fig. 3g). Because the N-terminal region harbours a expression of RNF8 did not trigger chromatin ubiquitination although Ub-binding motif19, we surmised that OTUB1 might display selec- its ubiquitination was OTUB1-sensitive (Supplementary Fig. 8b). tivity towards charged E2s. To test this possibility, we generated a These results indicated that OTUB1 suppresses DNA damage-induced UBC13(C87S) mutant to prepare a stable oxyester-linked version of poly-ubiquitination independently of its ability to cleave ubiquitin the UBC13,Ub intermediate24. The resulting UBC13:UBC13,Ub chains. mixture was subjected to OTUB1 pull-down assays and we found that OTUB1 had a marked preference for the charged E2 (Fig. 3g). Under OTUB1 interacts with E2 enzymes these conditions, retrieval of the charged E2 was entirely dependent on To gain insight on how OTUB1 inhibits RNF168-dependent ubiqui- the OTUB1 N terminus. The C23S mutant, which disrupts a reactive tination, the Tokyo- and Toronto-based authors independently cysteine in the N-terminal region of OTUB1 (ref. 19), behaved as wild- searched for OTUB1-interacting proteins by mass spectrometry21,22 type. Moreover, OTUB2 did not bind to the charged form of UBC13 (Supplementary Tables 2 and 3). In both sets of analyses, UBC13 (Fig. 3g). These results strongly suggested that ubiquitin-charged and E2 ubiquitin-conjugating enzymes of the UBE2D and UBE2E UBC13 is the target of OTUB1. families were found as major OTUB1-interacting proteins, along with ubiquitin (Fig. 3c). UBC13 and UBE2D2 were also independently OTUB1 directly inhibits UBC13 activity found as OTUB1-interacting proteins in a survey of the DUB inter- The direct interaction between UBC13 and OTUB1 spurred us to test actome23. Strikingly, the OTUB1-interacting E2s are related to each whether OTUB1 suppressed RNF168 E3 ligase activity in a fully

IP: Flag Myc–Ubiquitin ++++++ a b HA–RNF168 –+++++ GFP-OTUB1 Δ N ASA WT Input Empty (kDa) C91S Heavy chain Flag-OTUB1 (kDa) ASA Empty WT Empty Δ N 75-- ← C91S 50-S - ← -75 IB: Myc– H2AX-Ub4 37-- ←H2AX-Ub IB: Myc– -50 ubiquityl-H2AX ← 3 H2AX-Ub2 ubiquityl- -37 Acid IP 25-- ←H2AX-Ub 1 histones extraction Light chain -25 IB: Flag–H2AX IB: Histone H3 IB: Flag–OTUB1 IB: GFP–OTUB1 WCL Input IB: HA–RNF168 IB: Myc–Ubiquitin IB: HA–RNF168 e GST–pull down c d IP: Flag GST +––– OTUB1 GST–OTUB1 –+++ UBC13 +++– UBC13 UEV1a + – + + Flag– (kDa) ASA Δ N WT Empty OTUB2 C91S Input 50 IB: UBC13 Ponceau S 37 UBE2D2 Ubiquitin IB: RNF168 (GST IB: Flag– proteins) 25 OTUB1/OTUB2 IB: UBC13 OTUB1 UBE2D3 UBE2E3 g OTUB1 f OTUB1 GST– (Pcc1) Control WT C23S Δ N OTUB2 C91S UBC13 +++++++E2 input GST– 72 GST ASA Δ N WT GST C91S UBE2D1 UBE2E1 UBE2E2 OTUB2 55 (kDa) 43 proteins 50- IB: CB 34 Found by 37- ← GST 26 UBC13~Ub Toronto-based authors (2 replicates) 25- Toronto-based authors (1 replicate) ← IB: 20- 17 UBC13 Tokyo-based authors (unique peptide) (kDa) Tokyo-based authors (shared peptide) UBC13 15- ←UBC13~Ub IB: UBC13 Proteins from ref. 23 ←UBC13 Figure 3 | OTUB1 interacts with UBC13 to inhibit ubiquitin chain formation. indicated. d, 293T cells transfected with the indicated vectors were subjected a, b, 293T cells cotransfected with haemagglutinin (HA)–RNF168, to Flag immunoprecipitation. e–g, Recombinant UBC13 (e), UBC13/UEV1a Myc–ubiquitin, epitope tagged-OTUB1 and/or Flag–histone H2AX were (f), or a mixture of uncharged and ubiquitin-charged UBC13(C87S) subjected to Flag immunoprecipitation (IP) (a) or acid extraction of (UBC13,Ub) (g) were incubated with indicated GST-fusion proteins. CB, chromatin (b). c, The OTUB1 interaction network. Primary data are shown Coomassie blue. in the Supplementary Information. Proteins also found previously23 are 943 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010 recombinant system. Ubiquitination reactions were assembled with assay, UBC13 was first charged with biotinylated-ubiquitin in the UBC13 (in complex with UEV1a) and RNF168, in the presence or presence of OTUB1 or OTUB1DN and then reaction mixtures were absence of OTUB1. In this system, the UBC13–UEV1a complex incubated with UEV1a and an excess amount of unmodified ubiqui- formed low levels of ubiquitin chains independently of RNF168 tin to stimulate ubiquitin discharge25. We found that OTUB1 did not (Fig. 4a, lane 3). However, RNF168 greatly stimulated ubiquitination impact ubiquitin discharge. However, we observed that OTUB1 and (Fig. 4a, lane 5). RNF168 stimulated the synthesis of free ubiquitin OTUB1(C91S), but not OTUB1DN, inhibited di-ubiquitin synthesis chains because we were still able to detect ubiquitin chains follow- (Fig. 4e and Supplementary Fig. 13). Because OTUB1DN has DUB ing depletion of glutathione S-transferase (GST)–RNF168 with activity (Supplementary Fig. 3), this excludes the possibility that this glutathione-Sepharose (Fig. 4a, lanes 8 and 9). When increasing activity was responsible for the impaired generation of di-ubiquitin amounts of OTUB1 or OTUB1(C91S) were added to the reactions, (Fig. 4e). We probed these results further under slightly altered con- a notable inhibition of ubiquitin chain formation was observed ditions and found that OTUB1 inhibited, in a dose-dependent manner, (Fig. 4b and Supplementary Fig. 11). In a parallel to the in vivo the formation of di- but especially tri-ubiquitin linked through K63 situation, the OTUB1ASA mutant was unable to inhibit RNF168 in (Supplementary Fig. 14). This inhibition was also observed with this reconstituted assay (Fig. 4b, compare lanes 2 and 3). OTUB1(C91S) but, importantly, not with OTUB1DN or OTUB1ASA The above data indicated that OTUB1 may inhibit UBC13–UEV1a (Supplementary Fig. 14). Together, these results indicated that OTUB1 directly. We tested this possibility by assembling ubiquitination reac- specifically inhibits isopeptide bond formation between a donor ubi- tions in the presence of UBC13–UEV1a but in the absence of RNF168 quitin on UBC13 and an acceptor ubiquitin coordinated by UEV1a26. and observed that OTUB1 and OTUB1(C91S) suppressed UBC13- These results indicate that the non-canonical mode of action of dependent ubiquitination (Fig. 4c). These results indicate that OTUB1 during the DNA damage response is to inhibit the formation OTUB1 acts directly on UBC13 rather than via competition with of poly-ubiquitin chains by the RNF168–UBC13 complex. the E3. In support of this possibility, depletion of OTUB1 did not Because OTUB1 precipitated E2 enzymes of the UBE2D/E sub- result in an increased association of UBC13 with RNF168 (Fig. 4d). family, we examined whether OTUB1 bound and inhibited E2s of We next sought to determine the step in the ubiquitination reac- these families. Co-immunoprecipitation analyses confirmed the tion that was sensitive to OTUB1. Not surprisingly, OTUB1 did not interaction between OTUB1 and UBE2D2 (Fig. 4f). Next, based on inhibit the E1-dependent formation of UBC13,Ub (Supplementary our observation that OTUB1 binds more avidly to charged UBC13, Fig. 12). Next, we tested whether ubiquitin discharge from the we prepared an oxyester-linked charged version of UBE2D2. We UBC13,Ub intermediate was affected by OTUB1 (Fig. 4e). In this subjected the resulting equimolar UBE2D2:UBE2D2,Ub mixture

Glutathione a b GST–OTUB1 c sepharose GST–OTUB1 WT GST GST GST–OTUB1 depletion C91S GST OTUB1 WT C91S UBC13/UEV1a –

ASA + ++ Ubiquitin +++– +++ – + GST (kDa) only UBE1 – RNF168 9 9 0.9 1.8 0.9 1.8 4.5 9 (µM) + + + +++ + + (kDa) 250- UBC13/UEV1a + – +++ + + + + GST–RNF168 + + – +++ + + + 250- 150- Ubn (kDa) 150- 100- 250- 75- IB: FK2 100- IB: FK2 150- 75- 50- Ubn 100- 37- 75- 50- 37- 50- 25- 20- IB: FK2 37- 50- Ponceau S 37- 15- 25- (GST 10- 20- proteins) 50- 25- Ponceau S 15- Lane: 123 4 56789 (GST 37- 10- proteins) 150- 25- IB: RNF168 100- e Biotinylated ubiquitin (5 mM) Ubiquitin (240 mM) 150- UBC13, OTUB1 UEV1a IB: GST 100- UBE1, ATP EDTA ↓ ↓ Lane: 1 2 34567 89 (charge) (discharge) d –5 012 5 10 min Normal DTT (–) DTT (+) IP: RNF168 IgG Incubation time 0 1 2 5 10 0 12 5 10 10 10 (min) WT

ΔN (Marker) siRNA: (kDa) ← Control Control OTUB1 25- UBC13~Ub IB: RNF168 20- IP Streptavidin– ←Ub 15- ← 2 IB: UBC13 HRP 10- Ub1 Input IB: OTUB1 Ponceau S 50- (OTUB1) 37- f IP: Flag WCL Lane: 123 4 5678 9 10 11 12 g OTUB1 h TRAF6 – +++ + UBE2D2 ++ + + +

Flag– GST– N OTUB1 OTUB1 Empty m Control WT WT Δ OTUB2 (Pcc1) C91S E2 input OTUB1(C91S) 0 0 1.3 2.7 4.0 ( M) IB: Flag–OTUB1 UBE2D2 + – +++++ 250- GST 150- IB: UBE2D2 proteins IB: FK2 100- ← UBE2D2~Ub 75- CB ← UBE2D2 (kDa) ← Ponceau S 50- GST– OTUB1 ← Ub (GST 37- IB: UBE2D2 ← UBE2D2~Ub proteins) ←GST (UBCH5b) ← UBE2D2 25- Figure 4 | OTUB1 directly inhibits UBC13. a, b, Ubiquitination assays with Flag–OTUB1 and UBE2D2. g, Uncharged and Ub-charged UBE2D2(C85S) combinations of UBC13/UEV1a (E2), GST–RNF168 (E3) and wild-type or (UBE2D2,Ub) mixtures were subjected to pull-down assays with the mutant GST–OTUB1. c, Ubiquitination assays without RNF168. d, 293T indicated GST fusion proteins. h, Ubiquitination assays with TRAF6 (E3) cells transfected with control or OTUB1 siRNAs were subjected to RNF168 and UBE2D2 (E2) carried out in the absence or presence of recombinant immunoprecipitation and immunoblotting. e, UBC13 charge/discharge OTUB1(C91S). CB, Coomassie blue; WCL, whole-cell lysates. assay in the presence of OTUB1 or OTUB1DN. f, Co-immunoprecipitation of 944 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 ARTICLES to GST pull-down assays and found that OTUB1 and OTUB1(C91S), near-complete restoration of homologous recombination in cells but not OTUB2 nor OTUB1DN, preferentially bound to charged treated with the ATM inhibitor (Fig. 5b). We also observed that exo- UBE2D2 (Fig. 4g). Therefore, OTUB1 can directly bind to genous expression of OTUB1 or OTUB1(C91S) strongly inhibited UBE2D2 using a binding mode that mirrors that of the OTUB1– homologous recombination (Fig. 5c). This inhibition was probably UBC13 interaction. caused by a failure of BRCA1 to accumulate at DSB sites in OTUB1- Next, we assembled E3 ubiquitin ligase assays with UBE2D2 expressing cells (Fig. 5d). These observations indicated that OTUB1 is (UBCH5b), UBE2D3 (UBCH5c) or UBE2L3 (UBCH7). UBE2D2 a physiological modulator of DNA repair and that its depletion can and UBE2D3 are OTUB1-interacting E2s (Fig. 3c and Supplemen- mitigate the DNA repair defect associated with ATM inhibition. tary Fig. 9) whereas UBE2L3 was not found in the OTUB1 immunoprecipitates. The reactions were incubated with increasing amounts of Discussion OTUB1(C91S) (because wild-type OTUB1 has activity against We conclude that OTUB1 is an inhibitor of the DNA damage res- K48-linked chains). We found that OTUB1 inhibited UBE2D2- and ponse that suppresses RNF168- and UBC13-dependent ubiquitina- UBE2D3-dependent ubiquitination in a dose-dependent manner tion of chromatin. The physiological role of OTUB1 is probably to set (Fig. 4h and Supplementary Fig. 15). In contrast, UBE2L3-dependent a threshold for RNF168 activity (Supplementary Fig. 1). Indeed, ubiquitination was impervious to OTUB1 (Supplementary Fig. 15). OTUB1 depletion also potentiates ubiquitin-dependent DSB signal- These results suggest that in addition to UBC13, OTUB1 can inhibit ling at low radiation doses (Supplementary Fig. 17). Strikingly, E2 conjugating enzymes of the UBE2D/E subfamily. OTUB1 inhibits the DSB response independently of its catalytic activity. Interestingly, non-catalytic roles for DUBs are not un- OTUB1 depletion bolsters DSB repair precedented. For example, Ubp6 regulates proteasome-dependent Lastly, we investigated whether the depletion of OTUB1 could bolster degradation in a non-catalytic manner32. Moreover, a number of the response to DSBs. To do so, we pharmacologically disabled ATM DUB-family proteins such as USP39 and USP52 to 54 lack the necessary activity with its specific inhibitor KU55933 (ref. 27) and tested infrastructure for peptidase activity33,34, suggesting that they must pos- whether OTUB1 depletion rescued cellular phenotypes associated sess non-canonical functions. Therefore, non-catalytic roles for DUBs, with ATM inhibition. We first focused our attention on 53BP1 accu- perhaps owing to their ability to recognize ubiquitin, might be more mulation at DSBs since it is dependent on RNF168 but not entirely on pervasive than previously recognized. ATM28. Indeed, ATM inhibition leads to a reduction in 53BP1 focus UBC13 also acts with RNF8, upstream of RNF168, in the DSB size that is particularly manifest by the loss of large foci (Supplemen- response. Yet, RNF8 activity (as measured by RNF168 accumula- tary Fig. 16)29. Strikingly, the knockdown of OTUB1 in cells treated tion), seems impervious to OTUB1 in vivo. However, OTUB1 can with the ATM inhibitor rescued defective 53BP1 focus formation inhibit the synthesis of ubiquitin chains by RNF8 in vitro and can (Fig. 5a and Supplementary Fig. 16). inhibit RNF8 ubiquitination (Supplementary Figs 8b and 18). Although focus formation is a useful marker of the response to Therefore, it is possible that OTUB1 inhibits RNF8-dependent DSBs, the function of this cytological structure remains unclear. poly-ubiquitin chain formation in vivo but that these chains are Therefore, we tested whether OTUB1 depletion mitigated the DSB not critical for the DSB response. Indeed, we previously reported that repair defect of ATM-inhibited cells. To assess ATM-dependent DSB RNF168 interacts with mono- or di-ubiquitinated histones in res- repair, we monitored homologous recombination using the DR-GFP ponse to DNA damage3. Because OTUB1 is not an efficient inhibitor (direct repeat-green fluorescent protein) reporter30. In this assay, of mono-ubiquitination (Supplementary Fig. 14), these observations pharmacological inhibition of ATM reduced the efficiency of homo- indicate that the role of RNF8 in DSB signalling might be primarily to logous recombination31 (Fig. 5b). Knockdown of RAD51, that codes conjugate the first ubiquitin on substrates, which is then elongated by for a recombinase essential for homologous recombination, served as RNF168 in cooperation with UBC13. a positive control. Remarkably, depletion of OTUB1 resulted in a Lastly, there is currently a paucity of potential therapeutic strategies for the enhancement of the response to DSBs. Such agents could be useful to mitigate the unwanted effects of radiation therapy or may be a 50 b DR-GFP assay c DR-GFP assay 1.2 used as therapeutics in genome instability syndromes such as ataxia- 40 5 4 telangiectasia. A potentially attractive strategy to enhance the DSB 30 0.8 3 response might therefore be to selectively target the OTUB1–UBC13 20 cells (%) cells (%) 2 + + 0.4 interaction. 10 1 53BP1 foci (%) GFP GFP Cells with >10 large 0 0 0 METHODS SUMMARY KU55933: ––++–– ++KU55933: –+– – + OTUB1: siRNA: Control OTUB1 Control OTUB1 siRNA: Expression plasmids, proteins, antibodies and cell lines are described in Methods. Empty WT C91S No IR 2 Gy Plasmid transfections were carried out with Effectene (Qiagen) and siRNA trans- RAD51 Control Control OTUB1 OTUB1 d BRCA1 γ-H2AX GFP–OTUB1 BRCA1/γ-H2AX fections were carried out with DharmaFECT 1 (Thermo Fisher). Unless otherwise stated, siRNAs employed in this study were SMARTpools (Thermo Fisher). The siRNA#2 for OTUB1 was also purchased from Thermo Fisher (catalogue # 3 Gy D-021061-02). For immunofluorescence, cells were usually fixed with 2% paraformaldehyde ormethanol (at 220 uC),permeabilized and stained with antibodies. For immunoprecipitation, cells were lysed in TGN (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Tween 20, 0.3% NP40 supplemented with EDTA-free protease Figure 5 | Inhibition of OTUB1 rescues ATM inhibition. a, Quantification of and phosphatase inhibitor cocktails (Nacalai Tesque)). For acid extraction of his- large 53BP1 ionizing radiation-induced foci. (mean 6 s.d., N 5 3). U2OS tones, the nuclear fraction of cellswas extracted into 0.4 N H2SO4 and histones were cells transfected with the indicated siRNAs were treated with KU55933 or precipitated bythe addition of trichloroacetic acid. Invitro ubiquitination reactions DMSO, irradiated with 2 Gy and then processed for 53BP1 were performed as described previously3,25 with modifications detailed in Methods. immunofluorescence 1 h post-ionizing radiation. b, c, Cells transfected with the indicated siRNAs or transfected with OTUB1 expression vectors were Full Methods and any associated references are available in the online version of subjected to DR-GFP assay following treatment with KU55933 or DMSO the paper at www.nature.com/nature. 6 5 d (mean s.e.m., N 3). , U2OS cells transfected with a GFP–OTUB1 were Received 8 February; accepted 21 June 2010. irradiated with 3 Gy and processed for BRCA1 and c-H2AX immunofluorescence. Dashed line represents the outline of the nucleus. 1. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, Scale bar, 10 mm. 503–533 (2001). 945 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 466 | 19 August 2010

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Cell Biol. 10, 550–563 (2009). www.nature.com/nature. 13. Nicassio, F. et al. Human USP3 is a chromatin modifier required for S phase progression and genome stability. Curr. Biol. 17, 1972–1977 (2007). Acknowledgements We are grateful to T. Ikura, S. Olivarius, R. Sakasai, A. Shibata, 14. Shao, G. et al. The Rap80-BRCC36 de-ubiquitinating enzyme complex T. Oikawa, J. Unno, Y. Katuski, I. Imoto, S. Koyasu, R. Greenberg and the Core antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand Instrumentation Facility, Keio University School of Medicine for technical support breaks. Proc. Natl Acad. Sci. USA 106, 3166–3171 (2009). and reagents. We also thank R. Szilard and S. Angers for reading the manuscript. 15. Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon Work in the Nakada group in the Suda laboratory is supported by the Promotion of production. Science 318, 1628–1632 (2007). Environmental Improvement for Independence of Young Researchers, ‘Kanrinmaru 16. Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 Project’ from the Ministry of Education, Culture, Sports, Science and Technology downregulate NF-kB signalling. Nature 430, 694–699 (2004). (MEXT) Japan, Grant-in-Aid for Young Scientists (Startup 2009, 21870033) from 17. Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive MEXT, Takeda Science Foundation, Mitsubishi Pharma Research Foundation and regulator of Wnt-induced transcription with preference for binding and cleaving the Daiwa Anglo-Japanese Foundation. I.T. is supported by MEXT’s Distinctive K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008). University Education Support Program ‘Good Practice’. Work in the Natsume 18. Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated laboratory is supported by the New Energy and Industrial Technology deubiquitination. Biochem. J. 418, 379–390 (2009). Development Organization (NEDO). S.P. holds a studentship from the Boehringer 19. Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 Ingelheim Fonds. D.D. is the Thomas Kierans Chair in Mechanisms of Cancer linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009). Development and a Canada Research Chair (Tier 2) in Proteomics, Bioinformatics 20. Balakirev, M. Y., Tcherniuk, S. O., Jaquinod, M. & Chroboczek, J. Otubains: a new and Functional genomics. Work in the Durocher and Gingras laboratories is family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517–522 supported by grants MOP10703115 (DD) and MOP84314 (ACG) from the (2003). Canadian Institutes of Health Research. 21. Natsume, T. et al. A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal. Chem. 74, 4725–4733 Author Contributions S.N. and D.D. designed the experiments. S.N. performed (2002). most of the experiments described in the text. I.T. performed several experiments. 22. Goudreault, M. et al. A PP2A phosphatase high density interaction network M.M. and S.P. generated the inducible Flag–OTUB1 cell line. S.P. performed identifies a novel striatin-interacting phosphatase and kinase complex linked to immunofluorescence for UbK63, RNF168 and BRCA1 and carried out the ‘Toronto’ the cerebral cavernous malformation 3 (CCM3) protein. Mol. Cell. Proteomics 8, immunoprecipitation-tandem mass spectrometry (IP-MS/MS). L.O. carried out 157–171 (2009). the DR-GFP assays. Y.-C.J. carried out binding experiments with charged E2s and 23. Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human ubiquitination assays with UBE2D2 and UBE2L3. A.A.-H. carried out RNF168 deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009). immunoprecipitations. F.S. supervised Y.-C.J.; A.-C.G. analysed the ‘Toronto’ 24. Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2- IP-MS/MS data. S.-i.I. and T.N. performed and analysed the ‘Tokyo’ IP-MS/MS. Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific A.K. constructed plasmids and purified recombinant proteins. T.S. supervised I.T.; polyubiquitin chain formation. Nature Struct. Mol. Biol. 13, 915–920 (2006). S.N. and D.D. wrote the manuscript. 25. Petroski, M. D. et al. Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme. J. Biol. Chem. 282, Author Information Reprints and permissions information is available at 29936–29945 (2007). www.nature.com/reprints. The authors declare no competing financial interests. 26. VanDemark, A. P., Hofmann, R. M., Tsui, C., Pickart, C. M. & Wolberger, C. Readers are welcome to comment on the online version of this article at Molecular insights into polyubiquitin chain assembly: crystal structure of the www.nature.com/nature. Correspondence and requests for materials should be Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001). addressed to D.D. ([email protected]) or S.N. ([email protected]).

METHODS (1 mM) and recombinant OTUB1 proteins (18 mM) were assembled and incubated at 37 C. After 5 min, UEV1a (1 mM), ubiquitin (240 mM) and EDTA Cell culture and plasmid transfection. Cells were grown in Dulbecco’s modified u (10 mM) were rapidly mixed and incubated at 37 C for indicated time points. Eagle’s medium (DMEM) with 10% FBS. Transient and stable transfections were u Western blotting. Nitrocellulose membranes (GE Healthcare) were blocked performed using Effectene (Qiagen) following the manufacturer’s protocol. 21 with 5% Difco skim milk (BD) in TBST and stained with the indicated primary U2OS cells stably expressing OTUB1 were selected in 500 mgml G418 2 antibodies followed by horseradish peroxidase (HRP)-conjugated secondary (Nacalai Tesque) and maintained in 200 mgml 1 G418. HeLa cells were obtained antibodies. For biotin-streptavidin reactions, membranes were blocked with from the Health Science Research Resources Bank. U2OS cells were obtained Blocking One (Nacalai Tesque) and stained with HRP–streptavidin (DAKO). from ATCC. Supersignal West Dura (Pierce) or Chemi-Lumi One Super (Nacalai Tesque) Plasmids. The cDNAs for human OTUB1, RNF168 and ubiquitin were obtained were used for chemiluminescent HRP-conjugated antibody detection. from Open Biosystems. The cDNA for human H2AX was a kind gift from T. Chemiluminescent signals were detected with LAS4000 (Fuji Film). Ikura. The epitope-tagged versions of OTUB1, RNF168, ubiquitin and H2AX Immunoprecipitation. Typically, 10 cm dishes of 293T cells were grown and each were generated by subcloning the respective cDNAs into the pcDNA3-Flag, dish transfected with a total of 4–10 mg of indicated plasmids using Effectene pcDNA3-HA, pCMV-Myc (Clontech), pEGFP-C1 (Clontech), pGEXT4 (Qiagen). After 24 h post-transfection, cells were lysed in 0.7 ml of ice-cold (Promega) or pGEXP6 (Promega) as appropriate. The yellow fluorescent protein TGN buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Tween 20, 0.3% NP40 (YFP)–RNF8 expression vector was derived from that described previously6. supplemented with EDTA-free protease and phosphatase inhibitor cocktails Point mutations and deletions were generated by site-directed mutagenesis. (Nacalai Tesque)). Cell lysates were sonicated and then clarified by centrifugation Primers for site-directed mutagenesis were designed by The QuikChange at 4 uC for 30 min at 26,000g. Clarified cell lysates were pre-cleared using protein Primer Design Program (Stratagene). G-agarose (Sigma-Aldrich) and then subjected to immunoprecipitation with 20 ml RNA interference. siRNAs employed in this study were SMARTpools of anti-Flag (M2) affinity gel (Sigma-Aldrich) or anti-HA antibody. Immuno- (ThermoFisher) or individual siRNAs deconvolved from the SMARTpool. The precipitations were carried out for 1 h at 4 uC. Immunoprecipitates were washed SMARTpool (M-021061-00) directed against OTUB1 was employed in the four times with 1 ml of TGN buffer followed by two washes with ice-cold PBS. The experiments in Fig. 1b, c and Supplementary Table 1. The individual siRNA beads were then boiled in 60 mlof23 SDS sample buffer. duplex #2 (D-021061-02) targeting OTUB1 was employed in the most of experi- GST-pull down. A 50% slurry of glutathione Sepharose 4B beads (20 ml, ments. Non-targeting control siRNAs were purchased from Thermo Fisher or GE Healthcare) was equilibrated in assay buffer (50 mM Tris-HCl, pH 7.5, Sigma-Aldrich. Endoribonuclease-prepared siRNAs (esiRNAs) were produced 21 5 mM MgCl2, 100mM NaCl, 10% glycerol, 0.5 mg ml BSA and 5 mM b- exactly as described in ref. 35. All RNAi transfections were performed using mercaptoethanol). GST–OTUB1 (5 mg) was incubated with 5 ml of glu- DharmaFECT 1 (Thermo Fisher). tathione-Sepharose 4B in 100 ml of binding buffer (50 mM Tris-HCl, pH 7.5, Recombinant protein production. Expression of recombinant proteins in 21 5 mM MgCl2, 100mM NaCl, 10% glycerol, 0.5 mg ml BSA and 5 mM b- Escherichia coli strain BL21 was induced when bacterial cultures reached an mercaptoethanol) for 1 h. The beads were washed with binding buffer once A600 of 0.5 using 0.1 mM isopropyl-b-D-thiogalactopyranoside. Fusion proteins and incubated with 1 mg of recombinant UBC13 or UBC13/UEV1a complex were purified on glutathione-Sepharose 4B according to the batch method in100 ml of binding buffer for 1 h. Beads were washed with binding buffer four described by the supplier (GE Healthcare). PreScission protease (GE times and eluted with 25 ml of elution buffer (20 mM reduced glutathione in Healthcare) was used to remove the GST tag. Recombinant hexahistidine-tagged 50 mM Tris-HCl (pH 8.0)). For binding assays using ubiquitin-charged UBC13, UBE1, UBC13, UEV1a, UBC13/UEV1a, ubiquitin and OTUB1 were purchased 20 ml of 50% slurry of glutathione Sepharose 4B beads was equilibrated in assay from Boston Biochem. Biotinylated ubiquitin and recombinant hexahistidine- buffer (50 mM HEPES, pH 7.0, 100 mM NaCl, and 5 mM b-mercaptoethanol). tagged UBE2D2 and UBE2D3 were purchased from Biomol. Recombinant GST- GST–OTUB protein (20 mg) was added to the beads and incubated for 30 min at tagged RNF8 and TRAF6 were purchased from Abnova. 4 uC on a nutator. The beads were washed three times with 500 ml of the assay Antibodies. We used the following antibodies: OTUB1 (O9764, Sigma), 53BP1 buffer and mixed with 30 mg of E2 protein in a final volume of 50 ml assay buffer (clone 19, BD Biosciences; NB100-305, Novus Biologicals), K63-linked ubiquitin and then incubated for 2 h on ice. The beads were washed with 500 ml of assay chains (Apu3.A8, Genentech), MDC1 (AbD Serotec, AHP799), c-H2AX (clone buffer twice. The reaction was stopped with the addition of 10 mlof23 SDS JBW301, Upstate; 2577, Cell Signaling), histone H3 (ab1791, Abcam), conjugated loading buffer to the samples and heating at 90 uC for 10 min. 3 ubiquitin (clone FK2, Stressgen), RNF168 (ab58063, Abcam; RNF168-C ), ubi- Nuclear extracts and chromatin isolation. The procedure was performed as quitin (04-454, Upstate), GST (clone GS019, Nacalai Tesque), Flag (M2, Sigma; described previously36. F7425, Sigma), HA (clone HA-7, Sigma), myc (clone 9E10, Santa Cruz; 631206, DR-GFP assay. A HeLa clone carrying the DR-GFP homologous recombination Clontech), and GFP (GF090R, Nacalai Tesque) and BRCA1 (MS110 and MS13, reporter30 was used for analysis of homologous recombination. For analyses with Calbiochem). siRNA, cells were transfected with the indicated siRNA for 48 h. Cells were then Immunofluorescence microscopy. HeLa or U2OS cells were grown on No. 1 treated with DMSO or 10 mM KU55933 for 30 min, then transfected with 1 mgof glass coverslips. U2OS cells stably expressing OTUB1 were grown on poly-L- pCBASce, the I-SceI expression plasmid. The cells were placed back into media lysine 12 mm No. 1 glass coverslips (BD). Fixation and staining procedure were containing DMSO or 10 mM KU55933 for an additional 48 h before analysis. For described previously3. Confocal images were taken using an inverted micro- analyses involving OTUB1 plasmids, cells were co-transfected with 5 mg of the scopes (DMIRE2, Leica; Axiovert200M, Zeiss) with a spinning disk confocal indicated plasmids and 1 mg of pCBASce for 48 h before analysis. For all scanner (CSU10, Yokogawa) and equipped with a 363 oil or 340 water immer- described experiments, cells were collected via trypsinization, washed twice with sion lens, or an inverted microscope (TCS SP5, Leica) equipped with a 363 oil PBS, resuspended in 1 ml PBS and filtered. The proportion of GFP-positive cells immersion lens. Images were acquired using Volocity Software v. 4.1 was determined by flow cytometry on a BD FACSCalibur flow cytometer and (Improvision) or LAS AF (Leica). For quantification of the data obtained with analysed using FlowJo (Tree Star). immunostaining, a minimum of 300 cells (siRNA transfection) or 100 Flag- Histone acid extraction. Cells were lysed in nuclear preparation buffer (10 mM positive cells (Flag–OTUB1 overexpression) were analysed per experiment. Tris-HCl, pH 7.6, 150 mM NaCl, 1.5 mM MgCl2, 0.65% NP40). Nuclei were col- In vitro ubiquitination assay. Assays were set up in a total volume of 25 mlin lected by centrifugation (5 min, 920g,4uC). The nuclear pellet was extracted into 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 2 mM ATP, 1 mM DTT. UBE1 0.4 N H2SO4 for 1 h at 4 uC. The acid soluble fraction was collected after centrifu- (0.0125 mM; Boston Biochem), 0.4 mM of E2 (UBC13/UEV1a; Boston gation (5 min, 7,500g,4uC). Trichloroacetic acid was added to the supernatant to a Biochem), UBC13 (Boston Biochem) UBE2D2 or UBE2D3 (Biomol), ubiquitin final concentration of 20% and then incubated on ice for 1 h. Precipitates were (40 mM ubiquitin (Boston Biochem) or 10 mM biotinylated ubiquitin (Biomol)) collected by centrifugation (10 min, 15,000g,4uC). Precipitates were washed once and/or 0.2 mM recombinant E3 (GST–RNF168, GST–RNF8 (Abnova) or GST– with 0.1% HCl-acetone and twice with acetone and then resuspended in dH2O. TRAF6 (Abnova)) were incubated with various amounts of recombinant GST– Immunoprecipitation coupled with a direct nano-flow liquid chromatography- OTUB1. Reaction mixtures were incubated at 37 uC for 16 h. tandem mass spectrometry (Tokyo). Flag–OTUB1 was expressed in HEK293 cells UBC13–ubiquitin thioester bond formation assay. Assays were set up in a total and immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were volume of 25 ml in 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2,1mM eluted with a Flag peptide, digested with Lys-C endopeptidase (Achromobacter ATP, and 0.5 mM DTT. UBE1 (0.15 mM), 15 mM of UBC13 (Boston Biochem) protease I) and the cleaved fragments were directly analysed by a direct nano-flow and 12.5 mM of ubiquitin were incubated with various amount of recombinant liquid chromatography-tandem mass spectrometry (LC–MS/MS) system as previ- OTUB1 proteins (Boston Biochem). Reaction mixtures were incubated at 37 uC ously described21. Assays were repeated four times. for 5 min. Immunoprecipitation followed by tandem mass spectrometry (Toronto). UBC13 charge/discharge assay. Reactions containing UBE1 (0.04 mM), UBC13 Inducible Flag–OTUB1 and empty Flag control HEK293 cell lines were generated (2 mM), biotinylated-ubiquitin (5 mM), DTT (0.5 mM), MgCl2 (5 mM) and ATP by Flp-In T-REx technology (Invitrogen) as per the manufacturer’s protocol.

Flag affinity purification was performed on biological replicates of the bait personal communication ). Likely contaminants were removed from the raw list (OTUB1) and of the control (Flag alone) essentially as described previously22 of identified proteins. They included proteins that were detected in one of the with the following modifications. Baits and associated proteins were eluted with two control runs and the 55 most abundant proteins co-precipitating with the ammonium hydroxide, lyophilized in a SpeedVac, resuspended in 50 mM M2-agarose resin alone across multiple analyses (Datta et al., personal commu- ammonium bicarbonate (pH 8–8.3), and incubated at 37 uC with trypsin over- nication). To increase confidence in the interactions, only hits detected with 2 or night. The ammonium bicarbonate was evaporated, and the samples were resus- more unique peptides and with a ProteinPeptide probability .0.95 are reported pended in HPLC buffer A (3% ACN, 0.1% formic acid) and directly loaded onto in Supplementary Table 3. capillary columns packed in-house with Magic 5 mm, 100 A˚ , C18AQ. MS/MS data were acquired in data-dependent mode (over a 2-h acetonitrile 2–40% 35. Kittler, R., Heninger, A. K., Franke, K., Habermann, B. & Buchholz, F. Production of gradient) on a ThermoFinnigan LTQ equipped with a Proxeon NanoSource endoribonuclease-prepared short interfering RNAs for gene silencing in and an Agilent 1100 capillary pump. mammalian cells. Nature Methods 2, 779–784 (2005). Acquired RAW files were converted to mgf format, and searched with the 36. Me´ndez, J. & Stillman, B. Chromatin association of human origin recognition complex, Cdc6, and minichromosome maintenance proteins during the cell cycle: Mascot search engine (Matrix Sciences) against the human RefSeq database assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, (release 37; 38,097 entries searched) with a precursor ion mass tolerance of 3.0 8602–8612 (2000). and a fragment ion mass tolerance of 0.6. Methionine oxidation and glutamine 37. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to or asparagine deamidation were allowed as variable modifications, and trypsin estimate the accuracy of peptide identifications made by MS/MS and database specificity (with one missed cleavage allowed) was selected. search. Anal. Chem. 74, 5383–5392 (2002). PeptideProphet37 was run on each result set, and the resulting lists of peptides 38. Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for were assembled into proteins using ProteinProphet38. ProteinProphet outputs identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 were parsed into a relational database developed in-house (ProHits; Liu et al., (2003).

Waves on the surface of the Orion molecular cloud

Olivier Berne´1,2,Nu´ria Marcelino1 & Jose´ Cernicharo1

Massive stars influence their parental molecular cloud, and it has long molecular gas towards the far and near sides of the nebula (respectively been suspected that the development of hydrodynamical instabilities red and blue in Fig. 1b). The X-ray plasma bubble11 fills the southern can compress or fragment the cloud1,2. Identifying such instabilities cavity of molecular gas (Fig. 1b). In the infrared, the cloud surfaces has proved difficult. It has been suggested that elongated structures have ‘ripples’ in several regions. This is particularly obvious in the (such as the ‘pillars of creation’3) and other shapes arise because of southwest region, (Fig. 1c), where a series of five surprisingly regular instabilities4,5, but alternative explanations are available6,7.Onekey wavelets (hereafter ‘the ripples’) enshrouding an elongated molecular signature of an instability is a wave-like structure in the gas, which has cloud, subject to a strong velocity gradient (7–9 km s21 pc21; Figs 1c hitherto not been seen. Here we report the presence of ‘waves’ at the and 2a), are seen. surface of the Orion molecular cloud near where massive stars are A possibility would be that the CO structure results from the forming. The waves seem to be a Kelvin–Helmholtz instability that outflow of a young protostar and that the infrared emission comes arises during the expansion of the nebula as gas heated and ionized by from the associated Herbig–Haro objects12–14. However, we do not massive stars is blown over pre-existing molecular gas. find any evidence of an embedded protostar able to drive an outflow Because it is the closest massive-star-forming region (distance, in this region, either in the Spitzer IRAC/Multiband Imaging ,414 pc; ref. 8), the Orion nebula provides a unique opportunity to Photometer images or in the SIMBAD database. Nor do we find study, on small spatial scales and combining different wavelengths, the evidence of wings in the line profiles of 12CO emission (Fig. 2a), feedback action of the H II region and plasma on the cloud9,10. Figure 1 which are characteristic of outflows15. Examining the Spitzer shows the carbon monoxide (CO) millimetre map of the cloud, which Infrared Spectrograph (IRS) archival spectrum of the second ripple has a spatial coverage and angular resolution allowing morphological (Fig. 2c), we find no trace of highly ionized species ([Ne III], [Fe II]) comparison with high-angular-resolution mid-infrared and X-ray that would attest to the presence of a high-velocity Herbig–Haro images. shock12–14 (the [S III] emission detected here is spatially associated In the southern part of the nebula, the growing bubble of gas with the H II region). The comparison among the spatial distribution 12 ionized by massive stars (H II region) has pushed the surrounding of PAHs, the H2 0-0 S(1) pure rotational line intensity and CO

Figure 1 | Multiwavelength overview of the Orion a b 12 –1 CO –5–8 km s nebula. a, Spitzer mid-infrared image of the Orion 12CO 12–25 km s–1 MSX 8.3 μm nebula (courtesy of NASA JPL/Caltech/T. Megeath). b, Overlay of the Institut de Radioastronomie Millime´trique (IRAM) 30-m 12CO J 5 2-1 (where J stands for rotational Rippled clouds quantum number) maps of integrated emission in velocity between 25–8 km s21 (blue) and 12–25 km s21 (red), tracing the cold (10–100-K) molecular gas, and 8-mm emission from the Trapezium Midcourse Space Experiment (MSX) (1899 resolution; green), tracing dust and warm (100- 1,000-K) gas at the surface of clouds. The CO observations were performed using the IRAM heterodyne receiver array (HERA) multibeam H II region 24 12 X-ray bubble receiver , tuned at the frequencies of CO and 13CO J 5 2-1 (respectively 230.5 and 220.5 GHz) in each polarization. The spatial resolution of the maps is 1299 (0.025 pc) and the spectral resolution is 1 pc 0.4 km s21. c, Close-up view of the ripples in 12CO (2-1) emission at different velocities (blue) overlaid c 5.4 km s–1 5.8 km s–1 6.2 km s–1 6.8 km s–1 on the Spitzer Infrared Array Camera (IRAC) 3.6- mm map (attributed to emission of polycyclic aromatic hydrocarbons (PAHs); 2.599 resolution; green). In a and b, the solid contours delimit the 7.2 km s–1 7.6 km s–1 8.0 km s–1 diffuse soft-X-ray emission tracing the hot plasma 12CO detected by the X-ray Multi-Mirror Mission11,and IRAC 3.6 µm the dashed contours delimit the H II region.

1Centro de Astrobiologıá, CSIC/INTA, Carretera de Torrejoń a Ajalvir, km 4, 28850 Torrejoń de Ardoz, Spain. 2Leiden Observatory, Leiden University, PO Box 9513, NL-2300 RA Leiden, The Netherlands. 947 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a To Trapezium 123Ripple 12CO (2-1) 20 12CO (2-1) a Geometrical model Molecular * (K)

A 10 cloud T behind Ultraviolet photons 0 Plasma 0 5 10 15 0 5 10 15 0 5 10 15 V –1 V –1 V –1 LSR (km s ) LSR (km s ) LSR (km s ) H II region b

IRS cut 2 3 α 1 Molecular cloud

IRAC 3.6 µm 12CO 0.1 pc To observer c d East West 1.1 H (0-0) S(1) 2 [S III] H 12 b 180 PAH PAH 2 CO 0.0 0.1 0.2 0.3 )

–1 PAH emission Expected intensity variations Ripple summit 0.9 140 CO emission Ripple valley 100 0.7

H II region 60 0.5

Intensity (MJy sr HH2 shock 20 0.3 15 16 17 18 19 20 21 0 0.02 0.04 0.06 0.08 0.1 Intensity variations (normalized) Wavelength (µm) Distance (pc) c 0.0 0.1 0.2 0.3 IRAC 3.6 µm (PAH emission) Observed intensity variations Figure 2 | Infrared and millimetre observations of the ripples. a,IRAM IRAM 30 m (CO emission) HERA spectra of the 12CO and 13CO (2-1) lines at the three positions indicated in b. TA* is the antenna temperature and VLSR is the velocity relative to the local standard of rest. The red vertical long-dashed line indicates the velocity of ripple 1 and short-dashed lines indicate the velocities of ripples 2 and 3. b, General view of the ripples and surrounding environment, with the Spitzer IRAC 3.6-mm image in green and 12CO integrated over all velocities overlaid in blue. c, Spitzer IRS-LL2 spectra of the summit and valley of ripple 2 and the surrounding H II region. The LL2 spectrum of the Herbig–Haro 2 0.0 0.1 0.2 0.3 (HH2) shock is shown for comparison. d, East-to-west cut across a ripple, Distance (pc) showing the evolution of the line-integrated emission of PAH, H2 and Figure 3 | Geometrical model of the ripples. a 12 , Schematic of the geometric CO presented in the spectra. configuration. b, c, Expected PAH and 12CO emission (b) and PAH and 12CO emission observed with IRAM and IRAC (c). We assume that energy (2-1) emissions in an east-to-west cut across the ripples (marked ‘2’ collected in the IRAC 3.6-mm broadband filter is dominated by the 3.3-mm in Fig. 2b) shows a stratification with PAHs more to the east, followed band of PAHs. This is probably the case because the 16.4-mm PAH band is by H2 and then CO emission (Fig. 2d). This stratification is expected strongly detected in spectroscopy. The displacement of peak emission in ultraviolet-driven photodissociation regions that lie at the bound- between the two tracers can be well explained by a configuration in which the ary between H II regions formed by massive stars and molecular major axis of an elongated cloud forms an angle, a, of less than 90u with the 16,17 direction pointing to the Trapezium stars: the energy per unit of surface area gas , but not in shocks. Furthermore, the H2 S(1) intensity (,7 3 1028 Wm22 sr21) is compatible with the predictions of emitted by PAH molecules at the surface of the cloud, which depends on the ultraviolet illumination, is stronger on the upward side of the ripple and photodissociation-region models18 reproducing the conditions of 4 23 3 weaker on the downward, shadowed, part. However, the intensity of CO the observed region: dense gas (.10 cm ) highly irradiated (10 emission depends on the column density of gas in the line of sight, so it peaks times the interstellar standard radiation field). We therefore con- at the summit of the ripple. The observed CO intensity gives a gas column 21 22 clude that the observed periodic structure and emission are not the density of NH2 < 6 3 10 cm (see Supplementary Information for result of an outflow/Herbig–Haro shock but are instead consistent details), corresponding to an optical depth25 at 3.6 mmof,1022. This makes with a succession of photodissociation regions lying at the surface of it possible to see perfectly through the cloud in the mid-infrared. This an elongated, rippled, molecular cloud, shaped in a smooth process geometry is compatible with the ripples being in the blueshifted part of the (Fig. 3). cloud (Fig. 1), in front of the H II region, and with the velocity gradient due to the passage of the flow being from east to west. This configuration could also The rippled molecular cloud is subject to an important velocity 21 21 be an explanation for the non-detection of the plasma in X-rays, owing to gradient (7–9 km s pc ; Figs 1 and 2). This gradient cannot simply 11,26 21 extinction at this position . be due to the expansion of the H II region (that is ,3kms for a spherical bubble several parsecs in diameter, calculated on the basis of between fluids of different densities, whereas for the Kelvin–Helmholtz the difference between the mean velocities of the blue and red parts of instability to occur there must additionally be a velocity difference the southern cloud), so it must result from the acceleration of the between the fluids. In the present case, where density and velocity cloud by the passage of a flow of diffuse gas. We therefore propose gradients are present, the Kelvin–Helmholtz instability will dominate that the ripples have been formed by the mechanical interaction of (Supplementary Information). In addition to the hydrodynamical high-velocity plasma/gas, produced by massive stars, with the dense phenomenon, the effect of energetic radiation has to be taken into molecular gas, which has provoked hydrodynamical instabilities. The account. Far- and extreme-ultraviolet (respectively FUV and EUV) simplest non-trivial hydrodynamical instabilities able to explain the photons emitted by the massive stars will create a photo-ablation layer observed structures are Rayleigh–Taylor and Kelvin–Helmholtz at the surface of the cloud that will insulate the molecular cloud from instabilities19. The Rayleigh–Taylor instability occurs in an interface the shearing flow and prevent the instability from developing. 948 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

To verify that the development of a Kelvin–Helmholtz instability is Received 27 January; accepted 10 June 2010. possible, let us first consider a purely hydrodynamical case, where the 1. Tenorio-Tagle, G. & Bodenheimer, P. Large-scale expanding superstructures in insulating layer is assumed to be infinitely thin. Kelvin–Helmholtz galaxies. Annu. Rev. Astron. Astrophys. 26, 145–197 (1988). 2. Woodward, P. R. Shock-driven implosion of interstellar gas clouds and star instabilities with large spatial wavelengths above a value lKH will be removed by gravity19. The value of l depends on (Supplementary formation. Astrophys. J. 207, 484–501 (1976). KH 3. Hester, J. J. etal. HubbleSpace Telescope WFPC2 imaging ofM16:photoevaporation Information) the self gravity, gc, of the cloud; the density, nf, and velo- and emerging young stellar objects. Astron. J. 111, 2349–2360 (1998). city, vf, of the shearing gas; and the density, nc, of the sheared gas in the 4. Spitzer, L. Physical Processes in the Interstellar Medium 223–225 (Wiley- cloud. The CO observations allow us to estimate the cloud column Interscience, 1978). density, which, assuming a cylindrical geometry, constrains the density 5. Rodriguez-Franco, A., Martin-Pintado, J., Gomez-Gonzales, J. & Planesas, P. 4 23 211 22 Large-scale interaction of the H II region and the quiescent gas in Orion A. Astron. nc and gravity gc (respectively 10 cm and 3.5 3 10 ms )such Astrophys. 264, 592–609 (1992). that lKH can be estimated only from the parameters nf and vf. Similarly, 6. Pound, M. W. Molecular gas in the Eagle nebula. Astrophys. J. 493, L113–L116 (1998). the growth rate, vKH, of a Kelvin–Helmholtz instability of known spa- 7. Pound, M. W., Reipurth, B. & Bally, J. Looking into the Horsehead. Astron. J. 125, tial wavelength, l, solely depends on n and v (ref. 19 and 2108–2122 (2003). f f 8. Menten, K. M., Reid, M. J., Forbrich, J. & Brunthaler, A. The distance to the Orion Supplementary Information). The observed value, l 5 0.11 pc, sets a nebula. Astron. Astrophys. 474, 515–520 (2007). 21 minimum for lKH and the observed width of the CO lines (,2kms ), 9. Genzel, R., &. Stutzki, J. The Orion molecular cloud and star-forming region. Annu. and the height of the ripples (unlikely to be more than about 0.1 pc) Rev. Astron. Astrophys. 27, 41–85 (1989). implies a maximum growth rate of v < 2 3 1025 yr21. The analysis 10. Bally, J. in Handbook of Star Forming Regions: Vol. I, The Northern Sky (ed. Reipurth, B.) KH 459–473 (ASP Monogr. Publ. 4, Astronomical Society of the Pacific, 2008). of the evolution of lKH and vKH using a grid of realistic values of nf and 11. Gu¨del, M. et al. Million-degree plasma pervading the extended Orion nebula. vf shows that typical properties of either H II gas or X-ray plasma result Science 319, 309–311 (2008). in values for lKH and vKH that respect the observational constraints, 12. Neufeld, D. A. et al. Spitzer spectral line mapping of protostellar outflows. I. Basic and allow an instability to grow. data and outflow energetics. Astrophys. J. 706, 170–183 (2009). 13. Lefloch, B. et al. Warm molecular hydrogen and ionized neon in the HH 2 outflow. Now let us consider the effect of ultraviolet radiation. This will Astrophys. J. 590, L41–L44 (2003). allow the propagation of the instability to the entire cloud only if 14. Tappe, A., Lada, C. J., Black, J. H. & Muench, A. A. Discovery of superthermal the instability wavelength is greater than the thickness of the hydroxyl (OH) in the HH 211 outflow. Astrophys. J. 680, L117–L120 (2008). photo-ablation layer, which consists of a FUV-dominated sublayer 15. Bachiller, R. Bipolar molecular outflows from young stars and protostars. Annu. Rev. Astron. Astrophys. 34, 111–154 (1996). and an EUV-dominated sublayer (Supplementary Fig. 2). The 16. Tielens, A. G. G. M. et al. Anatomy of the photodissociation region in the Orion thickness of the FUV-dominated sublayer, estimated from the bar. Science 262, 86–89 (1993). PAH emission in the IRAC images, is probably low (,0.01 pc). 17. Berne´,O.et al. Mid-infrared polycyclic aromatic hydrocarbon and H2 emission as However, estimates show (Supplementary Information) that in the a probe of physical conditions in extreme photodissociation regions. Astrophys. J. neighbourhood of h1 Orionis C, the EUV-dominated sublayer is 706, L160–L163 (2009). 18. Kaufman, M. J., Wolfire, M. G., &. Hollenbach, D. J. [Si II], [Fe II], [C II], and H2 too large to allow the propagation of a Kelvin–Helmholtz instab- emission from massive star-forming regions. Astrophys. J. 644, 283–299 (2006). ility to the cloud. Given that the growth time for the observed 19. Chandrasekhar, S. Hydrodynamic and Hydromagnetic Stability Ch. XI (Int. Ser. instability is estimated to be .105 yr (ref. 19), it is possible that Monogr. Phys., Clarendon, 1961). 1 20. Ryutov, D. D., Kane, J. O., Pound, M. W. & Remingtom, B. A. Instability of an it formed before the birth of h OriC.Atthattime,theLyman ablatively-accelerated slab in the case of non-normal irradiation. Plasma Phys. continuum photon flux would have been significantly lower than Contr. Fusion 45, 769–781 (2003). today, and as a consequence so would have been the density and 21. Ryutov, D. D., Kane, J. O., Mizuta, A., Pound, M. W. & Remington, B. A. thickness of the EUV-dominated layer. With a much smaller insu- Phenomenological theory of the photoevaporation front instability. Astrophys. lating layer and in the absence of the X-ray plasma (which can only Space Sci. 307, 173–177 (2007). 1 22. Johnstone, D., Hollenbach, D. & Bally, J. Photoevaporation of disks and clumps by be driven by h Ori C), it would have been the expansion of dense nearby massive stars: application to disk destruction in the Orion nebula. H II gas produced by the population of older massive stars that Astrophys. J. 499, 758–776 (1998). resulted in the shearing of a pre-existing molecular clump, forming 23. Bally, J., Licht, D., Smith, N. & Walawender, J. Irradiated and bent jets in the Orion the Kelvin–Helmholtz instability at the position where we observe nebula. Astron. J. 131, 473–500 (2006). 24. Schuster, K.-F. et al. A 230 GHz heterodyne receiver array for the IRAM 30m the ripples. Because the surface of the cloud considered here is telescope. Astron. Astrophys. 423, 1171–1177 (2004). rippled and tilted, the later introduction of intense irradiation after 25. Weingartner, J. C. & Draine, B. T. Dust grain-size distributions and extinction in the birth of h1 Ori C would have resulted in a heterogeneous rocket the Milky Way, Large Magellanic Cloud, and Small Magellanic Cloud. Astrophys. J. acceleration20,21 of the clumps, possibly taking over from the 548, 296–309 (2001). 26. Wilms, J., Allen, A. & McCray, R. On the absorption of X-rays in the interstellar Kelvin–Helmholtz instability. medium. Astrophys. J. 542, 914–924 (2000). This selective photo-abrasion is now generating a train of molecu- Supplementary Information is linked to the online version of the paper at lar globules where the most eastern part of the elongated cloud is www.nature.com/nature. already detached from the rest of the cloud (Fig. 2b). The masses of the globules, close to one solar mass, imply that they can survive Acknowledgements We thank J. Bally and M. Pound for their comments, which 5 22 contributed to the improvement of the manuscript. We also acknowledge several 10 years of photo-ablation (Supplementary Information). B. Lefloc’h, J. Goicoechea and J. Martı´n-Pintado for discussions. D. Hochberg is Such a picture for the formation of the ripples is supported by the acknowledged for his reading of the manuscript. O.B. acknowledges C. Joblin for observation of bended outflows in the H II region23, attesting to the support. This work is based in part on observations made with the Spitzer Space run-over by ionized gas of pre-existing molecular gas hosting low- Telescope, which is operated by the Jet Propulsion Laboratory, California Institute of Technology under a contract with NASA. mass-star formation, and by the periodical distribution of clumps in the molecular filaments, which may result from similar Kelvin– Author Contributions O.B. conducted the scientific analysis and write-up of the Helmholtz-induced fragmentation5. Additional observations of paper. N.M. conducted the CO observations and analysis. J.C. initiated the project and was its principal investigator. periodic structures in the Orion nebula or other massive-star- forming regions at high angular and spectral resolution (in particular Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. at millimetre and radio wavelengths) could contribute to obtaining a Readers are welcome to comment on the online version of this article at clearer picture of the history of combined radiative/hydrodynamical www.nature.com/nature. Correspondence and requests for materials should be feedback of massive stars on their parental cloud. addressed to O.B. ([email protected]).

Enhancement of superconductivity by pressure-driven competition in electronic order

Xiao-Jia Chen1,2, Viktor V. Struzhkin1, Yong Yu1, Alexander F. Goncharov1, Cheng-Tian Lin3, Ho-kwang Mao1 & Russell J. Hemley1

Finding ways to achieve higher values of the transition temper- put in a neon environment in a gasket hole to ensure near-hydrostatic ature, Tc, in superconductors remains a great challenge. The super- conditions. As previous measurements appear to be limited to below conducting phase is often one of several competing types of 18 GPa (refs 13, 14), to our knowledge these are the highest-pressure electronic order, including antiferromagnetism and charge density magnetic measurements used for the detection of superconductivity waves1–5. An emerging trend documented in heavy-fermion1 and in HTSCs. 2 organic conductors is that the maximum Tc for superconductivity Figure 2a shows the representative temperature scans of an optim- occurs under external conditions that cause the critical temper- ally doped Bi2223 single crystal, of initial size 80 3 80 3 10 mm3,at ature for a competing order to go to zero. Recently, such competi- different pressures. For such a sample with sufficiently large dimen- tion has been found in multilayer copper oxide high-temperature sions, we obtained a very strong amplitude signal during the mea- superconductors (HTSCs3–5) that possess two crystallographically surements when keeping the sample signal in phase with the inequivalent CuO2 planes in the unit cell. However, whether the background magnetization vector. The superconducting transition competing electronic state can be suppressed to enhance Tc in is identified by the onset of deviation in the signal from the almost HTSCs remains an open question. Here we show that pressure- constant background on the high-temperature side. The supercon- driven phase competition leads to an unusual two-step enhance- ducting transition at 108 K is obtained at ambient pressure. It is clear ment of Tc in optimally doped trilayer Bi2Sr2Ca2Cu3O101d that at the pressure of 10.2 GPa the superconducting transition shifts (Bi2223). We find that Tc first increases with pressure and then to higher temperatures than at 1 atm (,0.1 MPa), but it moves back decreases after passing through a maximum. Unexpectedly, Tc to lower temperatures beyond that pressure. increases again when the pressure is further raised above a critical In Fig. 2b, we plot the signal phase of another Bi2223 sample, of value of around 24 GPa, surpassing the first maximum. The pres- initial size 50 3 70 3 10 mm3, cleaved from the same single crystal as in ence of this critical pressure is a manifestation of the crossover from our previous run. For such a small sample, our magnetic susceptibility the competing order to superconductivity in the inner of the three technique still can detect the superconducting transitions from the CuO2 planes. We suggest that the increase at higher pressures temperature scan of the phase, although the amplitude does not occurs as a result of competition between pairing and phase order- exhibit a signal of the same quality as that of the large sample ing in different CuO2 planes. We used Bi2223 single crystals in the present investigation. The structure of Bi2223 consists of two insulating Bi-O and Sr-O blocking a b Modulating coil layers intercalating in the c-axis direction with two structurally equi- Gasket valent outer CuO2 planes and one inner CuO2 plane, surrounded by Bi-O two Ca planes (Fig. 1a). The appearance of both outer and inner Sr-O CuO2 planes is common structural feature shared by all multilayer systems. The interlayer coupling between the CuO planes should Cu-O Excitation coil 2 Ca have an important role in the significant enhancement of Tc in a Cu-O Sample Diamond Signal coil Compensating coil 6 17 63 7,8 homologous series . Studies of the O and Cu NMR spectra of Ca cd Bi2223 show that the outer and inner CuO2 planes have distinctly Cu-O different physical properties. The imbalance of charge distribution Sr-O among the inner and outer CuO planes has been revealed by angle- 2 Bi-O resolved photoemission spectroscopy also9. Such an imbalance may account for the slight decline in T of multilayer compounds when c Bi2223 Ruby Neon 10,11 the number of CuO2 layers within a unit cell is greater than three . We chose pressure as an external parameter to tune the phase Figure 1 | Magnetic susceptibility measurement set-up. a, Schematic of the crystal structure of Bi Sr Ca Cu O . b, Schematic of the double- competition in Bi2223. We performed measurements of Tc in 2 2 2 3 101d Bi2223 using an improved magnetic susceptibility technique frequency modulation set-up for the diamond anvil cell. The sample, 12 together with ruby, is located inside the hole in a non-magnetic gasket. The described previously . The measurements were made at a range of coil system includes a signal coil, a compensating coil, a high-frequency pressures up to 36.4 GPa. Figure 1b, d shows the schematic diagram excitation coil and a low-frequency modulating coil. c, The sample and ruby of the double-frequency modulation set-up for a diamond anvil cell. in a neon environment in the gasket hole at 36.4 GPa and 80 K. d, A signal A photograph taken through the diamond windows of a single crystal coil wound around a diamond anvil and a compensating coil connected in at 36.4 GPa is shown in Fig. 1c. The crystal, together with ruby, was opposition.

1Geophysical Laboratory, Carnegie Institution of Washington, Washington DC 20015, USA. 2Department of Physics, South China University of Technology, Guangzhou 510640, China. 3Max-Planck-Institut fu¨r Festko¨rperforschung, D-70569 Stuttgart, Germany. 950 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a The measured behaviour of Tc as a function of pressure in optimally doped trilayer Bi2223 is plotted in Fig. 3. Below 24 GPa, Tc is non- 0.8 GPa monotonically dependent on pressure; this is reminiscent of other 100 nV observations in various optimally doped HTSCs13–16. As pressure is 1.5 GPa increased, Tc increases with a large initial positive slope of about 21 2.3 GPa 4 K GPa and then decreases after passing a maximum of 123 K at an optimal pressure of around 12 GPa. After that, Tc does not mono- tonously decrease with pressure as observed in most HTSCs13–16, but Amplitude 5.1 GPa instead increases above 25 GPa. On further compression, Tc signifi- 6 10.2 GPa cantly increases, reaching a high value of 136 10 K at 36.4 GPa. We could not measure the trend in Tc at higher pressures owing to the T c weakness of the signal. Nevertheless, Tc is much greater in the high- 15.4 GPa pressure regime than in the initial regime in this trilayer system. The 80 90 100 110 120 130 anomaly pressure, around 24 GPa, may be considered as a quantum Temperature (K) critical point where pressure suppresses a competing order and enhances superconductivity in Bi2223. Similar unusual pressure b 5 –5 effects have been observed in other multilayer systems17,18. The co- 2.3 GPa 20.4 GPa –10 0 existence of a competing phase with superconductivity has been shown in many multilayer HTSCs4,5. The suppression of competing –15 –5 order and inducement of superconductivity by pressure could be a generic phenomenon in other strongly correlated systems1. –20 –10 100 110 120 130 80 100 120 140 To confirm the superconducting transition temperatures obtained –5 0 by magnetic susceptibility, we also examined the temperature depend- 4.2 GPa 29.6 GPa –5 ence of Raman spectra under pressure. Strong superconductivity- –10 –10 induced phonon self-energy effects have been observed by Raman 19–22 –15 T –15 scattering in many multilayer copper oxides, including Bi2223 .As c –20 a result of these effects, phonon softening occurs when going from the –20 normal to the superconducting state if the phonon frequency is below 100 110 120 130 80 100 120 140 0 and in the vicinity of the maximum superconducting gap, 2D0.The 0 Phase (º) softening of phonon modes below 2D for T , T has been observed in –5 11.2 GPa 32.1 GPa 0 c –5 many HTSCs19–23. The angle-resolved photoemission spectroscopy –10 –10 21 measurements give a D0 value of about 37 meV (2D0 < 590 cm )for –15 –15 Bi2223 at ambient pressure24. Therefore, softening of phonon modes of –20 21 –20 this material below 590 cm should take place below the supercon- 100 110 120 130 80 100 120 140 ducting transition, though both the phonon frequencies and D0 are –5 0 pressure dependent (Supplementary Information). 17.1 GPa –5 36.5 GPa Figure 4a shows the Raman spectrum of a Bi2223 single crystal for –10 –10 –15 different temperatures at a pressure of 33.6 GPa. The oxygen-related 21 –15 –20 modes above 300 cm have clear asymmetric line shapes with char- –25 acteristic anti-resonances in the lower-frequency tails over the entire –20 100 110 120 130 80 100 120 140 160 temperature range considered. This observation indicates a strong Temperature (K)

Figure 2 | Magnetic susceptibility signals of Bi2Sr2Ca2Cu3O101d single crystals at various pressures. a, Typical amplitude records for a crystal, 140 with an initial size of 80 3 80 3 10 mm3, on warming. To obtain the strong Run 1 amplitude signal, we kept the sample signal in phase with the background. Run 2 3 b, Typical phase records for a crystal, with an initial size of 50 3 70 3 10 mm , 130 on warming. To obtain the strong phase signal, we kept the sample signal phase at 90u to the background phase during the measurements. The arrows (K) c indicate the superconducting transitions. T 120 (Supplementary Information). We thus kept the sample signal phase at 90u to the phase of the background magnetization vector during the 110 OP OP measurements to get optimal signals. At pressures below 20 GPa, the IP CO IP determined Tc is similar in behaviour to that determined in the first OP OP run. At 11.2 GPa, we obtained a Tc of 123 K, which is very close to the 100 value obtained at 10.2 GPa from the temperature scan of the ampli- 0 10203040 Pressure (GPa) tude (Fig. 2a). There is good agreement between the values of Tc determined for the two samples from the temperature scans of ampli- Figure 3 | Pressure dependence of Tc in optimally doped tude and phase. At higher pressures, the susceptibility data always have Bi2Sr2Ca2Cu3O101d. The symbols represent independent runs on the a discontinuity in slope at temperatures above the signal peak, changing samples cleaved from the same crystal. The triangle represents Tc at ambient from a ‘low-temperature’ slope to a ‘high-temperature’ slope. The onset pressure, and the two yellow bars are the Tc values derived from Raman measurements at 15.0 and 33.6 GPa. The dashed line indicates the boundary of signal deviation from the high-temperature background, where between the two pressure regimes. The insets show the pressure-driven magnetic flux completely enters the sample, is still taken to indicate transition of the inner CuO2 plane (IP) from the competing order (CO) to Tc. The particular feature of the signal on the low-temperature side of the superconducting state. OP, outer plane. The arrows indicate electron the onset (the low-temperature slope) could be related to strong non- spin; the up and down spins enclosed by ovals indicate condensed Cooper linear magnetic susceptibility in the superconducting state. pairs. Error bars, 1 s.d. 951 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a Torder and Tpair but also increases the hole concentration of the CuO2 33.6 GPa A1g(OBi) 28 plane . These effects together give a parabolic-like Tc–P behaviour A (O ,Ca,Sr) 1g Cu similar to that observed in the first regime of Bi2223 as well as in 13–16 B1g(OCu) T (K) many hole-doped HTSCs . In the high-pressure cycle beyond 190 24 GPa, the superconducting inner CuO2 plane is assumed to be still 145 in the underdoped region. However, the outer CuO2 planes have

Relative intensity 130 already been overdoped by the applied pressure. Higher Tcs are thus A1g(OSr) A1g(OCu,Ca) 111 expected in this system for different hole doping concentrations of 100 200 300 400 500 600 700 800 900 the CuO2 planes because a high pairing scale is derived from the Raman shift (cm–1) underdoped inner plane and a large phase stiffness from the overdoped bc outer ones. This indicates that the Tc of a given material can be increased by tuning the system to have both the underdoped and the A 390 1g overdoped components at the same time. The remarkable increase in T (K) Tc to 136 6 10 K at 36.4 GPa in the high-pressure regime comes from 145 387 the pressure-induced effective combination of the high pairing scale of ) the underdoped inner CuO2 layer with the large phase ordering of the –1 139 overdoped outer CuO2 layers. The natural appearance of an inhomo- 384 geneous charge distribution among the inner and outer CuO2 planes 130 makes trilayer Bi2223 an ideal system in which to obtain higher Tcs B1g 354 under pressure. We note that the enhancement of Tc was also observed Relative intensity 126 Frequency (cm in bilayers containing underdoped La22xSrxCuO4 films capped by a thin, heavily overdoped metallic layer of the same material29. 111 B1g(OCu) 351 METHODS SUMMARY 79 A1g(OCu,Ca,Sr) T c Magnetic susceptibility. For magnetic measurements, we used diamond anvil 348 cells fabricated from Cu-Be alloy, with the gaskets made from nonmagnetic Ni- 300 350 400 70 100 130 160 190 Cr alloy. The susceptibility of the metallic parts of the high-pressure cell is Raman shift (cm–1) Temperature (K) essentially independent of the external field. Applying a magnetic field to the Figure 4 | Raman spectra of a Bi2Sr2Ca2Cu3O101d single crystal at cell when it contains a sample will therefore change the signal coming from the 33.6 GPa. a, Representative Raman results in the frequency range of sample whereas the background arising from the surrounding diamagnetic parts 21 100–900 cm . b, Raman spectra of the B1g mode and the in-phase A1g mode, will remain nearly constant. This allows the signal from the sample to be sepa- and the corresponding fitted Fano profiles (red) plus a linear background. rated from that coming from the background. The signal changes abruptly in the The dashed blue lines indicate the positions of the modes. c, Temperature vicinity of the superconducting transition, allowing us to see these changes both dependences of the fitted frequencies of the B1g and in-phase A1g modes in the amplitude and the phase of the signal. (circles). The solid diamonds are the frequencies corrected for a small change Raman scattering. We typically used an argon ion laser at a wavelength of in pressure with temperature. The dashed line indicates Tc. The red lines are 514.5 nm for excitation of Raman spectra. The spectral resolution was set at guides to the eye. 2cm21. A single crystal of Bi2223 with dimensions of 30 3 30 3 10 mm3 was loaded with neon as the pressure medium in a diamond anvil cell. Synthetic, ultrapure interaction between the discrete phonon states and the electronic anvils were chosen to reduce diamond fluorescence. The incidence angle was 35u. We collected the scattered light along the normal to the crystal surface. continuum, as found for other multilayer systems at ambient pres- sure19–22. In Fig. 4b, we show the spectra of the mode corresponding Received 12 September 2009; accepted 17 June 2010. to the B 1g vibrations, in opposite directions, of the oxygen atoms in 1. Mathur, N. D. et al. Magnetically mediated superconductivity in heavy fermion the outer CuO2 planes and the mode associated with A1g(OCu,Ca,Sr) compounds. Nature 394, 39–43 (1998). in-phase vibrations of Ca and Sr with O in the outer CuO2 planes, and 2. Uji, S. et al. Magnetic-field-induced superconductivity in a two-dimensional 21 organic conductor. Nature 410, 908–910 (2001). the fits to these spectra. Both the B1g phonon at 350 cm and the in- 21 3. Mori, M. & Maekawa, S. Effect of antiferromagnetic planes on the phase A1g phonon at 380 cm exhibit abrupt changes across Tc and superconducting properties of multilayered high-Tc cuprates. Phys. Rev. Lett. 94, are well described by Fano profiles plus a linear background. On 137003 (2005). entering the superconducting state, both peaks decrease in frequency 4. Mukuda, H. et al. Uniform mixing of high-Tc superconductivity and 21 by approximately 6 cm . antiferromagnetism on a single CuO2 plane of a Hg-based five-layered cuprate. Figure 4c shows the temperature dependence of the frequencies of Phys. Rev. Lett. 96, 087001 (2006). 21 5. Chia, E. E. et al. Observation of competing order in a high-Tc superconductor using the B1g and A1g phonons at 350 and, respectively, 380 cm at femtosecond optical pulses. Phys. Rev. Lett. 99, 147008 (2007). 33.6 GPa. These two modes show the effect of a superconducting 6. Chen, X. J. & Lin, H. Q. Variation of the superconducting transition temperature of transition in accordance with the observations in many HTSCs19–23. hole-doped copper oxides. Phys. Rev. B 69, 104518 (2004); erratum 71, 109901 (2005). The softening in both the B1g mode and the in-phase A1g mode in 7. Trokiner, A. et al. 17O nuclear-magnetic-resonance evidence for distinct carrier Bi2223 demonstrates that the phonon frequencies are renormalized densities in the two types of CuO2 planes of (Bi,Pb)2Sr2Ca2Cu3Oy. Phys. Rev. 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Dependence of the superconducting transition 25–27 a dynamic inhomogeneity-induced pairing model (Supplemen- temperature on the type and number of CuO2 layers in Tl2Ba2Can-1CunO2n14-y. tary Information). This model predicts that Tc is roughly determined Phys. Rev. B 59, 4513–4523 (1999). 12. Gregoryanz, E. A. et al. Superconductivity in the chalcogens up to multimegabar by two characteristic scales: the pairing scale, Tpair, which charac- pressures. Phys. Rev. B 65, 064504 (2002). terizes pair formation, and the phase-ordering scale, Torder, which 13. Sadewasser, S., Schilling, J. S., Paulikas, A. P. & Veal, B. W. Pressure dependence of controls the stiffness of the system in response to phase fluctuations. Tc to 17 GPa with and without relaxation effects in superconducting YBa2Cu3Ox. The application of pressure not only leads to the increase of both Phys. Rev. B 61, 741–749 (2000). 952 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

14. Chen, X. J. et al. High-pressure phase diagram of Bi2Sr2CaCu2O81d single crystals. 27. Berg, E., Orgad, D. & Kivelson, S. A. Route to high-temperature superconductivity Phys. Rev. B 70, 214502 (2004). in composite systems. Phys. Rev. B 78, 094509 (2008). 15. Kubiak, R. et al. Pressure dependence of the superconducting transition 28. Chen, X. J., Lin, H. Q. & Gong, C. D. Pressure dependence of Tc in Y-Ba-Cu-O temperature of Bi- and Tl-based high-Tc superconductors. Physica C 166, 523–529 superconductors. Phys. Rev. Lett. 85, 2180–2183 (2000). (1990). 29. Yuli, O. et al. Enhancement of the superconducting transition temperature of

16. Gao, L. et al. Superconductivity up to 164 K in HgBa2Cam-1CumO2m121d (m51, 2, La2-xSrxCuO4 bilayers: role of pairing and phase stiffness. Phys. Rev. Lett. 101, and 3) under quasihydrostatic pressures. Phys. Rev. B 50, 4260–4263 (1994). 057005 (2008). 17. Berman, I. V. et al. Critical temperature of Tl Ba Ca Cu O at pressures of up to 2 2 2 3 x Supplementary Information is linked to the online version of the paper at 190 kbar. Pis’ma Z. Eksp. Teor. Fiz. 49, 668–671 (1989). www.nature.com/nature. 18. Tristan Jover, D. et al. Pressure dependence of the superconducting critical temperature of Tl2Ba2Ca2Cu3O101y and Tl2Ba2Ca3Cu4O121y up to 21 GPa. Phys. Acknowledgements We thank M. R. Beasley, E. Berg, Y. Chen, C. W. Chu, Rev. B 54, 10175–10185 (1996). R. E. Cohen, P. C. Dai, D.-L. Feng, E. Fradkin, T. Geballe, C. D. Gong, C.-Q. Jin, 19. Zhou, X. J., Cardona, M., Colson, D. & Viallet, V. Plane oxygen vibrations and their S. Kivelson, H.-Q. Lin, O. Millo, D. Orgad, S. Raghu, J. S. Schilling, Z.-X. Shen, L.-L. temperature dependence in HgBa2Ca2Cu3O81d single crystals. Phys. Rev. B 55, Sun, J. Tranquada, W.-F. Tsai, N.-L. Wang, H.-H. Wen, Z.-A. Xu, H. Yao, W.-G. Yin, 12770–12775 (1997). Z.-X. Zhao and J.-X. Zhu for discussions. This work was supported by the DOE 20. Hadjiev, V. G. et al. Strong superconductivity-induced phonon self-energy effects under grant no. DE-SC0001057, grant no. DE-FG02-02ER45955 and grant no. in HgBa2Ca3Cu4O101d. Phys. Rev. B 58, 1043–1050 (1998). DEFC03-03NA00144, and by Carnegie Canada. X.-J.C. acknowledges financial 21. Limonov, M., Lee, S., Tajima, S. & Yamanaka, A. Superconductivity-induced support from the NSFC under grant no. 10874046. resonant Raman scattering in multilayer high-Tc superconductors. Phys. Rev. B 66, 054509 (2002). Author Contributions X.-J.C., H.-k.M. and R.J.H. initiated the program and wrote the paper; X.-J.C., Y.Y. and V.V.S. performed the magnetic susceptibility 22. Kovaleva, N. H. et al. c-axis lattice dynamics in Bi-based cuprate superconductors. measurements; X.-J.C., A.F.G. and V.V.S performed the Raman scattering Phys. Rev. B 69, 054511 (2004). measurements; C.-T.L. synthesized the single crystals; X.-J.C., V.V.S., A.F.G., R.J.H. 23. Limonov, M. F., Rykov, A. I., Tajima, S. & Yamanaka, A. Superconductivity-induced and H.-k.M. analysed the data and discussed the results. All the authors read and effects on phononic and electronic Raman scattering in twin-free YBa Cu O 2 3 7-x commented on the manuscript. single crystals. Phys. Rev. B 61, 12412–12419 (2000). 24. Feng, D. L. et al. Electronic structure of the trilayer cuprate superconductor Author Information Reprints and permissions information is available at Bi2Sr2Ca2Cu3O101d. Phys. Rev. Lett. 88, 107001 (2002). www.nature.com/reprints. The authors declare no competing financial interests. 25. Emery, V. J. & Kivelson, S. A. Importance of phase fluctuations in superconductors Readers are welcome to comment on the online version of this article at with small superfluid density. Nature 374, 434–437 (1995). www.nature.com/nature. Correspondence and requests for materials should be 26. Kivelson, S. A. Making high-Tc higher: a theoretical proposal. Physica B 318, 61–67 addressed to X.J.C. ([email protected]), H.K.M. ([email protected]) or R.J.H. (2002). ([email protected]).

A strong ferroelectric ferromagnet created by means of spin–lattice coupling

June Hyuk Lee1,2, Lei Fang3*, Eftihia Vlahos2*, Xianglin Ke4*, Young Woo Jung3, Lena Fitting Kourkoutis5, Jong-Woo Kim6, Philip J. Ryan6, Tassilo Heeg1, Martin Roeckerath7, Veronica Goian8, Margitta Bernhagen9, Reinhard Uecker9, P. Chris Hammel3, Karin M. Rabe10, Stanislav Kamba8,Ju¨rgen Schubert7, John W. Freeland6, David A. Muller5,11, Craig J. Fennie5, Peter Schiffer4, Venkatraman Gopalan2, Ezekiel Johnston-Halperin3 & Darrell G. Schlom1

Ferroelectric ferromagnets are exceedingly rare, fundamentally has been shown experimentally and theoretically that such strains can interesting multiferroic materials that could give rise to new even stabilize systems in novel non-bulk phases, for example SrTiO3 in technologies in which the low power and high speed of field-effect ferroelectric phases17,18. electronics are combined with the permanence and routability of Recently a route to achieving simultaneously strong ferromagnetism voltage-controlled ferromagnetism1,2. Furthermore, the properties of and ferroelectricity in a single phase has been proposed9. This tech- the few compounds that simultaneously exhibit these phenomena1–5 nique makes use of a generic mechanism in which the electron spin are insignificant in comparison with those of useful ferroelectrics or couples to the lattice: ferromagnets: their spontaneous polarizations or magnetizations are 2~ 2{ v v0 l Si.Sj ð1Þ smaller by a factor of 1,000 or more. The same holds for magnetic- or electric-field-induced multiferroics6–8. Owing to the weak properties Here v is the frequency of an infrared-active phonon (lattice) mode, v0 is the bare phonon frequency, l is the macroscopic spin–phonon of single-phase multiferroics, composite and multilayer approaches N involving strain-coupled piezoelectric and magnetostrictive com- coupling constant and ÆSi Sjæ is the nearest-neighbour spin–spin cor- ponents are the closest to application today1,2. Recently, however, relation function. Such spin–lattice coupling normally leads to mag- 19 9 a new route to ferroelectric ferromagnets was proposed9 by which netocapacitance , but in theory this term, in conjunction with strain, magnetically ordered insulators that are neither ferroelectric nor could tune multiple ferroic order parameters simultaneously, resulting 9 ferromagnetic are transformed into ferroelectric ferromagnets in the emergence of new ground states . A simple model that captures using a single control parameter, strain. The system targeted, the essential physics of this tuning behaviour can be written as a first- order transition induced by a biquadratic coupling of lattice and mag- EuTiO3, was predicted to exhibit strong ferromagnetism (spontaneous magnetization, 7 Bohr magnetons per Eu) and strong ferro- netic order parameters: 22 2 2 4 4 electricity (spontaneous polarization, 10 mCcm )simultaneously APP AM M BPP BM M 2 2 9 F(M,P)~ z z z {jl’jM P under large biaxial compressive strain . These values are orders of 2 2 4 4 magnitude higher than those of any known ferroelectric ferromag- 2 2 4 4 net and rival the best materials that are solely ferroelectric or fer- APP ALL BPP BLL F(L,P)~ z z z zjl’jL2P2 romagnetic. Hindered by the absence of an appropriate substrate to 2 2 4 4 provide the desired compression we turnedto tensile strain. Here we Here F is the Landau free energy, P, M and L are the ferroelectric, show both experimentally and theoretically the emergence of a ferromagnetic and antiferromagnetic order parameters, respectively, multiferroic state under biaxial tension with the unexpected benefit and AP, BP, AM, BM, AL and BL are expansion coefficients. The sign and that even lower strains are required, thereby allowing thicker high- strength of the biquadratic coupling coefficient, l9, which is positive quality crystalline films. This realization of a strong ferromagnetic for antiferromagnetic order and negative for ferromagnetic order, ferroelectric points the way to high-temperature manifestations of originates in the spin–lattice coupling and is fundamental to the tuning this spin–lattice coupling mechanism10. Our work demonstrates behaviour. Such biquadratic magnetoelectric coupling, as well its that a single experimental parameter, strain, simultaneously con- change of sign under magnetic bias, was recently confirmed for 20 trols multiple order parameters and is a viable alternative tuning unstrained bulk EuTiO3 and was found to be large . The above model 11 9 parameter to composition for creating multiferroics. led to the prediction that (001) EuTiO3 would transform from its Using epitaxy and the mis-fit strain imposed by an underlying sub- paraelectric and antiferromagnetic unstrained ground state19 to a strate, it is possible to strain dielectric thin films to per cent levels—far simultaneously ferromagnetic and ferroelectric ground state for com- beyond where they would crack in bulk. Such strains are used to pressive strains exceeding 1.2%. enhance the mobility of transistors12 and increase superconducting13, Owing to the lack of appropriate substrates and the high strains 14,15 16 ferromagnetic and ferroelectric transition temperatures. In fact, it involved for those that do exist (for example, a commensurate EuTiO3

1Department of Materials Science and Engineering, Cornell University, Ithaca, New York 14853-1501, USA. 2Department of Materials Science and Engineering, Pennsylvania State University, University Park, Pennsylvania 16802-5005, USA. 3Department of Physics, Ohio State University, Columbus, Ohio 43210-1117, USA. 4Department of Physics and Materials Research Institute, Pennsylvania State University, University Park, Pennsylvania 16802, USA. 5School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA. 6Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA. 7Institute of Bio and Nanosystems, JARA-Fundamentals of Future Information Technologies, Research Centre Ju¨lich, D-52425 Ju¨lich, Germany. 8Institute of Physics ASCR, Na Slovance 2, 182 21 Prague 8, Czech Republic. 9Leibniz Institute for Crystal Growth, Max-Born-Straße 2, D-12489 Berlin, Germany. 10Department of Physics and Astronomy, Rutgers University, Piscataway, New Jersey 08854-8019, USA. 11Kavli Institute at Cornell for Nanoscale Science, Ithaca, New York 14853, USA. *These authors contributed equally to this work. 954 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a LSAT SrTiO3 DyScO3 Discussion 1), we find that the theoretical critical strain to reach the ferroelectric ferromagnetic ground state is lower in biaxial tension (e 510.75%) than in biaxial compression (e 521.2%). Lower FM AFM FM s s + + + strains are experimentally appealing as they allow thicker commen- FE PE FE surately strained films to be grown, provided that suitable substrates exist. Tensile strain has the added advantage that the ferroelectric –2.0 –1.5 –1.0 –0.5 00.5 1.0 1.5 2.0 polarization is in-plane, eliminating the importance of finite- 21,22 Biaxial strain, εs (%) size effects associated with depolarization fields . To assess the theoretical phase diagram, we prepare commensurate EuTiO3 films b Bulk unstrained c Strained on substrates in the same structural family (perovskites) with lattice EuTiO3 EuTiO3 film constants suitably close to that of EuTiO3 so films thick enough to test for ferroelectricity and ferromagnetism can be grown. Commensurate films reduce the complicating influence of dislocations on film properties23. Our approach is illustrated in Fig. 1b, c. Our experiments are performed on 22-nm-thick commensurate (001)-oriented EuTiO3 films grown by reactive molecular-beam epi- 24 DyScO3 substrate taxy on (001) (LaAlO3)0.29–(SrAl1/2Ta1/2O3)0.71 (LSAT), (001) SrTiO3 and (110) DyScO3 substrates. These substrates impart biaxial Figure 1 | Predicted effect of biaxial strain on EuTiO3 and our approach to strains of 20.9%, 0.0% and about 11.1%, respectively. The struc- imparting such strain in EuTiO3 films using epitaxy. a, First-principles tural, ferroelectric and ferromagnetic properties of these films (in epitaxial phase diagram of EuTiO3 strained from 22% (biaxial compression) to 12% (biaxial tension), calculated in 0.1% steps. Regions addition to multiple control samples including bare substrates) are with paraelectric (PE), ferroelectric (FE), antiferromagnetic (AFM) and measured to assess the predictions of Fig. 1a. ferromagnetic (FM) behaviour are shown. b, c, Schematic of unstrained bulk X-ray diffraction (Fig. 2a) and cross-sectional scanning transmis- EuTiO3 (b) and epitaxially strained thin-film EuTiO3 on the DyScO3 sion electron microscopy (STEM; Fig. 2b) reveal the films to be substrate (c), showing the in-plane expansion due to biaxial tension. commensurate, smooth and of high structural perfection. Cross- sectional chemical mapping of the interface between the EuTiO3 film film on a LaAlO3 substrate would have a biaxial strain of es < 22.9%), and the DyScO3 substrate at atomic resolution (Fig. 2b) demon- we are not able to experimentally achieve the biaxial compression strates that the interface between the film and the substrate is abrupt. required while maintaining a high-quality crystalline film. This leads Temperature-dependent second harmonic generation (SHG) us to extend the calculated strain phase diagram of (001) EuTiO3, measurements (Fig. 3a) indicate the paraelectric-to-ferroelectric 9 which previously considered only the effect of biaxial compression , transition temperature (Tc) of the films. Materials lacking inversion to include biaxial tension (Fig. 1a). By doing so (Supplementary symmetry exhibit SHG and ferroelectrics lack inversion symmetry.

a * 001

DyScO3 * +001

SrTiO3 Eu Ti * 001 Intensity (arbitraryunits) EuTiO3 LSAT

21 22 23 24 2θ (º)

DyScO3

Dy Sc

Figure 2 | Structural characterization by X-ray diffraction and STEM of 22- showing a coherent interface and a low density of defects in the EuTiO3 film. nm-thick commensurate epitaxial EuTiO3 films. a, h–2h X-ray diffraction Bottom: A-site and B-site elemental maps of the interface obtained by scans of EuTiO3 on DyScO3 (red), EuTiO3 on SrTiO3 (blue) and EuTiO3 on combining the Eu-M4,5 (green) and Dy-M4,5 (red) electron energy-loss LSAT (green) in the vicinity of the out-of-plane 001p EuTiO3 reflection, spectroscopy edges, and the Ti-L2,3 (yellow) and Sc-L2,3(blue) edges where the subscript refers to pseudocubic indices. Clear thickness fringes are extracted from two separate 256 3 256-pixel spectrum image acquisitions seen. The substrate peaks are denoted with asterisks. b, Annular dark-field (one for the Eu/Dy edges, the other for the Sc/Ti edges). Intermixing is and spectroscopic imaging of the EuTiO3-on-DyScO3 heterostructure limited to one to two atomic layers at the interface. characterized in a. Top: annular dark-field/STEM images of the structure 955 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

0º || S SS a b S mm2 fit 330º 30º 80 PP Cooling P mm2 fit 60º 60 300º Heating EuTiO3 on DyScO3

40 270º 90º || P

20 T c EuTiO3 on SrTiO3 0 240º 120º EuTiO on LSAT 3 T = 5 K 0100200300 210º 150º Temperature (K) 180º c d 150 30 units) (arbitrary Polarization EuTiO3 on DyScO3 1,600 100

SHG intensity (arbitrary units) 20 50 T c 10 0 800 Electric field || [001] DyScO –50 0 3 Dielectric constant

–100 Electric field || T –10 = 5 K [110] DyScO3 –150 0 –1.0 –0.5 0 0.5 1.0 0 100 200 300 Electric field (kV mm–1) Temperature (K)

Figure 3 | Commensurate EuTiO3 strained in biaxial tension at 11.1% on respectively, where the incidence plane is formed by the sample normal and DyScO3 is ferroelectric below Tc < 250 K. a, Temperature dependence of the direction of propagation of the incident light field. c, SHG hysteresis loop the SHG signal of EuTiO3 on DyScO3 (red), EuTiO3 on SrTiO3 (blue) and (top) and corresponding polarization loop (bottom) for EuTiO3 on DyScO3 EuTiO3 on LSAT (green). b, Experimental polar plots (points) and mm2 fit at 5 K. d, Dielectric constant versus temperature for a nearly commensurate (line) with analyser along Æ100æp directions for EuTiO3 on DyScO3 at 5 K. (es 511.1%) 100-nm-thick EuTiO3-on-DyScO3 film, determined by far- The P and S polarizations are in and perpendicular to the incidence plane, infrared reflectance spectroscopy.

Therefore, SHG activity is a necessary but insufficient condition for substrate. If a superconducting quantum interference device ferroelectricity. Only the EuTiO3-on-DyScO3 film exhibits an SHG (SQUID) magnetometer is used under typical measurement magnetic response, with Tc < 250 K. The absence of an SHG response from the fields, the paramagnetic response of the thick substrate swamps the EuTiO3 strained at es 520.9% (EuTiO3 on LSAT) and es 5 0.0% signal from the strained EuTiO3 film. For this reason, we use a com- (EuTiO3 on SrTiO3) indicates that EuTiO3 in these strain states is bination of the magneto-optic Kerr effect (MOKE) and SQUID mag- not ferroelectric. netometry. Exploiting the optical nature of the MOKE technique, the Having determined that the EuTiO3 film strained at es 511.1% probe is tuned to a wavelength between 690 nm and 750 nm to max- (EuTiO3 on DyScO3) is potentially ferroelectric, we ask whether it imize the absorption in the EuTiO3 epilayer while minimizing inter- satisfies additional ferroelectric criteria: a polar point group; a peak in actions with the substrate. This renders the measurement insensitive dielectric constant versus temperature at Tc; and changes in domain to the paramagnetic substrates as well as to the antiferromagnetic state populations when an electric field is applied. To establish the point of DyScO3 below its Ne´el temperature, TN 5 3.1 K (ref. 25). The group symmetry, we make SHG polar plots (Fig. 3b). The experimental MOKE response from all three EuTiO3 films and a bare DyScO3 data fit well with the mm2 polar point group model. This result is substrate is shown in Fig. 4a. The EuTiO3 strained at es 511.1% consistent with the first-principles calculations. We probe domain (EuTiO3 on DyScO3) has a clear ferromagnetic hysteresis loop, with dynamics by monitoring the SHG intensity as a function of applied sharp switching to full saturation, signifying that it is ferromagnetic, in in-plane electric field (Fig. 3c). contrast to the EuTiO3 with es 520.9% (EuTiO3 on LSAT) or We make an additional test of the ferroelectric phase transition by es 5 0.0% (EuTiO3 on SrTiO3). The temperature dependence of the measuring the dielectric constant as a function of temperature in a MOKE (Fig. 4b) shows that the Curie temperature of the sample strained EuTiO3-on-DyScO3 film. Owing to the low bandgap of strained at es 511.1% is TC 5 4.24 6 0.02 K (Supplementary EuTiO3 (0.9 eV; ref. 24) and associated leakage issues, the low- Discussion 3), which is slightly lower than 5.5 K, the Ne´el temperature frequency dielectric constant is extracted from far-infrared reflec- of unstrained EuTiO3 (refs 19, 24). tance measurements on a 100-nm-thick EuTiO3-on-DyScO3 film Because the MOKE measurements of ferromagnetism are not on an (Fig. 3d and Supplementary Discussion 2). A peak in dielectric con- absolute scale, we use SQUID measurements to quantify the spontan- stant as a function of temperature is clearly seen at a temperature eous magnetization of EuTiO3 strained at es 511.1%. These mea- comparable to that at which the onset of the phase transition revealed surements are made in nominally zero residual magnetic field to by SHG occurs (Fig. 3a). The dielectric anomaly is caused by an minimize the paramagnetic response of the substrate. The strained optical soft mode, which has a frequency minimum at Tc (Sup- EuTiO3 is cooled in a 100-Oe field, which is high enough to polarize plementary Fig. 1). This anomaly is additional evidence for the ferro- the EuTiO3 film yet small enough to minimize the residual magnetic electric phase transition in strained EuTiO3 on DyScO3. All these field during subsequent SQUID measurements. The observed mag- results are consistent with EuTiO3 strained at es 511.1% under- netization (Fig. 4b) is seen to rise at the same temperature (TC) as the going a paraelectric-to-ferroelectric transition at about 250 K. MOKE signal and follows it until the antiferromagnetic transition of Testing for ferromagnetism in strained EuTiO3-on-DyScO3 films the DyScO3 substrate occurs, at 3.1 K, where the substrate signal masks is complicated by the large paramagnetic response of the DyScO3 the magnetization of the strained EuTiO3 film. In agreement with the 956 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a Bulk EuTiO3 exhibits a decrease in its dielectric constant as it is EuTiO on DyScO 3 3 cooled through its antiferromagnetic transition19. Owing to the change 1.0 in the sign of the spin–spin correlation function in equation (1) when strained EuTiO3 becomes ferromagnetic, the dielectric constant of EuTiO on SrTiO EuTiO3 on DyScO3 should increase as the film is cooled through its 3 3 ferromagnetic transition. Such behaviour was observed (Fig. 4c), 0 (mrad) further corroborating the ferromagnetic transition in commensurately

Kerr Bare strained EuTiO on DyScO and its strong spin–lattice coupling.

θ 3 3 DyScO EuTiO3 on LSAT 3 Our results confirm the theorized mechanism9 and open the door to higher-temperature implementations of strong ferromagnetic –1.0 ferroelectrics10, which would allow for dramatic improvements in T = 2.0 K numerous devices and applications: magnetic sensors26, energy har- 27 –40 –20 0 20 40 vesting, high-density multistate memory elements , wireless power- Magnetic field,H (Oe) ing of miniature systems28, and tunable microwave filters, delay lines, 29,30 b phase shifters and resonators . 1.6 4 METHODS SUMMARY 3 1.8 K We performed first-principles density functional calculations using projector- augmented-wave potentials within spin-polarized GGA 1 U approximation as per Eu) 1.2 θ B 3 Remanent μ implemented in the Vienna Ab-initio Simulation Package (Supplementary

per Eu) 0 24 B

M ( T Discussion 1). We grew EuTiO3 thin films by reactive molecular-beam epitaxy , N μ 3.8 K

(mrad) from elemental europium and titanium sources at a substrate temperature of 2 M ( 0.8 28 EuTiO3 on 650 uC in a background partial pressure of molecular oxygen of 3 3 10 torr. –3 DyScO3 EuTiO3 films in three strain states (EuTiO3 on DyScO3, EuTiO3 on SrTiO3 and EuTiO on LSAT; see Supplementary Table 1), as well as bare substrates, were 1 –300 0 300 0.4 3 H characterized structurally by X-ray diffraction (Supplementary Discussions 5 Magnetization, (Oe) T and 6) and STEM; chemically by STEM/electron energy-loss spectroscopy, C Rutherford backscattering spectrometry (Supplementary Fig. 2) and X-ray 0 0 2345678 absorption spectroscopy (Supplementary Discussion 4); (iii) for ferroelectricity Temperature (K) by SHG (Supplementary Discussion 7) and far-infrared reflectance (Supplemen- tary Discussion 2); (iv) for ferromagnetism using the MOKE (Supplementary c 2.4 Discussion 3) and a SQUID (Supplementary Fig. 3); and (v) for the temperature dependence of capacitance (Supplementary Discussion 8).

Full Methods and any associated references are available in the online version of 2.3 the paper at www.nature.com/nature. Received 14 March; accepted 6 July 2010.

1. Eerenstein, W., Mathur, N. D. & Scott, J. F. Multiferroic and magnetoelectric materials. Nature 442, 759–765 (2006). 2.2 EuTiO3 on DyScO3 Capacitance (pF) Capacitance 2. Ramesh, R. & Spaldin, N. A. Multiferroics: progress and prospects in thin films. Nature Mater. 6, 21–29 (2007). 3. Rivera, J.-P. & Schmid, H. Electrical and optical measurements on nickel iodine Bare DyScO3 boracite. Ferroelectrics 36, 447–450 (1981). 2.1 4. Ikeda, N. et al. Ferroelectricity from iron valence ordering in the charge-frustrated 246810 system LuFe2O4. Nature 436, 1136–1138 (2005). Temperature (K) 5. Park, J. Y., Park, J. H., Jeong, Y. K. & Jang, H. M. Dynamic magnetoelectric coupling in ‘‘electronic ferroelectric’’ LuFe O . Appl. Phys. Lett. 91, 152903 (2007). Figure 4 | Magnetization and capacitance measurements showing that 2 4 6. Kimura, T. et al. Magnetic control of ferroelectric polarization. Nature 426, 55–58 EuTiO3 on DyScO3 is ferromagnetic below TC 5 4.24 6 0.02 K and that (2003). these two quantities are coupled. a, MOKE measurements at 2.0 K of 7. Lottermoser, T. et al. Magnetic phase control by an electric field. Nature 430, EuTiO3 on DyScO3 (red), EuTiO3 on SrTiO3 (blue), EuTiO3 on LSAT 541–544 (2004). (green) and bare DyScO3 substrate (gold). hKerr is the Kerr-induced 8. Kimura, T., Sekio, Y., Nakamura, H., Siegrist, T. & Ramirez, A. P. Cupric oxide as an polarization rotation in the optical probe beam and is proportional to the in- induced-multiferroic with high-Tc. Nature Mater. 7, 291–294 (2008). plane magnetization. b, Temperature dependence of the magnetization 9. Fennie, C. J. & Rabe, K. M. Magnetic and electric phase control in epitaxial EuTiO3 measured using both the MOKE and a SQUID. Inset, isothermal SQUID from first principles. Phys. Rev. Lett. 97, 267602 (2006). magnetization curves at T 5 1.8 and 3.8 K. The red data points with error 10. Lee, J. H. & Rabe, K. M. Epitaxial-strain-induced multiferroicity in SrMnO3 from first principles. Phys. Rev. Lett. 104, 207204 (2010). bars (representing 1s variations for both temperature and hKerr) show the temperature dependence of the remanent value of h for EuTiO on 11. Goto, T., Kimura, T., Lawes, G., Ramirez, A. P. & Tokura, Y. Ferroelectricity and Kerr 3 giant magnetocapacitance in perovskite rare-earth manganites. Phys. Rev. Lett. DyScO3, hRemanent. mB, Bohr magneton. c, Temperature dependence of the 92, 257201 (2004). capacitance of EuTiO3 on DyScO3 near TC. The capacitance of a bare 12. Nguyen, L. D., Brown, A. S., Thompson, M. A. & Jelloian, L. M. 50-nm self-aligned- DyScO3 substrate measured under the same conditions is also shown. gate pseudomorphic AlInAs/GaInAs high electron mobility transistors. IEEE Trans. Electron. Dev. 39, 2007–2014 (1992). 13. Bozovic, I., Logvenov, G., Belca, I., Narimbetov, B. & Sveklo, I. Epitaxial strain and MOKE results, clear hysteresis loops are observed below 4 K (Fig. 4b, superconductivity in La2–xSrxCuO4 thin films. Phys. Rev. Lett. 89, 107001 (2002). inset) and the spontaneous magnetization of the strained EuTiO is 14. Beach, R. S. et al. Enhanced Curie temperatures and magnetoelastic domains in 3 Dy/Lu superlattices and films. Phys. Rev. Lett. 70, 3502–3505 (1993). seen to be large, that is, several Bohr magnetons per Eu. Polarized 15. Fuchs, D. et al. Tuning the magnetic properties of LaCoO3 thin films by epitaxial X-ray measurements of the local moment connect the observed mag- strain. Phys. Rev. B 77, 014434 (2008). 21 netism with that of Eu (Supplementary Discussion 4). From the 16. Choi, K. J. et al. Enhancement of ferroelectricity in strained BaTiO3 thin films. combination of these results, and because the ferromagnetism occurs Science 306, 1005–1009 (2004). 17. Pertsev, N. A., Tagantsev, A. K. & Setter, N. Phase transitions and strain-induced well above the antiferromagnetic transition of the DyScO3 substrate, ferroelectricity in SrTiO3 epitaxial thin films. Phys. Rev. B 61, R825–R829 (2000). we conclude that the observed ferromagnetism is not correlated with 18. Haeni, J. H. et al. Room-temperature ferroelectricity in strained SrTiO3. Nature the magnetic ordering of the underlying substrate. 430, 758–761 (2004). 957 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

19. Katsufuji, T. & Takagi, H. Coupling between magnetism and dielectric properties Supplementary Information is linked to the online version of the paper at in quantum paraelectric EuTiO3. Phys. Rev. B 64, 054415 (2001). www.nature.com/nature. 20. Shvartsman, V. V., Borisov, P., Kleemann, W., Kamba, S., &. Katsufuji, T. Large off- diagonal magnetoelectric coupling in the quantum paraelectric antiferromagnet Acknowledgements The authors acknowledge discussions and interactions with M. D. Biegalski, D. H. A. Blank, C. B. Eom, M. B. Holcomb, M. Lezˇaic´, J. Mannhart, EuTiO3. Phys. Rev. B 81, 064426 (2010). 21. Junquera, J. & Ghosez, P. Critical thickness for ferroelectricity in perovskite L. W. Martin, D. V. Pelekhov, R. Ramesh, K. Z. Rushchanskii, N. Samarth, ultrathin films. Nature 422, 506–509 (2003). A. Schmehl, D. A. Tenne, J.-M. Triscone, D. Viehland and L. Yan. In addition, the 22. Fong, D. D. et al. Ferroelectricity in ultrathin perovskite films. Science 304, financial support of the National Science Foundation through grant DMR-0507146 1650–1653 (2004). and the MRSEC program (DMR-0520404, DMR-0820404 and DMR-0820414), 23. Chu, M.-W. et al. Impact of misfit dislocations on the polarization instability of and of the Czech Science Foundation (project no. 202/09/0682), is gratefully epitaxial nanostructured ferroelectric perovskites. Nature Mater. 3, 87–90 (2004). acknowledged. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under 24. Lee, J. H. et al. Optical band gap and magnetic properties of unstrained EuTiO3 films. Appl. Phys. Lett. 94, 212509 (2009). contract no. DE-AC02-06CH11357. 25. Ke, X. et al. Low temperature magnetism in the perovskite substrate DyScO3. Author Contributions The first-principles calculations were performed by C.J.F. and Appl. Phys. Lett. 94, 152503 (2009). K.M.R. The thin films were synthesized by J.H.L. and D.G.S. on single-crystal 26. Zhai, J. Y., Xing, Z. P., Dong, S. X., Li, J. F. & Viehland, D. Detection of pico-Tesla substrates including DyScO3 grown by M.B. and R.U. The films were characterized magnetic fields using magneto-electric sensors at room temperature. Appl. Phys. using the MOKE by L.F., Y.W.J., P.C.H. and E.J.-H.; by SHG by E.V. and V. Gopalan; Lett. 88, 062510 (2006). using a SQUID and by capacitance by X.K. and P.S.; by electron microscopy and 27. Gajek, M. et al. Tunnel junctions with multiferroic barriers. Nature Mater. 6, spectroscopy by L.F.K. and D.A.M.; by X-ray diffraction by J.H.L., J.W.K. and P.J.R.; by 296–302 (2007). X-ray absorption spectroscopy and X-ray magnetic circular dichroism by J.W.F.; by 28. Bayrashev, A., Robbins, W. P. & Ziaie, B. Low frequency wireless powering of Rutherford backscattering spectrometry by T.H., M.R. and J.S.; and by far-infrared microsystems using piezoelectric-magnetostrictive laminate composites. Sens. reflectance by V. Goian and S.K. D.G.S., C.J.F., J.W.F. and J.H.L. wrote the manuscript. Actuators A 114, 244–249 (2004). 29. Fetisov, Y. K. & Srinivasan, G. Electric field tuning characteristics of a ferrite- Author Information Reprints and permissions information is available at piezoelectric microwave resonator. Appl. Phys. Lett. 88, 143503 (2006). www.nature.com/reprints. The authors declare no competing financial interests. 30. Das, J., Song, Y.-Y., Mo, N., Krivosik, P. & Patton, C. E. Electric-field-tunable low Readers are welcome to comment on the online version of this article at loss multiferroic ferrimagnetic-ferroelectric heterostructures. Adv. Mater. 21, www.nature.com/nature. Correspondence and requests for materials should be 2045–2049 (2009). addressed to D.G.S. ([email protected]).

METHODS parameter, JH 5 1.0 eV, were used that give a reasonable account of the magnetic exchange constants which were extracted by mapping GGA 1 U calculations of EuTiO is a cubic perovskite oxide (space group, Pm33m) with a lattice constant 3 the total energy for different spin configurations at T 5 0 onto a classical a 5 3.905 A˚ at room temperature19,31. This oxide is the subject of this study Heisenberg model, E 52S J ÆS NS æ (in our notation, the energy per spin because its PE–AFM ground state lies very close to a phase transition shown spin ij ij i j bond is 2J). We note that, given the relatively small energies involved, electronic in Fig. 1a. Owing to this sensitivity, it is imperative to select a thin-film growth relaxations converged to less than 1027 eV and ionic relaxations were performed technique that can yield the intrinsic properties of EuTiO . Reactive molecular- 3 until residual forces reached less than 0.5 3 1023 eV A˚ 21. Phonon frequencies beam epitaxy24 (MBE), in contrast to other methods used for EuTiO film 3 were calculated using the direct method where each ion was moved by approxi- growth32–36, is so far the only technique that has been able to achieve the intrinsic mately 0.01 A˚ and also using density functional perturbation theory as imple- properties of unstrained EuTiO as-grown films. 3 mented in the Vienna Ab-initio Simulation Package. Calculations with the We grew EuTiO thin films by MBE in an EPI 930 MBE chamber. Molecular 3 GGA 1 U approximation overestimate the lattice constant by ,1%. We intro- beams of europium and titanium were generated using a conventional effusion s cell and a Ti-Ball titanium sublimation pump37, respectively. Each metal flux duce a shift of the zero of 33, the out-of-plane component of the stress, so that 13 22 21 s 5 5 ˚ (,1.5 3 10 atoms cm s ) was measured before growth using a quartz crystal 33 0 for the cubic structure at the experimental lattice constant, aexp 3.9 A. g ; 2 microbalance calibrated by Rutherford backscattering spectrometry measure- Thus, the correct cubic structure is obtained at mis-fit strain (a aexp)/ 5 ments. Europium and titanium were co-deposited onto the substrate under an aexp 0. 28 oxygen background partial pressure of 3 3 10 torr. This oxygen partial pressure 31. Brous, J., Fankuchen, I. & Banks, E. Rare earth titanates with a perovskite structure. was found to be optimal for the growth of single-phase EuTiO3 films with the Acta Crystallogr. 6, 67–70 (1953). 21 41 desired oxidation states of Eu and Ti . The substrate temperature was 650 uC 32. Wang, H.-H., Fleet, A., Brock, J. D., Dale, D. & Suzuki, Y. Nearly strain-free ˚ 21 and the growth rate was ,0.1 A s . In situ reflection high-energy electron dif- heteroepitaxial system for fundamental studies of pulsed laser deposition: EuTiO3 fraction images are consistent with the growth of smooth and epitaxial thin-film on SrTiO3. J. Appl. Phys. 96, 5324–5328 (2004). surfaces during deposition (Supplementary Fig. 4). Following deposition, the 33. Kugimiya, K., Fujita, K., Tanaka, K. & Hirao, K. Preparation and magnetic properties of oxygen deficient EuTiO thin films. J. Magn. Magn. Mater. 310, 2268–2270 EuTiO3 thin films were cooled under the same oxygen partial pressure in which 3–d they were grown. (2007). Substrates. (001) LSAT, (001) SrTiO and (110) DyScO substrates were selected 34. Chae, S. C. et al. Magnetic properties of insulating RTiO3 thin films. J. Electroceram. 3 3 22, 216–220 (2009). to induce biaxial strains of 20.9%, 0.0% and about 11.1% in EuTiO films, 3 35. Fujita, K., Wakasugi, N., Murai, S., Zong, Y. & Tanaka, K. High-quality respectively. These substrates are isostructural with EuTiO3 (all are perovskites), antiferromagnetic EuTiO3 epitaxial thin films on SrTiO3 prepared by pulsed laser are commercially available with high structural perfection and have lattice con- deposition and postannealing. Appl. Phys. Lett. 94, 062512 (2009). stants in the region of interest to test the predictions of Fig. 1a (ref. 38). In 36. Hatabayashi, K. et al. Fabrication of EuTiO3 epitaxial thin films by pulsed laser addition, SrTiO3 and LSAT are non-magnetic, which simplifies magnetic deposition. Jpn. J. Appl. Phys. 48, 100208 (2009). characterization. LSAT is technically tetragonal although its c/a structural distor- 37. Theis, C. D. & Schlom, D. G. Cheap and stable titanium source for use in oxide tion is small enough (0.9995) that we treat it is if it were cubic with a 5 3.869 A˚ (ref. molecular beam epitaxy systems. J. Vac. Sci. Technol. A 14, 2677–2679 (1996). ˚ 39). SrTiO3 is cubic at room temperature and has a lattice constant a 5 3.905 A 38. Schlom, D. G. et al. Strain tuning of ferroelectric thin films. Annu. Rev. Mater. Res. 37, 589–626 (2007). (ref. 40), which is nearly the same as the corresponding lattice constant in EuTiO3. 39. Steins, M., Doerschel, J. & Reiche, P. Crystal structure of aluminium lanthanum The (001) SrTiO3 substrate was treated in buffered HF for 30 s and annealed at 950 uC for 1 h to obtain TiO termination on the surface before EuTiO film strontium tantalum oxide, (La0.272Sr0.728)(Al0.648Ta0.352)O3. Z. Kristallogr. New 2 3 Cryst. Struct. 212, 77 (1997). growth41. DyScO is orthorhombic with lattice constants a 5 5.440 A˚ , 3 40. Hellwege, K.-H. & Hellwege, A. M. (eds) Landolt-Bo¨rnstein: Numerical Data and 5 ˚ 5 ˚ b 5.717 A and c 7.903 A (ref. 42). The (110) DyScO3 surface has a rectangular Functional Relationships in Science and Technology New Series, Group III, Vol. 16a surface mesh that strains EuTiO by 11.0% along the ½1110 in-plane 3 DyScO3 59 (Springer, 1981). direction and by 11.2% along the perpendicular ½001 in-plane direction. DyScO3 41. Koster, G. & Kropman, B. L. Rijnders, G. Blank, D. H. A. & Rogalla, H. Quasi-ideal The (110) DyScO3 substrate was annealed at 1,000 uC for 13 h in flowing oxygen at strontium titanate crystal surfaces through formation of strontium hydroxide. 1-atm pressure to promote a ScO2-terminated surface. Appl. Phys. Lett. 73, 2920–2922 (1998). Theory. In density functional theory, the failure of the generalized gradient 42. Velicˇkov, B. Kahlenberg, V. Bertram, R. & Bernhagen, M. Crystal chemistry of approximation (GGA) properly to capture the physics of strongly correlated GdScO3, DyScO3, SmScO3, and NdScO3. Z. Kristallogr. 222, 466–473 (2007). systems is well established. A widely accepted approach beyond GGA is the 43. Anisimov, V. I., Aryasetiawan, F. & Lichtenstein, A. I. First-principles calculations GGA plus Hubbard U (GGA 1 U) method43. We performed first-principles of the electronic structure and spectra of strongly correlated systems: the density functional calculations using projector-augmented-wave (PAW) poten- LDA1U method. J. Phys. Condens. Matter 9, 767–808 (1997). 44. Kresse, G. & Hafner, J. Ab initio molecular dynamics for liquid metals. Phys. Rev. B tials within the spin-polarized GGA 1 U approximation as implemented in the 44–47 47, 558–561 (1993). Vienna Ab-initio Simulation Package with a plane-wave cut-off of 500 eV 45. Blochl, P. E. Projector augmented-wave method. Phys. Rev. B 50, 17953–17979 and an 8 3 8 3 8 C-centred k-point mesh. The PAW potential for Eu treated the (1994). 7 2 6 2 4f 5s 5p 6s states as valence states. All calculations were performed with col- 46. Kresse, G. & Furthmuller, J. Efficient iterative schemes for ab initio total-energy 21 linear spins and without spin–orbit coupling. As expected for Eu , which lacks calculations using a plane-wave basis set. Phys. Rev. B 54, 11169–11186 (1996). orbital degrees of freedom, inclusion of spin–orbit coupling does not change the 47. Kresse, G. & Joubert, D. From ultrasoft pseudopotentials to the projector results. Values of the Eu on-site Coulomb parameter, U 5 5.7 eV, and exchange augmented-wave method. Phys. Rev. B 59, 1758–1775 (1999).

Near-simultaneous great earthquakes at Tongan megathrust and outer rise in September 2009

J. Beavan1, X. Wang1, C. Holden1, K. Wilson1, W. Power1, G. Prasetya1, M. Bevis2 & R. Kautoke3

The Earth’s largest earthquakes and tsunamis are usually caused great earthquakes on the subduction interface is not known. Related by thrust-faulting earthquakes on the shallow part of the subduc- questions are whether slip on the plate interface at largely uncoupled tion interface between two tectonic plates, where stored elastic subduction zones can ever generate damaging tsunamis, and to what energy due to convergence between the plates is rapidly released1,2. extent thrust earthquakes on the subduction interface and normal- The tsunami that devastated the Samoan and northern Tongan faulting earthquakes in the adjacent outer-rise region may be trig- islands on 29 September 2009 was preceded by a globally recorded gered by each other. The Mw 5 8 Samoa–Tonga earthquake of 29 magnitude-8 normal-faulting earthquake in the outer-rise region, September 2009 provides information on all three questions. where the Pacific plate bends before entering the subduction zone. The event was measured at teleseismic distances by the global Preliminary interpretation suggested that this earthquake was the seismic network and was interpreted from centroid moment tensor source of the tsunami3. Here we show that the outer-rise earth- (CMT) solutions as a normal-faulting earthquake in the outer rise9,10. quake was accompanied by a nearly simultaneous rupture of the Preliminary interpretations3 assumed this earthquake was the source shallow subduction interface, equivalent to a magnitude-8 earth- of the tsunami that caused major devastation and nearly 200 deaths in quake, that also contributed significantly to the tsunami. The sub- Samoa and northern Tonga. Displacements at continuous Global duction interface event was probably a slow earthquake with a rise Positioning System (GPS) stations in Samoa and central Tonga were time of several minutes that triggered the outer-rise event several also consistent with a normal-faulting event (Fig. 1, Supplementary minutes later. However, we cannot rule out the possibility that the Figs 1 and 4, and Supplementary Tables 1–6), although a ,15-mm normal fault ruptured first and dynamically triggered the subduc- displacement at Niue was inconsistent. In contrast to the teleseismic tion interface event. Our evidence comes from displacements of interpretations, automated inversion11, assuming a thrust-fault Global Positioning System stations and modelling of tsunami source, of tsunami wave data from ocean-bottom pressure sensors waves recorded by ocean-bottom pressure sensors, with support (Deep-ocean Assessment and Reporting of Tsunamis (DART) from seismic data and tsunami field observations. Evidence of the buoys) produced results consistent with a thrust event. Our own subduction earthquake in global seismic data is largely hidden efforts to model the DART data with a normal-faulting event were because of the earthquake’s slow rise time or because its ground unsuccessful. motion is disguised by that of the normal-faulting event. Following the earthquake, two pre-existing campaign GPS sites on Earthquake doublets where subduction interface events trigger the northern Tongan island of Niuatoputapu were re-observed by large outer-rise earthquakes have been recorded previously4, but Tongan surveyors. Instead of the ,80-mm displacement to the this is the first well-documented example where the two events southwest predicted by dislocation models of the normal-faulting occur so closely in time and the triggering event might be a slow earthquake, the observations showed that the island had moved earthquake. As well as providing information on strain release ,350 mm to the east (Figs 1 and 2). The measured ,100 mm of mechanisms at subduction zones, earthquakes such as this provide subsidence was also greater than the ,10 mm of subsidence expected a possible mechanism for the occasional large tsunamis generated from the normal-faulting earthquake. at the Tonga subduction zone5, where slip between the plates is The most straightforward explanation for the Niuatoputapu GPS predominantly aseismic6. data is that slip occurred on a patch of the subduction interface near The majority of slip in great subduction earthquakes occurs at ‘aspe- Niuatoputapu at some time between December 2005 and October rities’, that is, patches of the subduction interface that have remained 2009. From this, together with the tsunami waveform evidence for a tightly coupled together for some time while the surrounding regions thrust earthquake on 29 September 2009, we infer that the subduction have slipped aseismically7,8. In some cases, such as the Tonga and interface slip occurred within a few minutes of the outer-rise earth- Mariana trenches, almost all the slip occurs aseismically and there quake. This is supported by aftershocks (Fig. 1), which were located are few, if any, great earthquakes. The convergence rate between the predominantly west of the trench. For the thrust earthquake not to be Pacific plate and the Lau basin across the northern Tonga trench ranges obvious in global seismic records, it must have occurred shortly after from 200 to 250 mm yr21, increasing northwards6, so if the plate inter- the outer-rise event, such that its signals are disguised within those of face here engaged in the stick–slip behaviour characteristic of Chile or the earlier earthquake, and/or so slowly that its far-field signals are 12,13 Sumatra, Tonga would generate more Mw $ 8 megathrust earthquakes much smaller than expected (a ‘tsunami earthquake’) . than anywhere else on the Earth6. In fact, we cannot be sure that any of We generate dislocation models that fit all available GPS data using the few great historical earthquakes in this region were underthrusting two uniform-slip rectangular fault planes (Fig. 1, Supplementary Figs events located on the plate interface5. 2 and 3, and Supplementary Tables 3 and 5). We constrain the fault Whether elastic strains at largely uncoupled subduction zones such parameters of the outer-rise event to be close to those determined as Tonga can ever build up sufficiently to allow the production of from CMT inversions9,10, and the location of the thrust event to be

1GNS Science, PO Box 30368, Lower Hutt 5040, New Zealand. 2School of Earth Sciences, Ohio State University, Columbus, Ohio 43210, USA. 3Ministry of Lands, Survey, Natural Resources and Environment, PO Box 5, Nuku’alofa, Tonga. 959 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

13º 600 Observed NTPT NTRP Samoan Islands

Models Normal fault SAMO 400 14º FALE Both faults

100 mm ASPA East East 200 North North 15º Displacement (mm) 0 Niuatoputapu Mw = 7.9; avg. slip, 8.6 m Upwards Upwards NTPT Latitude south 16º 180º 190º –200 NTRP 10º 51425 Samoa 1996 2000 2004 2008 1996 2000 2004 2008 AFI ASPA Year Fiji NTPT VAVS NIUM MSVF Figure 2 | Observed displacements at the two campaign GPS sites on 17º Tonga Niuatoputapu showing their large eastward offset between 2005 and 51426 2009. A best-fit linear trend from the start of the record until December Mw = 8.0; avg. slip, 4.1 m 2005 is subtracted from each time series; the dashed lines show the 61s

30º uncertainty of the projected fit. The dotted lines show the predicted 0 50 100 54401 coseismic displacement from the two-fault model of Fig. 1. Error bars, 1s. km 18º this conclusion to be nearly independent of rupture velocity. For 186º 187º 188º 189º 190º comparison, the slow earthquake that preceded the great 1960 Longitude east Chilean earthquake16 had a rise time of ,300 s. Figure 1 | The 2009 Samoa–Tonga earthquake as a double event, with We use the fault planes, fault mechanisms and uniform-slip ampli- observed and modelled GPS displacements and fault-plane solutions. The tudes from a range of well-fitting GPS models to predict tsunami yellow arrows (with 95% confidence uncertainty ellipses) are observed waveforms at the DART buoys. For each model, we adjust the rise displacements at GPS sites. The black arrows show displacements from an time of the thrust fault and the time delay between the two ruptures outer-rise normal-fault dislocation model fitted to observations from to find parameters that both provide a good fit to the waveforms from continuous GPS sites only (that is, not NTPT or NTRP). The predictions of this model at GPS sites NTPT and NTRP are in strong disagreement with the the three nearest buoys and satisfy the timing constraints (Fig. 4). Fits observations. The red arrows are displacements for a model fitted to all GPS to the far-field DART data are shown in Supplementary Figs 7 and 8. data using two faults: a subduction interface thrust fault and an outer-rise In the model of Figs 1 and 4, the rupture starts simultaneously all normal fault. VAVS and NIUM, two sites located south of the region in the across the thrust fault (infinite rupture velocity). Models using a main figure, are well fitted by the two-fault model but NIUM is poorly fitted finite rupture velocity and various rupture initiation points on the by the single-fault model. The outlines of the uniform-slip rectangular fault thrust fault cause changes in the details but do not alter our conclu- planes projected to the surface are shown by yellow rectangles with sions. For the GPS model of Fig. 1, the approximately (0, 0) intercept associated lower-hemisphere focal mechanisms. See Supplementary Tables and –0.5 slope of the mis-fit contours in Fig. 4d show that rupture of 1, 3 and 5 for the displacement values and fault parameters. The red star the outer-rise normal fault begins when the subduction thrust rup- shows the epicentre of the main shock from the US Geological Survey Preliminary Determination of Epicentres (PDE) catalogue, and the open ture is about halfway through. The largest region of well-fitting para- grey circles are PDE epicentres for the first month of aftershocks. meter space is for models in which slow rupture on the subduction Bathymetry contours are shown from 1,000 to 6,000 m at 1,000-m intervals. interface precedes the normal-fault rupture by several minutes. Inset, locations of DART buoys whose data we fitted (red dots), seismic However, there is a small region with time delay .0 s and rise time stations (green squares) and some GPS sites including those not in the main ,100 s that allows a relatively fast rupture on the interface to follow figure (black triangles). The magenta lines are a representation of tectonic the normal-fault event. An alternative model is shown in Sup- plate and microplate boundaries. plementary Figs 4 and 6. Although a fast rupture on the subduction interface immediately near the subduction interface14. The GPS models are non-unique, but following the outer-rise earthquake cannot be ruled out, the seismic all viable models have the centre of the normal fault 60–115 km from and tsunami evidence are predominantly in favour of a slow thrust on the centre of the thrust in a generally northeasterly direction and the the subduction interface that precedes the outer-rise earthquake by moment magnitude of the thrust 0.0–0.16 units higher than that of several minutes. Eye-witness reports from Niuatoputapu suggest the the normal fault (Supplementary Tables 5 and 6). tsunami arrived about 7 min after major ground-shaking started. To investigate whether a slow earthquake could have occurred on This is consistent with a tsunami travel time to Niuatoputapu of the subduction interface before the outer-rise normal-fault earth- ,10 min from a subduction thrust event that started several minutes quake, we model the seismic signal that would be observed at the before the normal-fault event, although the subjectivity of time esti- closest seismic station, AFI on Samoa. Seismic radiation amplitude is mates in a crisis situation renders this inconclusive. proportional to the slip rate between the two sides of the fault surface We and other researchers17 documented tsunami run-up of several and depends to a lesser extent on rupture velocity, the rate at which metres along the southern coasts of Samoa and American Samoa, the rupture front propagates across the fault15. Instead of slip rate, we with .10 m observed locally. These heights are explicable by the here use the rise time, which is slip magnitude divided by slip rate. normal-faulting event alone as well as by our two-fault interpreta- Rise times of 100 s and less for a subduction interface thrust predict a tion. At Niuatoputapu (Supplementary Fig. 9), we documented flow clear signal that is not observed at AFI (Fig. 3), whereas rise times of heights as high as 15 m at the eastern end of the island and run-up of 200 s and more predict a signal below the observed AFI noise level. up to 6 m at various locations18. These cannot easily be explained by Therefore, if the subduction interface earthquake occurred before the the normal fault alone, but our two-fault models in which the thrust outer-rise earthquake, it had a rise time of ,200 s or more. We find fault adds about 25% to the Niuatoputapu tsunami amplitude can 960 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

AFI Vz 200 50 s a 100 s 150 s 0.02 DART 54401 200 s

) 100 –1 0.00

0 –0.02 Velocity (µm s 1.5 2.0 2.5 3.0 –100 b 0.10 DART 51246

–200 0.00 17:46 17:48 17:50 Time on 29 September 2009 Figure 3 | Observed seismic noise before onset of outer-rise earthquake arrivals at AFI compared with model subduction thrust seismograms. Red –0.10 Wave surface elevation (m) trace shows the observed AFI vertical-component velocity record (V ); the 0.5 1.0 1.5 2.0 z c onset of strong ground motion from the normal fault is at 17:48:34.5, and the 0.10 record is clipped after this time. The green, blue, brown and black traces DART 51245 show the calculated velocity for Mw 5 8 subduction interface thrust events starting 170 s before the normal fault with respective rise times of 50, 100, 150 and 200 s. The predicted thrust signal is at or below the noise level of the data for rise times longer than ,200 s. If the thrust event started after the 0.00 normal fault, its signal would be disguised within the high-amplitude waves from the normal fault. Supplementary Fig. 5 shows the horizontal components. –0.10 0.5 1.0 1.5 2.0 match the observations (Supplementary Fig. 10), especially if construc- Time after normal-fault origin time (h) tive interference occurred between the waves from the two tsunami d sources. 300 A number of explanations have been proposed for the triggering of 0.021 one earthquake by another, including static Coulomb stress changes and dynamic triggering due to strong ground-shaking from an earlier 200 0.02 earthquake19. In either case, the second fault must already be close to failure for triggering to operate. The Coulomb stress change favour- 0.019 ing normal faulting on the outer-rise fault due to the modelled slip on 100 0.014 the subduction interface shows an increase .0.25 MPa (Supplemen- 0.018 tary Fig. 11), so static stress triggering is a plausible explanation if the 0.017 0.013 0.016 thrust initiated first. The Coulomb stress change from the normal 0 fault is small and in the wrong sense to favour thrusting on the 0.015 subduction interface, which implies dynamic triggering of the sub- 0.012 duction thrust if the outer-rise event initiated first. In the 2006–2007 –100 0.01 0.013 4 Approx. Kuril subduction/outer-rise earthquake doublet , the subduction region thrust preceded the outer-rise earthquake by months, suggesting forbidden static stress triggering with some involvement of viscous or fluid flow. –200 by AFI

16,20 Time delay of thrust relative to normal fault (s) seismic There are several examples where slow slip deep on the subduc- 0.013 observations 0.011 tion interface may have triggered an earthquake on a shallower part of the interface after some time delay, but we believe this is the first –300 documented example where slip on the subduction interface may 0 100 200 300 400 500 600 have triggered an outer-rise earthquake with a delay of only a few Rise time of thrust fault (s) minutes. It would also be the first documented example where an Figure 4 | Observed and modelled tsunami wave elevations and mis-fit outer-rise earthquake dynamically triggered a great earthquake on contours. a–c, Observations (black) at the three nearest DART ocean- the subduction interface, if that was the order of events. Without the bottom pressure sensors, with predictions of the GPS two-fault model of GPS and tsunami waveform evidence, it is unlikely that the 2009 Fig. 1 (red) and the preliminary US Geological Survey outer-rise normal- earthquake doublet would have been recognized, so it is possible that fault model25 (green). The latter disagrees strongly with the data, especially at similar events have been recorded seismically in the past and have DART buoys 54401 and 51246. The two-fault predictions use a thrust-fault been classified as complex sources. rise time of 200 s, with the normal fault rupturing 105 s after the initiation of The linear motion of the Niuatoputapu GPS sites between 1996 and the thrust fault. Time zero is the origin time of the normal-fault event, and 1.5 h of data around the first tsunami arrival time are shown for each DART 2005 (Fig. 2) indicates that no other episodic events approaching the record. d, Contours show root-mean-squared mis-fit (in metres) of DART size of the 2009 event occurred over that time interval. The 1996–2005 21 sea-level data predicted by this GPS source model as a function of thrust- velocity of these sites relative to the Pacific plate, of 239 6 2mmyr faulting rise time and time delay between the initiation of the two events. See in azimuth 105 6 1u (1s uncertainties), is consistent with the velocity Supplementary Fig. 6 for the predictions of an alternative GPS model and measured from 1990 to 19926, suggesting continuous steady motion Supplementary Fig. 8 for the predictions at far-field DART buoys. 961 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010 since 1990. If the interface slip in the 29 September 2009 event was inundation limit was distinguished principally by the extent of dead or damaged ,4 m as modelled (Fig. 1), this is only about 16 yr of accumulated slip vegetation. We measured flow depths from features such as water marks in at the ,240 mm yr21 relative plate rate, suggesting that such events buildings, damage to structures and trees, and entrainment of debris. Flow should recur about every 16 yr if slip were uniform over the fault heights were estimated from flow depths by calibration against benchmarks or natural tide level indicators. surface and the interface were fully coupled. In reality, there could have been larger slip on one or more asperities of smaller dimension, Full Methods and any associated references are available in the online version of with aseismic slip already having occurred between the asperities, the paper at www.nature.com/nature. implying a repeat time between similar events much longer than Received 21 January; accepted 21 June 2010. 16 yr. Alternatively, the 2009 event could represent a surge of slip that will be followed by a slowing of motion over the next several years. 1. Kanamori, H. Rupture process of subduction-zone earthquakes. Annu. Rev. Earth This slowing could be recorded as a temporary decrease in the Planet. Sci. 14, 293–322 (1986). Niuatoputapu velocity relative to the Pacific plate. 2. US Geological Survey. Largest Earthquakes in the World Since 1900. Æhttp:// earthquake.usgs.gov/earthquakes/world/10_largest_world.phpæ (2010). Severely damaging tsunamis originating from the Tonga trench are 3. US Geological Survey. Poster of the Samoa Islands Region Earthquake of 29 rare in the historical record5,21. The moment release in the 2009 September 2009 - Magnitude 8.0. Æhttp://earthquake.usgs.gov/earthquakes/ earthquake is of similar size to the 1917, 1919 and 1948 earthquakes eqarchives/poster/2009/20090929.phpæ (2009). and somewhat smaller than the 1865 earthquake5. Only one (1917) 4. Ammon, C. J., Kanamori, H. & Lay, T. A great earthquake doublet and seismic stress transfer in the central Kuril islands. Nature 451, 561–565 (2008). is in the northern Tonga area near Samoa, and this has been 5. Okal, E. A., Borrero, J. & Synolakis, C. E. The earthquake and tsunami of 1865 5 interpreted as a normal-faulting earthquake. A report (http:// November 17: evidence for far-field tsunami hazard from Tonga. Geophys. J. Int. www.samoaobserver.ws/index.php?option5com_content&view5 157, 164–174 (2004). article&id514751:1917-earthquake-revisited&catid51:latest-news& 6. Bevis, M. et al. Geodetic observations of very rapid convergence and back-arc Itemid550) on the 1917 event suggests similarities to the 2009 event, extension at the Tonga arc. Nature 374, 249–251 (1995). 7. Thatcher, W. Order and diversity in the modes of circum-pacific earthquake including the majority of tsunami damage occurring along the south recurrence. J. Geophys. Res. 95, 2609–2623 (1990). coast, but it is not possible to infer from available records whether the 8. Pacheco, J. F. & Sykes, L. R. Seismic moment catalog of large shallow earthquakes, 1917 earthquake might have involved rupture on the subduction 1900–1989. Bull. Seismol. Soc. Am. 82, 1306–1349 (1992). interface. The 2009 earthquake demonstrates that the Tonga subduc- 9. US Geological Survey. USGS Centroid Moment Tensor Solution. Æhttp:// neic.usgs.gov/neis/eq_depot/2009/eq_090929_mdbi/neic_mdbi_cmt.htmlæ tion interface is capable of generating damaging tsunamis, although in (2009). 2009 it was the outer-rise event that was the principal source of near- 10. US Geological Survey. Global CMT Project Moment Tensor Solution: Samoa field tsunami damage. Islands Region. Æhttp://neic.usgs.gov/neis/eq_depot/2009/ The fault sources we use for the tsunami and seismic modelling are eq_090929_mdbi/neic_mdbi_hrv.htmlæ (2009). necessarily approximate because of the limited constraints imposed 11. National Oceanographic and Atmospheric Administration. DART Buoy Comparison with Modelled Results. Æhttp://nctr.pmel.noaa.gov/ by the GPS data and our assumption of uniform slip. The fact that samoa20090929/dart-comparison-1.pngæ (2010). our simple model provides a good fit to GPS displacements and 12. Kanamori, H. Mechanism of tsunami earthquakes. Phys. Earth Planet. Inter. 6, tsunami waveform data is evidence that our basic interpretation is 346–359 (1972). correct, but we expect that modelling of very long-period seismic 13. Satake, K. & Tanioka, Y. Sources of tsunami and tsunamigenic earthquakes in data that takes into account both rupture sources will provide subduction zones. Pure Appl. Geophys. 154, 467–483 (1999). 14. Bonnardot, M.-A., Re´gnier, M., Ruellan, E., Christova, C. & Tric, E. Seismicity and improved understanding of this earthquake doublet. state of stress within the overriding plate of the Tonga-Kermadec subduction Our study provides an example of how a GPS-measured displace- zone. Tectonics 26, doi:10.1029/2006TC002044 (2007). ment at a site landwards of a trench can be diagnostic of a potentially 15. Aki, K. & Richards, P. G. Quantitative Seismology: Theory and Methods Vol. II tsunamigenic subduction thrust earthquake, even if the earthquake is 799–849 (Freeman, 1980). 16. Cifuentes, I. L. & Silver, P. G. Low-frequency source characteristics of the great too slow to generate significant seismic waves. This is an argument 1960 Chilean earthquake. J. Geophys. Res. 94, 643–663 (1989). for including GPS stations with data recorded and analysed in near 17. Dominey-Howes, D. & Thaman, R. UNESCO-IOC International Tsunami Survey real time as one component of operational tsunami detection Team Samoa, Interim Report of Field Survey 14th-21st October 2009. Misc. Report systems22–24. No. 2, 26–27 (Aust. Tsunami Res. Centre, Univ. New South Wales, 2009). 18. Wilson, K. J. et al. Post-Tsunami Survey of Niuatoputapu Island, Tonga, Following the 30th September 2009 South Pacific Tsunami (GNS Science Report 2009/71, METHODS SUMMARY Institute of Geological and Nuclear Sciences, 2009). GPS data and modelling. We estimated coseismic displacements and their 19. Freed, A. M. Earthquake triggering by static, dynamic and postseismic stress uncertainties at GPS stations from pre- and post-earthquake data, correcting transfer. Annu. Rev. Earth Planet. Sci. 33, 335–367 (2005). for interseismic motion where necessary, and modelled them assuming uniform 20. Linde, A. T. & Sacks, I. S. Slow earthquakes and great earthquakes along the slip on rectangular fault planes. Several solutions close to the minimum mis-fit Nankai trough. Earth Planet. Sci. Lett. 203, 265–275 (2002). were selected as input for tsunami modelling. 21. Frohlich, C. et al. Huge erratic boulders in Tonga deposited by a prehistoric Tsunami modelling. Tsunami generation, propagation, run-up and inundation tsunami. Geology 37, 131–134 (2009). were simulated using the COMCOT model, with the GPS model providing the 22. Sobolev, S. V. et al. Tsunami early-warning using GPS-shield arrays. J. Geophys. Res. 112, doi:10.1029/2006JB004640 (2007). initial sea-floor displacement. Bathymetry with 2-arcmin spatial resolution was 23. Song, Y. T. Detecting tsunami genesis and scales directly from coastal GPS used to calculate tsunami evolution and wave profiles at DART buoy locations. stations. Geophys. Res. Lett. 34, doi:10.1029/2007GL031681 (2007). Tsunami run-up and inundation were computed using nested grids to increase 24. Blewitt, G. et al. GPS for real-time earthquake source determination and tsunami spatial resolution near shore, high-resolution bathymetry and topography, and a warning systems. J. Geod. 83, 335–343 (2009). simple representation of bottom friction. 25. US Geological Survey. Finite Fault Model. Æhttp://earthquake.usgs.gov/ We divided the thrust plane into 120 subfaults that each rupture with a given earthquakes/eqinthenews/2009/us2009mdbi/finite_fault.phpæ (2009). rise time, starting either simultaneously or as determined by the velocity of Supplementary Information is linked to the online version of the paper at rupture from some point on the plane. The root-mean-squared mis-fit with www.nature.com/nature. DART observations was plotted as a function of rise time and time delay between the two sources. Acknowledgements We thank the following agencies for their publicly available Seismic data and modelling. Velocity records at seismic station AFI, less than data: New Zealand GeoNet, Geoscience Australia, the US National Geodetic 200 km from the hypocentre, were filtered from 0.001 to 0.5 Hz and compared Survey and the International GNSS Service (for GPS data); the NOAA (for DART data); and the USGS and the IRIS Data Management Center (for seismic data). We with predicted signals from a slow earthquake on the subduction interface, thank the governments of Tonga, Samoa and Niue, and the Niue Meteorological assuming a range of rise times, to provide constraints on thrust-fault rise time Service, for supporting GPS data collection in their countries in partnership with and the time delay between ruptures. GNS and Ohio State University; ‘A. Pongi, M. Archbold, D. Matheson and Tsunami field observations. We measured tsunami run-up, inundation dis- R. Williams for their contributions to GPS observations at Niuatoputapu; tance and flow depths using GPS observations and a laser rangefinder. The D. Caccamise and R. Smalley for their efforts with the Samoan high-rate GPS data; 962 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

M. Chadwick, J. Haines and D. Agnew for discussions; and L. Wallace for a review. effects. M.B. analysed high-rate GPS data. J.B., X.W., C.H., M.B., K.W., G.P. and The Coulomb stress modelling used software GNSTRESS2 by R. Robinson. W.P. were responsible for the data interpretation and all authors discussed the results and commented on the manuscript. Author Contributions J.B. processed and modelled the GPS data. X.W. modelled the DART and tsunami run-up data with support from G.P. and W.P. C.H. modelled Author Information Reprints and permissions information is available at the seismic data. K.W. and W.P. made field observations of the tsunami effects in www.nature.com/reprints. The authors declare no competing financial interests. Samoa and on Niuatoputapu. R.K.’s team at the Tonga MLSNRE has been Readers are welcome to comment on the online version of this article at instrumental in the collection of GPS data from Niuatoputapu, including the critical www.nature.com/nature. Correspondence and requests for materials should be post-earthquake observations, and they also made field observations of tsunami addressed to J.B. ([email protected]) or X.W. ([email protected]).

27 METHODS displacements are computed using Okada’s elastic finite fault theory. These serve GPS data analysis and modelling. GPS daily position solutions are analysed with as bottom boundary disturbances and become the source of tsunami generation. BERNESE v5.0 software26 in a network solution using double-differenced data To model the rupture, the entire thrust-fault plane is further divided into 120 from stations in Australia, New Zealand and Pacific islands. IGS final orbits and subfault segments, each with the same slip amount and orientation. The uplift earth orientation parameters are held fixed. We use IGS relative antenna phase motion of each subfault segment rises from zero to its full value linearly over a patterns, ocean load estimates from Topex model 7.1 as calculated by Onsala given rise time, and its starting time is determined by the velocity of rupture Space Observatory, and a global ionosphere model from the University of Bern. starting from the centre or one of the corners of the thrust-fault plane. We model Zenith delays are estimated hourly and tropospheric tilt estimated daily using rise times from 0 to 600 s, and find that the efficiency of tsunami generation falls Niell mapping functions. Ambiguities are fixed as far as possible using the off quickly as the rise time increases beyond 300 s. Several initiation points and a BERNESE software’s quasi-ionosphere-free strategy. Daily solutions are trans- wide range of rupture velocities are investigated, but we report only results for formed to the ITRF2000/IGb00 reference frame using a three-parameter Helmert the rupture starting simultaneously all across the fault (infinite rupture velocity), transformation onto a set of Australian and Pacific IGS stations. because the rupture velocity is a second-order effect in comparison with the rise For continuous stations, the coseismic offsets are estimated by subtracting a time. The normal fault is modelled with a zero rise time and infinite rupture linear trend, averaging two days of data before and two days after the earthquake velocity. The preferred rise time of thrust faulting and the optimal time delay and taking their difference. Uncertainties are estimated from the scatter in these between the two faults are identified by minimizing the root mean square of data. Time series for cGPS stations VAVS, NIUM and FALE are calculated during residuals between numerical results and the measurements at the three closest routine processing by the NZ GeoNet project and may be viewed at http:// DART buoys over a time window of 45 min around the first tsunami arrival. www.geonet.org.nz/resources/gps/timeseries/. For campaign stations, we make Seismic data and modelling. We analyse data from a velocity sensor at seismic linear fits to the pre-earthquake data (1996–2005 for NTPT; 2000–2005 for station AFI, located less than 200 km from the epicentre. The recorded seismo- NTRP), extend the fits to the time of the post-earthquake data and take the grams are corrected for instrument response and band-pass-filtered from 0.001 difference between the observed and predicted positions. Uncertainties are based to 0.5 Hz using a causal Butterworth filter. The low filter bandwidth is realistic on the scatter in the post-earthquake GPS campaign data and the uncertainty in considering the size of the modelled events, the uncertainties linked to the one- the linear fit to the pre-earthquake data projected to the time of the earthquake. dimensional crustal model and the large source–receiver distance. A signal with The dislocation models use Okada’s27 equations for uniform slip on a rect- high amplitudes in the low-frequency range is also expected if the thrust event angular fault in an elastic half-space. We undertake a Monte Carlo search for the has a slow rise time. lowest-mis-fit models, constraining the normal-fault mechanism to be close to Synthetic seismograms are computed using the discrete-wavenumber the solution from the Global CMT project10, the strike of the thrust fault to be AXITRA code29 for a subduction thrust event with a uniform slip distribution, close to the strike of the trench and its dip to be 16u as determined from a fit to a constant rupture velocity and rise time, and source characteristics defined by seismicity14. For each model, the fault locations, dimensions and mechanism are the GPS model. A variety of rupture velocities are tested but the variation in the chosen randomly within a constrained range, and the best-fit slip magnitudes are amplitude of predicted seismograms is small so we use an infinite rupture velo- determined by inversion (see Supplementary Information for details). Out of city (the whole plane starts to slip simultaneously). Rise times of 50, 100, 150 and 5,000,000 models tested, 137 have reduced x2 values between 0.26 and 0.5 (these 200 s are tested. For illustrative purposes, we start the thrust rupture 170 s before values suggest that our coseismic displacement uncertainties are overestimated the origin time of the outer-rise event to see if the predicted signal from the thrust by a factor of 1.5–2). We tested about ten of these models and found that the could be detected above the noise level of the observed data in the minutes before DART data were better fitted when the slip magnitude on the thrust was rela- the normal-fault arrivals at AFI. Using the delay between thrust-fault initiation tively high in comparison with that on the normal fault. We selected several such and the time at which the predicted signal first exceeds the noise level at AFI, we models for detailed tsunami waveform and run-up modelling, and we present plot a ‘forbidden region’ of parameter space on Fig. 3. two illustrative examples in the paper, one in the main text (model A; Fig. 1) and Field observations. We make measurements of the tsunami properties where it one in Supplementary Information (model B; Supplementary Fig. 4). impacted the coastline on Samoa, American Samoa and Niuatoputapu18, using a High-rate GPS data from American Samoa (ASPA), Niue (NIUM) and Vava’u real-time kinematic GPS and a laser rangefinder. The observations on Samoa and (VAVS) were searched for any evidence of a displacement signal arriving earlier American Samoa are made as part of one of several international teams studying than the normal-fault arrivals. This was unsuccessful, in part because sufficient the event. Tsunami run-up is the vertical height above mean sea level that the pre-event high-rate data were not downloaded before being automatically tsunami reached at its inland limit. The inundation distance is the horizontal deleted from the NIUM and VAVS GPS receivers, and in part because the pre- distance from the shoreline to the inland limit of flow. The inland limit of the dicted signals at the three sites are 15 mm at most. tsunami flow is distinguished in the field by the obvious line between dead and Tsunami waveform and run-up modelling. Tsunami generation and trans- healthy vegetation. The dispersal of debris and damage to built and natural Pacific propagation, as well as the run-up and inundation at Niuatoputapu features could also be used as a guide to inundation extent, along with eyewitness and Samoa, are simulated using an in-house numerical model, COMCOT observations. Tsunami flow height (or maximum water level) is the elevation of (Cornell Multi-grid Coupled Tsunami model), which solves shallow-water the tsunami flow above mean sea level; flow depth is the vertical distance between equations in spherical and/or Cartesian coordinates and has been applied to the ground surface and the height of the tsunami flow. Tsunami flow depths are study the impacts of several historical and recent tsunami events, including estimated from features such as water marks inside buildings, damage to struc- the 2004 Indian Ocean tsunami28. Bathymetry with 2-arcmin spatial resolution tures (for example the levels to which windows were broken), gouge marks from (from the NGDC ETOPO2 database) is used to calculate tsunami evolution and entrained debris on the exterior of buildings and up tree trunks, broken branches obtain computed time-history records of wave profiles at DART buoy locations. and vegetation stripping on trees, and the presence of debris caught in tree To determine tsunami run-up heights, a dynamically two-way-coupled system branches. Each of these indicators has varying degrees of reliability and in some of nested grids is used to reduce grid spacing gradually to 1 arcsec (about 28 m) at cases only constrains a minimum or maximum flow depth. Measured elevations Niuatoputapu and the southeast end of Upolu Island, Samoa. These high- are calibrated to mean sea level to give flow heights through the observation of resolution topographic/bathymetric data are interpolated from contour maps natural tide level markers or by occupying established geodetic benchmarks. and navigation charts. Manning’s formula (n 5 0.013) is implemented to estimate the bottom friction effect in the run-up and inundation calculation. Additional 26. Dach, R., Hugentobler, U., Fridez, P. & Meindl, M. Bernese GPS Software Version 5.0 (Astronomical Institute, Univ. Bern, 2007). roughness of the land surface, including vegetation effects, was not accounted for; 27. Okada, M. Surface deformation due to shear and tensile faults in a half-space. Bull. including these effects could significantly attenuate the flow height and run-up Seismol. Soc. Am. 75, 1135–1154 (1985). predictions on Niuatoputapu because great damage was evident where the 28. Wang, X. & Liu, P. L.-F. An analysis of 2004 Sumatra earthquake fault plane tsunami struck in the unpopulated, forested areas of the eastern and northern mechanism and Indian Ocean tsunami. J. Hydraul. Res. 44, 147–154 (2006). coastline, where ‘swathes of mature forest were completely destroyed, debris piles 29. Coutant, O. Expression of the Green’s Functions in Cylindrical Coordinates used with a of trees and vegetation were built up on land and in the lagoon, the shoreline Reflectivity Method. Tech. Report (Laboratoire de Ge´ophysique Interne et was significantly scoured and the land surface was stripped of soil’18. Sea-floor Tectonophysique, 1994).

The 2009 Samoa–Tonga great earthquake triggered doublet

Thorne Lay1, Charles J. Ammon2, Hiroo Kanamori3, Luis Rivera4, Keith D. Koper5 & Alexander R. Hutko6

Great earthquakes (having seismic magnitudes of at least 8) usually The Samoa–Tonga great earthquake sequence began on 29 September involve abrupt sliding of rock masses at a boundary between 2009, when the Mw 5 8.1 Samoa earthquake (17 h 48 min 11 s coordi- tectonic plates. Such interplate ruptures produce dynamic and nated universal time (UTC), 15.51 uS, 172.03 uW; ref. 11) occurred off- static stress changes that can activate nearby intraplate aftershocks, shore of the northern portion of the Tonga subduction zone (Fig. 1), as is commonly observed in the trench-slope region seaward of a about 170 km south of Samoa and 190 km southwest of American great subduction zone thrust event1–4. The earthquake sequence Samoa. Tsunami waves several metres high swept onto all sides of addressed here involves a rare instance in which a great trench- Tutuila, with localized run-ups as large as 12 m (ref. 12). Tsunami slope intraplate earthquake triggered extensive interplate faulting, arrivals were widely recorded by deep-ocean assessment and reporting reversing the typical pattern and broadly expanding the seismic and of tsunamis (DART) buoys and tide gauges around the Pacific13. The tsunami hazard. On 29 September 2009, within two minutes of the last regional seismic event to produce as large a tsunami in Samoa was initiation of a normal faulting event with moment magnitude 8.1 in on 25 June 1917 (ref. 14), associated with an Mw < 8.3 event with the outer trench-slope at the northern end of the Tonga subduction estimated location of 15.5u S, 173u W (ref. 15). The rapidly conver- zone, two major interplate underthrusting subevents (both with ging northern Tonga subduction zone is not known to have great moment magnitude 7.8), with total moment equal to a second great underthrusting earthquakes, despite having high levels of smaller- earthquake of moment magnitude 8.0, ruptured the nearby sub- magnitude seismicity; there are uncertainties in the mechanism, duction zone megathrust. The collective faulting produced tsunami seismic moment, and precise location of the 1917 event (attempts waves with localized regions of about 12 metres run-up that to relocate the event16 indicate that it may have been under the trench- claimed 192 lives in Samoa, American Samoa and Tonga. Overlap slope, but the location uncertainty is very large). The 2009 Samoa of the seismic signals obscured the fact that distinct faults separated event, with a seismic moment that places it among the four largest by more than 50 km had ruptured with different geometries, known trench-slope events, thus appears similar to the 1933 Sanriku with the triggered thrust faulting only being revealed by detailed and 1977 Sumbawa earthquakes, with the Tonga subduction zone seismic wave analyses. Extensive interplate and intraplate after- megathrust apparently having weak frictional coupling. shock activity was activated over a large region of the northern Several facts indicate that the actual situation is not clear-cut. Semi- Tonga subduction zone. automated analyses of the seismic waves for the 29 September 2009 Great extensional faulting events in the shallow outer trench-slope event yielded unusual inconsistency in point-source focal mechanism region seaward of subduction zones are relatively rare, with the three determinations. The global centroid moment tensor (GCMT)17 solu- largest-known events being the 1933 Sanriku, Japan, earthquake tion obtained from world-wide surface-wave ground motions for (moment magnitude Mw 5 8.4), the 1977 Sumbawa, Indonesia, dominant periods of about 200 s indicated a predominantly earthquake (Mw 5 8.3), and the 2007 Kuril Islands earthquake normal-fault solution (Fig. 1), but with a significant non-double (Mw 5 8.1). These huge intraplate earthquakes all had relatively high couple component that suggests either geometric complexity of the stress drop and strong tsunamigenesis, with ruptures extending faulting or some interference effect. Our analysis of longer-period through the oceanic crust and at least 15–20 km into the uppermost (200–1,000 s) ground motions called the W-phase18 (Supplemen- mantle4–6. These extensive fractures of the oceanic lithosphere are a tary Fig. S1), gave a predominantly double-couple normal faulting result of plate bending and slab-pull forces5,7,8. The deep faulting solution (Fig. 1) with strike w 5 152u, dip d 5 67u and rake l 5277u, 21 probably facilitates hydration of the lithosphere before subduction, and a seismic moment of M0 5 1.5 3 10 Nm. The significant dif- enabling intermediate-depth and deep earthquakes once the plate ference between the W-phase and GCMT solutions is unusual8,18. subducts9,10. The Sanriku and Sumbawa events occurred seaward of Several moderate-size trench-slope extensional events in 2009 pre- subduction zones for which the largest interplate thrust events are ceded the mainshock (Fig. 1), with the 30 August 2009 (Mw 5 6.6) small, suggesting weak frictional coupling of the megathrusts, event (Fig. 1) having very consistent W-phase and GCMT normal- whereas the 2007 Kuril Islands event occurred about 60 days after faulting solutions, so the discrepancies in models for the great event 4,8 an adjacent great (Mw 5 8.3) interplate thrust event . Intuitively, a cannot be attributed to regional wave propagation effects and are great trench-slope extensional event would not be expected to pre- probably due to source complexity. Attempts to model the tsunami cede a large adjacent thrust if slab-pull stresses are important in waves recorded at NOAA buoys using either of the seismic wave loading the trench-slope, because a locked interplate fault would tend point-source faulting geometries failed to match detailed first-arrival to shelter the up-dip region from the slab-pull1–3. Large trench-slope polarities and timing13,19,20. Aftershocks occurred over a widespread events before large interplate ruptures are infrequent and usually area to the west of the epicentre, within the Tonga block and/or along involve compressional, rather than extensional faulting1,3. the Tonga subduction zone plate boundary megathrust; only a

1Department of Earth and Planetary Sciences, University of California Santa Cruz, Santa Cruz, California 95064, USA. 2Department of Geosciences, The Pennsylvania State University, 440 Deike Building, University Park, Pennsylvania 16802, USA. 3Seismological Laboratory, California Institute of Technology, MS 252-21, Pasadena, California 91125, USA. 4Institut de Physique du Globe de Strasbourg, UMR7516, Universite´ de Strasbourg/CNRS, France. 5Department of Earth and Atmospheric Sciences, Saint Louis University, 3642 Lindell Boulevard, St Louis, Missouri 63108, USA. 6US Geological Survey, NEIC, MS 966 Box 2504, DFC, Denver, Colorado 80225, USA. 964 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

–13° M 10 m of normal-faulting displacement through the crust and upper- 8 w 7 6 5 most mantle under the outer trench slope. The largest aftershock likely Savaii Samoa to be on the mainshock fault (Fig. 1) occurred on 19 October 2009 M = 6.0 M = 8.0 1 Oct 09 10 Oct 09 19a Oct 09 Upolu w w (Mw 5 6.0), and we estimate a hypocentral depth of ,24 km below the –14° American Samoa ocean bottom for this event (Supplementary Fig. 4), supporting quite Tutuila 23 Apr 09 deep rupture of the lithosphere during the mainshock. 14 Oct 09 30 Aug 09 26 Jun 09 24 Paciﬁc An initial report of an additional ‘hidden’ triggered thrust event plate with Mw 5 7.9 on the megathrust 110 s after the initiation of normal 19b Oct 09 –15° 29 Sept 09 (1) faulting motivated further investigation using seismic signals ranging 1 Nov 09 29 Sept 09 from 1-s-period P-waves to .1,000-s-period free oscillations. We W-phase 1 found clear evidence of radiation complexity in broadband (period 29 Sept 09 (2) 4 Oct 09 of 30–500 s) short-arc (R1) Rayleigh-wave source time functions 2 (STFs), processed to remove propagation effects assuming normal –16° 3 GCMT 2 Oct 09 29 Sept 09 (3) fault radiation (Fig. 2). The R1 STFs show two strong pulses with Tonga Tonga 17 Oct 09 negative amplitudes about 70 s and 110 s into the signals, with maxi- block subduction zone mum amplitudes to the east and west. These can be accounted for by –175° –174° –173° –172° –171° –170° radiation from two thrust subevents. A waveform modelling search over locations, timing and strength of the secondary events provided –6,000 –4,000 –2,000 0 2,000 90 20 32 the locations of events 2 (15.75u S, 172.25u W) and 3 (16u S, Ocean depth (m) Elevation (m) Slip (m) 172.25u W) shown in Fig. 1, and the synthetic predictions in Fig. 2. Figure 1 | The Samoa–Tonga great earthquake doublet region. The great Detailed search results are presented in Supplementary Fig. 6. (Mw 5 8.1) 29 September 2009 Samoa earthquake ruptured an outer trench- Back-projections of short period (0.3–5 s) P waves from regional slope normal fault (blue mechanism and circle labelled 1) with the indicated networks in Australia, southern Asia, Japan (Hi-Net and F-Net), fault plane and slip distribution and co-seismically triggered two initially Alaska and North America (USArray) were performed to locate unrecognized major (Mw 5 7.8, Mw 5 7.8) thrust fault subevents (blue secondary sources of radiation. In this procedure, teleseismic record- mechanisms and circles labelled 2 and 3) on the megathrust. The dashed ings are aligned for a grid of possible source locations and summed to white oval indicates the probable zone of underthrusting slip. The blue star indicates the centroid location estimated for event 3 from regional surface- detect any radiation from each position as a function of time. This wave modelling. Grey circles indicate the locations and magnitudes of procedure reveals loci of secondary radiation as shown in Fig. 3 for shallow (depth ,100 km) earthquakes from 1 January 1973 up to the 2009 the F-Net data. The trench-slope normal fault radiation is clearly great earthquake sequence, including three moderate-size (Mw 5 5–6.6) separate from bursts of energy at 52 s, 91 s and 118 s, with the first trench-wall/outer-rise extensional events between April and August 2009 for which GCMT17 solutions (grey beachballs) are indicated (the M scaling of w a all GCMT solutions is indicated in the top right beachball inset). Red circles indicate earthquake epicentres and magnitudes (top left inset gives the 300 scaling) for the mainshock and aftershocks. The W-phase and GCMT point- source solutions (inset blue beachball mechanisms) for the mainshock and GCMT solutions for the larger aftershocks (red beachballs) are shown. The Negative secondary Negative secondary 200 arrivals arrivals white arrows indicate the direction of motion of the Pacific plate relative to the Australian plate at 8.4–8.6 cm yr21 (from the global plate motion model NUVEL-1; see the plate motion calculator at http://sps.unavco.org/ Time (s) 100 crustal_motion/dxdt/model/), with back-arc spreading increasing the convergence rate relative to the Tonga block to values of ,25 cm yr21. (On 19 October 2009, two events, a and b, occurred.) 0

0 30 60 90 120 150 180 210 240 270 300 330 360 handful of aftershocks were on either possible fault plane of the main- Azimuth (º) shock point-source solutions (Fig. 1). Many of the larger western b aftershocks have GCMT thrust fault solutions (Fig. 1), clearly indi- 300 cating that, in this instance, the trench-slope event activated moderate-size faulting on the megathrust, rather than the typical reverse behaviour. Remote triggering of activity has been observed for 200 other large events21,22, but there is no documented prior instance of a trench-slope event initiating such extensive activity on a megathrust. Time (s) 100 For the 1933 Sanriku earthquake, two groups of aftershocks with distinct shear (S) wave to compressional (P) wave differential times and frequency spectra were interpreted as aftershocks on the mainshock 0 fault plane and triggered events on the megathrust boundary23. The initial 60–100 s of the source process can be well explained by 0 30 60 90 120 150 180 210 240 270 300 330 360 Azimuth (º) normal faulting in the trench slope. Inversion of teleseismic broadband P waves and horizontally polarized S waves (SH waves) with periods of Figure 2 | Observed and modelled R1 STFs. a, Observed R1 STFs obtained 1 to 200 s for a finite-source model indicates a fault orientation with by deconvolving the data with Green’s functions for the normal fault w 5 144u, d 5 65u and l 5286u, generally similar to the W-phase geometry using a water-level deconvolution. Positive amplitudes are shaded. orientation (Supplementary Figs 2 and 3), with a seismic moment of We note the strong negative amplitudes in the azimuth windows 80–130u b 3 21 and 260–310u, with two pulses being clearly evident towards the east. , The 1.8 10 Nm, constrained by the composite fault modelling dis- timing and amplitude of the thrust faulting is modelled with synthetics cussed below. After about 12 s of relatively low-amplitude seismic wave constructed for a three-source model involving an initial normal fault radiation, the main rupture duration lasted about 50 s, with predomi- consistent with the body-wave model for the first 64 s of rupture and two nantly bilateral rupture expansion on a fault plane dipping steeply secondary thrust subevents with parameters constrained from regional towards the southwest (Fig. 1). The model indicates approximately modelling and a search on location and onset time. 965 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

r Figure 3 | P-wave back-projection using the Japanese F-Net stations. The 1.0 top trace is the stack power spatially integrated over the grid as a function of time, with red lines indicating the times of the spatial snapshots. Snapshots 0.5 from the continuous back-projection (see Supplementary movie 1) indicating intervals in which the normal fault radiation dominates (during 0.0 the first 50 s, typified by the 25 s frame), and when secondary events on the Normalized powe –20 0 20 40 60 80 100 120 140 megathrust or in the upper plate occur (frames at 52 s, 91 s and 118 s). At Time (s) 52 s, there is simultaneous radiation from the normal fault and the megathrust. The event at 118 s is probably in the upper plate, and although it –14º t = 25 s t = 52 s is strong in the short-period radiation, it is not strong at long periods. The full animations for F-Net and five other networks are shown together in the Supplementary Information. –15º provided by normal and thrust faulting aftershocks were used to –16º model 25–100-s-period regional Rayleigh and Love waves, providing good time resolution of the arrivals from the normal and thrusting –17º Thrust 1 subevents (Fig. 4a). Signals from 12 azimuthally distributed regional stations indicate secondary arrivals with varying focal mechanisms and timing (additional modelling is shown in Supplementary Fig. 8). –14º t = 91 s t = 118 s The best resolution is for the second thrust event; its estimated location is identified by the blue star in Fig. 1 (16.0u S, 172.4u W). Guided –15º by the back-projections and regional wave modelling, an iterative waveform inversion method was applied to broadband (1 to 200 s) –16º P and SH waves, confirming the locations of the subevents to the southwest of the normal fault radiation (Supplementary Fig. 9). A Thrust 2 –17º fault model inversion for the second source confirmed the subevent Aftershock geometry and seismic moment (Supplementary Fig. 10). –174º –172º –170º –174º –172º –170º For more-distant surface waves, the discrete arrivals from the subevents cannot be resolved, but the composite radiation still pro- Beam power from back-projection of F-Net P waves vides bounds on the relative timing, mechanisms and moments of the normal and thrust components. Figure 4b shows an example of two corresponding to the megathrust locations of the subevents modelling very-long-period (714–1,000 s) global Rayleigh waves, found using the R1 STFs. Full animations of the back-projections indicating how the W-phase solution fails to match the phase and from all of the regional networks are provided in the Supplementary amplitude of the data at station CTAO, but the sum of the normal Information. fault event plus two thrust subevents matches both very well. Extensive waveform modelling of surface waves further con- Additional station results are presented in Supplementary Figs 11 strained the composite source process. Empirical Green’s functions and 12. Modelling of phase and amplitudes of intermediate-period a Figure 4 | Surface wave modelling for simple 2 PPT 33–200 s 29 September 2009 0 and composite models. a, The regional R1 arrival –2 2 PPT 33–200 s at station PPT is modelled by shifting and )

6 2 T2 0 summing STF-filtered recordings at the same –2 Shift = 89 s station from the 19 October 2009 normal faulting 0 2 T1 aftershock (N1) and the 10 October 2009 thrust 0 –2 Shift = 45 s faulting aftershock (T1, T2). The shift is the –2 relative alignment of the N1, T1 and T2 traces in 2 N1 0 seconds. The observed (black) and the summed

Digital counts (10 –2 Shift = 0 s predicted (red) waveforms for station PPT are 2 Sum of N1+T1+T2 –4 0 Sum of N1+T1+T2 superimposed on the right. b, Ultralong period –2 (774–1,000 s) Rayleigh-wave motions at station 0 200 400 600 800 1,000 0 200 400 600 800 1,000 b CTAO (black) are compared to predictions for CTAO the W-phase solution (red, on left) and for the 0.03 0.03 normal-plus-two-thrust-subevent composite 0.02 0.02 model (red, on right). w 5 257.2u, D 5 39.9u. 0.01 0.01 Station instrument 00; long-period vertical 0.00 0.00 motion. c, Intermediate-period (200–1,000 s) (mm) –0.01 –0.01 Love-wave motions at station PFO (black) are –0.02 –0.02 compared to predictions for the W-phase solution (red, on left) and for the normal-plus- Ground displacement –0.03 –0.03 0 1,000 2,000 3,000 4,000 0 1,000 2,000 3,000 4,000 two-thrust-subevent composite model (red, on c PFO right). w 5 46.4u, D 5 72.1u. Station instrument 2.0 2.0 00; long-period tangential motion. Additional 1.5 1.5 waveform comparisons are shown in 1.0 1.0 Supplementary Figs 8, 11, 12 and 13. 0.5 0.5 0.0 0.0 (mm) –0.5 –0.5 –1.0 –1.0 –1.5 –1.5

Ground displacement –2.0 –2.0 0 1,000 2,000 3,000 4,000 0 1,000 2,000 3,000 4,000 Time after the origin time (s)

(200–1,000 s) global Rayleigh and Love waves provides further the thrust subevents. Back-projection of short-period P waves from various constraints on the composite radiation, particularly for the source networks26,27 to a surface grid in the source region imaged the space-time history orientations and relative moments. Figure 4c shows the Love wave at of radiation from the normal fault in the outer trench-slope as well as bursts of station PFO, which is poorly matched by the W-phase solution, energy from the megathrust associated with high-frequency radiation from but well modelled by the composite source. Additional waveform triggered events. Using recordings for both normal faulting and thrust faulting aftershocks, regional Rayleigh- and Love-wave signals in the period range modelling results are shown in Supplementary Fig. 13. Spheroidal 25–100 s were forward-modelled using the empirical Green’s function method; and toroidal mode analysis extended this modelling to even longer the small event signals were convolved with STFs appropriate for larger events periods, with similar results; the composite normal-plus-thrust and then shifted and summed to match the mainshock waveforms. Differential model improves the fit of observations near predicted nodes for arrival times of the signals from the thrust and normal faulting were used to the W-phase solution significantly (Supplementary Fig. 14). locate their relative centroids. Iterative and linearized broadband body-wave Each seismic wave bandwidth and wave type contributes to the inversions further constrained the faulting properties of the triggered thrusts. resolution of the location, timing and strength of the secondary The composite three-source models were adjusted in absolute moment and fault sources on the megathrust. Overall, we resolve two large thrust earth- geometry to match ultralong-period Rayleigh-wave amplitude and phase and quake subevents located on the shallow megathrust plane about intermediate-period Love- and Rayleigh-wave amplitudes at globally distributed 50 km southwest of the trench-slope faulting (Fig. 1), which ruptured stations. Free oscillation spectra for stations at nodes of the normal fault radi- from ,49–89 s and ,90–130 s after the mainshock initiation. The ation were used to confirm the relative strength of the thrust components. first of these appears to overlap slightly in time the last part of the Full Methods and any associated references are available in the online version of normal faulting. The thrust faulting geometries are consistent with the paper at www.nature.com/nature. thrust aftershock mechanisms, and have geometries with w 5 185u, d 5 29u, l 5 90u, consistent with the Pacific plate underthrusting in Received 23 February; accepted 24 May 2010. the Tonga subduction zone (Fig. 1). The seismic moments of the two 20 1. Christensen, D. H. & Ruff, L. J. Seismic coupling and outer rise earthquakes. thrusts subevents are estimated to be 5.2 3 10 Nm (Mw 5 7.8) and J. Geophys. Res. 93, 13421–13444 (1988). 20 5.3 3 10 Nm (Mw 5 7.8). Together these events have a seismic 2. Dmowska, R., Rice, J. R., Lovison, L. C. & Josell, D. Stress transfer and seismic phenomena in coupled subduction zones during the earthquake cycle. J. Geophys. moment magnitude of Mw 5 8.0; equal to a great earthquake on the megathrust. Res. 93, 7869–7884 (1988). 3. Lay, T., Astiz, L., Kanamori, H. & Christensen, D. H. Temporal variation of large The Samoa–Tonga great earthquake sequence lacks any known intraplate earthquakes in coupled subduction zones. Phys. Earth Planet. Inter. 54, precedent; essentially, a great outer trench-slope event seaward of a 258–312 (1989). putatively weakly coupled megathrust induced large and widespread 4. Ammon, C. J., Kanamori, H. & Lay, T. A great earthquake doublet and seismic seismic failure of the megathrust. The timing favours triggering of the stress transfer cycle in the central Kuril islands. Nature 451, 561–566 (2008). thrust activity, because it overlaps the normal faulting in time. It is 5. Kanamori, H. Seismological evidence for a lithospheric normal faulting; the Sanriku earthquake of 1933. Phys. Earth Planet. Inter. 4, 289–300 (1971). reasonable to infer that the megathrust had significant strain accu- 6. Lynnes, C. S. & Lay, T. Source process of the great 1977 Sumba earthquake. mulation and was not totally frictionally decoupled. Given the lack of J. Geophys. Res. 93, 13407–13420 (1988). recorded great underthrusting events in the northern Tonga subduc- 7. Taylor, M. A. J., Zheng, G., Rice, J. R., Stuart, W. D. & Dmowska, R. Cyclic stressing tion zone, one possibility is that the shallow region of the megathrust and seismicity at strong coupled subduction zones. J. Geophys. Res. 101, normally undergoes episodic creep events rather than fast seismic 8363–8381 (1996). 8. Lay, T. et al. The 2006–2007 Kuril Islands great earthquake sequences. ruptures, but high strain rates following the trench-slope failure drove J. Geophys. Res. 114, B11308, doi:10.1029/2008JB006280 (2009). 25 a conditionally stable frictional environment to seismic failure . 9. Ranero, C. R., Villasen˜or, A., Phipps Morgan, J. & Weinrebe, W. Relationship Alternatively, moderate-size patches of unstable friction that would between bend-faulting at trenches and intermediate-depth seismicity. Geochem. Geophys. Geosyst. 6, Q12002, doi: 10.1029/2005GC000997 (2005). have released in thrust events of Mw , 7.0, characteristic of the largest prior events, were driven to fail simultaneously. Long-term geodetic 10. Faccenda, M., Gerya, T. V. & Burlini, L. Deep slab hydration induced by bending- related variations in tectonic pressure. Nature Geosci. 2, 790–793 (2009). observations of continuous deformation on the megathrust are 11. Earthquake Hazards Program. Magnitude 8.1—Samoa Islands region. Æhttp:// needed to help constrain its frictional behaviour. The large thrusts earthquake.usgs.gov/eqcenter/eqinthenews/2009/us2009mdbi/#detailsæ are southwest of the normal fault, so the megathrust region immedi- (US Geological Survey, 2009). ately down-dip of the trench-slope event may have undergone prior 12. Jaffe, B. Surprises from the deadly September 29, 2009, Samoa tsunami. Æhttp:// creep, allowing slab-pull to load the trench-slope environment (and soundwaves.usgs.gov/2009/12/æ (US Geological Survey, 2009). 13. NOAA Center for Tsunami Research. Tsunami event—September 29, 2009 loading the adjacent megathrust region to the south). In addition, Samoa. http://nctr.pmel.noaa.gov/samoa20090929/ (2009). because the region is near a tear in the Pacific plate, lateral loading 14. Iida, K., Cox, D. C. & Pararas-Carayannis, G. Preliminary catalog of tsunamis of the stress in the trench-slope region could be a factor. Certainly, the occurring in the Pacific Ocean. Data Report 5, HIG-67–10, 261 (Hawaii Institute of widespread aftershock activity, much of which involves underthrust- Geophysics, University of Hawaii, 1967). ing, but some of which appears to be in the upper Tonga block (Figs 1 15. Gutenberg, B. & Richter, C. F. Seismicity of the Earth and Associated Phenomena 2nd edn (Princeton University Press, 1954). and 3), indicates that dynamic and static stress changes within the 16. Okal, E. & Kirby, S. H. Large earthquakes and tsunamis at the Samoa corner in the subducting plate had wide-ranging effects across the region. The context of the 2009 Samoa event. Eos 90 (52), (Fall Meet. Suppl.), abstr. regional triggering phenomenon adds complexity to the seismic and U21E–2182 (2009). tsunami hazard assessment and warning procedures for this region as 17. Ekstro¨m, G. & Nettles, M. Global Centroid-Moment-Tensor Project. Æhttp:// well as for other regions of putatively weakly coupled megathrusts www.globalcmt.org/æ (National Science Foundation, 2009). 18. Kanamori, H. & Rivera, L. Source inversion of W-phase: speeding up seismic where large intraplate events may occur and drive underthrusting tsunami warning. Geophys. J. Int. 175, 222–238 (2008). ruptures. 19. Fujii, Y. & Satake, K. Samoa Islands Tsunami on Sep. 29, 2009. http:// iisee.kenken.go.jp/staff/fujii/Samoa/tsunami.html (2009). METHODS SUMMARY 20. Tonini, R., Pagnoni, G., Armigliato, A. & Tinti, S. The 29th September Samoa A point-source representation of the total seismic radiation from the earthquake islands tsunami: preliminary simulations based on the first focal mechanisms sequence was determined by inversion of 200–1,000-s-period three-component hypotheses and implications of uncertainties in tsunami early warning strategies. Eos 90(52), (Fall Meet. Suppl.), abstr. U21E–2185 (2009). W-phase ground motions. A finite-fault slip model for the ,60-s-long outer 21. Hill, D. P. et al. Remote seismicity triggered by the M7.5 Landers, California trench-slope failure was determined using linearized inversion of teleseismic earthquake of June 28, 1992. Science 260, 1617–1623 (1993). P-wave and SH-wave ground displacements in the period range 1 to 200 s. 22. Velasco, A. A., Hernandez, S., Parsons, T. & Pankow, K. Global ubiquity of dynamic The triggered thrust faulting was detected and constrained using several analyses. earthquake triggering. Nature Geosci. 1, 375–379 (2008). Long-period Rayleigh-wave STFs, obtained by correcting for propagation 23. Umino, N., Kono, T., Hasegawa, A. & Tamura, Y. Revisiting the 1933 off Sanriku effects, were modelled with a grid search for locations and strengths of secondary earthquake (M8.1) by using smoked-paper seismographs. Ann. Meet. Seismol. Soc. thrust events, providing estimates of relative locations and seismic moments of Jpn abstr. [in Japanese] (2007). 967 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

24. Li, X., Shao, G. & Ji, C. Rupture process of the 2009 Mw 8.1 Samoa earthquake Research Institutions for Seismology (IRIS) Data Management System (DMS) and constrained by joint inverting teleseismic body, surface waves and local strong the F-Net and Hi-Net data centres were used to access the data. C. Ji kindly shared motion. Eos (Fall Meet.) abstr. U21D–03 (2009). details of his model. Z. Duputel wrote the W-Phase software version used in this 25. Scholz, C. Earthquakes and friction laws. Nature 391, 37–42 (1998). study. We thank H. Savage and E. Brodsky for discussions of frictional conditional 26. Ishii, M., Shearer, P. M., Houston, H. & Vidale, J. E. Extent, duration and speed of stability. This work was supported by NSF grant EAR0635570 and USGS Award the 2004 Sumatra-Andaman earthquake imaged by the Hi-net array. Nature 435, Number 05HQGR0174. 933–936 (2005). 27. Xu, Y., Koper, K. D., Sufri, O., Zhu, L. & Hutko, A. R. Rupture imaging of the Mw 7.9 12 Author Contributions All authors contributed equally to the analysis and May 2008 Wenchuan earthquake from back projection of teleseismic P waves. preparation of this paper. Geochem. Geophys. Geosyst. 10, Q04006, doi: 10.1029/2008GC002335 (2009). Supplementary Information is linked to the online version of the paper at Author Information Reprints and permissions information is available at www.nature.com/nature. www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at Acknowledgements This work made use of GMT and SAC software and www.nature.com/nature. Correspondence and requests for materials should be Federation of Digital Seismic Networks (FDSN) seismic data. The Incorporated addressed to T.L. ([email protected]).

METHODS around the known fault location and the P-wave travel times were computed from each grid location to each station in the network using standard Earth model Several seismological procedures were applied to characterize the overall radi- AK135 (ref. 32). The differential travel times between grid and station locations ation from the source region for the great Samoa-Tonga triggered doublet, are then applied as shifts to the data (which have been initially cross-correlated and including point-source representations of the faulting, finite-fault models shifted so that the first arrival is predicted by the Earth model), aligning any inferred from inversion of teleseismic sigals, back-projection of short-period P 26,27 features in the signals originating at a given location at whatever time lag they waves from various networks to a target grid in the source region, and may appear in the signal. Because the source radiation image is smeared in space extensive modelling of regional and teleseismic body waves, surface waves, and time by partial constructive interference from grid locations around the true and normal modes. point of coherent radiation, there are artefacts in the images. In general these streak W-phase inversion. Long-period (200–1,000 s) ground motions comprising the the true source image along the azimuth from the source region to the network, W-phase (very-long-period fundamental-mode and overtone energy super- with too-early apparent radiation at greater distance than the true source location imposed on body-wave arrivals) from 33 stations (totalling 57 channels, 29 of and too-late apparent radiation at closer distances. Points of peak constructive which were vertical components) were inverted for a point-source moment tensor interference are the probable true source locations, and distinct or continuous loci following the procedure of ref. 18, including a search over centroid location. A of strong radiation indicate radiation from a continuous fault or possibly from source depth of 18 km was assumed, and the moment tensor at the optimal discrete sources, such as early aftershocks. Ideally, one might use data from all centroid (215.16u, 2172.07u, Supplementary Fig. 1) has Mrr 521.055, azimuths tosuppress the streaking effects and find optimal source locations, but we Mhh 5 0.470, Mww 5 0.584, Mrh 520.359, Mrw 5 0.950, Mhw 520.614 (where have found that combining images from different azimuths is problematic owing subscripts r, h and w are polar coordinates for the tensor dipole and couple terms 21 to azimuthal variations in Green’s functions leading to incompatible imaging of of the moment tensor) all in units of 10 Nm. This solution is close to a double features formed from coherent diving or depth phases. couple (Fig. 1), and has a best nodal plane with strike w 5 152u, dip d 5 67u and 21 P-wave data from six regional networks were collected to provide back- rake l 5277u, and a seismic moment of 1.50 3 10 Nm (Mw 5 8.05). Centroid projection images for various azimuths from the Samoa–Tonga region. These time shift and half-duration values of 24 s were specified. consist of 22 stations from Australia, 34 stations from east Asia, 73 stations from P- and SH-wave finite fault inversion. We inverted teleseismic P and SH waves the Japanese F-Net, 558 stations from the Japanese Hi-Net, 56 stations from the filtered in the passband 1–200 s to estimate details of the slip distribution for the Alaska region, and 519 stations from USArray. For a given network, the raw traces initial, normal faulting portion of the seismic radiation. Given the uncertainty in were aligned and normalized with a multi-channel cross-correlation (MCCC) the fault orientation, we first performed many finite-source inversions using algorithm33 using a 15-s-long window that started 5 s before the theoretical teleseismic P waves for which first arrival times could be accurately picked, using P-wave arrival time. The small offsets in time required for proper alignment a grid search over fault plane strike and dip, with rake being allowed to vary on account for travel-time perturbations created by the three-dimensional structure 28,29 the fault . The body-wave Green functions were computed for simple layered of the Earth. We assumed that these offsets were valid for the entire source region. oceanic source and continental receiver models. The sub-fault STFs were para- The amplitude scaling factors account for site effects, differences in seismometer meterized as six overlapping triangles of variable strength with 2-s half-durations gain, and the radiation pattern. The source region was gridded in increments of and 2-s lag offsets, giving total subfault rupture durations of 14 s. The fault model 0.1u in latitude and longitude, and 1.0 s in time. The depth was fixed at the grid spacing was 5 km along dip and 15–35 km along strike for models with nominal value of 18 km. For each grid point, a sliding 10-s-long window was used 21 varying rupture velocity, Vr 5 1.0–3.5 km s . We used a hypocentral depth of to compute mean-square power from beams formed with fourth-root stacking. 18 km, 12 km below the ocean floor. Modelling regional R1 and G1 using empirical Green’s functions. Regional P R1 STF modelling. Rayleigh-wave STFs were obtained by deconvolving the waves, short-arc Rayleigh waves (R1) and short-arc Love waves (G1) were modelled observed vertical component short-arc Rayleigh waves (R1) with point-source for the period range 25–100 s to resolve the presence and timing of secondary thrust 21 synthetic seismograms spanning the group velocity window from 8.00 km s to events. Recordings at stations PPT, RAR, SNZO, CTAO, MSVF, HNR, TARA, 21 30 2.75 km s computed for the PREM structure with corrections for aspherical KNTN, MIDW, KIP, and XMAS for normal fault (19 October 2009) and thrust 31 phase velocity heterogeneity . The fundamental-mode synthetics were com- fault (10 October 2009) aftershocks both with seismic moments of 1.0 3 1018 Nm puted assuming the United States Geological Survey (USGS) epicentre, a source were used as empirical Green’s functions for the corresponding components of depth of 15 km and the P-wave inversion double couple fault geometry. motion for the 29 September 2009 recordings. The signals from the normal fault Deconvolutions were first performed using an iterative procedure with a posi- aftershock were convolved with STFs predicted for the finite-source model of the 2 2 tivity constraint and include a Gaussian filter (exp(2v /4a ), with a 5 0.1) to mainshock, while the thrust fault aftershock signals were convolved with a suite of emphasize periods longer than 30 s. Only signals that fitted the convolutional triangle source functions of varying duration. Amplitude-scaled and time-shifted model (comparison of original and reconstructed waveforms) at or better than sums of two-source (one normal, one thrust) and three-source (one normal, two 90% of the signal power were retained. Small onset-time shifts were applied to thrust) signals were matched to the observations, to constrain the relative timing the STFs to account for unmodelled aspherical structure phase delays. The final and strength of secondary sources (Fig. 4a and Supplementary Fig. 8). The differ- R1 STFs were binned and stacked for comparison with the finite source model. ential arrival times were then used to locate the centroids of the normal fault and The spectra of the R1 STFs for 167 stations were stacked to give the low-fre- thrust fault events relatively. This procedure provides satisfactory fits to the 25–100- quency spectrum in Supplementary Fig. 5, while the geometrical average of 33 s-period pass band using two thrusting subevents with 40-s-duration triangle P-wave ground velocity spectra from stations at epicentral distances of 60u to 90u source functions. Shorter-duration source functions are suggested by the presence gave the high-frequency spectrum. The seismic moment of the STFs is probably of un-modelled short-period energy, but the empirical Green’s function method biased slightly high by the positivity constraint in the deconvolutions, so we scaled appears to break down at a period of about 20 s, probably because of strong crustal the moment to the moment estimate from composite source modelling. heterogeneity and offset of the true faulting location from the position of the two When the R1 signals are deconvolved using a frequency-domain water-level small empirical Green’s function sources. The relative amplitudes of the sources in procedure without positivity constraint, it becomes clear that there are strong the three-source models were allowed to vary from station to station, because the negative-amplitude features in the resulting STFs after about 50 s into the signals, focal mechanism and position of theactual faulting probably differs from that of the indicating the inadequacy of the normal faulting Green’s function for the later empirical Green’s function events, but matching the phase of the interfering arrivals portion of the seismic radiation. The water-level deconvolutions are plotted as a provides good relative arrival times for the appropriately scaled arrivals. The dif- function of azimuth from the normal fault epicentre in Fig. 2. Note the strong ferential arrival times for all of the modelled stations with stable results (in some negative amplitudes as azimuths to the east and west, and the dovetail involving cases the empirical Green’s function events are nodal or the mainshock traces are two negative pulses particularly evident towards the east. This is suggestive of clipped), were used to invert for relative locations of the centroids of the normal- two later thrust mechanism subevents for which the assumption of a normal fault and thrust-fault radiation. faulting polarity Green’s function gives rise to the negative amplitudes in the Modelling body waves from thrust events. A variable focal mechanism inver- STFs. sion29 for spatially distributed sources was performed using the relative locations Back-projections of teleseismic P waves from regional networks. Back- of the normal faulting and thrust faulting indicated by the R1 STFs, P-wave back- projection of teleseismic P waves involves minimal assumptions about the source projections, and regional waveform modelling. A three-subevent model indi- location of energy arriving across a seismic network, so it is ideal for detecting cates that two secondary thrust sources occurred about 54 s and 105 s after the radiation from secondary sources that are not independently located because event onset, located to the southwest on the megathrust (Supplementary Fig. 9). their arrivals are immersed in signals from an initial event. We use back-projection The first thrust event overlaps the modelled normal faulting radiation, as indi- of many short-period P waves both to constrain the spatial extent of the normal cated by the back-projections (Fig. 3), so it is hard to model body-waves directly, faulting rupture process and to detect any secondary off-fault sources of short- but the second source is well enough isolated that it can be directly modelled by a period radiation. A spatial grid of possible source radiation locations was assumed finite-source inversion (Supplementary Fig. 10). This inversion has high noise

levels owing to the earlier arrivals, but provides support for the seismic moment model with no aspherical corrections, because those are small for periods greater and STF used in the R1 STF and regional waveform modelling. than 200 s. The excellent match in phase and amplitude supports the composite Modelling long-period R1 and G1 ground motions. The superposition of three source model, and yields strong constraints on the seismic moment of the nor- events with varying focal mechanism, timing, and centroid locations within a mal faulting and thrust faulting components. two-minute interval implies that the long-period (.150 s) seismic waves must involve strong interference, affecting point-source representations retrieved 28. Hartzell, S. H. & Heaton, T. H. Inversion of strong ground motion and teleseismic from the W-phase and GCMT moment tensor inversions and associated esti- waveform data for the fault rupture history of the 1979 Imperial Valley, California, mates of seismic moment. The occurrence of normal and thrust faulting with earthquake. Bull. Seismol. Soc. Am. 73, 1153–1184 (1983). rotated geometries produces complex patterns of motion such as the presence of 29. Kikuchi, M. & Kanamori, H. Inversion of complex body waves. Bull. Seismol. Soc. strong Love waves at azimuths towards which the normal fault radiation of Love Am. 81, 2335–2350 (1991). waves is nodal. Interference between the events also causes point-source solu- 30. Dziewonski, A. M. & Anderson, D. L. Preliminary reference Earth model. Phys. tions to match some signals poorly, as shown for W-phase predictions for very- Earth Planet. Inter. 25, 297–356 (1981). 31. Boschi, L. & Ekstro¨m, G. New images of the Earth’s upper mantle from long-period Rayleigh waves in Fig. 4b and Supplementary Fig. 11. We modelled measurements of surface-wave phase velocity anomalies. J. Geophys. Res. 107, S1, the composite radiation of three-event sequences, adjusting the fault geometries doi: 10.1029/2000JB000059 (2002). and seismic moments of the secondary events to fit global distributions of ultra- 32. Kennett, B. L. N., Engdahl, E. R. & Buland, R. Constraints on seismic velocities in the long-period (714–1,000 s) Rayleigh waves (Supplementary Fig. 12) and long- earth from travel times. Geophys. J. Int. 122, 108–124 (1995). period (.200 s) R1 and G1 signals (Supplementary Fig. 13), matching the phase 33. VanDecar, J. C. & Crosson, R. S. Determination of teleseismic phase arrival times and amplitude of the observations of the same station distribution as used in the using multi-channel cross correlation and least squares. Bull. Seismol. Soc. Am. 80, W-phase inversion. This procedure used normal mode sums for the PREM 150–169 (1990).

Promiscuity and the evolutionary transition to complex societies

Charlie K. Cornwallis1*, Stuart A. West2, Katie E. Davis3 & Ashleigh S. Griffin1*

Theory predicts that the evolution of cooperative behaviour is societies more generally, the challenge is to test whether promiscuity favoured by low levels of promiscuity leading to high within-group rates explain why an individual with the potential to breed chooses to relatedness1–5. However, in vertebrates, cooperation often occurs help instead. between non-relatives and promiscuity rates are among the highest There are a number of reasons why monogamy may be less important recorded. Here we resolve this apparent inconsistency with a phylo- in the evolution of cooperative breeding in vertebrates. First, direct genetic analysis of 267 bird species, demonstrating that cooperative benefits of cooperation that increase reproductive success and do not breeding is associated with low promiscuity; that in cooperative depend on kinship have been suggested to have a greater role in verte- species, helping is more common when promiscuity is low; and that brates12–14. Second, strict lifetime monogamy is relatively rare in verte- intermediate levels of promiscuity favour kin discrimination. brates. Two of the highest promiscuity rates ever recorded are in Overall, these results suggest that promiscuity is a unifying feature cooperative breeders: the superb fairy-wren, Malurus cyaneus, and the across taxa in explaining transitions to and from cooperative Australian magpie, Gymnorhina tibicen (Supplementary Information). societies. Third, it has been argued that cooperative breeding will actually lead The increase in complexity from simple replicating molecules to to higher rates of promiscuity, because helpers release females from complex animal societies has involved approximately eight major the need to secure paternal care from her mate through paternity evolutionary transitions6,7. Almost all of these transitions have required assurance15. the problem of cooperation to be solved, allowing independently rep- We exploit the extensive literature on birds to test whether rates of licating entities to reproduce cooperatively as a group. The problem we female promiscuity have influenced the evolutionary transitions to and address is what causes the transition from independent breeding, from cooperative breeding. Specifically, we use promiscuity data to where individuals pursue their own selfish interests, to cooperative compare the potential kin-selected benefits of helping across bird breeding, where individuals forgo their own reproduction to help species, and ask whether this helps explain the distribution of coopera- others. tive breeding. There is wide unexplained diversity in both promiscuity 1,2 It has been proposed that a key factor driving the transition from and cooperation across bird species12,13,16–19 (Fig. 2). We collected solitary individuals to complex animal societies is the number of males ab that a female mates with (the ‘monogamy hypothesis’; Fig. 1). 0.5 Monogamy/low promiscuity Offspring Consider an individual that faces the decision of either dispersing to High intragroup relatedness breed independently or remaining in its family (natal) group to help 0.4 raise siblings. If the mother of this individual is monogamous (mated with only one male), then the focal individual will share half of its 0.3 3–5 Independent Cooperative Siblings genes with both its potential offspring and its siblings (Fig. 1b). In breeding breeding 0.2 this case, helping to raise a sibling is equivalent to raising offspring Relatedness, r from the point of view of passing genes to the next generation. Any 0.1 small ecological benefit of cooperation could then tip the balance in 1,2,4,5,8 0.0 favour of helping to raise siblings . In contrast, if the mother of High promiscuity 2 4 6 8 10 12 14 16 18 20 our focal individual mates with multiple males, relatedness to siblings Low intragroup relatedness Promiscuity (no. of mates) will be lower than relatedness to its own offspring and selection for Figure 1 | The monogamy hypothesis. a 4,5,9 , Monogamy, or low levels of cooperative breeding will be weaker (Fig. 1b). promiscuity, leads to high relatedness in family groups that favours the The monogamy hypothesis has the potential to be of general transition to cooperative societies (increasing the relatedness term, r,in importance, across all sexually reproducing organisms1,2,10. It builds Hamilton’s rule3, which states that cooperation will be favoured if rb . c, on kin selection theory to suggest that a single variable, the rate of where b is the benefit in terms of reproductive success to the recipient of aid promiscuity, could have a key role in determining when the evolu- and c is the cost to the performer of a cooperative behaviour). High levels of tionary transition to cooperative animal societies occurs, because of promiscuity lead to a low relatedness in family groups that favours the loss of its influence on relatedness (Fig. 1). Support for the monogamy cooperative breeding. b, Promiscuity and relatedness. Female promiscuity hypothesis comes from analyses of eusocial insects, which have shown (number of mates) is plotted against the mean genetic relatedness between potential helpers and either their siblings or their offspring. An individual is that strict lifetime monogamy is the ancestral state of transitions to 5 11 always related to its offspring with r 0.5. In contrast, as the number of males societies with sterile worker castes . However, the irreversible evolu- its mother mates with increases, the relatedness to siblings decreases from tion of sterile castes has occurred in very few species—it is more r 5 0.5 to r 5 0.25 (full-siblings to half-siblings). Across 11 species of commonly the case that non-breeding helpers retain reproductive cooperative breeders, we found this expected negative relationship between potential. To understand evolutionary transitions to cooperative helper–offspring relatedness and female promiscuity (Supplementary Fig. 1).

1Edward Grey Institute, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. 2Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. 3Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. *These authors contributed equally to this work. 969 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

ab Although these results are consistent with the monogamy hypothesis, they do not demonstrate that the transition to cooperative breeding is more likely to occur in less promiscuous species. To test this prediction, we examined whether cooperative breeding and promiscuity are correlated through evolutionary time and found support for the predicted negative correlation (multi-response BPMM20: r 520.63 (phylogenetic correlation), CI 520.86 to 20.33, P 5 0.001, Supplementary Table 5; continuous-time Markov models of correlated versus independent evolution21: P 5 0.02, Supplementary Table 6). cd As well as predicting transitions to cooperative breeding, the monogamy hypothesis predicts that transitions from cooperative to non- cooperative breeding will be more likely in more promiscuous species. In the species that we examined, cooperative breeding evolved 33 times and has been lost 20 times (Fig. 4a). Examining transitions to cooperative breeding, we found that promiscuity was lower in non- cooperative ancestors of cooperative breeders than in non-cooperative ancestors of non-cooperative species (BPMM: cooperative: b 5 0.01, CI 5 0.001 to 0.08; non-cooperative: b 5 0.19, CI 5 0.07 to 0.34; Figure 2 | Making sense of the diversity. Helping may be facultative, as in P 5 0.001; Fig. 4b and Supplementary Table 7). Furthermore, the rate the Seychelles warbler, Acrocephalus sechellensis28 (a), or obligate: the chicks of transition to cooperative breeding was over twice as high for ances- in the nest of the white-winged chough, Corcorax melanorhamphos (b), have tors with low promiscuity as for ancestors with high promiscuity no chance of survival if their parents do not receive assistance from helpers. (Supplementary Table 6). Examining the loss of cooperative breeding, Promiscuity can be high in cooperative breeders but low in species without we found that cooperative ancestors of non-cooperative species tended cooperative care—cooperative breeding is unknown in seabirds such as the to be more promiscuous than cooperative ancestors of only coopera- puffin, Fratercula arctica (c), which are either strictly monogamous or have tive species, but this result was not significant (BPMM: cooperative: extremely low rates of promiscuity. In contrast, the superb fairy-wren, Malurus cyaneus (d), is a cooperative breeder with one of the highest extra- b 5 0.02, CI 5 0.005 to 0.16; non-cooperative: b 5 0.08, CI 5 0.01 to pair paternity rates ever recorded15. To make helping pay, the benefits have 0.24; P 5 0.07; Fig. 4b and Supplementary Table 7). Overall, these to outweigh the costs of breeding independently, and this depends on results show that transitions to cooperation were associated with low ecological as well as genetic factors. (Photos courtesy M. Hammers (a), N. promiscuity, and suggest that increases in promiscuity have led to the Beck (b), S. Patrick (c), G. Dabb (d).) breakdown of cooperation (Fig. 4b, c). available data on promiscuity rates in birds (267 species) and defined Although our results show that high levels of promiscuity favour the species as either cooperative or non-cooperative depending on the loss of cooperative breeding, there are many promiscuous, coopera- presence of non-breeding helpers in family groups (Supplementary tive species (Supplementary Table 15). However, relatedness between Table 15). helpers and beneficiaries can be increased by kin discrimination 1,3,22 Consistent with the monogamy hypothesis, we found that the level of (directing aid preferentially towards relatives (Supplementary promiscuity (the percentage of broods in the population containing one Information)). There is no evidence to suggest that birds are able to or more extra-group offspring; Supplementary Information) was sig- discriminate kinship between chicks in a brood using genetic cues, but nificantly lower in cooperatively breeding species (Fig. 3a and there is evidence to suggest they can discriminate kinship between Supplementary Table 2). Overall, promiscuity rates were three times broods, using behavioural cues such as vocalizations and breeder greater in non-cooperative species than in cooperative species (Bayesian turnover16,23,24. By measuring promiscuity as the percentage of nests 20 phylogenetic mixed model (BPMM): cooperative: Nspecies 5 35, para- containing extra-group young, we can make predictions about the meter estimate (b) 5 0.05, credible interval (CI) 5 0.02 to 0.17; non- strength of selection for kin discrimination across species with differ- cooperative: Nspecies 5 232, b 5 0.15, CI 5 0.05 to 0.30; P 5 0.004; ent levels of promiscuity. When promiscuity rates are very high or very Fig. 3a and Supplementary Tables 2 and 3). low, variance in relatedness between broods will be low and we predict that selection to adjust helping effort with respect to kinship will be ab 30 relatively weak23,24. In contrast, with intermediate levels of promis- 100 cuity, variation in relatedness will be highest and we predict that 25 80 selection for kin discrimination will be greater. This prediction was 20 supported by our data, as we found a dome-shaped relationship across 60 species between the strength of kin discrimination and promiscuity 15 rates (Nspecies 5 15, b 520.31, CI 520.67 to 0.01, P 5 0.03; Fig. 5 40 10 and Supplementary Tables 8 and 15). Promiscuity (%) Promiscuity Promiscuity is also predicted to influence the frequency of coopera- Cooperative nests (%) 20 5 tion in cooperative breeders. Although we have treated all cooperative 0 0 breeders equally in the above analyses, the extent of cooperation varies Cooperative Non-cooperative 0 20406080 across species. In some species, such as the white-winged chough, Breeding system Promiscuity (%) Corcorax melanorhamphos, reproductive success is completely Figure 3 | Promiscuity and cooperation. a, Rates of promiscuity (percentage dependent on the presence of helpers, whereas in other species coop- of broods with one or more offspring sired by an extra-group male) in eration seems to be facultative, with some pairs breeding successfully cooperative and non-cooperative species. Promiscuity was significantly higher without helpers (Supplementary Table 15). As promiscuity increases, in non-cooperative than in cooperative species (Supplementary Tables 2 and a greater ecological benefit is required for kin selection to favour 3). Data shown, mean 6 s.e. b, The relationship between levels of cooperation (population with the lowest percentage of nest with helpers throughout the cooperation, so we predict that the presence of helpers will be less 1,2,4,5 species range) and promiscuity in cooperative species. Helpers were present in frequent in more promiscuous cooperative species . In agreement a lower percentage of nests in species with higher rates of promiscuity with this, we found that more promiscuous cooperative breeding (Supplementary Table 9). The line is the log-linear regression curve. species had a lower percentage of nests attended by helpers 970 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a us

Taeniopygia guttata s a Passer domesticus u c Anthus spinoletta

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us sechellensis Loxioides bail alus bistrigi C l L Se roica s Turdoides squamiceps Serin ar Card C aga rut cit u tus Oe alcari usilla C rmi ilus Ca nd h a melanopogon inaceus C droica c na rub Geothlypi e p p Paradoxornis webbianus Ve n a bula oni i enicl t D landra Zosterops lateralis r Dendroica s n o imus i eto delli gal s De r ca audacu S so lla arit T Wil il chonyx oryzi u nobaenus ceps Ca za s c rdoides bicolo W ri a schusi terus beriza citrinellaiz Doli r orgiana Ic be e odia wichensis Ta Em dramuramus m el nd Tachycinetac albi Progne subis Em a ge h Ripari m cophrys ycineta bicolor Emb Spizella p collis spiz lus saleu i Delichon urbicum Ammommod a lb Petrochelidon ariel a riparia r A a Melo ercu a Aegithalos caudatus Melospizass trichichi malis alis Psal Hirundo rustica linea Pa Zono ater triparus minimus nco hye Panurus biarmicus Zonotriu J pilo criss vacea nalis Pi ardi Alauda arvensis olothrus a oli c Remiz pendulinus M sis Agelaius phoeniceus en LophophanesBaeolophus cristatus bicolor irang nalis caerulea P ens aric Cardi scand Poecile caroline Passerina za s cost asserina cyanea Poecile atricapillus P arina ac Geospi a j Poecile montanus Ramphocelu nsis Volatini don CyanistePeripars caeruleus ae Phainopepta europaeala nitensucotis us at Sit Troglodytes udovicianusgriseus s Parus humilis er ryothorus le Petroica goodenovii Th us nuchali Parus major Thryothorus l h Lanius bu Campylorhynchuslorhync Gymnorhina tibicen Campy Pachycephala pectocephalusralis Ramphociotornisnclus superbus brachyurus Aphelocoma ultramarina Lampr Aphelocoma coerulescens Sturnus vulgaris Sturnus unicolor Ca nia svecica locitta formosa Lusci Cyanocorax morio Ficedula hypoleuca Perisoreus canadensis Ficedula albicollis Phoenicurus phoenicurus Perisoreus ubecula infaustus Erithacus r Corvus corone Corvus brachyrhynchos Turdus merula Turdus grayi Coloeus monedula Turdus mig ratorius cichla mustelina Pica pica Hylo Lanius collurio Sialia sialis s Lanius minor Sialia currucoide Sialia mexicana Lanius ludovicianus Tinamus major Grallina cyanoleuca Dromaius novaehollandiae Corcorax melanorhamphos Struthio camelus Struthidea cinerea Cygnus atratus Vireo solitarius Branta leucopsis Vireo olivaceus Chen rossii Chen c Notiomystis cincta Anas strepaerulescenser Anas platyrhynchosa Philesturnus carunculatus Hymenolaimus malacor PomatostomusAcanthiza temporali pusillas Alectura lathami Lagopus lagopus Sericornis frontalis hynchos melanocephala Lagopus leucura Manorina Centrocercus urophasianus Petroica australis s Lyrurus tetrix Mele Manorina melanophrys a Malurus coronatu Grus gris gallopav Malurus cyaneus Porphyr canadensis Porphyr o Malurus splendens io p Tribo orphyrio phoebe Gallinula io Stipiturus malachurus ns nyx mortierihochstett Coragyps atratus CercomacraSayornis tyrannina chloropus e Gav i ri T ia immer r halassarche EmpidonaxEmpidonax viresce traillii Thalassarch Thalassarche ca avogaste eiri Phoe m Pyrocephalus rubinus Diomedea e elano Tyrannus tyrannus b e ch aenia fl Oceanod astria El a epops rys p kus mont Oc ostomahri up F irroratau s ElaeniaT occhiriquensisp tylus u e t U lmaranites oceanicus a dac Thalass romaxul tri us Puffinus ans s us leucorhoa Colaptes auratusborealis Calo glaci ide Sp oi es mediorus v Eu h n ca Pico e ectr t a s Eudyptedyptess pachnisc schyrhl enuir ant lis Dendrocopos majormici s P Picoidcopos ygoscelis a is a for Pygos us d o rc rus Frega iomedeas tica ndro h tris e pes Sul u D lo novaeguineaebullockoidpatagonues Sul mbo asalis a celi aner arius P ta e s cano P h a granti geli d pus is s ld Mel Dace Moni h al dactylatr mi n H a a ade t ti Merops rpus passerinu l n arcti o Char a a crocorax o y r glan V ematopus ostralcroco liae n F Cuculu s C as r c yanoliseus o tropusApus grillii a anel cus h C n C h Chara ad u iter gentil Laru haradri ara b a s Ce p Larus occidentalis lu e rax ari Ris r nschi S d s ch i c Chrysococcyx b s St us Clamat St te eus Fratercula arctica s r a Ce s ius semip galapagoensis U er d Acci Aquila heliaca ctua U rbo A erco rna a hiati er J c Ired C Tryngites subruficollis rius u o r i s r n a l cora anu l i pphus g t s le i e tote alidr a l rid Falco naumannico eleonorae c a nsis columbarius morine aalg hirundo peregrinus a ale c ipa alle ra o ac s u Bute alco tinnunculu n eg Fal r lis Asio otus mvia l F a riu i i a cops asio s m us a tyl x r jaca u s elegans Tyto alba al thene no ra andrin Falco sparverius Strix aluco e s Falco poleucos s macc Falco A u a matus ry ll t

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bc30 Descendants 15 Non-cooperative 25 Both Cooperative 10

20 5

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5 –10 Ancestral promiscuity (%) Ancestral promiscuity

0 (%) Change in female promiscuity –15 Non-cooperative Cooperative Coop. to Coop. to Non-coop. Non-coop. Ancestor coop. non-coop. to coop. to non-coop. Evolutionary transition Figure 4 | Promiscuity and the transition to and from cooperative cooperative descendants (dark-grey bars), or both non-cooperative and breeding. a, The phylogenetic distribution of cooperative breeding and cooperative descendants (light-grey bars). Non-cooperative ancestors that ancestral rates of female promiscuity. Red species labels and branches lead to cooperative descendants had lower promiscuity than those that represent cooperative breeding, and black species labels and branches produced non-cooperative descendants (Supplementary Table 7). Similarly, indicate non-cooperative species. Blue circles indicate ancestral values of cooperative ancestors that produced non-cooperative descendants were rates of promiscuity (larger circles correspond to higher promiscuity). more promiscuous than those that produced cooperative descendants Petroica are a poorly constrained group and have been split in the phylogeny. (Supplementary Table 7). c, The changes in promiscuity associated with The resulting transitions make no difference to the results (Supplementary transitions to and from cooperative breeding. Rates of promiscuity Tables 11–14). b, Promiscuity in non-cooperative and cooperative ancestral decreased during transitions to cooperation, but decreased when species that gave rise to only non-cooperative descendants (black bars), only cooperation broke down. Data shown, mean 6 s.e. of ancestral values.

2 (Nspecies 5 30, b 520.15, CI 520.29 to 20.02, P 5 0.01; Fig. 3b and across species, linking research on kin selection and sexual selection . Supplementary Table 9). However, our results also demonstrate how differences between taxa Overall, our results support the hypothesis that promiscuity is a influence the consequences of promiscuity. In eusocial insects, mul- unifying explanatory variable in the transition to cooperative societies tiple mating is a derived state, evolving after the appearance of sterile 971 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

1.0 7. Queller, D. C. Relatedness and the fraternal major transitions. Phil. Trans. R. Soc. Lond. B 355, 1647–1655 (2000). 0.8 8. Queller, D. C. & Strassmann, J. E. Kin selection and social insects. Bioscience 48, 165–175 (1998). 0.6 9. Hamilton, W. D. Altruism and related phenomena, mainly in social insects. Annu. Rev. Ecol. Syst. 3, 193–232 (1972). 0.4 10. West, S. A. & Gardner, A. Altruism, spite, and greenbeards. Science 327, 1341–1344 (2010). 0.2 11. Hughes, W. O. H., Oldroyd, B. P., Beekman, M. & Ratnieks, F. L. W. Ancestral 0.0 monogamy shows kin selection is key to the evolution of eusociality. Science 320,

Kin discrimination 1213–1216 (2008). –0.2 12. Cockburn, A. Evolution of helping behavior in cooperatively breeding birds. Annu. Rev. Ecol. Syst. 29, 141–177 (1998). –0.4 13. Hatchwell, B. J. & Komdeur, J. Ecological constraints, life history traits and the evolution of cooperative breeding. Anim. Behav. 59, 1079–1086 (2000). 0204060 80 14. Clutton-Brock, T. Breeding together: kin selection and mutualism in cooperative Promiscuity (%) vertebrates. Science 296, 69–72 (2002). Figure 5 | Kin discrimination and rates of promiscuity. There was a 15. Mulder, R. A., Dunn, P. O., Cockburn, A., Lazenby-Cohen, K. A. & Howell, M. J. Helpers significant quadratic relationship between kin discrimination (correlation liberate female fairy-wrens from constraints on extra-pair mate choice. Proc. R. Soc. between relatedness and help provided) and promiscuity (measured as in Lond. B 255, 223–229 (1994). Fig. 3a), indicating that kin discrimination was strongest in species with 16. Krebs, J. R. & Davies, N. B. Behavioural Ecology: An Evolutionary Approach 291–317 (Blackwell Scientific, 1993). intermediate rates of promiscuity (Supplementary Table 8). 17. Bennett, P. M. & Owens, I. P. F. Evolutionary Ecology of Birds (Oxford Univ. Press, 2002). castes, and has not led to the loss of eusociality1,2,11,25. In contrast, high 18. Arnold, K. E. & Owens, I. P. F. Cooperative breeding in birds: the role of ecology. promiscuity can lead to the complete or partial loss of cooperative Behav. Ecol. 10, 465–471 (1999). 19. Arnold, K. E. & Owens, I. P. F. Cooperative breeding in birds: a comparative test of breeding in birds. This effect may be ameliorated by kin discrimina- life history hypothesis. Proc. R. Soc. Lond. B 265, 739–745 (1998). tion, which increases average relatedness between helpers and bene- 20. Hadfield, J. D. & Nakagawa, S. General quantitative genetic methods for ficiaries, and can thereby stabilize cooperation even when females comparative biology: phylogenies, taxonomies, and multi-trait models for mate with multiple males (Figs 3b, 4b and 5). More generally, our continuous and categorical characters. J. Evol. Biol. 23, 494–508 (2010). results provide empirical support for E. O. Wilson’s statement that 21. Pagel, M. & Meade, A. Bayesian analysis of correlated evolution of discrete 26 characters by reversible-jump Markov chain Monte Carlo. Am. Nat. 167, 808–825 ‘‘sex is an antisocial force in evolution’’ by showing how promiscuity (2006). reduces relatedness and hence disrupts selection for cooperation in 22. Griffin, A. S. & West, S. A. Kin discrimination and the benefit of helping in family groups. cooperatively breeding vertebrates. Science 302, 634–636 (2003). 23. Cornwallis, C. K., West, S. A. & Griffin, A. S. Routes to indirect fitness in METHODS SUMMARY cooperatively breeding vertebrates: kin discrimination and limited dispersal. J. Evol. Biol. 22, 2445–2457 (2009). We collected data on female promiscuity and whether species were cooperative or 24. Gardner, A., West, S. A. & Buckling, A. Bacteriocins, spite and virulence. Proc. R. non-cooperative breeders from literature searches (Supplementary Information). Soc. Lond. B 271, 1529–1535 (2004). Our definition of cooperative breeding excluded polyandrous species such as the 25. Boomsma, J. J. & Ratnieks, F. L. W. Paternity in eusocial Hymenoptera. Phil. Trans. dunnock, Prunella modularis, where an additional breeding adult may attend the R. Soc. Lond. B 351, 947–975 (1996). nest27 (Supplementary Information), as the monogamy hypothesis only makes 26. Wilson, E. O. Sociobiology: The New Synthesis 155 (Harvard Univ. Press, 1975). predictions about transitions to cooperation in family groups1,2. We carried out 27. Davies, N. B. Dunnock Behaviour and Social Evolution 117–130 (Oxford Univ. Press, our analyses using BPMMs with Markov chain Monte Carlo estimation20 using 1992). ‘MCMCglmm’ in R, version 2.10.1(see Methods for all software references). 28. Komdeur, J. Importance of habitat saturation and territory quality for evolution of cooperative breeding in the Seychelles warbler. Nature 358, 493–495 (1992). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Received 11 May; accepted 7 July 2010. Acknowledgements We thank J. Hadfield for statistical advice, K. Boomsma, S. Nakagawa and B. Sheldon for comments and discussion, M. Nelson-Flower and 1. Boomsma, J. J. Kin selection versus sexual selection: why the ends do not meet. P. Brennan for access to unpublished data, and the ERC and Royal Society for Curr. Biol. 17, R673–R683 (2007). 2. Boomsma, J. J. Lifetime monogamy and the evolution of eusociality. Phil. Trans. R. funding. The compilation of our data set was made possible by access to the Soc. B 364, 3191–3207 (2009). collections of the Alexander Library. 3. Hamilton, W. D. The genetical evolution of social behaviour. I. J. Theor. Biol. 7, 1–16 Author Contributions All authors contributed extensively to the work presented in (1964); The genetical evolution of social behaviour. II. J. Theor. Biol. 7, 17–52 (1964). this paper. 4. Charnov, E. L. Evolution of eusocial behaviour: offspring choice or parental parasitism? J. Theor. Biol. 75, 451–465 (1978). Author Information Reprints and permissions information is available at 5. Charnov, E. L. Kin selection and helpers at the nest: effects of paternity and www.nature.com/reprints. The authors declare no competing financial interests. biparental care. Anim. Behav. 29, 631–632 (1981). Readers are welcome to comment on the online version of this article at 6. Maynard Smith, J. & Szathmary, E. The Major Transitions in Evolution 6–10 www.nature.com/nature. Correspondence and requests for materials should be (Freeman, 1995). addressed to A.S.G. ([email protected]).

METHODS Data on relatedness and kin discrimination. We analysed how promiscuity affected the strength of kin discrimination and the relatedness between helpers Data collection and the phylogenetic tree. Data on extra-group paternity. All and breeders in cooperative species. First, we measured the strength of kin species used in the analyses are listed in Supplementary Table 15 and we use Latin discrimination for each of the cooperative breeders in our data set for which names listed in the International Ornithological Congress master list, version 2.3 this data was available. This measure captures the extent to which helpers in a (December 2009; http://www.worldbirdnames.org/). We searched for all published species preferentially direct aid towards related young. Specifically, we measured data on extra-pair paternity data in birds by entering the following search terms the effect size (r) of relatedness on either the probability of becoming a helper or into the Web of Science (http://thomsonreuters.com/products_services/science/ the amount of help provided. Second, we collected genetic data on the related- science_products/a-z/web_of_science) on 18 March 2010: keywords ‘‘extra-pair ness between helpers at the nest and breeders. Further details on how this data paternity OR extra pair paternity OR extra-pair fertilization OR extra pair fertiliza- were collected are described in refs 22, 23. tion OR extra-pair fertilization OR extra pair fertilization OR extrapair’’; title Avian supertree. Full details of the avian supertree we used for our analyses will ‘‘parentage’’ AND topic ‘‘birds’’; title ‘‘mating system’’ AND ‘‘birds’’; all references be published elsewhere (K.E.D. and R. D. M. Page, submitted), so here we that cite refs 29, 30, which are major reviews on extra-pair paternity in birds. Two describe the methods in brief. The supertree was created from a total of 966 bird statistics on female promiscuity are frequently published and are highly correlated: trees published between 1976 and 2008 and covering 6,219 species. The supertree the percentage of chicks fathered by extra-pair fathers and the percentage of broods was created using the Imperial College supercomputer CX2. CX2 is an SGI ICE withoneormoreextra-pairchicks(r 5 0.93, N 5 272, P , 0.0001). 8200EX with 122 nodes. Each node has two quad-core 2.93-GHz Intel X5570 We collected data on the percentage of broods in the population containing (Nehalem) processors and 24 GB of RAM. The analysis was run on 64 cores over extra-group offspring (hereafter referred toas female promiscuity), as this provides 72 h using TNT33 with the option xmult level 5 10, an aggressive search strategy a population-wide measure of the level of female promiscuity. Furthermore, we devised to find the shortest trees in as little time as possible. TNT found a single choose to use extra-group paternity rather than extra-pair paternity for coopera- MPT of length 26,242. tive species, as this removes the possibility of the results being influenced by We pruned the full supertree (6,219 species) to include only species for which individuals gaining parentage in the nest where they were helping. In species we had data on female promiscuity (267 species), which we then used for all without a pair bond—for example species without male care, lekking species analyses. and parasitic species—we used data on the proportion of nests containing chicks Statistical analyses. General techniques. We used BPMMs with Markov chain fathered by more than one male. Monte Carlo estimation using ‘MCMCglmm’20 in R, version 2.10.134. Flat non- When there were multiple studies presenting data on promiscuity for a single informative priors with a uniform low degree of belief across all parameters were species, we calculated the mean value of all studies weighted by sample size for set in all analyses (for specific details see Supplementary Information). Residual use in subsequent analyses (Supplementary Table 15). In cases where we found variance cannot be calculated for binary traits, and in analyses of binary traits (for multiple papers presenting the same set of paternity data, from the same study example cooperation: 0 5 non-cooperative, 1 5 cooperative) residual variance population and over the same years, to avoid duplication we used information was therefore fixed to 1 (ref. 35). Parameter estimates reported are modes from only one of these papers to calculate the species mean. from the posterior distribution with 95% lower (lower CI) and upper (upper We collected data on 280 species. However, for 7 species only estimates of the CI) credible intervals. Reported P values testing differences between levels (for number of extra-group offspring were available (that is, no data were available on example non-cooperative versus cooperative breeders) are the numbers of itera- the number of broods with extra-group paternity), and for 6 species the number of tions when one level is greater or less than the other divided by the total number of broods analysed for paternity were not reported. Therefore, we included 267 iterations. Reported P values for correlations (for example between promiscuity species in our analyses. To verify that excluding species did not change our results, and the probability of being a cooperative breeder) are the numbers of iterations we also analysed data on all species (N 5 276), which gave almost identical results where the correlation is greater or less than zero divided by the total number of (Supplementary Tables 2 and 3). Of the 267 species used in our analyses, 13 species iterations. were not in the supertree. However, for all 13 species there was a congener in the We examined the convergence of models in three ways using the ‘coda’ R supertree that was not already in our data set. We therefore set the position of the package36. First, we used the run length diagnostic of ref. 37 to estimate the 13 missing species in the supertree to their closely related congeners. burn-in period and total run length for all parameters in each analysis. Second, Data on breeding system. We categorized species as either cooperative or non- we used the diagnostics of refs 38, 39 to assess the convergence of each analysis. cooperative. There is a wide diversity of cooperative care for offspring in birds; The convergence diagnostic of ref. 38 calculates z-scores from mean parameter however, the monogamy hypothesis makes predictions specifically about selection estimates plus/minus standard errors generated from the first 10% and the last to help in family groups. Therefore, we only classed species as cooperative if helpers 50% of the chain38.Ifz-scores follow an asymptotically standard normal distri- did not breed orhad zero-to-limited opportunities tobreed; typically, these helpers bution, the samples are considered to be drawn from a stationary distribution38. would be retained natals. This definition of cooperative breeding discounts many The diagnostic of ref. 39 examines whether the sampled output comes from a polyandrous species that are often thought of as ‘cooperative breeders’, such as stationary distribution, using the Crame´r/von Mises test. Third, we examined dunnocks and the Galapagos hawk23 (Supplementary Table 15). We did not define plots of the sampled output to check visually that the chain had mixed properly. species as cooperative unless there were helpers present in at least 10% of the nests Testing how promiscuity influences the evolution of cooperation. We performed in any part of their range31,32; neither did we define species as cooperative if there six analyses to examine how promiscuity influences the evolution of cooperative was only limited or anecdotal evidence of helpers being present or if the breeding breeding. status of helpers was ambiguous. That said, very few of the species classed as non- (1) Testing the relationship between promiscuity and the relatedness between cooperative fall into this grey area; an example is the buff-breasted wren potential helpers and breeders. We tested whether relatedness between breeders (Supplementary Table 15). Species were classified blind as cooperative or non- and potential helpers (response variable) across cooperative breeding species was cooperative, without knowledge of extra-group paternity data. We researched all negatively related to female promiscuity (explanatory variable) using BPMMs species in our cooperative breeder set, to determine the breeding status of helpers. with a binomial distribution and a logit link function (Supplementary Fig. 1). Often the most accurate information (using genetic markers) came from the We ran two analyses. First, we analysed the relationship between relatedness and paternity studies we used to measure promiscuity, but we also researched species female promiscuity treating species as independent data points (Supplementary more widely to obtain a general picture of the breeding system for each species; Table 1). Second, we repeated the analysis taking into account the phylogenetic additional sources of information used are cited in Supplementary Table 15. relationships between species by specifying a phylogenetic covariance matrix (see Classifying species as either cooperative or non-cooperative is inevitably refs 20, 35 for details). crude, as in our set of cooperative breeders there is a wide diversity of breeding (2) Testing the association between cooperative breeding and promiscuity. We system—some species, such as the apostlebird and the white-winged chough, are tested whether female promiscuity (response variable) was lower in cooperative obligate cooperative breeders (never known to reproduce successfully without species compared to non-cooperative species (explanatory variable) using helpers), whereas others are only cooperative in certain areas of their range, for BPMMs with a binomial distribution and a logit link function (Supplementary example the carrion crow. We sought to capture some of this diversity by search- Table 2). We accounted for differences in samples sizes between studies by ana- ing the literature for data on the percentage of nests with helpers for each lysing female promiscuity (response variable) as the total number of broods with cooperative breeder in our data set. We aimed to determine the upper and lower one or more extra-group offspring versus the number of broods with only within- limits on the proportion of nests with helpers, throughout a species range, and a group chicks, rather just than analysing percentages, which disregard sample sizes. point estimate for the study population used in each study from which we We ran all analyses twice, once treating species as independent data points and extracted data on paternity (Supplementary Table 15). We found the most once taking into account phylogenetic relationships between species. informative of these measures to be the lower limit, as this clearly distinguishes (3) Phylogenetic tests of correlated evolution between cooperative breeding between facultative and obligate cooperative breeders. and promiscuity. We tested whether there was a negative phylogenetic correlation

between female promiscuity and cooperative breeding using multi-response variable, z-transformed) in relation to female promiscuity (explanatory variable) BPMMs. We fitted the probability of being cooperative (0 5 non-cooperative, and weighted each data point by the inverse variance associated with each study, 1 5 cooperative) as a binary response and the rate of female promiscuity (number which we approximated by 1/(n 2 3) where n is the sample size of the study41.We of broods with extra-group chicks versus number of broods with only within- again ran the analysis twice, once treating species as independent data points and group chicks) as a binomial response. To establish the strength of the correlation once taking into account phylogenetic relationship between species (Sup- between the evolution of cooperation and levels of female promiscuity, three plementary Table 8). analyses were performed. First, we analysed species as independent data points, (6) Female promiscuity and levels of cooperation. We tested whether variation allowing the phenotypic correlation between cooperation and female promiscuity in the percentage of cooperative nests (binomial response variable) was nega- to be estimated (Supplementary Table 4). Second, we estimated the phylogenetic tively correlated with levels of promiscuity (explanatory variable) using a correlation between the two variables by specifying an unstructured phylogenetic BPMM. We ran the analysis twice, once treating species as independent data variance–covariance matrix for the two traits (MCMCglmm code 5 us(trait): points and once taking into account phylogenetic relationships between species animal)20,35 (Supplementary Table 5). This estimates variance attributable to (Supplementary Table 9). phylogenetic history in both traits and the covariance between the traits that arises Variables confounding the relationship between promiscuity and coopera- owing to shared ancestry between species. These techniques are analogous to those tion. There are variables that correlate with both promiscuity and cooperative used in quantitative genetics to estimate genetic correlations between traits breeding and may therefore potentially confound any relationship between 20,35 through pedigrees . Finally, we fitted a phylogenetic variance–covariance cooperative breeding and promiscuity. Two of the most widely established vari- matrix for the two traits but constrained the phylogenetic covariance between ables that correlate with both cooperative breeding and promiscuity are longev- cooperation and female promiscuity to be zero (MCMCglmm code 5 idh(trait): ity and breeding range19,29,30. We checked whether the relationship between 20,35 animal), therefore assuming independent evolution of the two traits .Wethen cooperative breeding and female promiscuity was still present after controlling 35 compared the deviance information criteria (DIC ) of the model estimating for these variables. correlated evolution between the traits with the analysis where the evolution of First, we gained data on species longevity from The Animal Ageing & cooperation and female promiscuity were assumed to be independent. Longevity Database42 (http://genomics.senescence.info/species/). We were able (4) The influence of female promiscuity on transitions to and from cooperation. to obtain information on the maximum longevity for 184 species (16 coopera- We tested whether evolutionary transitions to and from cooperative breeding were tive, 168 non-cooperative) for which we had data on female promiscuity and associated with different levels of female promiscuity in two different ways. cooperative breeding. We found that the difference in female promiscuity 21,40 First, we used the BayesTraits DISCRETE module with maximum- between cooperative and non-cooperative species was not confounded by lon- likelihood estimation to estimate transition rates to and from cooperative breed- gevity; in fact, the negative relationship between cooperative breeding and pro- ing under high and low levels of promiscuity. DISCRETE can only examine miscuity was stronger after controlling for variation in longevity (BPMM of transitions between binary traits and therefore we classified female promiscuity female promiscuity (response variable) with breeding system (cooperative and as low (less than or equal to the median) or high (greater than the median). We non-cooperative) and longevity (covariate) as fixed effects (posterior mode tested whether models allowing correlated evolution of cooperative breeding and (95% CI)): cooperative breeders 5 0.01 (0.002, 0.15) versus non-cooperative female promiscuity had a significantly higher log likelihood than models assum- 40 breeders 5 0.12 (0.04, 0.43), P 5 0.001; longevity 520.02 (20.05, 0.03), ing independent evolution of traits, using log-likelihood ratio tests with degrees P 5 0.26). of freedom equal to the difference in the number of parameters estimated Second, cooperative breeders tend to be more prevalent nearer the equator, (Supplementary Table 6). Furthermore, using log-likelihood ratio tests we tested where female promiscuity tends to be lower30. We therefore tested whether any whether individual transition rates were significantly different from zero, com- differences in female promiscuity between cooperative and non-cooperative paring models where individual transition rates were restricted to zero with breeders were influenced by latitude by entering breeding range, as listed in the models where transition rates were estimated21,40. International Ornithological Congress master list, version 2.3, as an explanatory Second, because of the restrictions of having to use binary traits in DISCRETE, variable (fixed factor). We found that the relationship between promiscuity and we examined whether transitions to and from cooperative breeding were influ- cooperation still remained even after controlling for breeding range (BPMM of enced by female promiscuity using a different approach. Using data on whether female promiscuity (posterior mode (95% CI)): cooperative breeders 5 0.08 species were cooperative or non-cooperative (response variable), we reconstructed (0.03, 0.33) versus non-cooperative breeders 5 0.23 (0.09, 0.60), P 5 0.002; states of cooperation for ancestral species using a BPMM with 7,100,000 iterations, DIC of model including breeding range 5 19115.1; DIC of model excluding 100,000 burn-in iterations and a thinning interval of 1,000 iterations20. For each breeding range 5 19124.1). node, this gave posterior modes of being non-cooperative or cooperative. For all nodes, the posterior modes were either greater than 0.9999, and were classed as 29. Griffith, S. C., Owens, I. P. F. & Thuman, K. A. Extra pair paternity in birds: a review cooperative, or less than 0.0001, and classed as non-cooperative. Furthermore, of interspecific variation and adaptive function. Mol. Ecol. 11, 2195–2212 (2002). the model correctly predicted cooperative and non-cooperative breeding for all 30. Spottiswoode, C. & Moller, A. P. Extrapair paternity, migration, and breeding contemporary species. This analysis revealed that there were 33 transitions from synchrony in birds. Behav. Ecol. 15, 41–57 (2004). non-cooperative to cooperative breeding and 20 transitions from cooperative to 31. Cockburn, A. Prevalence of different modes of parental care in birds. Proc. R. Soc. B non-cooperative breeding. 273, 1375–1383 (2006). We used the information from ancestral reconstruction of cooperative breed- 32. Hatchwell, B. J. The evolution of cooperative breeding in birds: kinship, dispersal ing to classify nodes of the tree according to the descendant species they gave rise and life history. Phil. Trans. R. Soc. B 364, 3217–3227 (2009). to, which fell into the following categories: (i) non-cooperative nodes that gave 33. Goloboff, P. A., Farris, J. S. & Nixon, K. C. TNT, a free program for phylogenetic analysis. Cladistics 24, 774–786 (2008). rise to only non-cooperative direct descendants; (ii) non-cooperative nodes that 34. R Development Core Team. R: A Language and Environment for Statistical gave rise to both non-cooperative and cooperative direct descendants; (iii) non- Computing (R Foundation for Statistical Computing, Vienna) Æhttp://www. cooperative nodes that gave rise to only cooperative direct descendants; (iv) r-project.orgæ (2010). cooperative nodes that gave rise to only non-cooperative direct descendants; 35. Hadfield, J. D. MCMC methods for multi-response generalised linear mixed (v) cooperative nodes that gave rise to both non-cooperative and cooperative models: the MCMCglmm R package. J. Stat. Softw. 33, 1–22 (2010). direct descendants; (vi) cooperative nodes that gave rise to only cooperative 36. Plummer, M., Best, N., Cowles, K. & Vines, K. Convergence diagnosis and output direct descendants. We analysed whether classifications (i)–(vi) (explanatory analysis for MCMC. R News 6, 7–11 (2006). variable) were associated withdifferences in female promiscuity(responsevariable), 37. Raftery, A. E. & Lewis, S. M. One long run with diagnostics: Implementation to test whether transitions to and from cooperative breeding were preceded by strategies for Markov chain Monte Carlo. Stat. Sci. 7, 493–497 (1992). differences in female promiscuity. Although data on female promiscuity is only 38. Geweke, J. in Bayesian Statistics (eds Bernado, J. M., Berger, J. O. Dawid, A. P. & available for the tips of the tree, phylogenetic mixed models allow ancestral values Smith, A. F. M.) 169–194 (Clarendon, 1992). 39. Heidelberger, P. & Welch, P. D. Simulation run length control in the presence of an for female promiscuity to be estimated and it is therefore possible to test whether initial transient. Oper. Res. 31, 1109–1144 (1983). there were differences in female promiscuity among categories (i)–(vi) (Sup- 40. Pagel, M. Inferring the historical patterns of biological evolution. Nature 401, plementary Table 7). 877–884 (1999). (5) Meta-analysis of promiscuity and kin discrimination. Finally, we tested 41. Raudenbush, S. W. in The Handbook of Research Synthesis (eds Cooper, H. & whether female promiscuity was related to the strength of kin discrimination in Hedges, L. V.) 301–321 (Russell Sage Foundation, 1994). cooperative species using a random-effects Bayesian phylogenetic meta- 42. de Magalha˜es, J. P. & Costa, J. A database of vertebrate longevity records and analysis20. We analysed variation in the effect size of kin discrimination (response their relation to other life-history traits. J. Evol. Biol. 22, 1770–1774 (2009).

An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis

Matthew P. R. Berry1, Christine M. Graham1*, Finlay W. McNab1*, Zhaohui Xu6, Susannah A. A. Bloch3, Tolu Oni4,5, Katalin A. Wilkinson2,4, Romain Banchereau9, Jason Skinner6, Robert J. Wilkinson2,4,5, Charles Quinn6, Derek Blankenship7, Ranju Dhawan8, John J. Cush6, Asuncion Mejias10, Octavio Ramilo10, Onn M. Kon3, Virginia Pascual6, Jacques Banchereau6, Damien Chaussabel6 & Anne O’Garra1

Tuberculosis (TB), caused by infection with Mycobacterium of expression-level and statistical filters and hierarchical clustering tuberculosis, is a major cause of morbidity and mortality world- (Supplementary Fig. 2a, b(i), Supplementary Table 3 and Methods). wide. Efforts to control it are hampered by difficulties with We then applied the 393-transcript list to two independent cohorts diagnosis, prevention and treatment1,2. Most people infected with (UK test set; South African validation set). Hierarchical clustering of M. tuberculosis remain asymptomatic, termed latent TB, with a transcriptional profiles showed patients with active TB cluster inde- 10% lifetime risk of developing active TB disease. Current tests, pendently of latent TB and healthy controls, in both intermediate however, cannot identify which individuals will develop disease3. (London) and high-burden (South Africa) regions, with a significant The immune response to M. tuberculosis is complex and incom- association between cluster and study group (Fisher’s exact test: pletely characterized, hindering development of new diagnostics, P 5 0.00001365, UK (Fig. 1a); P 5 5.79 3 10210, South Africa therapies and vaccines4,5. Here we identify a whole-blood 393 tran- (Fig. 1b)). This was independent of ethnicity, age or gender (Sup- script signature for active TB in intermediate and high-burden plementary Fig. 2b(ii), c, d). The transcriptional profiles of 10–25% settings, correlating with radiological extent of disease and revert- of patients with latent TB (5/21 test set, 3/31 validation set) clustered ing to that of healthy controls after treatment. A subset of patients with patients with active TB (Fig. 1a, b). The k-nearest neighbour with latent TB had signatures similar to those in patients with class prediction, using the 393-transcript list, gave a sensitivity of active TB. We also identify a specific 86-transcript signature that 61.67%, specificity of 93.75% and an indeterminate rate of 1.9% discriminates active TB from other inflammatory and infectious for the test set (Supplementary Table 4), with five patients with latent diseases. Modular and pathway analysis revealed that the TB sig- TB classified as active TB and four patients with active TB mis- nature was dominated by a neutrophil-driven interferon (IFN)- classified. In the validation set the sensitivity was 94.12%, specificity inducible gene profile, consisting of both IFN-c and type I IFN-ab 96.67% and indeterminate rate 7.8%. The UK patients were of diverse signalling. Comparison with transcriptional signatures in purified ethnicity, potentially infected with different M. tuberculosis lineages, cells and flow cytometric analysis suggest that this TB signature suggesting the signature may be independent of bacterial clade, reflects changes in cellular composition and altered gene expres- although molecular typing was not available. The proportion of sion. Although an IFN-inducible signature was also observed in latent patients having a transcriptional signature similar to that of whole blood of patients with systemic lupus erythematosus (SLE), active TB was equal to the expected frequency of patients at risk of their complete modular signature differed from TB, with increased progression to active disease3, potentially identifying patients with abundance of plasma cell transcripts. Our studies demonstrate a latent TB with sub-clinical active disease or higher burden latent hitherto underappreciated role of type I IFN-ab signalling in the infection. pathogenesis of TB, which has implications for vaccine and thera- Four out of 21 patients with active TB in the test set, also mis- peutic development. Our study also provides a broad range of tran- classified by class prediction, clustered with healthy controls and scriptional biomarkers with potential as diagnostic and prognostic patients with latent TB (filled circle, hash symbol, and filled square tools to combat the TB epidemic. and diamond in Fig. 1a), demonstrating molecular heterogeneity that Blood transcriptional profiling has improved diagnosis and could reflect clinical variance. To address this, radiographic extent of understanding of disease pathogenesis6–9. Such a comprehensive disease was assessed by three physicians, blinded to clinical diagnosis unbiased survey will provide insights into the immunopathogenesis and transcriptional profile (Supplementary Fig. 3)10. The median of TB, leading to advances in control of this complex disease. ‘molecular distance to health’11, a composite of the number of tran- Genome-wide transcriptional profiles were generated from blood scripts in a profile that significantly differ from the healthy control from patients with active TB (before treatment), patients with latent baseline, and the degree of that difference, was significantly higher for TB and healthy controls (Supplementary Fig. 1, and Supplementary those with advanced disease than for those with minimal or no disease Tables 1 and 2). A distinct 393-transcript signature was defined in (Fig. 1c). We show for the first time that the transcriptional signature patients with active TB (training set, London), using a combination in blood correlates with extent of disease in patients with active TB,

1Division of Immunoregulation, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. 2Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. 3Department of Respiratory Medicine, St. Mary’s Hospital, Imperial College Healthcare NHS Trust, London W2 1NY, UK. 4Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa. 5Division of Medicine, Wright Fleming Institute, Imperial College, London, W2 1PG, UK. 6Baylor Institute for Immunology Research/ANRS Center for Human Vaccines, INSERM U899, 3434 Live Oak Street, Dallas, Texas 75204, USA. 7Institute for Health Care Research and Improvement, Baylor Health Care System, Dallas, Texas 75206, USA. 8Department of Radiology, St Mary’s Hospital, Imperial College Healthcare NHS Trust, London W2 1NY, UK. 9UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA. 10Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205, USA. *These authors contributed equally to this work. 973 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a The test set (UK) Latent individuals cluster c 54 Participants with active TB # 0.1

P<0.001 3,000 P<0.01 1.0 2,000 P<0.001

5.0 Normalized expression 1,000 distance to health

Weighted molecular Weighted 0

Minimal diseaseModerate No disease diseaseAdvanceddisease Modal extent of radiographic Study group disease b The validation set (South Africa) Latent individuals cluster 54 Participants with active TB (and Σ) Σ

d P 2,000 <0.0001

1,500

1,000

500 distance to health Weighted molecular Weighted 0 0212 Study group Active Time post-treatment (months) Latent Control Study group

Figure 1 | A distinct whole-blood 393-gene transcriptional signature of disease and the median ‘molecular distance to health’ compared between active TB. The 393 transcripts differentially expressed in whole blood of groups (Methods) (Kruskal–Wallis analysis of variance, Dunn’s multiple patients with active and latent TB and healthy controls. a, Test set. comparison, P , 0.0001). d, Patients with active TB at 0, 2 and 12 months b, Validation set (South Africa) profiles, ordered by hierarchical clustering after initiation of anti-mycobacterial treatment. The median ‘molecular (Spearman correlation with average linkage) creating a condition tree, upper distance to health’ for each time point was compared (Friedman’s repeated horizontal edge of heatmap; study grouping (clinical phenotype) are the measures test, Dunn’s multiple comparison). Horizontal bars, median, 5th coloured blocks at each profile base. Heatmap rows, genes; columns, and 95th percentiles. participants. c, Profiles were grouped according to radiographic extent of and reflects changes at the site of disease. The transcriptional signature The modular TB signature revealed decreased abundance of was diminished in patients with active TB after 2 months, and com- B-cell (Module, M1.3) and T-cell (M2.8) transcripts and increased pletely extinguished by 12 months after treatment, with ‘molecular abundance of myeloid-related transcripts (M1.5 and M2.6). The largest distance to health’ at 12 months significantly lower than at baseline proportion of transcripts changing in a given module in TB was within pretreatment (Fig. 1d and Supplementary Fig. 4), reflecting radio- the IFN-inducible module (M3.1; 75–82% of IFN-module transcripts graphic improvement. Thus the blood transcriptional signature of (Fig. 3a and Supplementary Fig. 7)). Because a type I IFN-inducible patients with active TB could be used to monitor efficacy of treatment, signature, linked with disease pathogenesis, has been demonstrated in and is reflective of the host response to infection with M. tuberculosis. peripheral blood mononuclear cells from patients with SLE13,14,we The 393-gene active TB signature may reflect common inflammatory compared whole-blood modular signatures from patients with other responses evoked during many diseases. We therefore identified a TB- diseases. Patients with SLE demonstrated over-representation of the specific 86-gene whole-blood signature through analysis of signifi- IFN-inducible module (M3.1 (Fig. 3a) quantified in Supplementary cance12, compared with patients with other bacterial and inflammatory Fig. 8), but displayed a plasma-cell-related module absent in TB diseases (Supplementary Fig. 5, and Supplementary Tables 5 and 6). (M1.1 (Fig. 3a and Supplementary Fig. 8)). The blood modular sig- This 86-gene signature was then tested against patients normalized to nature from patients with group A Streptococcus or Staphylococcus their own controls from seven independent data sets by class prediction infection, or Still’s disease, showed minimal to no change in the (k-nearest neighbours) (Fig. 2a). Sensitivities in the TB training and IFN-inducible module (M3.1) but marked over-representation of the validation sets were 92% and 90% respectively, distinguishing active TB neutrophil-related module (M2.2), distinguishing these diseases from from other diseases with a pooled specificity of 83% (Supplemen- TB (Fig. 3a and Supplementary Fig. 8). Thus the IFN-inducible sig- tary Table 7). As with the 393-gene signature, this 86-gene signature nature is not common to all inflammatory responses, but is preferen- was diminished in response to treatment (Fig. 2b) and reflected the tially induced during some diseases, potentially reflecting protection or same heterogeneity in identical samples from patients (Supplemen- pathogenesis. Although SLE and TB share common inflammatory tary Fig. 6). components such as an IFN-inducible response, the overall pattern To identify functional components of the transcriptional host res- of transcriptional changes (Fig. 3a) and their amplitude (Supplemen- ponse during active TB, we used a modular data-mining strategy, using tary Fig. 8) distinguishes one disease from another. sets of genes that are coordinately expressed in different diseases and The TB blood-transcriptional signature could represent altered defined as specific modules, often demonstrating coherent functional cell composition or changes in gene expression in discrete cellular relationships through unbiased literature profiling7. The blood modu- populations. Percentages of B cells, and of T cells carrying the CD4 lar signature of patients with active TB compared with healthy controls and CD8 antigens, assessed by flow cytometry, were significantly (filtering out only undetected transcripts, a 5 0.01, in at least two indi- diminished in patients with active TB, with reduced numbers of total viduals) was similar in all three TB data sets (Fig. 3a and Supplementary and central memory T cells carrying the CD4 antigen (Fig. 3b and Fig. 7), confirming the reproducibility of the transcriptional signature. Supplementary Fig. 9a, b), in keeping with previous studies15. That 974 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

ab Healthy Active tuberculosis patients controls 86 Genes 86 Genes

Test TrainingSA Strep Staph Still’s SLE pSLE 0212 Time post-treatment (months)

TB Figure 2 | A distinct whole-blood 86-gene transcriptional signature of Staphylococcus (Staph; n 5 40; control 5 12), Still’s disease (Still’s; n 5 31; active TB is distinct from other diseases. a, Comparison of 86-gene control 5 22), Adult (SLE; n 5 29; control 5 16) and paediatric SLE (pSLE; signature in patients with TB and other diseases normalized to their own n 5 49; control 5 11) patients. b, Expression levels of 86 gene signatures controls; TB (training, n 5 13; control, n 5 12), TB (SA, n 5 20; after 2 and 12 months of treatment in patients with TB. (Scale as in Fig. 1.) control 5 12), group A Streptococcus (Strep; n 5 23; control 5 12),

a TB test set (UK) Adult SLE Paediatric SLE 1 2 3 4 5 67 8 9 10 11 1234567 891011 1234567 891011 M1 M1 M1 M2 M2 M2 M3 M3 M3

Group A Streptococcus Staphylococcus Still’s disease 1 2 3 4 5 67 8 9 10 11 1234567 891011 1234567 891011 M1 M1 M1 M2 M2 M2 M3 M3 M3

Neutrophils 1234567 891011 Plasma cells Platelets Erythrocytes Over-represented M1 T-cells Under-represented B-cells M2 Interferon 1009080706050 40 30 20 Myeloid lineage Probe sets P < 0.05 (%) M3 Ribosomal proteins Inflammation Probe representation in module Functional interpretation Cytotoxic cells Undetermined No module

b CD4+ T cells CD8+ T cells B cells (CD19+CD20+) c Monocytes (CD14+) Inflammatory monocytes Neutrophils (CD16+) + + 15 (CD14 CD16 ) 20 ***8 8 8 100 15 6 ** 80 10 6 6 60 10 4 4 4 5 40 5 2 2 2 20 0 0 0 0 0 0 ) Percentage ) Percentage –1 1.6 * 0.8 0.8 –1 0.6 0.6 6 ml ml * 6 1.2 0.6 0.6 6 0.4 0.4 4 0.8 0.4 0.4 0.2 0.2 2 0.4 0.2 0.2 0 0 0 0 0 0 Cell number (10 Cell number (10

Active Active Active Active Active Active Control Control Control Control Control Control Figure 3 | Whole-blood transcriptional signature of active TB reflects shown by colour-coded grid. Whole blood (test set patients with active TB distinct changes in cellular composition and gene expression. a, Gene and controls) analysed by flow cytometry for (b) CD31CD41 T cells, expression (disease versus healthy controls) of TB (test set) and different CD31CD81 T cells and CD191CD201 B cells, and (c) CD141 monocytes, diseases mapped within a pre-defined modular framework. Spot intensity CD141CD161 inflammatory monocytes and CD161 neutrophils. Error (red, increased; blue, decreased) indicates transcript abundance. Functional bars, median; **P , 0.01, *P , 0.05, Mann–Whitney U-test. interpretations previously determined by unbiased literature profiling 975 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010 the reduction in T-cell transcripts revealed by the modular analysis viral infections but may be detrimental during bacterial19, including (Fig. 3a) resulted from changes in cell numbers in the blood, was mycobacterial, infections20,21. Absence of IFN-ab signalling in mice further confirmed because expression of these transcripts in purified improved outcome after infection with highly virulent20–22, but not T cells from the same individuals did not differ between patients with less virulent, strains of M. tuberculosis23. Highly virulent strains of M. TB and healthy controls (Supplementary Fig. 9c). In contrast, the tuberculosis induce higher levels of type I IFNs20. There are reports of increase in myeloid transcripts (M1.5, M2.6 (Fig. 3a and Sup- TB reactivation during IFN-a treatment for hepatitis D viral infec- plementary Fig. 7)) in the blood of patients with active TB was not tion24. The increase in type I IFN-ab-inducible transcripts in the accounted for by changes in monocytes (CD141, CD162) or neu- blood of patients with active TB (Fig. 4c), correlating with disease trophils (CD161, CD142) although inflammatory monocytes severity, provides the first data in human disease to support a role for (CD141, CD161) were increased (Fig. 3c and Supplementary Fig. type I IFNs in the pathogenesis of TB. These IFN-inducible tran- 10a), as in other diseases16. Increased abundance of myeloid transcripts were overexpressed in purified blood neutrophils and to a scripts was less pronounced in purified monocytes (CD141) lesser extent monocytes, but not T cells carrying the CD4 and CD8 (Supplementary Fig. 10b), which suggests involvement of other cells. antigens, from patients with active TB, compared with healthy con- Pathway analysis confirmed IFN signalling as the most significantly trols (Fig. 4d; top to bottom: OAS1, IFI6, IFI44, IFI44L, OAS3, IRF7, over-represented pathway in the 393-gene signature (Fisher’s exact test, IFIH1, IFI16, IFIT3, IFIT2, OAS2, IFITM3, IFITM1, GBP1, GBP5, Benjamini–Hochberg correction for multiple testing, P , 0.0000001 STAT1, GBP2, TAP1, STAT1, STAT2, IFI35, TAP2, CD274, SOCS1, (Supplementary Fig. 11)). Genes downstream of both IFN-c and type CXCL10, IFIT5). Neutrophils are the predominant cell type infected IIFN-ab receptor signalling were significantly over-represented in with rapidly replicating M. tuberculosis in patients with TB25. blood from patients with active TB (Fig. 4a–c). IFN-a2 and IFN-c Evidence from genetically susceptible mice suggests that neutrophils proteins were not elevated in serum from patients with active TB, contribute to pathology during infection with M. tuberculosis26. Our although the IFN-inducible chemokine CXCL10 (IP10) was signifi- studies support a role for neutrophils in the pathogenesis of TB, cantly increased (Supplementary Fig. 11c–e). which may result from over-activation by IFN-c and type I IFNs. Although IFN-c is protective during immune responses to intra- Earlier microarray studies, limited by small numbers of patients cellular pathogens, including mycobacteria4,17,18, the role of type I and custom microarrays, reported a small number of genes in blood IFN-ab is less clear. Type I IFN signalling is crucial for defence against associated with TB27,28. Here we provide the first complete description

Interferon signalling ab

Complex Cytokine/growth factor Enzyme Cytoplasm Kinase Peptidase Phosphatase Transcription regulator Transmembrane receptor Transporter Unknown Relations Over-represented transcripts Nucleus

c Whole blood d Separated cells Neutrophils Monocytes CD4 CD8 IFN-inducible genes

Control Active Control Active Control Active Control Active Control Active

0.1 1 5 Figure 4 | Interferon-inducible gene expression in active TB. Canonical signalling. Transcript abundance of representative IFN-inducible genes in pathway of Ingenuity pathways analysis for interferon signalling; symbol active TB from (c) whole blood and (d) separated blood leucocyte indicates gene function (legend on right). Transcripts over-represented in population. Transcript abundance/expression is normalized to the median test set patients with active TB shaded red. a, Type II IFN-c. b, Type I IFN-ab of the healthy controls. 976 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS of the human blood transcriptional signature of TB. The signature of 18. Flynn, J. L. & Chan, J. Immunology of tuberculosis. Annu. Rev. Immunol. 19, 93–129 (2001). active TB, observed in 10–20% of patients with latent TB, may identify 19. Decker, T., Muller, M. & Stockinger, S. The yin and yang of type I interferon activity those individuals who will develop active disease, facilitating targeted in bacterial infection. Nature Rev. Immunol. 5, 675–687 (2005). preventative therapy, but longitudinal studies are needed to assess 20. Manca, C. et al. Hypervirulent M. tuberculosis W/Beijing strains upregulate type I this. That the TB signature is dominated by type I IFN-signalling IFNs and increase expression of negative regulators of the Jak-Stat pathway. J. and reflects extent of lung disease, may indicate the process leading Interferon Cytokine Res. 25, 694–701 (2005). 21. Ordway, D. et al. The hypervirulent Mycobacterium tuberculosis strain HN878 to disease susceptibility. These data improve our understanding of the induces a potent TH1 response followed by rapid down-regulation. J. Immunol. fundamental biology of TB and may offer future leads for diagnosis 179, 522–531 (2007). and treatment. 22. Manca, C. et al. Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with METHODS SUMMARY induction of IFN-alpha/beta. Proc. Natl Acad. Sci. USA 98, 5752–5757 (2001). 23. Cooper, A. M., Pearl, J. E., Brooks, J. V., Ehlers, S. & Orme, I. M. Expression of the Whole blood was collected into Tempus tubes (Applied Biosystems) from patients nitric oxide synthase 2 gene is not essential for early control of Mycobacterium as follows: those with active TB (confirmed by culture for M. tuberculosis); those tuberculosis in the murine lung. Infect. Immun. 68, 6879–6882 (2000). with latent TB (defined by a positive tuberculin-skin test (TST) (London)) and/or 24. Telesca, C. et al. Interferon-alpha treatment of hepatitis D induces tuberculosis apositiveM. tuberculosis antigen-specific IFN-c release assay (IGRA); healthy exacerbation in an immigrant. J. Infect. 54, e223–e226 (2007). controls (recruited in London; TST/IGRA negative). RNA was extracted from 25. Eum, S. Y. et al. Neutrophils are the predominant infected phagocytic cells in the whole blood and purified (by Dynabeads, Invitrogen) neutrophils, monocytes airways of patients with active pulmonary tuberculosis. Chest 137, 122–128 (2010). 26. Eruslanov, E. B. et al. Neutrophil responses to Mycobacterium tuberculosis infection and T cells carrying the CD4 and CD8 antigens, and genome-wide transcriptional in genetically susceptible and resistant mice. Infect. Immun. 73, 1744–1753 profiles were generated using Illumina HT12 V3 Beadarrays, and analysed using (2005). 11 Genespring GX (see Methods). Calculations of ‘molecular distance to health’ , 27. Jacobsen, M. et al. Candidate biomarkers for discrimination between infection transcriptional modular analysis7 and analysis of significance12 were performed as and disease caused by Mycobacterium tuberculosis. J. Mol. Med. 85, 613–621 previously described. Pathway analysis was performed using Ingenuity (Ingenuity (2007). Systems). Multiplex serum protein measurement was performed using the 28. Mistry, R. et al. Gene-expression patterns in whole blood identify subjects at risk Milliplex Multi-Analyte Profiling System by Millipore UK. Flow cytometry was for recurrent tuberculosis. J. Infect. Dis. 195, 357–365 (2007). performed on a Beckman Coulter Cyan using Summit software version 3.02, Supplementary Information is linked to the online version of the paper at followed by FlowJo analysis. www.nature.com/nature. Full Methods and any associated references are available in the online version of Acknowledgements We thank the patients and volunteer participants. We thank the paper at www.nature.com/nature. D. Kioussis (MRC National Institute for Medical Research (NIMR)) and D. Young (NIMR) for discussion and input. We thank N. Baldwin (Baylor Institute for Received 23 March; accepted 2 June 2010. Immunology Research (BIIR)) for advice and support on bioinformatics analysis, Q.-A. Nguyen (BIIR) and colleagues for providing technical assistance with 1. Dye, C., Floyd, K. & Uplekar, M. in Global Tuberculosis Control: Surveillance, microarray processing, and S. Caidan (NIMR), J. Wills (NIMR) and S. Phillips (BIIR) Planning, Financing Ch. 1, 17–37 (World Health Organization, 2008). for help and advice with sample storage and transport. We thank the TB service at 2. Kaufmann, S. H. & McMichael, A. J. Annulling a dangerous liaison: vaccination Imperial College Healthcare NHS Trust, B.M. Haselden and the TB service at strategies against AIDS and tuberculosis. Nature Med. 11, S33–S44 (2005). Hillingdon Hospital, Uxbridge, UK. We also thank H. Giedon and R. Seldon for help 3. Barry, C. E. III et al. The spectrum of latent tuberculosis: rethinking the biology and in laboratory analyses, and Y. Hlombe for recruitment of patients and follow-up in intervention strategies. Nature Rev. Microbiol. 7, 845–855 (2009). South Africa. A. Rae (NIMR), T. Dipucchio (BIIR) and K. Palucka (BIIR) provided 4. Cooper, A. M. Cell-mediated immune responses in tuberculosis. Annu. Rev. advice on flow cytometry. We thank G. Hayward for help depositing the microarray Immunol. 27, 393–422 (2009). data. We thank J. Brock (NIMR) for help with graphics. M.P.R.B. was supported by 5. Young, D. B., Perkins, M. D., Duncan, K. & Barry, C. E. III. Confronting the scientific an MRC career development fellowship and a grant from the Dana Foundation obstacles to global control of tuberculosis. J. Clin. Invest. 118, 1255–1265 (2008). Program in Human Immunology. The research was funded by the Medical 6. Ardura, M. I. et al. Enhanced monocyte response and decreased central memory T Research Council, UK, MRC Grant U117565642 and The Dana Foundation Program cells in children with invasive Staphylococcus aureus infections. PLoS ONE 4, e5446 in Human Immunology. A.O’G., C.M.G. and F.W.McN. are funded by the Medical (2009). Research Council, UK. V.P. is supported by National Institutes of Health (NIH) R01 7. Chaussabel, D. et al. A modular analysis framework for blood genomics studies: AR050770-01, NIH P50 ARO54083 and NIH 1 U19 AI082715-01. The work of J.B., application to systemic lupus erythematosus. Immunity 29, 150–164 (2008). D.C. and V.P. is supported by the Baylor Health Care System Foundation and the 8. Pascual, V., Chaussabel, D. & Banchereau, J. A genomic approach to human NIH (U19 AIO57234-02, U01 AI082110, P01 CA084512). autoimmune diseases. Annu. Rev. Immunol. 28, 535–571 (2010). Author Contributions M.P.R.B., D.C., O.M.K and A.O’G. designed the study on TB 9. Ramilo, O. et al. Gene expression patterns in blood leukocytes discriminate with input from J.B. and R.J.W. and for other diseases with input from V.P. and O.R.; patients with acute infections. Blood 109, 2066–2077 (2007). M.P.R.B., S.A.A.B., T.O., K.A.W., J.J.C., A.M., R.B. and O.M.K. recruited, sampled and 10. Falk, A. & O’Connor, J. B. in Diagnosis Standards and Classification of Tuberculosis, collected data about patients; M.P.R.B., R.B., A.M. and C.M.G. processed whole vol. 12 (eds Falk, A. et al.), 68–76 (National Tuberculosis and Respiratory Disease blood for microarray experiments with help from J.S.; C.G. performed blood-cell Association, 1969). subset separations and processing for microarray experiments with help from J.S.; 11. Pankla, R. et al. Genomic transcriptional profiling identifies a candidate blood M.P.R.B., C.M.G. and Z.X. performed microarray data analysis, with advice and biomarker signature for the diagnosis of septicemic melioidosis. Genome Biol. 10, input from J.S., D.C. and V.P.; M.P.R.B. and Z.X. performed Ingenuity, modular and R127.1–R127.22 (2009). ‘molecular distance to health’ analyses; M.P.R.B. performed multiplex serum 12. Allantaz, F. et al. Blood leukocyte microarrays to diagnose systemic onset juvenile analyses; F.W.McN. performed flow cytometry analysis; D.C., V.P. and A.O’G. idiopathic arthritis and follow the response to IL-1 blockade. J. Exp. Med. 204, supervised data analysis; M.P.R.B. and D.B. performed statistical analysis; M.P.R.B., 2131–2144 (2007). S.A.A.B., R.D. and O.M.K performed analyses of radiology; A.O’G. and M.P.R.B. 13. Baechler, E. C. et al. Interferon-inducible gene expression signature in peripheral wrote the manuscript, with early input from C.M.G., F.W.McN., J.B., D.C. and J.S., blood cells of patients with severe lupus. Proc. Natl Acad. Sci. USA 100, 2610–2615 and subsequently all authors provided advice and approved the final manuscript. (2003). 14. Bennett, L. et al. Interferon and granulopoiesis signatures in systemic lupus Author Information All microarray data are deposited in GEO under accession erythematosus blood. J. Exp. Med. 197, 711–723 (2003). numbers GSE19491, GSE19444, GSE19443, GSE19442, GSE19439, GSE19435 and 15. Beck, J. S., Potts, R. C., Kardjito, T. & Grange, J. M. T4 lymphopenia in patients with GSE 22098. Some of the work has been submitted as US patent application PCT active pulmonary tuberculosis. Clin. Exp. Immunol. 60, 49–54 (1985). 371: Blood Transcriptional Signature of Mycobacterium Tuberculosis Infection: 16. Auffray, C., Sieweke, M. H. & Geissmann, F. Blood monocytes: development, Serial No: 12/602,488. Reprints and permissions information is available at heterogeneity, and relationship with dendritic cells. Annu. Rev. Immunol. 27, www.nature.com/reprints. The authors declare no competing financial interests. 669–692 (2009). Readers are welcome to comment on the online version of this article at 17. Casanova, J. L. & Abel, L. Genetic dissection of immunity to mycobacteria: the www.nature.com/nature. Correspondence and requests for materials should be human model. Annu. Rev. Immunol. 20, 581–620 (2002). addressed to A.O’G. ([email protected]).

METHODS more than 48,000 probes. The arrays were then washed, blocked, stained and scanned on an Illumina BeadStation 500 following the manufacturer’s protocols. Participant recruitment and patient characterization. The local Research Illumina BeadStudio version 2 software (Illumina) was used to generate signal Ethics Committees (REC) at St Mary’s Hospital, London, UK (REC 06/ intensity values from the scans. Q0403/128) and the University of Cape Town, Cape Town, South Africa (REC Separated cells isolation and RNA extraction. Whole blood was collected in 012/2007) approved the study. All participants were older than 18 years and gave EDTA. Neutrophils (CD151), monocytes (CD141) and T cells carrying the CD4 written informed consent. Participants were recruited from St Mary’s Hospital and CD8 antigens were isolated sequentially using Dynabeads according to and Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, manufacturer’s instructions. RNA was extracted from whole blood (59 Prime UK, Hillingdon Hospital, the Hillingdon Hospitals NHS Trust, Uxbridge, UK, PerfectPure Kit) or separated cell populations (Qiagen RNeasy Mini Kit) and and the Ubuntu TB/HIV clinic, Khayelitsha, Cape Town, South Africa. Patients stored at 280 uC until use. were prospectively recruited and sampled before any anti-mycobacterial treat- Microarray data analysis. For normalization, Illumina BeadStudio version 2 ment was started, but only included in the final analysis if they met the full software was used to subtract background and scale average signal intensity for clinical criteria for their relevant study group. A subset of patients with active each sample to the global average signal intensity for all samples. A gene expres- TB recruited into the first cohort recruited in London was also sampled at 2 and sion analysis software program, GeneSpring GX version 7.3.1 (Agilent 12 months after the start of therapy. Patients who were pregnant, immuno- Technologies, hereafter referred to as GeneSpring), was used to perform further suppressed or who had diabetes or autoimmune disease were ineligible and normalization. All signal intensity values less than 10 were set to equal 10. Next, excluded from this study. In South Africa, all participants had routine HIV per-gene normalization was applied, by dividing the signal intensity of each testing using the Abbott Determine HIV1/2 rapid antibody assay test kit probe in each sample by the median intensity for that probe across all samples (Abbott Laboratories), and patients with positive HIV tests were excluded. except for Fig. 4c, d and Supplementary Figs 9c and 10b, where signals are Patients with active TB were confirmed by laboratory isolation of M. tuberculosis normalized to the median of each control group. These normalized data were on mycobacterial culture of a respiratory specimen (either sputum or broncho- used for all downstream analyses except the assessment of molecular distance to alveolar lavage fluid) with sensitivity testing performed by the Royal Brompton health detailed below. Hospital Mycobacterial Reference Laboratory, London, UK, or the Reference Using GeneSpring, all transcripts were filtered first to select detected tran- Labratory of the National Health Laboratory Service, Groote Schuur Hospital, scripts: those called ‘present’ in greater than 10% of all samples. Present calls were Cape Town, South Africa. In the UK, patients with latent TB were recruited from selected if the signal precision was less than 0.01. The remaining transcripts were those referred to the TB clinic with a positive TST, and a positive result using an filtered to select the most variable probes: those that had a minimum of twofold IGRA. Participants in South Africa with latent TB were recruited from indivi- expression change compared with the median intensity across all samples, in duals self-referring to the voluntary testing clinic at the Ubuntu TB/HIV clinic, greater than 10% of all samples. and IGRA positivity alone was used to confirm the diagnosis, irrespective of TST We next performed unsupervised analysis, using hierarchical clustering and result (although this was still performed). Healthy control participants were class discovery. This approach aims to create an unbiased grouping of samples on recruited from volunteers at the MRC National Institute for Medical Research, the basis of their molecular profiles, independently of any other phenotypic or Mill Hill, London, UK. To meet the final criteria for study inclusion, healthy clinical classification. Transcripts meeting the filtering criteria are then subjected volunteers had to be negative by both TST and IGRA. 29 to hierarchical clustering using GeneSpring. For hierarchical clustering of genes, Tuberculin skin testing. This was performed according to the UK guidelines we used a clustering algorithm based upon Pearson correlation, creating a ver- using 0.1 ml (2 tuberculin units) tuberculin purified protein derivative (RT23, tical dendrogram of genes, where transcripts with a similar expression pattern Serum Statens Institute). A positive TST was termed $6 mm if BCG (bacille across all samples are grouped together. The distances between branches of the Calmette–Gue´rin) unvaccinated or $15 mm if BCG vaccinated, as per the UK tree relate to the similarity of the expression patterns, and the distance between 30 national guidelines . clusters is determined by the average of the distance between all points in each IFN-c release assay testing. The QuantiFERON Gold In-Tube Assay (Cellestis) cluster, known as average linkage. The vertical expression profiles so generated was performed according to the manufacturer’s instructions. can then be subjected to the same hierarchical clustering algorithm, now group- Total and differential leucocyte counts. Two millilitres of whole blood were ing individual participants into horizontally presented clusters on the basis of the collected into Terumo Venosafe 5ml K2-EDTA tubes (Terumo Europe). similarity of their expression profiles. For this stage, we base the clustering Samples were then analysed within 4 h using the Nihon Kohden MEK-6400 algorithm on Spearman’s rank correlation. By examining the cluster member- Automated Hematology Analyser (Nihon Kohden). ship we can assess both whether the samples are grouping according to known Assessment of radiographic extent of disease. Plain chest radiographs were factors (clinical diagnosis, demographic features) and discover if there are obtained for all patients recruited in London as digital images and graded by three unknown subclasses within the data set. independent clinicians, blinded to the transcriptional profiles and the clinical Supervised analysis was performed using statistical filtering and class com- data, using a modified version of the classification system of the US National parison. The aim of the supervised analysis is to identify transcripts that are Tuberculosis and Respiratory Disease Association10. This system characterizes the differentially expressed between study groups and that might serve as classifiers radiographic extent of disease into ‘minimal’, ‘moderately advanced’ or ‘far or yield insight into immunopathogenesis: that is, class comparison. The filtered advanced’ stages, according to criteria based upon the density and extent of lesions list of transcripts generated for unsupervised analysis was used as the starting and presence of absence of cavitation. We modified the system for use in our study point for the supervised analysis: that is, those transcripts that were both detected so that it also included a classification of ‘No disease’, and accounted for the and had at least a twofold change in expression compared with the median, in presence of pleural disease or lymphadenopathy. The system was then converted greater than 10% of all samples. Using GeneSpring, these transcripts were then into a decision tree to aid classification (Supplementary Fig. 3a). tested using the Kruskal–Wallis test for comparisons across all study groups, RNA sampling, extraction and processing for microarray analysis. Three with a 5 0.01. Adjustment for multiple testing was applied using the Benjamini– millilitres of whole blood were collected into Tempus tubes (Applied Hochberg false discovery rate set at 1%. Lists of transcripts generated in this way Biosystems), vigorously mixed immediately after collection, and stored between were then used for hierarchical clustering as described above. Interpretation of 220 and 280 uC before RNA extraction. RNA was isolated from training set functional roles of individual transcripts was established by searching the data- samples using 1.5 ml whole blood and the PerfectPure RNA Blood Kit (5 base at the National Center for Biotechnology Information gene database at PRIME). Test and validation (South Africa) set samples were extracted from http://www.ncbi.nlm.nih.gov/sites/entrez?db5gene. 1 ml of whole blood using the MagMAX-96 Blood RNA Isolation Kit (Applied For class prediction, we used one of the tools available within GeneSpring. The Biosystems/Ambion) according to the manufacturer’s instructions. Two and a prediction model used the k-nearest neighbours algorithm, with 10 neighbours half micrograms of isolated total RNA was then globin-reduced using the and a P value ratio cutoff of 0.5. All genes from the 393 transcript list were used GLOBINclear 96-well format kit (Applied Biosystems/Ambion) according to for the prediction. The prediction model was refined by cross-validation on the the manufacturer’s instructions. Total and globin-reduced RNA integrity was training set, with the one active outlier excluded. This model was then used to assessed using an Agilent 2100 Bioanalyser showing a quality of RNA integrity predict the classification of the samples in the independent test and validation number of 7–9.5 (Agilent Technologies). RNA yield was assessed using a sets. Where no prediction was made, this was recorded as an indeterminate NanoDrop 1000 spectrophotometer (NanoDrop Products, Thermo Fisher result. Sensitivity, specificity and 95% confidence intervals were determined Scientific). Biotinylated, amplified antisense complementary RNA (cRNA) tar- using GraphPad Prism version 5.02 for Windows. P values were determined gets were then prepared from 200 to 250 ng of the globin-reduced RNA using the using two-sided Fisher’s exact test. Illumina CustomPrep RNA amplification kit (Applied Biosystems/Ambion). ‘Molecular distance to health’ was performed as previously described11. It aims Seven hundred and fifty nanograms of labelled cRNA was hybridized overnight to convert transcript abundance values into a representative score indicating the to Illumina Human HT-12 V3 BeadChip arrays (Illumina), which contained degree of transcriptional perturbation of a given sample compared with a healthy

baseline. This is performed by determining whether the expression values of a tubes, reference 454098, Greiner BioOne; or BD 4-ml vacutainer tubes, reference given sample lie inside or outside two standard deviations from the mean of the 368975; Becton Dickinson). Tubes were centrifuged at 2000g for 5 min at room healthy controls. temperature and the serum portion extracted and frozen at –80 uC pending Additional functional analysis of differentially expressed genes was performed analysis. Analysis was performed by multiplexed cytokine bead-based immuno- using Ingenuity pathways analysis (Ingenuity Systems, www.ingenuity.com). assay by Millipore UK using the Milliplex Multi-Analyte Profiling system. The Canonical pathways analysis identified the pathways from the Ingenuity pathways serum levels of 63 cytokines, chemokines, soluble receptors, growth factors, analysis that were most significantly represented in the data set. The significance of adhesion molecules and acute phase proteins were measured in this way in each the association between the data set and the canonical pathway was measured sample. Samples were assayed for levels of MMP-9, C-reactive protein, serum using Fisher’s exact test to calculate a P value representing the probability that the amyloid A, EGF, eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF, GM-CSF, association between the transcripts in the data set and the canonical pathway was GRO, IFN-a2, IFN-c, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IL-1a, explained by chance alone, with a Benjamini–Hochberg correction for multiple IL-1b, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, CXCL10 (IP10), MCP-1, testing applied. The program can also be used to map the canonical network and MCP-3, MIP-1 a, MIP-1b, PDGF-AA, PDGF-AB/BB, RANTES, soluble CD40 overlay it with expression data from the data set. ligand, soluble IL-2RA, TGF-a, TNF-a, VEGF, MIF, soluble Fas, soluble Fas 7,11 Transcriptional modular analysis was performed as described previously . ligand, tPAI-1, soluble ICAM-1, soluble VCAM-1, soluble CD30, soluble In the context of the present study, because the modular framework was derived gp130, soluble IL-1RII, soluble IL-6R, soluble RAGE, soluble TNF-RI, soluble using Affymetrix HG U133A&B GeneChips, it was necessary to translate the TNF-RII, IL-16, TGF-b1, TGF-b2 and TGF-b3. probes comprising the modules into their equivalents on the Illumina platform. Flow cytometry. Two hundred microlitres of whole blood (collected in Sodium- RefSeq identities were used to match probes between the Affymetrix HG U133 Heparin tubes) per staining panel were incubated with the appropriate antibodies and Illumina HT-12 V3 platforms. Unambiguous matches were found for 2,071 for 20 min at room temperature in the dark. Red blood cells were then lysed using out of the 5,348 Affymetrix probe sets, and these were used in the present BD FACS lysing solution (BD Biosciences), incubating for 10 min at room tem- modular analysis. The matching probes were preserved in their original modules. perature in the dark. Cells were spun down and washed in 2 ml FACS buffer (PBS/ To present the global transcriptional changes graphically, for the disease group as BSA/azide) before being fixed in 1% paraformaldehyde. Samples were then run on a whole versus the healthy control group as a whole, spots are aligned on a grid, a Beckman Coulter Cyan using Summit software version 3.02. Analysis was per- with each position corresponding to a different module based on their original formed using FlowJo version 8.7.3 for Macintosh (Tree Star). Gating strategies definition. Spot intensity indicates the percentage of differentially expressed used are set out in Supplementary Figs 9a and 10a. Flow cytometric data are transcripts changing in the direction shown, from the total number of transcripts presented as dot plots (Fig. 3 and Supplementary Fig. 9b). Where appropriate, detected for that module, whereas spot colour indicates the polarity of the pooled flow cytometry data were tested for significance using the Mann–Whitney change (red, overrepresented; blue, underrepresented). rank sum U-test. All antibodies were purchased from BD Pharmingen or Caltag Significance analysis was performed as previously described12. Transcriptional Laboratories (Invitrogen) except for CD45RA, which was purchased from changes in whole blood were evaluated through statistical group comparison Beckman Coulter. performed systematically for active TB (test set), Staphylococcus infection, Still’s Statistical analysis. Molecular distance to health and modular framework ana- disease, and adult and paediatric SLE versus their respective healthy controls, lysis calculations were performed using Microsoft Excel 2003. Statistical analysis which allowed the normalization of each disease group to its own matched of continuous variables and correlation analysis was performed using GraphPad healthy control group, thus avoiding biological or technical confounding factors. Prism version 5.02 for Windows (GraphPad Software). Analysis of categorical A TB-specific whole-blood signature composed of 86 genes was identified variables was performed using SPSS version 14 for Windows. (Supplementary Fig. 5 and Supplementary Tables 5 and 6, P , 0.01) that was not in the four other data sets (P . 0.05) using a Mann–Whitney U-test with 29. Salisbury, D., Ramsay, M. & Noakes, K. in Immunization against Infectious Disease Benjamini–Hochberg false discovery rate correction for multiple testing. Class 3rd edn 391–408 (HMSO, 2006). prediction was performed using k-nearest neighbours algorithm, as before. 30. National Institute for Health and Clinical Excellence. Tuberculosis. Clinical Multiplex serum protein measurement. One to four millilitres of blood were Diagnosis and Management of Tuberculosis, and Measures for its Prevention and collected into serum clot activator tubes (either Greiner BioOne 1-ml vacuette Control (Royal College of Physicians, 2006).

Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells

Paola Bonfanti1,2{, Ste´phanie Claudinot1,2, Alessandro W. Amici1,2, Alison Farley3, C. Clare Blackburn3 & Yann Barrandon1,2

The thymus develops from the third pharyngeal pouch of the or double positive cells (16%). After serial passaging, less than 2% of anterior gut and provides the necessary environment for thymo- the cells were positive for K8/18, while all were positive for K5/14, a poiesis (the process by which thymocytes differentiate into mature situation maintained in serial cultivation (Supplementary Fig. 1a). T lymphocytes) and the establishment and maintenance of self- These data indicate that clonogenic TECs originate from both the tolerance1–3. It contains thymic epithelial cells (TECs) that form a cortex and the medulla but that serial cultivation predominantly complex three-dimensional network organized in cortical and selected for a medullary phenotype. Clonogenic TECs were then medullary compartments, the organization of which is notably different from simple or stratified epithelia4. TECs have an essential ab role in the generation of self-tolerant thymocytes through expression of the autoimmune regulator Aire5,6, but the mechanisms involved in the specification and maintenance of TECs remain unclear7–9. Despite the different embryological origins of thymus and skin (endodermal and ectodermal, respectively), some cells of 10–12 the thymic medulla express stratified-epithelium markers , 4 105 79.3 15.1 10 interpreted as promiscuous gene expression. Here we show that 19.3 70.1 104 the thymus of the rat contains a population of clonogenic TECs 103

3 that can be extensively cultured while conserving the capacity to EGFP 10 EpCAM 102 integrate in a thymic epithelial network and to express major 102 0 histocompatibility complex class II (MHC II) molecules and Aire. –102 These cells can irreversibly adopt the fate of hair follicle multi- –102102 103 104 105 0 103 104 105 EpCAM UEA potent stem cells when exposed to an inductive skin microenviron- c + ment; this change in fate is correlated with robust changes in gene 70 K5/K14 d O expression. Hence, microenvironmental cues are sufficient here to 60 K8/K18 EpCAMTECsSkin HF cells3T3 H 2 50 DP Six1

sorted – 40 re-direct epithelial cell fate, allowing crossing of primitive germ + 30 13 day 0 20 Hoxa3 layer boundaries and an increase in potency . Percentage 10 K8/18 0 Pax1 TECs were isolated from embryonic, post-natal or adult thymus EpCAM Ker5/14 obtained from wild-type or enhanced green fluorescent protein Pax9 (EGFP) rats14 and cultured in conditions used in human cell therapy15; 100 Foxn1 80 under these conditions, TECs formed progressively growing colonies 60 Plet1 cultured – cultured + (refs 16, 17 and Y.B. and H. Green, unpublished data). Embryonic day 7 40 Aire

Percentage 20 TECs were labelled with a fluorescent anti-EpCAM antibody and K5/14 K8/18 0 Actb sorted. (EpCAM, epithelial cell adhesion molecule.) EpCAM2 cells EpCAM 1 never formed colonies, whereas 0.1–0.5% of the EpCAM1 cells did Figure 1 | Clonogenic TECs maintain thymic identity in culture. a, EpCAM 1 1 TECs can form colonies in culture. Fluorescence-activated cell sorting (Fig. 1a). Moreover, both EpCAM UEA-1 (medullary TEC) and 1 2 1 2 (FACS) analysis illustrates the distribution of EpCAM and EpCAM cells EpCAM UEA-1 (cortical TEC) sorted cells gave rise to colonies from E17 EGFP1 thymi. Only a fraction of EpCAM1 sorted cells (0.5%) (Fig. 1b), consistent with previous observations that separate progeni- formed colonies (50 cells out of 104 sorted cells). b, Clonogenic cells derive 18,19 tors exist for the cortical and medullary compartments . (UEA-1, from both EpCAM1UEA1 and EpCAM1UEA2 populations. FACS plot Ulex Europaeus agglutinin-1.) More than 90% of cultured TECs in a shows the distribution of UEA1 and UEA2 cells in the EpCAM1 sorted cells. colony expressed p63 (Supplementary Fig. 1a), a transcription factor c, EpCAM1 sorted cells were cytocentrifuged and immunostained for K5/14 critical for maintenance of the proliferative potential of TECs17.We and K8/18 as were 7-day-old colonies (scale bars, 50 mm). Percentage of TEC investigated whether clonogenic TECs had a keratin pattern indicative expressing K5/14, K8/18 or both (DP) was evaluated in three independent experiments. Data are mean 6 s.d. d, Cultured EGFP1 TECs maintain of a cortical (K8/18) or a medullary (K5/14) identity. Immediately 1 1 thymic identity. EpCAM TECs were sorted and mRNAs extracted, as were after sorting, 60% of the EpCAM cells stained positively for K5/14, cultured EGFP1 TECs (embryonic day 16, E16, P1 and P7), skin (P0) and whereas 20% were K8/18 positive; the remainder were double positive cultured hair follicle multipotent stem cells (P5). Transcripts were detected (Fig. 1c). After a week of cultivation, 77% of the colonies only con- by RT–PCR (35 cycles) in EpCAM-sorted thymic cells and in subcultured tained K5/141 cells, whereas others contained only K8/181 cells (7%) TECs.

1Laboratory of Stem Cell Dynamics, School of Life Sciences, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2Department of Experimental Surgery, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland. 3Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, School of Biological Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK. {Present address: Diabetes Research Center, Vrije Universiteit Brussel (VUB), B-1090 Brussel, Belgium. = 978 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

passaged every six or seven days while maintaining 10–20% colony- a Mouse or rat forming efficiency over passages. We randomly cloned20,21 100–150 embryo Thymus single TECs from first or second passage cultures. Cells cloned with dissection an efficiency of 15–20%, and could be expanded for more than 50 doublings (Supplementary Fig. 1b) while maintaining a normal diploid karyotype (2n 5 42). Most importantly, cultured TECs Cell dissociation expressed several genes important for TEC identity including key transcriptions factors (Eya1, Six1, Pax1, Pax9, Foxn1 and Hoxa3) Cell aggregate Expansion and Plet-1 (placental-expressed transcript-1) encoding the cell sur- in vitro face antigen recognized by the monoclonal antibody MTS24 (ref. 22; Rat thymus 104 Fig. 1d). MHC-class II and Aire expression were undetectable in b cultured TECs (as in hair follicle stem cells) by immunochemistry. 103

Colonies of TECs had a different phenotype from colonies of hair 102 follicle stem cells (Supplementary Fig. 1c), even if they expressed CD8 markers of terminal epidermal differentiation—such as involucrin 101 or LEKTI, a serine protease inhibitor encoded by Spink5 (Sup- 100 100 101 102 103 104 plementary Fig. 1d), and several other genes linked to epidermal, muco- CD4 sal or hair follicle differentiation—as revealed by PCR with reverse transcription (RT–PCR) analysis (Supplementary Fig. 1e and 1f). We investigated the ability of cultured TECs to contribute to thymic morphogenesis using a whole-organ re-aggregation assay19,23–26 (Fig. 2a). Implanted aggregates consistently organized in structures that closely resembled a thymus (Fig. 2b), containing CD41CD81 GFP Ab K5 GFP K5 GFP positive lymphocytes of mouse origin within an epithelial network, Hoechst GFP Hoechst even if the donor carrier cells were of rat origin (Fig. 2b), indicating c that the implants had some thymic functionality as expected23. Clusters of EGFP1 cells were identified in several areas and were predominantly localized in the medulla structure of the reconstituted thymi (Fig. 2b). A few EGFP1 cells were also identified in other areas, in particular in the network lining the edge of a lobula (Fig. 2b); 2 2 GFP MHCclassII MHCclassII interestingly, the latter cells were K5/14 and K8/18 . Clonal cultured Hoechst Hoechst GFP TECs also consistently integrated in the medullary-like area (15/24 transplants) (Supplementary Table 1). MHC-class II expression was evident in the cultivated TECs engrafted in reconstituted thymi when revealed by an antibody specific to rat MHC-class II and confocal microscopy (Fig. 2c). Aire expression was also readily detected in some, but not all, GFP-labelled TECs (Fig. 2c). Most importantly, AIRE AIRE AIRE GFP stem cells isolated from hair follicles, footpad, vagina and oesophagus Hoechst GFP Hoechst 1 only formed cyst-like structures containing limited number of EGFP d cells (Fig. 2d and Supplementary Table 1) and never expressed MHC- class II when incorporated in reconstituted thymi (not shown). We then tested the capacity of clonogenic TECs to participate in the formation of hair follicles and epidermis in a short-term recon- 27 stitution assay (Supplementary Fig. 2a). After 2–3 weeks, aggregates GFP GFP Ab had generated EGFP1 epidermis and hair follicles with a 10% Hoechst Hoechst Hoechst frequency when cells were taken from thymus but with a 100% Figure 2 | Cultured thymic epithelial cells can incorporate into a thymic frequency when cells were from hair follicles (Supplementary network and express MHC class II and Aire. a, Schematic representation of 1 Fig. 2b). Most importantly, EGFP TECs were detected in all epi- the thymic aggregation assay. Right panel, morphology of a chimaeric rat/ dermal and hair follicle layers (Supplementary Fig. 2c), clearly indi- mouse aggregate at grafting (insert) and 5 weeks post-transplantation under cating that clonogenic TECs were able to generate skin lineages in the kidney capsule of an athymic mouse. b, Incorporation of cultured EGFP1 response to skin morphogenetic signals. We next investigated if TECs TEC cells into thymic aggregates. Upper-left panel, histology showing could participate in long-term hair follicle renewal using a functional medulla and cortical areas (haematoxylin/eosin staining); scale bar, 100 mm. assay developed for multipotent stem cells of the hair follicle28 Upper-middle panel, flow cytometric analysis of T cells recovered from a rat/ 1 rat thymus with the presence of mouse CD4 and CD8 single- and double- (Fig. 3a). Long-term participation of EGFP TECs in epidermal positive T cells. Upper-right panel, gross appearance of a chimaeric rat/rat renewal and hair follicle regeneration (hair cycle) was observed for thymic aggregate, showing several areas containing EGFP1 TECs from clone several months in 18% of the grafts (10 of 56) in 10 independent PRE16Cl11; scale bar, 500 mm. Lower-left panel, EGFP1 cells immunostained transplants (Fig. 3b and Supplementary Table 2); cultured multi- with anti-GFP antibody. Lower-middle panel, confocal microscopy potent hair follicle stem cells (HF) and tracheal epithelial cells demonstrating that most integrated EGFP1 cells adopted a medulla (pseudo-stratified epithelium) were used as positive and negative phenotype (K51). Lower right panel, integrated EGFP1 TECs are lining a 1 thymic lobule and do not express K5. Scale bars, 20 mm. c, High-resolution controls, respectively. Next, we recovered EGFP colony-forming 1 cells from four of six independent long-term skin grafts confocal microscopy demonstrating that EGFP cells integrated into a 3D (Supplementary Fig. 3), indicating that thymus-derived epithelial thymic network express MHC-class II and Aire. Upper panels, co-localization of rat MHC-class II (red) and EGFP1 (green); scale bar, 20 mm. Lower panels, cells could retain growth capability for a long time in a skin micro- co-localization of the nuclear protein Aire (red) and EGFP (green); scale bar, environment. These skin-recovered cells (subsequently referred as 10 mm. d, Cultured HF multipotent stem cells do not integrate into a sr1-TECs) were cultured for three to four passages before being developing thymic microenvironment. Immunostaining against GFP protein transplanted into developing mouse skin. They were involved in (red) evidences EGFP1 (green) area composed mostly of enucleated cells; epidermal renewal and were incorporated at a high frequency (23 scale bar, 25 mm. Nuclei are counterstained with Hoechst 33342 (blue). 979 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a Single First skin Graft Second skin Graft cell TECs transplantation harvest sr-TECs transplantation harvest

0 8 50 167 191 337 days

In vitro In vivo In vitro In vivo

bcFirst skin transplantation d Second skin transplantation

GFP GFP

1 2345 6 Epid. 1 19 days HF HF

789101112 HF SG 13 14 15 16 17 18

HFs * 19 20 * 146 days X 119 days * 75 days

Epid. SG

K14 Rat DNA GFP GFP 113 days 81 days Mouse DNA Hoechst Hoechst

Figure 3 | Thymic epithelial cells have skin potency. a, Schematic probe (green), excluding fusion between mouse and rat cells. Nuclei are representation of a serial-transplantation strategy. b, Cultured EGFP1 TECs counterstained with Hoechst 33342 (blue). Lower-right panel, FISH on a contribute to sebaceous glands (SG), epidermis and cycling HFs for several cryosection (8 mm) of a skin serial transplant with a sr1-TEC clone: rat probe months. EGFP1 TECs contribute to HF (clone PRP7Cl10 119 days post- (orange) and mouse probe (green) show rat nuclei in HF structures and transplantation (PT), scale bar, 500 mm), to HF and epidermis (clone excluded in vivo fusion between donor rat and recipient mouse cells. FISH PRE16Cl11, 75 days PT), to epidermis (clone PRE16Cl11, 113 days PT) and to probes are detailed in Supplementary Information. Scale bar, 25 mm. sebaceous glands (clone PRP1Cl11, 81 days PT). Scale bars, 100 mm. Asterisk d, Cultured sr1-TECs contributed to several EGFP1 HF, SG and epidermis indicates location of hair bulb. c, EGFP1 cells were isolated from the grafts (146 days PT); asterisk locates hair bulbs; scale bars, 500 and 50 mm. At higher and cultured again; sr1-TECs have a diploid karyotype (n 5 42, XX). Lower- magnification (lower panels) a transversal section of a HF shows contribution left panel, FISH of sr1-TECs labelled with a rat probe (orange) and a mouse of cultured TECs to all layers of the HF structure. Scale bar, 20 mm. of 24) into hair follicles that cycled for more than 100 days (Fig. 3d We next performed a genome-wide expression screen on clono- and Supplementary Table 2). genic TECs, sr1-TECs and clones recovered after a second skin trans- Unexpectedly, the sr1-TECs also renewed the epidermis in sharp plantation (subsequently referred as sr2-TECs, Supplementary Fig. 3). contrast to bona fide HF multipotent stem cells that can only generate Individual clonal populations behaved differently; after the first round epidermis for 3 weeks or after injury in this assay28. To eliminate the of transplantation, some sr1-TECs clustered closely with TECs while possibility that transplanted rat TECs had fused with cells of the others clustered close to hair follicles; this pattern was maintained recipient mouse, we performed a two-colour fluorescent in situ after the second round of skin transplantation, indicating that addi- hybridization (FISH) using mouse (green) and rat (orange) genomic tional exposure to skin microenvironmental cues had little impact on DNA as probes (Fig. 3c); no double-labelled cells were detected, thus reprogramming (Fig. 4c). This pattern correlated with functional eliminating fusion. Furthermore, a karyotype analysis revealed that capacity: sr1-TEC no. 218 efficiently integrated into the thymic net- 93% of the EGFP1 TECs cultured after the first round of skin trans- work in the reconstitution assay and clustered close to thymic epithe- plantation had a normal rat chromosomal count (Fig. 3c). We next lial cells; in contrast, sr1-TEC no. 217, which both incorporated into examined the ability of sr1-TECs to integrate into a thymic epithelial the thymic network and generated epidermis and sebaceous glands in network in our reconstitution assay (18 of 28) (Supplementary the thymic assay, clustered closer to hair follicle; and sr1-TEC no. 57, Table 1). sr1-TECs did not perform homogeneously; one clone which did not integrate into thymus clustered very close to hair follicle (no. 57) behaved similarly to HF cells and it did not integrate into (Fig. 4c). Accordingly, robust change in expression of genes was a thymic microenvironment, whereas two other clones (nos 217 and observed, including downregulation of transcription factors import- 218) integrated into the thymic epithelial network and expressed ant to TECs (for example, Pax1, Pax9 and Eya1), and upregulation of MHC class II antigens and Aire (Fig. 4a and b). In addition, one epidermal markers (for example, Lhx2, galectin 7 and claudins; clone (no. 217) gave also rise to stratified epithelium and sebaceous Supplementary Tables 3 and 4). RT–PCR demonstrated that thy- glands (Fig. 4b). Together, these data suggest that the recovered mus-specific genes, like Six1, Pax9, Hoxa3, Foxn1 and Plet-1, were clones were primed towards the HF fate to different extents. consistently expressed in cultured sr1-TECs (not shown), whereas 980 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a b Epid.-like SG capability to form skin, as revealed by our assay, as they naturally express markers of skin differentiation in vivo. This would suggest the Thy existence of a generic program of stratification and that skin deter- K14 GFP GFP GFP mination is independent of primary germ line origin, as TECs are of Hoechst Hoechst Hoechst endodermal and not of ectodermal origin. The question then arises as to why TECs need a stratification program while forming a complex three-dimensional network, the organization of which is strikingly different from stratified epithelia. Second, TECs may improve their MHCII K14 ability to become multipotent keratinocyte stem cells following K14 AIRE GFP GFP GFP GFP GFP Hoechst Hoechst microenvironmental reprogramming. The robust changes in gene expression in response to exposure to skin morphogenetic signals c Cluster with minimized variance d favour this hypothesis, as does the fact that the TECs, in which the 0246 transcriptome clusters closest to hair follicle stem cells, have lost their Height 218

1 thymic epithelial competence. Third, TECs may express a unique 218 Pax1 mRNA Pax9 mRNA 347 2.5 1.5 epithelial program, spanning from simple to stratification, which 2 347 2 1.5 1 PRP1Cl11 primes them for subsequent reprogramming by morphogenetic signals. 1 0.5 PRP1Cl11 0.5 Whether TECs can respond to a microenvironment other than skin will PRE16Cl11 Fold change 0 0 PRE16Cl11 be of interest. TECs TECs TECs PRP4mobulk r- s HF cells sr-TECs HF cells TECs sr-TECs PRP4mobulk PRP4moCl09 Eya1 mRNA Foxn1 mRNA METHODS SUMMARY PRP4moCl09 8 287 1 EGFP Sprague–Dawley rats (green rat CZ-004’’ SDTgN (act-EGFP)obsCZ-004) 22 6 287 0.8 14 0.6 217 4 were from Japan SLC . Hair follicle and thymic epithelial cells were cultured onto 1 0.4 21,28 217 0.2 2 a feeder layer of lethally irradiated 3T3-J2 cells as described . Single cells were

354 Fold change 0 0 20

sr-TECs isolated as described and clones were expanded once a week. Thymic aggregates 354 ECs TECs TECs 26 907 sr-T HF cells sr-TECs HF cells were performed as described . Briefly, thymi were dissected from either E13.5/ 1 907 57 E14.5 wild-type mouse embryos or E15/E16 rat embryos and dissociated into a 1 57 Gal7 mRNA Gata3 mRNA single-cell suspension and used to aggregate cultured TECs obtained from EGFP 194 2.5 1.5 2 rats, in approximately 1:4 ratio (cultured TECs/donor carrier cells),and then sr-TECs 194 2 1 YR219bulk 1.5 implanted under the kidney capsule of athymic mice. Skin aggregates were per- YR219bulk 1 0.5 0.5 formed as described27. Briefly, mass or clonal cultures of TECs were obtained from 54 Fold change 0 0 54 embryos (PRE16) or from newborn EGFP rats (PRP1) and were aggregated ex YR219Cl07 TECs TECs sr-TECs sr-TECs HF cells HF cells HF cells YR219Cl07 vivo with skin cells obtained from E14.5 wild-type mouse embryos and implanted YR219P5RC under the kidney capsule of athymic mice. Cultured hair follicle multipotent stem YR219P5RC cells were used as a control. For long-term skin reconstitution assay, cultured Figure 4 | Fate of skin-recovered thymic epithelial cells. a, b, sr-TEC clones TECs or hair follicle multipotent stem cells were grafted as described28. Gene display a non-homogeneous behaviour when incorporated into thymic profiling were performed at the Lausanne DNA Array Facility (DAFL) using rat aggregates. a, sr1-TECs no. 218 integrated into the epithelial thymic network Affymetrix chips (Rat Gene ST 1.0 Array). Chrombios (Raubling, Germany) and contributed to the medullary compartment; scale bar, 100 mm. Lower performed karyotyping and FISH analyses. Detailed methods and procedures panels, several EGFP1 cells (green) expressed K14; scale bars, 25 mm. including primers for PCR analysis, antibodies for flow cytometry and immuno- b, Upper panels, sr1-TECs no. 217 integrated into a thymic network, but also staining are provided in Supplementary Information. generated an epidermis-like structure (Epid-like or ORS-like structure) and m Full Methods and any associated references are available in the online version of sebaceous glands (SG); K14 immunostaining (red); scale bars, 100 m. the paper at www.nature.com/nature. Lower right panel, K14 staining of SG; scale bar, 20 mm. A third clone (clone 57) did not integrate at all (not shown). Lower-left and middle panels, Received 3 August 2009; accepted 7 June 2010. expression of Aire and rat MHC-class II antigens; scale bars, 10 mm. c, Microarray analysis of cultured TECs and HF cells before and after skin 1. Manley, N. R. & Blackburn, C. C. A developmental look at thymus organogenesis: transplantation; cluster with minimized variance based on 119 transcripts where do the non-hematopoietic cells in the thymus come from? Curr. Opin. differentially expressed between HF cells and TECs (P,0.001 and a fold Immunol. 15, 225–232 (2003). 2. Boehm, T. Thymus development and function. Curr. Opin. Immunol. 20, 178–184 change higher than 4). sr-TECs clustered closer to TECs, to HF or very close (2008). to hair follicle after the first transplantation (1), and maintained the same 3. Anderson, G., Lane, P. J. & Jenkinson, E. J. Generating intrathymic pattern after the second skin transplantation (2). Cluster correlates with the microenvironments to establish T-cell tolerance. Nature Rev. Immunol. 7, capacity of the sr-TECs to incorporate into a thymic network. d, Relative fold 954–963 (2007). changes for Pax1, Pax9, Eya1, Foxn1, Gal7 and Gata3 transcripts (Q-PCR). 4. van Ewijk, W. et al. Thymic microenvironments, 3-D versus 2-D? Semin. Immunol. Data show mean 6 s.e.m. (TECs: n 5 3; sr-TECs: n 5 4 and HF cells: n 5 2). 11, 57–64 (1999). sr-TECs represent the mean of sr1- and sr2-TECs. 5. Kyewski, B. & Klein, L. A central role for central tolerance. Annu. Rev. Immunol. 24, 571–606 (2006). 6. Abramson, J., Giraud, M., Benoist, C. & Mathis, D. Aire’s partners in the molecular Pax1 expression was undetectable, a finding confirmed by quantitat- control of immunological tolerance. Cell 140, 123–135 (2010). ive PCR (Fig. 4d). 7. Blackburn, C. C. et al. One for all and all for one: thymic epithelial stem cells and Our results demonstrate that embryonic and postnatal thymi regeneration. Trends Immunol. 23, 391–395 (2002). 8. Holla¨nder, G. et al. Cellular and molecular events during early thymus contain a subpopulation of clonogenic TECs that can function as development. Immunol. Rev. 209, 28–46 (2006). epidermal stem cells and as multipotent hair follicle stem cells when 9. Mathis, D. & Benoist, C. Aire. Annu. Rev. Immunol. 27, 287–312 (2009). exposed to an inductive skin microenvironment, unambiguously 10. Laster, A. J., Itoh, T., Palker, T. J. & Haynes, B. F. The human thymic demonstrating an increase in potency13. Recent reports indicate that microenvironment: thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation. Differentiation 31, cortical and medullary TECs derive from a common bipotent pre- 67–77 (1986). 18,29 cursor or that the thymus may contain a small population of 11. Heid, H. W., Moll, I. & Franke, W. W. Patterns of expression of trichocytic and epithelial stem cells17,30; our results favour the latter, as suggested epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles. by the extensive self-renewal of cultured TECs and their capacity to Differentiation 37, 137–157 (1988). 12. Hale, L. P. & Markert, M. L. Corticosteroids regulate epithelial cell differentiation incorporate into a three-dimensional epithelial network while and Hassall body formation in the human thymus. J. Immunol. 172, 617–624 expressing MHC-class II and Aire. Several mechanisms could explain (2004). why TECs can acquire skin potency. First, TECs may have an inherent 13. Smith, A. A Glossary of stem-cell biology. Nature 441, 1060 (2006). 981 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

14. Ito, T., Suzuki, A., Imai, E., Okabe, M. & Hori, M. Bone marrow is a reservoir of 27. Moscona, A. Rotation-mediated histogenetic aggregation of dissociated cells. A repopulating mesangial cells during glomerular remodeling. J. Am. Soc. Nephrol. 12, quantifiable approach to cell interactions in vitro. Exp. Cell Res. 22, 455–475 (1961). 2625–2635 (2001). 28. Claudinot, S., Nicolas, M., Oshima, H., Rochat, A. & Barrandon, Y. Long-term 15. Ronfard, V., Rives, J. M., Neveux, Y., Carsin, H. & Barrandon, Y. Long-term renewal of hair follicles from clonogenic multipotent stem cells. Proc. Natl Acad. regeneration of human epidermis on third degree burns transplanted with Sci. USA 102, 14677–14682 (2005). autologous cultured epithelium grown on a fibrin matrix. Transplantation 70, 29. Rossi, S. W., Jenkinson, W. E., Anderson, G. & Jenkinson, E. J. Clonal analysis 1588–1598 (2000). reveals a common progenitor for thymic cortical and medullary epithelium. Nature 16. Sun, T. T., Bonitz, P. & Burns, W. H. Cell culture of mammalian thymic epithelial 441, 988–991 (2006). cells: growth, structural, and antigenic properties. Cell. Immunol. 83, 1–13 (1984). 30. Rodewald, H. R. Thymus organogenesis. Annu. Rev. Immunol. 26, 355–388 (2008). 17. Senoo, M., Pinto, F., Crum, C. P. & McKeon, F. p63 Is essential for the proliferative Supplementary Information is linked to the online version of the paper at potential of stem cells in stratified epithelia. Cell 129, 523–536 (2007). www.nature.com/nature. 18. Bleul, C. C. et al. Formation of a functional thymus initiated by a postnatal epithelial progenitor cell. Nature 441, 992–996 (2006). Acknowledgements We are grateful to M. Nicolas and A. Smith for discussions 19. Hamazaki, Y. et al. Medullary thymic epithelial cells expressing Aire represent a and support, M. Okabe for supplying EGFP rats, J. Roberts, M. Garcia and R. Teisanu unique lineage derived from cells expressing claudin. Nature Immunol. 8, 304–311 for help with cell sorting, J. C. Sarria and T. Laroche for confocal imaging, and (2007). K. Harsman, O. Hagenbu¨chle and S. Pradervand from the DAFL for microarray 20. Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different analyses. Y.B was supported in this work by the Swiss National Science Foundation capacities for multiplication. Proc. Natl Acad. Sci. USA 84, 2302–2306 (1987). (grant 3100A0-104160), the Juvenile Diabetes Research Foundation, the EPFL and 21. Rochat, A., Kobayashi, K. & Barrandon, Y. Location of stem cells of human hair the CHUV. C.B. was supported by a Leukaemia Research Fund grant. The European follicles by clonal analysis. Cell 76, 1063–1073 (1994). Union (EU) supported Y.B. and C.B. through the sixth (EuroStemCell) and seventh 22. Depreter, M. G. et al. Identification of Plet-1 as a specific marker of early thymic (EuroSyStem, OptiStem) framework programmes. A.W.A. was supported by a epithelial progenitor cells. Proc. Natl Acad. Sci. USA 105, 961–966 (2008). fellowship from the Heiwa Nakajima Foundation. 23. Anderson, G., Jenkinson, E. J., Moore, N. C. & Owen, J. J. MHC class II-positive Author Contributions P.B., C.C.B. and Y.B. contributed to the design of the epithelium and mesenchyme cells are both required for T-cell development in the experiments and the interpretation of the results, S.C., A.W.A., A.F. and P.B. thymus. Nature 362, 70–73 (1993). performed experiments, and Y.B. and P.B wrote the paper. 24. Gill, J., Malin, M., Hollander, G. A. & Boyd, R. Generation of a complete thymic microenvironment by MTS24(1) thymic epithelial cells. Nature Immunol. 3, Author Information Raw and normalized microarray data are accessible through 635–642 (2002). the NCBI Gene Expression Omnibus public (http://www.ncbi.nlm.nih.gov/geo) 25. Bennett, A. R. et al. Identification and characterization of thymic epithelial data base (series record GSE21686). Reprints and permissions information is progenitor cells. Immunity 16, 803–814 (2002). available at www.nature.com/reprints. The authors declare no competing financial 26. Sheridan, J. M., Taoudi, S., Medvinsky, A. & Blackburn, C. C. A novel method for interests. Readers are welcome to comment on the online version of this article at the generation of reaggregated organotypic cultures that permits juxtaposition of www.nature.com/nature. Correspondence and requests for materials should be defined cell populations. Genesis 47, 346–351 (2009). addressed to Y.B. ([email protected]).

METHODS mouse (OF1) and incubated in 2% EDTA at 37 uC for 120 min. After rinsing in medium supplemented with 10% BS, the epidermis was gently separated from Animals. Sprague–Dawley rats, which constitutively expressed EGFP (green rat the dermis to form a small pocket with the help of a 30-gauge needle. Donor cells CZ-004’’ SDTgN (act-EGFP)obsCZ-004), were supplied by Japan SLC14. Wild- (5 3 105) were then carefully injected into the pocket and allowed to attach at type Sprague–Dawley (OFA) rats were obtained from Iffa Credo. Athymic (Swiss room temperature for 1 h. Grafts were kept overnight at 4 C before being trans- Nu2/2) and OF1 mice were supplied by Charles River Breeding Laboratories. All u planted onto the back of athymic mice with their dermal side facing the mouse animals were maintained in a 12 h light cycle providing food and water ad libitum. fascia, stitched in place and covered with a skin flap to prevent drying. Flaps Rats were killed by CO inhalation, whereas mice were killed by intra-peritoneal 2 underwent necrosis or were manually opened a few days later, so the graft (i.p.) injection of pentobarbital. Experiments were conducted in accordance with became air-exposed. In rare cases, flaps were removed after 12–15 days. the EU Directive (86/609/EEC) for the care and use of laboratory animals and that Transplanted cells remained on athymic mice for the duration of the experiment, of the Swiss Confederation. Mating of adult female and male rats (age .11 weeks) usually several months. was carried out overnight. Time-pregnant rats were killed by CO inhalation and 2 RT–PCR. Total RNA was isolated from rat TE cultured cells or from dissected uteri with embryos were removed by dissection. thymic lobes using the TRIzol reagent (Invitrogen) according to the manufac- Cell dissociation. Thymic lobes were removed from embryos at E16 and E17 and turer’s instructions. Total RNA was extracted using TRIzol reagent (Invitrogen) from postnatal rats at P0, P1, P7, P12, P32 and P137. Dissected thymic lobes were and amplified using the OneStep RT–PCR kit (Qiagen) or reverse transcribed harvested and kept in cold Hank’s Balanced Salt Solution (HBSS, Gibco– using the Superscript III enzyme with random primers (Invitrogen) and amp- Invitrogen) complemented with 10% Fetal Bovine Serum (FCS, Gibco– lified with the GoTaq PCR reagent kit (Promega) for 35 or 39 cycles. 1 mgof Invitrogen) during dissection. Single lobes were placed in individual 35 mm 21 21 total RNA was reverse-transcribed as detailed in the SuperscriptTM III reverse Petri dishes and washed in HBSS without Ca and Mg twice before being 21 transcriptase kit (Invitrogen). Random primers were used at 100 ng ml dissociated in 0.05% trypsin solution containing 0.1% EDTA at 37 uC. Skin grafts (Invitrogen), the dNTPs mix at 0.5 mM (Qiagen) and the RNasin RNase inhibitor containing GFP-positive hair follicles were carefully dissected from the back of at 2 U ml21 (Promega). PCR products were resolved byagarose gel electrophoresis, recipient nude mice and dissociated in 30 ml of 0.05% trypsin containing 0.1% stained with ethidium bromide, and visualized under ultraviolet light. b-actin EDTA at 37 uC with magnetic stirring for 60 to 120 min. control for equal loading was used throughout the experiments. Primers are listed Cell culture. Hair follicle and TECs were cultured onto a feeder layer of lethally in Supplementary Table 3 and were designed to span at least one intron using 21,28 irradiated 3T3-J2 cells as described . 3T3 cells were cultured in Dulbecco-Vogt Primer3 software. modification of Eagle’s Medium (DMEM, Gibco–Invitrogen) supplemented Quantitative PCR. For Q-PCR, cDNAs were diluted 10 times and 1 ml was with 10% bovine serum (BS) (Hyclone), fed every 2–3 days and serially passed ampli?ed with the Light-Cycler FastStart DNA Master SYBR Green I kit (Roche once a week for a maximum of 12 weeks. 3T3 cells used as feeder layer were Diagnostics), in a 12.5 ml total reaction volume; 1.15 ml of the FastStart Reaction irradiated (60 Gy) and plated at a density of 2.5 3 104 cells cm22. Epithelial cells mix SYBR Green I, 4 mM MgCl2 and 0.5 mM of each primer were included in the were cultured in cFAD medium consisting of a 3:1 mixture of DMEM and Ham’s reaction. Rat primers were designed using the Light-Cycler Probe Design program F-12 medium (Gibco–Invitrogen). Supplements were as follows: 10% FCS from Roche (temperature of annealing set at 60 uC) and Microsynth. The Q-PCR (Gibco), hydrocortisone 0.4 mgml21 (Calbiochem, VWR Int.), 1026 M cholera 21 29 program consisted of hot start enzyme activation at 95 uC for 15 min, 40 cycles of toxin (Sigma,), 5 mgml insulin (Sigma), 2 3 10 M 3,39,5-triiodo-L-thyronin amplification at 95 uC for 10 s, 55 uCor60uC for 5 s and 72 uC for 6 s. Finally, to (T3) (Sigma). Recombinant human epidermal growth factor rhEGF (QED) was 21 obtain the melting curve, the analysis at 65 uC for 15 min was followed with a added at the first feeding (10 ng ml ). All cultures were incubated at 37 uCina cooling cycle to reach 40 uC for 30 s. For the data analysis, the rat hypoxanthine- 10% CO2 atmosphere, and the medium was changed twice a week. Cultures were guanine phosphoribosyltransferase (HPRT) housekeeping gene was used as passaged once a week. internal control. Induction values were calculated using the Roche RelQuant 31 Single cell isolation. Single cells were isolated as described . Briefly, individual analysis software. List of primers is given in Supplementary Table 4. cells were aspirated into a Pasteur pipette under a Zeiss Axiovert inverted micro- Microarrays. Quantitative analysis of RNA expression was performed using scope using a 103 objective and immediately inoculated into a 35 mm Petri dish Affymetrix gene chip cDNA microarrays (Affymetrix). Cultivated cells sorted already containing lethally irradiated 3T3 cells. Cultures were fed every 3–4 days on the basis of GFP expression were used to prepare total RNA using the RNeasy with cFAD medium. Clones were passaged and expanded once a week. Mini Kit (Qiagen). RNA quality was measured using the Agilent Bioanalyzation Colony-forming efficiency. Hundred cells were plated onto a 60 mm Petri dish system (Agilent Technologies), to ensure the integrity of the RNA. cDNA syn- containing lethally irradiated 3T3 cells and cultured for 12 days as described thesis, hybridization to the Affymetrix GeneChip Rat Expression Array 230 2.0 before they were fixed in formalin and stained in 1% rhodamine-B. Colonies (31,042 gene-level probe sets) or Rat Gene ST 1.0 Array (27,342 gene-level probe were scored under a dissecting microscope. sets) and analysis was performed by the Lausanne DNA Array Facility (DAFL) Thymic aggregates. Thymic aggregates were obtained following a compaction using standard protocols. To identify differentially expressed genes from each protocol26, modified as follows. Briefly, E13.5/E14.5 mouse embryos (OF1) or group, P values were calculated using Bioconductor limma package32 and probe E16/E17 rat embryos (SD) were harvested from the uteri under sterile condi- sets with a false discovery rate33 ,0.05 were considered significant. tions, transferred in 100 mm Petri dishes containing 10% BS-DMEM medium Immunostaining. Immunohistochemistry (IHC) and immunocytochemistry and stored on ice. Thymic lobes were removed from the embryonic chests and (ICC) were performed on tissue sections or cultured cells. For tissue sections, immerged into cold HBSS supplemented with 10% FCS. Subsequently, the lobes entire tissue was embedded in OCT compound (Tissue-Tek), frozen in 240 uC 21 21 were washed in HBSS without Ca and Mg and transferred into a solution methyl-butane. Sections (8-mm) were cut on a Leica CM3000 cryostat. Cells were 21 containing collagenase/dispase (1 mg ml ) and 0.05% DNase for 30 min. The cultured onto glass coverslips. Tissues or cells were fixed in 4% paraformalde- lobes were then dissociated in a trypsin solution (0.05%) containing 0.1% EDTA hyde. Aspeci?c sites were blocked with 5% goat serum/PBS (for extracellular until a single cell suspension was obtained. Embryonic thymic cells were mixed epitopes) or 5% goat serum/PBS/0.1% triton (for intracellular epitopes). with cultured thymic cells at different ratios: 6–8 dissociated embryonic lobes Tissues or slides were incubated with primary antibodies diluted in blocking 5 5 5 with 10 to 2 3 10 cultured TECs or HF cells expressing GFP; 10 mouse fibro- buffer overnight at 4 uC. Secondary antibodies were Alexa-Fluor568 or Alexa- blasts were added as carrier cells as described23. To obtain aggregates, cells were Fluor488 conjugated (Molecular Probes, Invitrogen) and used at 1:400 dilution. aspirated into a 200 ml pipette and centrifuged at 1,300 r.p.m. for 3 min (ref. 26). Nuclei were stained with Hoechst 33432 (1026 M). The DABCO solution Aggregates were then transferred on a 0.8 mm Isopore membrane filter (Sigma) with glycerol (Merck) in PBS was used for mounting. The following (Millipore) and incubated at 37 uC for 24 h before being grafted under the kidney primary antibodies were used: rabbit anti-keratin 5 (1:500; Abcam), rabbit anti- capsule of athymic mice. keratin 14 (1:500; Covance), mouse anti-LEKTI (1:100; Zymed), mouse anti- Skin aggregates. Aggregates were obtained as described27 with modifications. keratin 18 (1:100; Progen), mouse anti-p63 (1:50; Dako), mouse anti-involucrin Briefly, the skin was dissected under sterile conditions from the back of E13.5 and (1:100; Lab Vision), guinea pig anti-keratin 8 (1:100; Neomarkers); mouse anti- E14.5 mouse embryos and dissociated in 0.05% trypsin containing 0.1% EDTA MHCclassII (1:400; Abcam); rabbit anti-GFP (1:400; Abcam); rabbit anti-Ki67 at 37 uC until a single cell suspension was obtained. Dissociated cells were filtered (1:400; Abcam); rabbit anti-pankeratin (1:400; Dako); goat anti-Aire D-17 through a 70 mm cell-strainer and counted. 4 3 105 skin cells were mixed with (1:100 Santa Cruz); rabbit anti-Aire M300 (1:100; Santa Cruz); chicken anti- (1–2) 3 105 cultured TECs or HF cells and plated in individual 96-well V-bottom GFP (1:500; Abcam); mouse anti-rat MHC Class II (1:200; eBiosciences). plate. Cells were aggregated in the plate by centrifugation (1,300 r.p.m., 10 min). Histology. Cryosections of thymic aggregates (8 mm) were stained with Aggregates were kept at 37 uC in a 10% CO2 atmosphere for 24 h before being haematoxylin-eosin. grafted under the kidney capsule of athymic mice. Karyotyping and FISH analyses. Chrombios (Raubling) performed karyotype Skin morphogenetic assay. Cultured TECs or hair follicle multipotent stem cells and FISH analyses. Rat genomic DNA was labelled with carboxytetramethyl- were grafted as described28. Briefly, a piece of skin was obtained from a newborn rhodamine-dUTP (red fluorescence) by degenerate oligonucleotide primer

PCR, and mouse satellite DNA was labelled with FITC-dUTP (green fluor- washed in HBSS and resuspended in HBSS containing the AlexaFluor 647- escence). 8 mm tissue sections were incubated at 50 uC for 1 h and then pepsin conjugated goat anti-mouse IgG1 antibody (Molecular Probes, Invitrogen). 2 treated (200 ml 0.01 N HCl/2 ml pepsin (7.5 u ml 1)) at 37 uC for 12 min. Sections Cells were finally incubated at 4 uC for 15 min before they were washed with were washed in 23 SSC for 20 min, then dehydrated in graded ethanol and dried ice-cold HBSS. Proper isotype control (mouse IgG1) was used as a negative at 50 uC. For FISH, 3 mg of each mouse and rat genomic DNA were used for control. Cells were resuspended in HBSS with 10% FCS, filtered using a 70-mm labelling by a standard nick translation procedure (90 min, 15 uC). Rat DNA was cell strainer (Millipore), and sorted by flow cytometry using a fluorescence- labelled with TAMRA-dUTP, and mouse DNA with FITC-dUTP at the concen- activated cell sorter (FACS) DIVA (BD Biosciences). Clean separation between tration of 20 mM for each dUTP. 300 ng of each probe was combined, precipi- EpCAM1 and EpCAM2 cell populations were confirmed by second FACS ana- tated and resolved in 15 ml hybridization buffer (50% formamide, 23 SSC, 10% lysis. Dead cells were excluded by addition of propidium iodide (Sigma) and dextran sulphate). Probes and sections were denatured together on a hot plate gating on the negative cells. Fluorescent staining for Ulex europaeus agglutinin I (72 uC) for 8 min. Hybridization was performed at 37 uC in a humid chamber (UEA-1) was performed using biotinylated UEA-1 (1:800, Vector Laboratories) overnight. Sections were then washed in 23 SSC at room temperature for 10 min and secondary reagent was Streptavidin-PE/Cy7 (1:100; Biolegend). followed 70 uC for 1 min in 0.43 SSC/Tween, before they were stained with Image acquisition and processing. Images were acquired under inverted fluor- DAPI. Sections were examined with a Axioplan II fluorescence microscope escence microscope Axiovert 200 or the Leica Sp2 confocal upright and inverted (Zeiss) equipped with a black-and-white CCD camera. Image capture was per- microscopes. Confocal images and three-dimensional reconstruction were pro- formed with SmartCapture software (Digital Scientific) using appropriate fluor- cessed using Imaris 6.2 software. escence filters (ChromaTechnology). Flow cytometry. Thymi were obtained from rats and dissociated into single cells 31. Barrandon, Y. & Green, H. Cell size as a determinant of the clone-forming ability of as described above. Cells were resuspended in cold HBSS supplemented with human keratinocytes. Proc. Natl Acad. Sci. USA 82, 5390–5394 (1985). 10% FCS and were kept on ice. Cells were centrifuged at 4 uC for 10 min at 32. Smyth, G. K. Linear models and empirical Bayes methods for assessing differential 1,500 r.p.m., resuspended in HBSS containing 10% FCS and blocked at 4 uC expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, article 3 (2004). for 15 min. Cells were then incubated with a mouse anti-EpCAM antibody 33. Hochberg, Y. & Benjamini, Y. More powerful procedures for multiple significance (1:100, Biovendor) for 30 min on ice, centrifuged at 1,100 r.p.m. for 10 min, testing. Stat. Med. 9, 811–818 (1990).

Trans-acting small RNA determines dominance relationships in Brassica self-incompatibility

Yoshiaki Tarutani1{, Hiroshi Shiba1, Megumi Iwano1, Tomohiro Kakizaki2{, Go Suzuki3, Masao Watanabe4, Akira Isogai1 & Seiji Takayama1

A diploid organism has two copies of each gene, one inherited from However, the molecular mechanisms underlying this monoallelic each parent. The expression of two inherited alleles is sometimes de novo methylation have yet to be elucidated. biased by the effects known as dominant/recessive relationships, sRNAs are known to be important in gene silencing14,15. For instance, which determine the final phenotype of the organism. To explore small interfering RNAs (siRNAs) regulate target genes not only by the mechanisms underlying these relationships, we have examined degrading or sequestering the target transcripts but also by inducing the monoallelic expression of S-locus protein 11 genes (SP11), the de novo methylation of the homologous DNA region by RNA- which encode the male determinants of self-incompatibility in directed DNA methylation. The methylated 59 regions of recessive Brassica. We previously reported that SP11 expression was mono- SP11 alleles show little homology with those of dominant SP11 alleles allelic in some S heterozygotes, and that the promoter regions of (Supplementary Fig. 2), whereas the SP11 59 sequences in either domi- recessive SP11 alleles were specifically methylated in the anther nant or recessive S haplotypes are relatively conserved1,2,16.Wethere- tapetum1–3. Here we show that this methylation is controlled by fore speculated that monoallelic methylation at recessive SP11 trans-acting small non-coding RNA (sRNA). We identified inverted promoters in S heterozygotes might occur in trans by the action of genomic sequences that were similar to the recessive SP11 promo- dominant SP11-allele-specific sRNAs localized within or around the ters in the flanking regions of dominant SP11 alleles. These S locus, and that these sRNAs might mediate the methylation of the sequences were specifically expressed in the anther tapetum and recessive SP11 alleles only, not the alleles with which they are associated. processed into 24-nucleotide sRNA, named SP11 methylation To address this hypothesis, we performed in silico searches for possible inducer (Smi). Introduction of the Smi genomic region into the sRNAs with sequence similarity to the target methylated regions from recessive S homozygotes triggered the methylation of the promoter the 76-kilobase (kb) and 75-kb SP11 genomic regions of the dominant 5,17 of recessive SP11 alleles and repressed their transcription. This is S9 and S12 haplotypes, respectively . As a result, the sequences with an example showing sRNA encoded in the flanking region of a homology to the target methylated regions, tentatively named SP11- dominant allele acts in trans to induce transcriptional silencing methylation-inducing region (SMI), were found 3.6 and 3.0 kb down- of the recessive allele. Our finding may provide new insights into stream of the 39 untranslated region of S9-S-locus glycoprotein (SLG) the widespread monoallelic gene expression systems. and S12-SLG, respectively, within the S-locus region in which the Self-incompatibility systems have been exploited in many angio- recombination was shown to be suppressed (Fig. 1a)18.TheSMI sperms in the balance between reproductive assurance and inbreed- sequences suggested that these putative sRNAs that were highly similar ing depression4. In most species the self/non-self recognition in to the target-methylated regions could be processed from the predicted self-incompatibility is controlled by a large number of haplotypes imperfect stem-loop precursors (Fig. 1b). Results from genomic PCRs (S1, S2,…,Sn) at the S locus. The self-incompatibility response occurs showed that the corresponding structures found in S9 and S12 haplo- when pollen and pistil share the same S haplotype. In Brassica, each S types were also present in other dominant S8 and S52 haplotypes haplotype encodes the pollen-borne ligand named SP11 (also named (Supplementary Fig. 3). 5–7 S-locus cysteine-rich (SCR)) and its stigmatic receptor, S-receptor To examine whether the S9-SMI region is actually expressed in kinase (SRK)8,9; their S haplotype-specific interaction triggers a self- anthers, we performed 59 and 39 rapid amplification of complementary incompatibility response in the stigma epidermis10,11. SP11 is DNA ends (RACE) PCR on polyadenylated RNA samples prepared expressed in the anther tapetum, a sporophytic tissue. The self- from anthers of S9S9 homozygotes. A 2.0-kb transcript that was pre- incompatibility phenotype in pollen is therefore determined by the dicted to be 59-capped and polyadenylated, and to form an imperfect dominance relationships between the two S haplotypes carried by the stem-loop structure, was identified (Supplementary Fig. 4). Reverse- plant (Supplementary Fig. 1). On the basis of these relationships, the transcriptase-mediated polymerase chain reaction (RT–PCR) analysis S haplotypes in Brassica have been broadly classified into two groups: showed that this transcript was present in the anthers at early stages of pollen-dominant S haplotypes (class I) and pollen-recessive S haplo- development when the tapetum cells were fully intact, but not in 2,3,12,13 types (class II) . Pollen-dominant S haplotypes (such as S8, S9, stigmas or leaves (Fig. 2a). S12 and S52 in Brassica rapa) are generally co-dominant, and they are We then screened a sRNA cDNA library from anther tissues of S9S9 almost always dominant over recessive S haplotypes (such as S44, S60, homozygotes by using a biotinylated oligonucleotide probe specific 3,13 S40 and S29) .InS heterozygotes with dominant and recessive for the target-methylated region of the S40-SP11 promoter, and S haplotypes, the expression of the recessive SP11 allele is silenced sequenced the clones obtained. We obtained a 24-nucleotide sRNA as a result of tapetum-specific de novo cytosine methylation in its presumably derived from the identified S9-SMI transcript, desig- 1 promoter region just before the initiation of SP11 transcription . nated Smi, which showed nearly perfect homology to the S40-SP11

1Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan. 2Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan. 3Division of Natural Science, Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashiwara, Osaka 582-8582, Japan. 4Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. {Present addresses: Department of Integrated Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411- 8540, Japan (Y.T.); Vegetable Breeding Research Team, National Institute of Vegetable and Tea Science, Ano, Mie 514-2392, Japan (T.K.). 983 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a a b S 9

5 kb Anther Stigma Leaf S -SMI SLG SP11 SRK 9 transcript S12 GAPA

P b T S P T 9 U G C A G G -UUU c AUGCA UAAUCGUUUUGU ACU UUUUACAC U AA CAUAGAA U + UACGU AUUAGCAAAACA UGA AAAAUGUG A UU GUAUCUU U -Smi+ -Smi G U U C U - UGGG 9 9 /S / /S / S 60 S 60 S 60 S 60 S 60 S 60 S 9 -SmiT10A+ S 9 -SmiT10A+ S 9 9 12 9 60 60 9 60 60 U G C A G - UU S S 60 S S S S 60 S S S 26 nt S 26 nt AUGCA UAAUCGUUUUGU ACU UUUUACAC UAAA CAUA AAAU G UACGU AUUAGCAAAACA UGA AAAAUGUG AUUU GUAU UUUA U 22 nt 22 nt G U U C - C GG 100 25736 100 8 48 49 5S rRNA 5S rRNA c S Smi 5′ AUGUUUACGUGUAAAAUAGUUACA 3′ 9- (class I) Figure 2 | Expression analyses of Smi.a, RT–PCR analysis of S9-SMI S Smi 5′ AUGUUUACGUGUAAAAUAGUUACA 3′ 7 52- (class I) transcripts. RNAs from anthers (mixtures of stages 2–4) , stigmas and leaves were subjected to RT–PCR. Expression of the glyceraldehyde-3-phosphate S SP11 –158 AATGTTTACGTGTGAAATAGGCAATT –133 40- S SP11 –119 AATGTTTACGTGTGAAATAGGCAATT –94 dehydrogenase A-subunit gene (GAPA) was used as a control. b, In situ 44- S SP11 –120 ATTGTTTACGTGTAAAATAGGCAATT –95 hybridization for S9-Smi. Left: sections of anther (stage 2) from S9S9 60- S -SP11 –118 AATGTTTACGTGTGAAATAGGCAATT –93 homozygote were probed with labelled S9-Smi antisense probe. Right: a 29 competition experiment using a tenfold excess of unlabelled probe is shown S Smi 5′ AUGUUUACGAGUAAAAUAGUUACA 3′ 60- (class II) as a control. Scale bar, 20 mm. T, tapetum cells; P, pollen grains. c, Small- S Smi 5′ AUGUUUACGAGUAAAAUAGUUACA 3′ 29- (class II) RNA gel-blot analysis. Small RNAs from anthers (mixtures of stages 1–3) of S S homozygote, S S homozygote, S -Smi transformant (S S /S - Figure 1 | Smi sequences homologous to the methylated region of the 9 9 60 60 9 60 60 9 Smi1) and S -SmiT10A transformant (S S /S -SmiT10A1) were recessive SP11 alleles. a, Location of SMI in the S-locus regions of the 9 60 60 9 hybridized with locked nucleic-acid-modified dominant (left) and recessive dominant S and S haplotypes. Asterisks denote positions of SMI. b,Stem- 9 12 (right) Smi antisense probes. Ethidium bromide-stained 5S rRNA is shown loop precursors predicted from SMI sequences. Sequences homologous to the as a loading control. Numbers below blots represent quantified and target-methylated regions are underlined. c, Sequence alignment of the normalized signal intensities. nt, nucleotides. identified dominant (class I: S9 and S52) Smi sequences, the methylated recessive SP11 59 regions and the identified recessive (class II: S60 and S29) Smi To confirm that dominant Smi triggers monoallelic DNA methyla- sequences. The box indicates the highlyhomologous region between dominant tion of recessive SP11 promoters, we generated transgenic B. rapa Smi sequences and recessive SP11 alleles (18 out of 19 nucleotides identical). expressing dominant S -Smi. We first amplified 3.2-kb and 1.7-kb Red, mismatched bases compared with dominant Smi sequences in the box. 9 genomic sequences containing the S9-Smi (Supplementary Fig. 4b), 59 methylated region (2157 to 2139, where the translation start site and introduced them into B. rapa cv. Osome, an S52S60 heterozygous 9,20 is at 11) (18 out of 19 nucleotides; Fig. 1c). S9-Smi was also highly hybrid line susceptible to Agrobacterium-mediated transformation . homologous to the target methylated region of the other recessive One and two transgenic plants containing the 3.2-kb and 1.7-kb trans- SP11 alleles (18 out of 19 nucleotides for S29-SP11, S60-SP11 and S44- genes were obtained, respectively. These transformants showed the S52 SP11; Fig. 1c). Furthermore, a sRNA identical to S9-Smi was also self-incompatibility phenotype in pollen, as was observed in the obtained from the other dominant S52S52 homozygotes. In situ mother plant Osome (S52 is dominant over S60), suggesting that Smi hybridization analysis showed that S9-Smi existed predominantly did not affect the dominant (class I) self-incompatibility phenotype. in anther tapetum cells at the early uninucleate pollen stage (stage 2 They were then pollinated with pollen from recessive S homozygotes 7 in our classification ) (Fig. 2b). It is plausible that initiation of Smi (S44S44, S60S60, S40S40 and S29S29) and subsequently self-pollinated and transcription occurs before monoallelic methylation in the promoter selected for various recessive S homozygotes carrying the S9-Smi region of the recessive SP11 allele, which becomes highly methylated transgene. Because the progenies of the three transgenic plants in the latter half of stage 2 (ref. 1). obtained showed the same phenotype irrespective of the transgene Contrary to our expectation, a homologous sequence to Smi from size, hereafter we show only the data for the two transgenic plants dominant S haplotypes was also found in recessive S60 haplotypes carrying the 1.7-kb S9-Smi transgene (S9-Smi1 1 and 2). All of the 9 kb downstream of S60-SP11 (Supplementary Fig. 5a). The asso- segregants with S9-Smi transgenes (S9-Smi1), but none of those ciated 24-nucleotide sRNA, S60-Smi, was obtained from anthers of without transgenes (S9-Smi-null), became fully self-compatible irre- the S60S60 homozygote. S60-Smi contained a base substitution spective of the S haplotypes (Supplementary Fig. 6), suggesting that (URA) at position 10, a site known to be important for the S9-Smi transgene interfered with the self-incompatibility of all microRNA (miRNA)-mediated cleavage of target transcripts19. recessive S-homozygotes tested. To investigate the site of self-incom- This substitution decreased its similarity to the target methylated patibility interference, reciprocal pollinations were performed region (17 out of 19 nucleotides; Fig. 1c) compared with the Smi between wild-type S60S60 homozygotes and the plants carrying the variant from the dominant S haplotypes. Corresponding sequences S9-Smi transgene (S60S60/Smi1). The self-incompatibility pheno- were also found in other recessive S40S40 and S29S29 homozygotes type of the stigmas was not altered in the S60S60/S9-Smi1 plants (Supplementary Fig. 5b), but not in S44S44 homozygote. To verify (Supplementary Fig. 7), whereas their pollen grains lost the S60 self- the expression levels of the dominant and the recessive Smi variants, incompatibility phenotype and became compatible with the stigmas we performed small RNA gel-blot analyses. As shown in Fig. 2c, the of S60S60 homozygotes (Fig. 3a). The pollen grains of the S9-Smi- dominant S9-Smi was highly expressed in anthers, whereas the reces- null segregants (S60S60/Smi-null), however, did not change the self- sive S60-Smi was hardly detected. These data led us to propose that a incompatibility phenotype (Supplementary Fig. 7). The loss of the highly expressed dominant Smi with high similarity to its target self-incompatibility phenotype in pollen was also observed in other induces promoter methylation and repressed expression of the recessive S44S44, S40S40 and S29S29 homozygotes carrying the S9-Smi recessive SP11 alleles. transgene (Supplementary Tables 1–3). 984 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS a states of recessive S -SP11 in S S /S -Smi1 transformants and Pollen 60 60 60 9 S S S S S -Smi+ S S S -SmiT10A S60S60/S9-Smi-null segregants with the use of bisulphite sequencing. Stigma 60 60 60 60/ 9 1 60 60/ 9 + We examined 400 base pairs of the S60-SP11 59 region, which had been shown to be highly methylated in the dominant/recessive S hetero- zygotes1. Significant cytosine methylation was observed at all CpG, S S 60 60 CpNpG and CpNpN sequence contexts, spreading from the Smi homologous region in S60S60/S9-Smi1 plants, whereas a similar PT methylation profile was not detected in the S9-Smi-null segregants (Fig. 3b). The methylation profile of the S60-SP11 59 region in S60S60/S9-Smi1 plants was nearly identical to that observed in b 1 100 S52S60 heterozygotes . Analyses of S29S29, S40S40 and S44S44 homozy- S S gotes carrying the S9-Smi transgene also showed significant increases 60 60 in DNA methylation at the SP11 promoter, which was accompanied 0 100 by a decrease in SP11 transcript levels (Supplementary Tables 1–3 and S S 60 60/ Supplementary Fig. 8). Together, these results indicate that Smi from a S -Smi 9 -null dominant S haplotype represses recessive SP11 transcription by trig- 0 gering monoallelic de novo methylation in its promoter in dominant/ 100 S S recessive S heterozygotes. 60 60/ S -Smi+ 9 1 A recent report has suggested that DNA methylation of miRNA 0 target loci in moss is dependent on miRNA dosage21. To determine 100 S S whether the inability of recessive S60-Smi to induce silencing of its 60 60/ S -Smi+ 9 2 own S60-SP11 also depends on its expression level, we generated 0 transformants carrying S9-Smi transgenes with a recessive-type point S S 100 Methylated cytosine (%) 60 60/ mutation at the tenth nucleotide position (S9-SmiT10A). Pollen S -SmiT10A+ 9 grains from S60S60 homozygotes carrying the S9-SmiT10A transgene 0 (S60S60/S9-SmiT10A1) did not lose the S60 self-incompatibility 100 phenotype, even though the expressed recessive Smi was much higher S S 52 60 than the endogenous S60-Smi in S60S60 homozygotes and almost 0 comparable to the dominant Smi in S60S60/S9-Smi1 transformants (Figs 2c and 3a). Furthermore, the expression level of S -SP11 and –400 –1 60 S SP11 ′ the methylation profile of the S -SP11 59 region in S S /S - 60- 5 region 60 60 60 9 SmiT10A1 transformants were similar to those in S60S60 homozy- Figure 3 | Effect of S9-Smi transgene on self-incompatibility phenotype and gotes (Fig. 3b and Supplementary Table 4). These results suggest that DNA methylation. a, Representative results of pollination tests. Stigmas of the inability of recessive Smi to repress expression of the recessive S60S60 homozygote were pollinated with pollen from S60S60 homozygote, S9- SP11 alleles is due to the one-base mismatch to the target at nucleo- Smi transformant (S S /S -Smi1 1) or S -SmiT10A transformant (S S / 60 60 9 9 60 60 tide position 10. S9-SmiT10A1). A bundle of pollen tubes (PT) indicates compatible pollination (arrow). b, S60-SP11 DNA methylation profile in the anther Here, we have shown that Smi located in the flanking region of tapetum. Tapetum DNA was extracted from anthers (mixtures of stages 3–5) a dominant SP11 allele acts in trans to control the pollen self- of S60S60 homozygote, null segregants (S60S60/S9-Smi-null), S9-Smi incompatibility dominant/recessive relationship through transcrip- transformants (S60S60/S9-Smi1 1 and S60S60/S9-Smi1 2), S9-SmiT10A tional gene silencing of the recessive SP11 allele. This is different from transformant (S60S60/S9-SmiT10A1) and S52S60 heterozygotes and the well-known dominant/recessive process, in which dominant and subjected to bisulphite sequencing analysis. Percentages of methylation at all recessive alleles encode ‘functional’ and ‘dysfunctional’ proteins, cytosine residues in the S -SP11 59 region (nucleotides 2400 to 21) are 60 respectively. Rather, the mechanism identified seems to be similar shown in the histograms. The results are from 30 cloned sequences. Black, CpG; blue, CpNpG; red, CpNpN (where N is A, T or C). Green, the region to that of paramutation, which governs non-Mendelian inheritance homologous to Smi. in maize, although whether DNA methylation directly causes silencing remains controversial22–25. Recent studies have suggested the dynamic We performed real-time PCRs to quantify S60-SP11 transcript nature of DNA methylation in plant and animal development26.We levels in S60S60/S9-Smi1 plants and found that the expression levels have reported the first example of trans-acting sRNAs controlling 25 of S60-SP11 were decreased (9–50) 3 10 -fold compared with wild- Mendelian inheritance in dominant/recessive relationships. type S60S60 plants and S60S60/S9-Smi-null segregants (Table 1). These We have previously described linear dominance relationships data confirm that the loss of the self-incompatibility phenotype in between pollen-recessive S haplotypes1,3. Methylation patterns of pollen resulted from the repression of S60-SP11 expression by the S9- recessive SP11 alleles in pollen-recessive S heterozygotes (plants Smi transgene. carrying two pollen-recessive S haplotypes, with one dominant over To examine whether DNA methylation was involved in this repres- the other) were nearly identical to those found in pollen-dominant sion, we extracted anther tapetum DNA and analysed the methylation and pollen-recessive S heterozygotes. Recently, the intergenic regions of recessive SP11 genes have been shown to contain some short Table 1 | Characterization of the S60S60 homozygotes carrying S9-Smi repetitive sequences with high sequence similarity to the target Pollen parent (anther) S60-SP11 expression* Pollen-tube growth on S60S60 stigma{ methylated region of the other recessive SP11 genes27. It is therefore S60S60 1.0 6 0.35 ,5 (incompatible) possible that structural genomic differences around the repetitive S60S60/S9-Smi-null 1.4 6 0.30 ,5 (incompatible) 25 regions of the recessive SP11 alleles might also mediate dominance S60S60/S9-Smi1 1 (8.9 6 2.7) 3 10 .200 (compatible) 25 relationships between them. S60S60/S9-Smi1 2 (51 6 2.5) 3 10 .200 (compatible) Recent studies reported that autosomal genes frequently show allele- * RNA from anthers (stages 3–5) of each plant was subjected to quantitative real-time PCR. The results (means 6 s.d., n 5 3) are shown relative to the S60-SP11 expression level of wild-type specific expression, although the underlying molecular mechanisms 28–30 S60S60 homozygotes. are unknown . This widespread monoallelic expression contributes { Pollen grains collected from each plant were used to pollinate stigmas of S60S60 homozygotes. The data represent the number of pollen tubes penetrating into the stigma. At least 12 to diversity in gene expression and phenotypic variation. Our find- pollinations were monitored for each cross. ing that sRNA acts in trans in heterozygous genomes to regulate 985 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010 transcriptional gene silencing through DNA methylation provides new 17. Shiba, H. et al. Genomic organization of the S-locus region of Brassica. Biosci. Biotechnol. Biochem. 67, 622–626 (2003). insights into monoallelic transcriptional control. 18. Casselman, A. L. et al. Determining the physical limits of the Brassica S locus by recombinational analysis. Plant Cell 12, 23–33 (2000). METHODS SUMMARY 19. Franco-Zorrilla, J. M. et al. Target mimicry provides a new mechanism for Genomic sequences homologous to the methylated regions of recessive SP11 regulation of microRNA activity. Nature Genet. 39, 1033–1037 (2007). alleles were identified in silico. Expression of this genomic region was examined 20. Shiba, H. et al. Alteration of the self-incompatibility phenotype in Brassica by by RT–PCR and in situ hybridization. Smi clones were obtained from anther transformation of the antisense SLG gene. Biosci. Biotechnol. Biochem. 64, 1016–1024 (2000). small RNA cDNA libraries enriched by the target recessive SP11 promoter 21. Khraiwesh, B. et al. Transcriptional control of gene expression by microRNAs. Cell region. Expression levels of Smi were evaluated by small RNA gel-blot analyses. 140, 111–122 (2010). The function of dominant Smi was examined by introducing S9-Smi genomic 22. Alleman, M. et al. An RNA-dependent RNA polymerase is required for regions into recessive S homozygous plants. Cytosine methylation of recessive paramutation in maize. Nature 442, 295–298 (2006). SP11 59 regions were analysed by bisulphite sequencing with genomic DNA 23. Erhard, K. F. Jr et al. RNA polymerase IV functions in paramutation in Zea mays. isolated from anther tapetum. Science 323, 1201–1205 (2009). 24. Stam, M., Belele, C., Dorweiler, J. E. & Chandler, V. L. Differential chromatin Full Methods and any associated references are available in the online version of structure within a tandem array 100 kb upstream of the maize b1 locus is the paper at www.nature.com/nature. associated with paramutation. Genes Dev. 16, 1906–1918 (2002). 25. Hale, C. J., Stonaker, J. L., Gross, S. M. & Hollick, J. B. A novel Snf2 protein Received 10 September 2009; accepted 24 June 2010. maintains trans-generational regulatory states established by paramutation in maize. PLoS Biol. 5, e275 (2007). 1. Shiba, H. et al. Dominance relationships between self-incompatibility alleles 26. Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA controlled by DNA methylation. Nature Genet. 38, 297–299 (2006). methylation patterns in plants and animals. Nature Rev. Genet. 11, 204–220 2. Shiba, H. et al. The dominance of alleles controlling self-incompatibility in Brassica (2010). pollen is regulated at the RNA level. Plant Cell 14, 491–504 (2002). 27. Kakizaki, T. et al. Comparative analysis of the S-intergenic region in class-II S 3. Kakizaki, T. et al. Linear dominance relationship among four class-II S haplotypes haplotypes of self-incompatible Brassica rapa (syn. campestris). Genes Genet. Syst. in pollen is determined by the expression of SP11 in Brassica self-incompatibility. 81, 63–67 (2006). Plant Cell Physiol. 44, 70–75 (2003). 28. Guo, M. et al. Allelic variation of gene expression in maize hybrids. Plant Cell 16, 4. Darwin, C. The Effects of Cross and Self Fertilisation in the Vegetable Kingdom 1707–1716 (2004). (Murray, 1876). 29. Wang, J., Valo, Z., Smith, D. & Singer-Sam, J. Monoallelic expression of multiple 5. Suzuki, G. et al. Genomic organization of the S locus: identification and genes in the CNS. PLoS ONE 2, e1293 (2007). characterization of genes in SLG/SRK region of S9 haplotype of Brassica campestris 30. Milani, L. et al. Allele-specific gene expression patterns in primary leukemic cells (syn. rapa). Genetics 153, 391–400 (1999). reveal regulation of gene expression by CpG site methylation. Genome Res. 19, 1–11 6. Schopfer, C. R., Nasrallah, M. E. & Nasrallah, J. B. The male determinant of self- (2009). incompatibility in Brassica. Science 286, 1697–1700 (1999). Supplementary Information is linked to the online version of the paper at 7. Takayama, S. et al. The pollen determinant of self-incompatibility in Brassica www.nature.com/nature. campestris. Proc. Natl Acad. Sci. USA 97, 1920–1925 (2000). 8. Stein, J. C., Howlett, B., Boyes, D. C., Nasrallah, M. E. & Nasrallah, J. B. Molecular Acknowledgements We thank Q. A. Ngo and P. Kaothien-Nakayama for critical cloning of a putative receptor protein kinase gene encoded at the self- comments and discussion; K. Nakashima for helpful comments; and H. Ichikawa, incompatibility locus of Brassica oleracea. Proc. Natl Acad. Sci. USA 88, 8816–8820 T. Ohnishi, F. Yamamoto, M. Kawashima and R. Nagai for technical assistance. This (1991). work was supported by the Program for Promotion of Basic Research Activities for 9. Takasaki, T. et al. The S receptor kinase determines self-incompatibility in Brassica Innovative Biosciences (to S.T.) from the Bio-oriented Technology Research stigma. Nature 403, 913–916 (2000). Advancement Institution (BRAIN), by Grants-in-Aid for Creative Scientific 10. Kachroo, A., Schopfer, C. R., Nasrallah, M. E. & Nasrallah, J. B. Allele-specific Research (to A.I., M.W. and S.T.) from the Japan Society for the Promotion of receptor-ligand interactions in Brassica self-incompatibility. Science 293, Science (JSPS), and by Grants-in-Aid for Scientific Research on Priority Area (to 1824–1826 (2001). H.S., M.W. and S.T.) and Grants-in-Aid for the Global Center of Excellence Program 11. Takayama, S. et al. Direct ligand–receptor complex interaction controls Brassica to NAIST from the Ministry of Education, Culture, Sports, Science and Technology self-incompatibility. Nature 413, 534–538 (2001). (MEXT). 12. Nasrallah, J. B. & Nasrallah, M. E. Pollen–stigma signaling in the sporophytic self- Author Contributions Y.T. designed and performed the experiments, and wrote incompatibility response. Plant Cell 5, 1325–1335 (1993). the paper. H.S., A.I. and S.T. initiated the study, provided advice regarding the 13. Hatakeyama, K., Watanabe, M., Takasaki, T., Ojima, K. & Hinata, K. Dominance experiments, and wrote the paper. M.I. performed the in situ hybridization. T.K., relationships between S-alleles in self-incompatible Brassica campestris L. Heredity G.S. and M.W. provided plant materials and performed the sequence analyses of 80, 241–247 (1998). the S locus. 14. Carthew, R. W. & Sontheimer, E. J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009). Author Information Sequence data have been deposited in GenBank under 15. Voinnet, O. Origin, biogenesis, and activity of plant microRNAs. Cell 136, accession numbers GQ473178–GQ473187 and GQ847769. Reprints and 669–687 (2009). permissions information is available at www.nature.com/reprints. The authors 16. Shiba, H. et al. A pollen coat protein, SP11/SCR, determines the pollen S-specificity declare no competing financial interests. Readers are welcome to comment on the in the self-incompatibility of Brassica species. Plant Physiol. 125, 2095–2103 online version of this article at www.nature.com/nature. Correspondence and (2001). requests for materials should be addressed to S.T. ([email protected]).

urea. The small RNAs were transferred by overnight capillary blotting in 20 3 SSC METHODS 1 Identification of genomic sequences homologous to methylated regions of to Hybond N membranes (GE Healthcare) and then crosslinked. Locked recessive SP11 alleles. The sequences were identified by in silico homology nucleic-acid (LNA)-modified probes complementary to dominant Smi searches that compared methylated promoter regions of recessive SP11 alleles (59-tGtaActAttTtaCacGtaAacAt-39, capital letters, LNA; lower-case letters, and S-locus genomic regions of the dominant S and S haplotypes, using DNA) and recessive Smi (59-tGtaActAttTtaCtcGtaAacAt-39, capital letters, 9 12 32 GENETYX-MAC v. 15 (Genetyx). LNA; lower-case letters, DNA) were end-labelled with [c- P]ATP with the use of T4 polynucleotide kinase (TaKaRa). Hybridization was performed in Identification of SMI regions from S8, S52, S44, S40 and S29 haplotypes. Genomic PCRs were performed using primers designed from genomic sequences ULTRAhyb-Oligo Hybridization Buffer (Ambion) at 37 uC. Membranes were then washed twice at 37 C with 2 3 SSC, 0.1% SDS for 15 min. Hybridization containing S9-SMI, S12-SMI and S60-SMI regions (see Supplementary Table 5). u Amplified products were cloned with a TOPO TA Cloning Kit (Invitrogen) and signals were detected with a BAS-2500 bioimaging analyser (Fuji Film). sequenced. Hybridization signals were quantified with ImageJ software (National Institutes Identification of S9-SMI transcript. Total RNA was extracted from the anthers of Health) and normalized to 5S rRNA. (stages 2–4), stigmas and leaves of B. rapa S9S9 homozygotes with an RNeasy Transgenic plants. Genomic sequences (3.2 and 1.7 kb) containing S9-Smi were Plant Mini Kit (Qiagen) in accordance with the manufacturer’s protocol. 59 and amplified with specific primers (see Supplementary Table 5) and genomic DNA 39 RACEs were performed with a GeneRacer Kit (Invitrogen) in accordance with from S9S9 homozygotes. A point mutation was introduced into S9-Smi at posi- the manufacturer’s protocol. For RT–PCR, cDNAs were synthesized with tion 10 with the use of specific primers (see Supplementary Table 5). These SuperScript III (Invitrogen) in accordance with the manufacturer’s protocol. products were cloned into the pBI121 vector and introduced into Primers used for RACE–PCR and RT–PCR are listed in Supplementary Table 5. Agrobacterium tumefaciens strain EHA105. The hypocotyl transformation of Screening of small RNA cDNA. Small RNAs were extracted from anthers (stages B. rapa S52S60 heterozygotes (cv. Osome) with Agrobacterium harbouring these 2 and 3) of B. rapa S9S9, S52S52, S29S29 and S60S60 homozygotes by using a constructs and the subsequent generation of transgenic plants were performed 9,20 mirVana miRNA Isolation Kit (Ambion). Small RNAs were then separated on as described previously . S52S60 heterozygotes carrying S9-Smi transgenes were denaturing 15% polyacrylamide gel with 8 M urea, and short RNAs (18–26 pollinated with pollen from recessive S homozygotes (S44S44, S60S60, S40S40 and nucleotides in length) were excised with a Small RNA Gel Extraction Kit S29S29). Selected recessive S heterozygotes (S44S60, S60S40 and S60S29) with trans- (TaKaRa). These short RNAs and the miRCat (Integrated DNA Technology) genes were subsequently self-pollinated to generate recessive S homozygotes with were used to synthesize small RNA cDNA in accordance with the manufacturer’s or without a transgene. To confirm the presence of S9-Smi transgenes, leaf pieces protocol. Screening of S9S9 and S52S52 small RNA cDNA was performed with from the transgenic plants were analysed in PCRs using transgene-specific primers. 59-biotinylated oligonucleotide probes (S40SP11-metF and S40SP11-metR) Pollination assay. Pollination assays were performed as described previously2. designed for both strands of the target methylated region of the recessive S40- Quantitative real-time PCR. Quantitative real-time PCRs were performed as SP11 promoter. Screening of S29S29 and S60S60 small RNA cDNA was performed described previously1 with a QuantiFast SYBR Green PCR Kit (Qiagen). Actin1 with 39-biotinylated oligonucleotide probes S29-SmiRNA and S60-SmiRNA, was used as an endogenous reference gene. Primers used for real-time PCRs are respectively. Each probe was mixed and hybridized overnight with small RNA listed in Supplementary Table 5. cDNA in 5 3 standard saline citrate (SSC) at 35 uC. The probes were then puri- Analysis of DNA methylation. The nuclei from anther tapetum cells were iso- fied with glutathione-Sepharose 4B (GE Healthcare) and washed twice at 37 uC lated from flower buds (stages 3–5) with a previously described method1. The with 2 3 SSC. Finally, hybridized small RNA cDNA was eluted in Tris-EDTA DNA was modified with bisulphite by using a Methylamp DNA Modification Kit (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 65 uC. For sequencing, the cDNAs (Epigentek) in accordance with the manufacturer’s protocol. The modified S29- obtained were amplified and cloned with a TOPO TA Cloning Kit (Invitrogen). SP11, S40-SP11, S60-SP11 and S44-SP11 were amplified with specific primers (see Probes and primers are listed in Supplementary Table 5. Supplementary Table 5). Amplified products were cloned with a TOPO TA In situ hybridization. The anthers at stage 2 of B. rapa S9S9 homozygotes were Cloning Kit (Invitrogen) and sequenced. As a control for complete bisulphite collected. Digoxigenin-labelled and unlabelled antisense S9-Smi oligo-DNA conversion, the B. rapa homologue of Arabidopsis thaliana PHAVOLUTA was 9 9 (5 -TGTAACTATTTTACACGTAAACAT-3 ) probes were obtained from the used31. At least five clones were sequenced from each plant. Bisulphite conver- m Nihon Gene Research Laboratory. In situ hybridization to 10- m-thick sion levels were more than 99% in all cases (data not shown). Paraplast (Sigma-Aldrich) sections of formaldehyde-fixed anther was performed as described previously2. 31. Bao, N., Lye, K. W. & Barton, M. K. MicroRNA binding sites in Arabidopsis class III Small RNA gel-blot analyses. Small RNAs (7 mg) extracted from anthers (stages HD-ZIP mRNAs are required for methylation of the template chromosome. Dev. 1–3) of each plant were separated on denaturing 15% polyacrylamide gel with 8 M Cell 7, 653–662 (2004).

Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases

Yannick Jacob1*, Hume Stroud2*, Chantal LeBlanc1, Suhua Feng3, Luting Zhuo1, Elena Caro2, Christiane Hassel1, Crisanto Gutierrez4, Scott D. Michaels1 & Steven E. Jacobsen2,3

Multiple pathways prevent DNA replication from occurring more defects as they undergo both mitosis and endoreduplication (genome than once per cell cycle1. These pathways block re-replication by duplication without mitosis), which is responsible for the widespread strictly controlling the activity of pre-replication complexes, polyploidy observed in mature leaf tissue9. Nuclei were extracted which assemble at specific sites in the genome called origins. from mature rosette leaves of wild-type Columbia (Col), atxr5, atxr6 Here we show that mutations in the homologous histone 3 and atxr5 atxr6 plants, stained with propidium iodide, and analysed lysine 27 (H3K27) monomethyltransferases, ARABIDOPSIS for DNA content. Col, atxr5 and atxr6 all showed well-resolved TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead populations of 2C, 4C, 8C, 16C and 32C nuclei (Fig. 1a). Thus, atxr5 to re-replication of specific genomic locations. Most of these loca- and atxr6 single mutations do not have an impact on DNA replica- tions correspond to transposons and other repetitive and silent tion in leaves. In contrast, the 8C and 16C peaks for atxr5 atxr6 were elements of the Arabidopsis genome. These sites also correspond much broader and skewed to the right, indicating that many 8C and to high levels of H3K27 monomethylation, and mutation of the 16C nuclei have higher DNA contents than the corresponding wild- catalytic SET domain is sufficient to cause the re-replication type nuclei (Fig. 1a). The distribution of nuclei between endoredu- defect. Mutation of ATXR5 and ATXR6 also causes upregulation plication levels was similar between wild type and atxr5 atxr6 double of transposon expression and has pleiotropic effects on plant mutants (Fig. 1b), suggesting that the primary defect in atxr5 atxr6 development. These results uncover a novel pathway that prevents plants is in the fidelity of S-phase progression rather than the number over-replication of heterochromatin in Arabidopsis. of rounds of endoreduplication. This is in contrast to mutations We previously characterized two redundant histone methyltrans- previously reported to affect the level of endoreduplication, but ferase genes, ATXR5 and ATXR6, and demonstrated that the ATXR5 not S-phase fidelity10. and ATXR6 proteins show H3K27 monomethylation (H3K27me1) Our previous work has shown that ,65% of atxr5 atxr6 nuclei activity in vitro, and that an atxr5 atxr6 double mutant shows a show significant decondensation of constitutive heterochromatin reduction of H3K27me1 in vivo2. The atxr5 atxr6 double mutant (that is, chromocentres) (Fig. 1c)2. Consistent with the flow cytome- shows pleiotropic defects in plant development, including smaller try results showing that the DNA content phenotype of atxr5 atxr6 misshapen leaves2. Overexpression of ATXR5 or ATXR6 also causes mutants is observed most strongly in 8C and 16C nuclei, we found, by morphological defects and male sterility3. Furthermore, the double microscopic analysis of sorted nuclei, that the heterochromatic mutant displays reactivation of the expression of a variety of both decondensation defect was also more extreme in these higher ploidy DNA transposons and retrotransposons2. Notably, atxr5 atxr6 did nuclei (Fig. 1c, d). As a control, we also analysed the DNA content of not disturb DNA methylation or histone H3K9 dimethylation decrease in dna methylation1 (ddm1) plants, which also show very (H3K9me2, a key repressive histone modification correlated with strong chromocentre decondensation defects, as well as reduced DNA methylation4–6), indicating that ATXR5 and ATXR6 act by DNA methylation and massively reactivated transposons11–13. The means of a novel pathway to maintain gene silencing. Previous work flow cytometry profile from extracted nuclei from ddm1-2 leaves also suggested that ATXR5 and ATXR6 show links with DNA rep- was similar to that of wild-type plants (Fig. 1a). These results show lication. ATXR5 and ATXR6 expression is regulated by the cell cycle, that the aberrant flow cytometry profiles observed in atxr5 atxr6 with expression peaking just before DNA replication3, and ATXR6 mutants are not simply a result of chromatin decondensation defects expression is strongly co-regulated with CDT1, ORC2 and other DNA and/or transposon derepression. replication proteins7. In addition, ATXR5 and ATXR6 contain PCNA- To test the hypothesis that there is indeed extra DNA in atxr5 atxr6 interacting-protein (PIP) motifs and have been shown to interact with mutants, we used an Illumina Genome Analyser II to sequence geno- the two PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) mic DNA from sorted nuclei (2C, 4C, 8C and 16C) of both wild-type proteins in Arabidopsis (AtPCNA1 and AtPCNA2)3. PCNA interacts and atxr5 atxr6 plants. A total of 84.9 million uniquely mapping 36- with DNA polymerase and serves as a general loading platform for nucleotide reads were mapped to the Arabidopsis thaliana genome, many proteins involved in diverse processes occurring at chromatin8. allowing up to two mismatches. We examined the distribution of Here we show that ATXR5 and ATXR6 are critical factors that act in a genomic DNA across all chromosomes by plotting the density of reads novel pathway to suppress DNA re-replication, especially in hetero- in non-overlapping 100-kilobase (kb) bins. For wild type, the ratio of chromatic regions of the Arabidopsis genome. 4C, 8C and 16C to 2C sequence reads was uniform across the genome, To determine whether ATXR5 and/or ATXR6 have a role in DNA showing that the genome is uniformly endoreduplicated in wild-type replication, we analysed the DNA content of leaf nuclei by flow Arabidopsis (Fig. 1e). However, in atxr5 atxr6 mutants, we observed an cytometry. Leaves are well suited for assessing DNA replication enrichment of reads in the pericentromeric heterochromatin in 4C,

1Department of Biology, Indiana University, 915 East Third Street, Bloomington, Indiana 47405, USA. 2Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA. 3Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California 90095, USA. 4Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Nicolas Cabrera 1, Cantoblanco, Madrid 28049, Spain. *These authors contributed equally to this work. 987 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a Mature leaves Col atxr5 atxr6 3.52.4 atxr5 atxr6 ddm1-2 600 3.0 4.4 2.7 4.4 4.3 3.3 400 3.8 5.2 4.8 4.7 5.4 6.2 6.6 14.8 200 6.8 7.1 14.9 7.1 0

Number of nuclei 2 4 8 16 32C 2 4 8 16 32C 2 4 8 16 32C 2 4 8 16 32C 2 4 8 16 32C DNA content b c d

30 80 20 Col 60 40 10 Nuclei (%) 20 0 0 2C 4C 8C 16C 32C atxr5 atxr6 2C 4C 8C 16C 2C 4C 8C 16C Decondensation (%) DNA content ef Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 100 100 80 80 60 60 40 40 TE (kb) 20 TE (kb) 20 Length of 0 Length of 0 1 1 0.5 0.5 0 0 (4C/2C) (4C/2C) 2 –0.5 2 –0.5

log –1 log –1 1 1 0.5 0.5 0 0 (8C/2C) (8C/2C) 2 –0.5 2 –0.5 log –1 log –1 1 1 0.5 0.5 0 0 (16C/2C) (16C/2C) 2 –0.5 2 –0.5 log –1 log –1 0110 20 30 0110 20 0 0200 0200 10 20 30 (Mb) 01102030 0110 20 0 020010200 02030 (Mb) Figure 1 | Heterochromatic DNA is over-produced in atxr5 atxr6 mutants. replicates were analysed. White bars represent wild type and black bars a, Flow cytometry profiles of Col, atxr5, atxr6, atxr5 atxr6 and ddm1-2 represent atxr5 atxr6. Error bars indicate one standard deviation. e, DNA is plants. Three-thousand gated events are plotted. The number above each replicated uniformly in wild-type nuclei during endoreduplication. The log2 peak (robust CV) indicates the number of fluorescence intensity units that ratios of genomic DNA Illumina reads from wild-type 4C versus 2C, 8C enclose the central 68% of nuclei for that endoreduplication level. versus 2C and 16C versus 2C are plotted across the chromosomes in 100-kb- b, Quantification of nuclei at each ploidy level for samples in panel a; Col, sliding windows. Plots of transposable element (TE) abundance (kb of black; atxr5, white; atxr6, grey; atxr5 atxr6, crosshatched. c,49,6-diamidino- transposon sequence per 100 kb genomic DNA) indicate pericentromeric 2-phenylindole (DAPI) staining of sorted nuclei from Col and atxr5 atxr6 regions. f, Similar analysis with atxr5 atxr6 mutants showing an increased leaves. Scale bar, 10 mm. d, Chromocentre decondensation occurs mainly in proportion of reads in pericentromeric heterochromatin in higher ploidy 8C and 16C nuclei. Thirty nuclei of each ploidy level from three biological nuclei.

8C and 16C compared to 2C, indicating that heterochromatin is over- (Fig. 2b). These small regions of over-replication were highly enriched replicated in 4C, 8C and 16C nuclei compared to 2C nuclei (Fig. 1f), in transposons and other repeat elements. Using the BLOC algorithm and that the over-replication is more severe in nuclei with higher (see Methods)14, we identified 407 sites of over-replication in the arms of ploidy levels. atxr5 atxr6 chromosomes (Supplementary Table 1). The over-replic- A comparison of the distribution of reads from atxr5 atxr6 ating regions were relatively small: 94% of regions were smaller than mutants with wild type showed that even in the 2C nuclei, atxr5 atxr6 25 kb, with a median size of 10.4 kb (Supplementary Fig. 2a). Most mutants show over-replication of pericentromeric heterochromatin, (80%) overlapped with previously defined H3K9me2 regions, a mark although to a lower extent than in nuclei of higher ploidy levels that is strongly correlated with DNA methylation and gene silencing6 (Supplementary Fig. 1a). Relative to wild type, atxr5 atxr6 mutants (Supplementary Fig. 2b). Thus, the regions that over-replicate in atx- showed a 2.9%, 11.0%, 29.1% and 28.4% increase in reads mapping r5 atxr6 mutants primarily consist of transposons and silent elements of to pericentromeric heterochromatin in 2C, 4C, 8C and 16C nuclei, the Arabidopsis genome.Over-replicationwasconfirmed byperforming respectively (Supplementary Fig. 1b). Sites of over-replication were quantitative polymerase chain reaction (qPCR) on defined sites well correlated at the different ploidy levels. For instance, the Pearson (Supplementary Fig. 3). Elements that are transcriptionally reactivated correlation between 8C and 16C atxr5 atxr6 nuclei was 0.84, indi- in atxr5 atxr6 mutants (TSI, Ta3, CACTA)2 were found to be over- cating that the same sites are over-replicating (Supplementary Fig. replicated, indicating a positive correlation between transposon react- 1c). These results show that atxr5 atxr6 mutants show over-replica- ivation and over-replication. tion of pericentromeric heterochromatin in both mitotic and endo- Re-replication is a well-known mechanism by which DNA is known cycling cells, with progressively stronger defects observed in nuclei to over-replicate and results when DNA replication is initiated from with higher ploidy levels. an origin multiple times during a single S phase1. Presumably because To examine over-replication in atxr5 atxr6 mutants at higher resolu- recently replicated chromatin is less compact, secondary replication tion, sequence reads were grouped and analysed in 200-base-pair non- forks move faster than primary forks, and collisions of the multiple overlapping bins. We found that over-replication of pericentromeric forks result in successively smaller fragments of DNA reiteratively heterochromatin is the result of the over-replication of many densely produced from the origin15 (Fig. 2c). This model predicts that spaced, but distinct, loci (Fig. 2a). We also observed localized over- sequences in the centre of the origin will be the most highly over- replication in small regions of the euchromatic arms of chromosomes replicated and that over-replication should drop off symmetrically on 988 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

either side of the origin. To determine if over-replication in the atx- a 2 r5 atxr6 mutant is consistent with re-replication, plots of sequencing atxr5 atxr6 0 reads averaged over the over-replicated regions were generated log (16C/2C) 2 –2 (Fig. 2d). In contrast to wild type, in which sequencing reads were uniformly distributed, atxr5 atxr6 mutants showed a bilaterally sym- atxr5 atxr6 log (8C/2C) metrical distribution of reads, with the highest density of reads in the 2 centre of the over-replicated regions (Fig. 2d). These results indicate atxr5 atxr6 that the extra DNA in atxr5 atxr6 mutants is a result of repeated log2(4C/2C) replication from defined sites. We next examined whether chromatin or naked DNA is being re- H3K9me2 replicated in atxr5 atxr6 mutants. To test this, we performed chro- TE (+) matin-immunoprecipitation of unmodified histone H3 followed by TE (–) Illumina sequencing (ChIP-seq) on wild type and atxr5 atxr6 PCG (+) mutants. Compared with wild type, H3 ChIP-seq reads in atxr5 atxr6 mutants were enriched in the pericentromeric heterochromatin to a 13,700 13,800 13,900 (kb) PCG (–) similar extent as was the input genomic DNA (Fig. 2e and Supplementary Fig. 2c). This result indicates that chromatin (DNA and associated histones) is re-replicated in atxr5 atxr6 mutants. Our b data also indicate that the re-replicated DNA is properly methylated. 2 atxr5 atxr6 If the re-replicated DNA was unmethylated, the per cent methylation 0 log2(16C/2C) in atxr5 atxr6 mutants would be predicted to be lower than in wild –2 type. However, we have previously shown that the per cent DNA atxr5 atxr6 methylation in atxr5 atxr6 mutant leaves (where re-replication was log (8C/2C) 2 observed) is the same as in wild type2, which suggests that the re- atxr5 atxr6 replicated DNA is properly methylated. Furthermore, to determine log2(4C/2C) whether re-replicated DNA was stably associated with the chromosomes, we performed qPCR on size-fractionated DNA (Sup- H3K9me2 plementary Fig. 4). We found that the extra DNA could be detected TE (+) in the high-molecular mass DNA fraction, indicating that at least part TE (–) of the re-replicated DNA is stably associated with the chromosome. Being associated with chromosomes, rather than being extrachromo- PCG (+) somal fragments, may help to explain the stability of re-replicating 7,040 7,080 18,960 18,980 17,820 17,860 (kb) PCG (–) DNA fragments present in 3–4-week-old leaf cells. Because ATXR5 and ATXR6 catalyse H3K27me1 (ref. 2), we wanted to examine whether the spatial distribution of H3K27me1 overlaps with re-replicating regions. Immunolocalization indicates c d atxr5 atxr6 atxr5 e WT log2( atxr6 Chr 3 0.014 0.8 /WT) 100 that H3K27me1 is a heterochromatic mark enriched in chromocen- 2,16–18 0.012 0.6 80 tres . A detailed global map of H3K27me1, however, has not been 0.4 2C 0.010 60 0.2 40 reported. We therefore profiled H3K27me1 genome-wide using 0.008 0 20 ChIP-seq. Consistent with the re-replication of pericentromeric het- 0.014 0.8 Length of TE (kb) 0 0.012 0.6 erochromatin in atxr5 atxr6 (Fig. 1f), we found that H3K27me1 was 0.4 2 0.6 4C 0.010 strongly enriched in pericentromeric heterochromatin (Fig. 3a). We

0.2 /WT) 0.4 0.008 0 0.2 also observed H3K27me1 in the coding regions of protein-coding 0.014 0.8 0 –0.2 0.6 genes and found that the amount of H3K27me1 was anticorrelated

0.012 atxr5 atxr6 (

Input gDNA:log –0.4

8C 0.4 0.010 with gene expression levels (Fig. 3b and Supplementary Fig. 5a). 0.2 2 0.6 0.008 Together, these results support a role for H3K27me1 in gene silencing. 0 0.4 /WT) 0.014 0.8 0.2 To gain additional evidence for a correlation between H3K27me1 0.012 0.6 0 0.4 –0.2 and re-replication in atxr5 atxr6 mutants, we examined the dispersed

16C 0.010 atxr5 atxr6

0.2 ( –0.4 H3 ChlP-seq:log re-replicating regions in the arms of the chromosomes. We found 0.008 0 0 10 20 (Mb) –5 50 –5 0 5 –5 0 5 that H3K27me1 ChIP-seq reads were significantly enriched in these Distance from centre of re-replicating regions (kb) regions compared to randomly selected control regions (permuta- tion test, P , 1026) (Fig. 3c). In addition, plots of the ratio of Figure 2 | Increased heterochromatic DNA in atxr5 atxr6 mutants is H3K27me1 to H3 ChIP-seq reads averaged over these re-replicating consistent with re-replication of chromatin. a, Genome browser view of a region of pericentromeric heterochromatin. Pericentromeric regions showed strong enrichment of H3K27me1, confirming a pos- heterochromatin contains densely spaced, ,10-kb over-replicating sites. itive correlation of H3K27me1 with sites that re-replicate in atx- Data are represented as log2 ratios (16C/2C, 8C/2C or 4C/2C) in 200-bp bins. r5 atxr6 mutants (Fig. 3d and Supplementary Fig. 5b). H3K9me2 microarray data6, TAIR8 protein-coding gene (PCG) and Given that H3K27me1 levels correlate with the re-replicated transposable element (TE) tracks are also shown on the plus (1) or minus regions of atxr5 atxr6 mutants, an interesting question concerns (2) strand of the genome. b, Genome browser view of examples of over- the mechanism by which the spatial distribution of H3K27me1 is replication in the arms of chromosomes. Three over-replicating regions are established. ATXR5 and ATXR6 both contain PHD domains, which shown. c, Model for DNA re-replication (ref. 22). d, Distribution of Illumina have been shown in multiple species to mediate interactions with reads in re-replicating regions. Plots of the average number of sequence methylated or unmethylated forms of histone H319. We performed reads 65 kb relative to the centre of over-replicating regions in atxr5 atxr6 in vitro binding assays with various H3 peptides using GST-tagged mutants, wild type, or the atxr5 atxr6 mutants/wild type log2 ratio (plotted in 100-bp bins). e, Histone content in re-replicating regions is higher in PHD domains of ATXR5 and ATXR6. The PHD domains of ATXR5 atxr5 atxr6 mutants. Log2 ratios of H3 ChIP-seq reads and input genomic and ATXR6 bound strongly to an unmethylated peptide correspond- DNA reads in atxr5 atxr6 mutants relative to wild type, plotted over ing to amino acids 1–21 of H3 (Fig. 3e). This binding was unaffected chromosome 3 in 100-kb sliding windows. by mono-, di-, or trimethylation at H3K9; however, binding was 989 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

a a Q92A, I95A, 2 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 ATXR6 L49W F98A, F99A Y243N N C 1 PHD PIP SET 0 b

(H3K27me1/H3) 16 2 –1 0110 20 30 0110 20 0 0200 0200 102030(Mb) log 12 bdTop 10% 50–70% c 10–30% 70–90% 0.2 0.9 8 0 30–50% 90–100% 0.8 0

Robust CV 4 0.7 –0.2 0 0.6 (H3K27me1/H3) (H3K27me1/H3) –0.4 c 2 2

–1 H3K27me1/H3 100 log log 0.5 –20 –10 0 +10 +20 (kb) –2 kb 0% 100% +2 kb Region Distance from centre of 80 Transcribed region re-replicating regions ef 60 –0.8 0–0.40.4 0.8 K4me0 K4me2 40 K4me1 K4me3 0% inputo peptide3 1–213K4me13K4me23K4me33K9me13K9me23K9me3 –0.50 –0.4 1 N H H H H H H H 20 ATXR5 (PHD) –0.5 Decondensed –0.54 (%) chromocentres 0 ATXR6 (PHD) –0.6 d (H3K27me1/H3)

2 –0.58 1.6 –0.7 log –2 –1 0 1 2 Outside Inside 1.2 region (kb) region (kb) 0.8 Figure 3 | Genome-wide mapping of H3K27me1 and anticorrelation with expression

Relative TSI 0.4 H3K4 methylation. a, H3K27me1 is enriched in heterochromatin. The log2 ratios of H3K27me1 reads to H3 ChIP-seq reads in wild type are plotted 0 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 b Col across the chromosomes (1 to 5) in 100-kb sliding windows. , H3K27me1 is ATXR6 anticorrelated with gene expression level. H3K27me1 ChIP-seq reads atxr5/6 PHD mut. SET mut. PIP mut. normalized to H3 ChIP-seq reads averaged over TAIR8 protein-coding Figure 4 | Functional PHD and SET domains and the PIP motif are required genes. The bodies of genes are scaled. Three-week-old wild-type plants were for the regulation of DNA replication by ATXR6.a, Structure of ATXR6. used for both ChIP-seq and RNA-seq. c, H3K27me1 is significantly enriched The domains (below) and point mutations (above) made to generate ATXR6 at sites of re-replication in the arms. Reads per base pair in re-replicating mutants are represented. b–d, Normal DNA replication as indicated by regions were calculated for both H3K27me1 and H3 ChIP-seq reads, and the robust CV (b), chromatin condensation (c) and TSI gene silencing (d)is ratio was calculated (black bar). Random regions with a similar distribution rescued in transgenic atxr5 atxr6 plants expressing wild-type ATXR6, but as re-replicating regions were generated 100,000 times and the same not PHD, SET, or PIP mutants. All phenotypes were scored on the same four calculation was performed. The mean value obtained from random regions representative transgenic lines (n . 20) generated from each construct. are shown (white bar) and the error bars represent the standard deviation. Error bars indicate one standard deviation. d, H3K27me1 is enriched in over re-replicating regions. The log2 ratio of H3K27me1 to H3 reads is plotted 620 kb relative to the centre of re- replicating regions of atxr5 atxr6 mutants. Data were plotted in 400-bp bins and smoothed by taking the moving average over six bins. e, Pull-down assay designed to disrupt the activity of each functional element (Fig. 4a). using purified GST-tagged PHD domains of ATXR5 and ATXR6 and Yeast-two-hybrid analysis and in vitro histone-peptide-binding and biotinylated H3 peptides with different methylated lysines. Interaction between the peptides and the GST–PHD domains was visualized by western methyltransferase assays were used to confirm disruption of the PIP- blot using a GST antibody. f, Analysis of the relationship between motif, PHD-domain and SET-domain activities, respectively H3K27me1 and H3K4 methylation. The log2 ratio of H3K27me1 to H3 is (Supplementary Fig. 6). Analysis of T1 plants transformed with each plotted over the boundaries of all H3K4me0/-me1/-me2/-me3 regions in the of the mutated ATXR6 constructs showed that the re-replication genome. Data are shown in 200-bp bins, and smoothed by taking the moving phenotype was never rescued by constructs containing the mutated average over 62 bins. The scale for the plots over H3K4me0 is in blue, and PIP motif, PHD domain or SET domain (Fig. 4b) (n . 20). These the scale for the others is in black. results show that the PIP motif, PHD domain and SET domain are all required for ATXR6 activity and indicate that depletion of H3K27me1 in the atxr5 atxr6 double mutant is probably responsible strongly reduced by increasing levels of H3K4 methylation. Thus, the for the re-replication phenotype. Consistent with this interpretation, PHD domains of ATXR5 and ATXR6 bound most strongly to H3 we found that the restoration of H3K27me1 levels also required the unmethylated at K4 (H3K4me0). Consistent with the hypothesis that wild-type PIP motif, PHD domain and SET domain (Supplementary binding of the ATXR5 and ATXR6 PHD domains to H3K4me0 chro- Fig. 7). Furthermore, only the wild-type construct rescued chromatin matin is helping to guide H3K27 monomethylation activity, we decondensation and loss of gene silencing defect seen in atxr5 atxr6 observed a strong anticorrelation between H3K4 methylation20 and mutants (Fig. 4c, d). These results indicate that the three functional H3K27me1 within genes and in the genome at large (Fig. 3f and elements of ATXR6 contribute to the prevention of re-replication, Supplementary Fig. 5c). chromatin decondensation and loss of gene silencing. Because loss of ATXR5 and ATXR6 leads to lower levels of Our results indicate that ATXR5 and ATXR6 are components of a H3K27me1 (ref. 2), it is possible that depletion of this mark is causing novel pathway required to suppress re-replication in Arabidopsis. re-replication in atxr5 atxr6 mutants. One prediction from this Notably, most of the re-replicating sites in atxr5 atxr6 mutants corre- model is that the PHD and SET domains of ATXR5 and ATXR6 spond to silent heterochromatin, which is composed mostly of trans- would be essential to prevent re-replication, as they are responsible poson sequences. It is tempting to speculate that the ATXR5/ATXR6 for binding and methylating H3, respectively (Fig. 4a). To investigate system may have evolved to suppress excess DNA replication of this, we first created a genomic construct that expresses ATXR6 under transposon sequences that would otherwise result in transposon its own promoter and confirmed that it can rescue (.95% of T1 reactivation. Conversely, transposons are remarkable in requiring transformed plants analysed) the re-replication phenotype of atx- both the typical repressive modifications such as H3K9me2 and r5 atxr6 mutant plants (Fig. 4b). We then made PIP-, PHD- and DNA methylation, as well as the novel ATXR5/ATXR6 H3K27me1 SET-mutant ATXR6 constructs by inserting point mutations pathway for transcriptional suppression. 990 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

METHODS SUMMARY 14. Pauler, F. M. et al. H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal FACS was used to generate flow cytometry profiles of leaf nuclei from Col and chromosome. Genome Res. 19, 221–233 (2009). atxr5 atxr6 and to sort nuclei based on endoreduplication level (2C, 4C, 8C and 15. Gomez, M. Controlled rereplication at DNA replication origins. Cell Cycle 7, 16C). Genomic DNA isolated from sorted nuclei was sequenced using an 1313–1314 (2008). 21 Illumina Genome Analyser II. SeqMap was used to map sequencing reads to 16. Fuchs, J., Demidov, D., Houben, A. & Schubert, I. Chromosomal histone the Arabidopsis genome. DNA from ChIP using H3 and H3K27me1 antibodies modification patterns—from conservation to diversity. Trends Plant Sci. 11, was sequenced and analysed in a similar fashion. In vitro binding assays used 199–208 (2006). biotinylated H3 peptides (Millipore, Billerica). 17. Lindroth, A. M. et al. Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J. 23, 4146–4155 Full Methods and any associated references are available in the online version of (2004). the paper at www.nature.com/nature. 18. Mathieu, O., Probst, A. V. & Paszkowski, J. Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis. EMBO J. 24, Received 23 March; accepted 24 June 2010. 2783–2791 (2005). Published online 14 July 2010; corrected 19 August 2010 (see full-text HTML version 19. Musselman, C. A. & Kutateladze, T. G. PHD fingers: epigenetic effectors and for details). potential drug targets. Mol. Interv. 9, 314–323 (2009). 1. Arias, E. E. & Walter, J. C. Strength in numbers: preventing rereplication via 20. Zhang, X., Bernatavichute, Y. V., Cokus, S., Pellegrini, M. & Jacobsen, S. E. multiple mechanisms in eukaryotic cells. Genes Dev. 21, 497–518 (2007). Genome-wide analysis of mono-, di- and trimethylation of histone H3 lysine 4 in 2. Jacob, Y. et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required Arabidopsis thaliana. Genome Biol. 10, R62 (2009). for chromatin structure and gene silencing. Nature Struct. Mol. Biol. 16, 763–768 21. Jiang, H. & Wong, W. H. SeqMap: mapping massive amount of oligonucleotides to (2009). the genome. Bioinformatics 24, 2395–2396 (2008). 3. Raynaud, C. et al. Two cell-cycle regulated SET-domain proteins interact with 22. Davidson, I. F., Li, A. & Blow, J. J. Deregulated replication licensing causes DNA proliferating cell nuclear antigen (PCNA) in Arabidopsis. Plant J. 47, 395–407 fragmentation consistent with head-to-tail fork collision. Mol. Cell 24, 433–443 (2006). (2006). 4. Jackson, J. P., Lindroth, A. M., Cao, X. & Jacobsen, S. E. Control of CpNpG DNA Supplementary Information is linked to the online version of the paper at methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, www.nature.com/nature. 556–560 (2002). 5. Malagnac, F., Bartee, L. & Bender, J. An Arabidopsis SET domain protein required Acknowledgements We thank G. Lambert and D. Galbraith for assistance with for maintenance but not establishment of DNA methylation. EMBO J. 21, flow cytometry; Y. Bernatavichute for assistance with ChIP experiments; and 6842–6852 (2002). M. Pellegrini and S. Cokus for advice on data analyses. Y.J. was supported by a 6. Bernatavichute, Y. V., Zhang, X., Cokus, S., Pellegrini, M. & Jacobsen, S. E. fellowship from Le Fonds Que´be´cois de la Recherche sur la Nature et les Genome-wide association of histone H3 lysine nine methylation with CHG DNA Technologies (FQRNT). S.F. is a Howard Hughes Medical Institute Fellow of the methylation in Arabidopsis thaliana. PLoS ONE 3, e3156 (2008). Life Science Research Foundation. Research in the Michaels’ laboratory was 7. Obayashi, T., Hayashi, S., Saeki, M., Ohta, H. & Kinoshita, K. ATTED-II provides supported by grants from the National Institutes of Health (GM075060), the coexpressed gene networks for Arabidopsis. Nucleic Acids Res. 37, D987–D991 Indiana METACyt Initiative of Indiana University, and the Lilly Endowment, Inc. (2009). C.G. was supported by grants from the Spanish Ministry of Science and Innovation 8. Moldovan, G. L., Pfander, B. & Jentsch, S. PCNA, the maestro of the replication (BFU2009-9783 and CSD2007-57B). S.E.J. is an investigator of the Howard fork. Cell 129, 665–679 (2007). Hughes Medical Institute. 9. Galbraith, D. W., Harkins, K. R. & Knapp, S. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol. 96, 985–989 (1991). Author Contributions S.D.M., S.E.J. and C.G. directed the research. Y.J., H.S., C.L., 10. Caro, E., Desvoyes, B., Ramirez-Parra, E., Sanchez, M. P. & Gutierrez, C. S.F., L.Z., E.C. and C.H. performed experiments. H.S. analysed data. H.S., Y.J., S.E.J. Endoreduplication control during plant development. SEB Exp. Biol. Ser. 59, and S.D.M. prepared the manuscript. 167–187 (2008). 11. Jeddeloh, J. A., Stokes, T. L. & Richards, E. J. Maintenance of genomic methylation Author Information Sequencing files have been deposited at GEO (accession requires a SWI2/SNF2-like protein. Nature Genet. 22, 94–97 (1999). codes GSE22411 and GSE21673). Reprints and permissions information is available 12. Fransz, P., ten Hoopen, R. & Tessadori, F. Composition and formation of at www.nature.com/reprints. The authors declare no competing financial heterochromatin in Arabidopsis thaliana. Chromosome Res. 14, 71–82 (2006). interests. Readers are welcome to comment on the online version of this article at 13. Soppe, W. J. et al. DNA methylation controls histone H3 lysine 9 methylation and www.nature.com/nature. Correspondence and requests for materials should be heterochromatin assembly in Arabidopsis. EMBO J. 21, 6549–6559 (2002). addressed to S.D.M. ([email protected]) or S.E.J. ([email protected]).

METHODS Yeast two-hybrid assay. pDEST22-AtPCNA1 was co-transformed with each of the two pDEST32-ATXR6 vectors in Saccharomyces cerevisiae (strain MaV203, Plant material. atxr5 (SALK_130607) and atxr6 (SAIL_240_H01) in the Invitrogen). As controls, pDEST22-AtPCNA1 and pDEST32-ATXR6/ATXR6(pip) Columbia genetic background were obtained from the Arabidopsis Biological were also used for co-transformation with empty pDEST32 and pDEST22 vectors, Resource Center. The ddm1-2 seeds have been described previously11,23. Plants 22 21 respectively. Five independent colonies from each transformation were selected and were grown under cool-white fluorescent light (,100 mmol m s ) under streaked on SC –Leu –Trp plates. The colonies were then replica plated on SC –Leu – long-day conditions (16 h of light followed by 8 h of darkness). Trp plates containing various concentrations (25, 50 or 100 mM) of 3-amino-1,2,4- Flow cytometry. Plant tissue was chopped with a razor blade in 500 mlof triazole (3AT), and grown for 3–4 days at 30 C. Galbraith buffer (45 mM MgCl , 20 mM MOPS, 30 mM sodium citrate, 0.1% u 2 Chromatin immunoprecipitation. ChIP was performed on crosslinked 3-week- Triton X-100) containing 20 mg of RNase A. The lysate was filtered through a old plants as previously described6 using anti-H3 (Abcam 1791) and anti- 40 mm cell strainer (BD Falcon) and propidium iodide was added to a final 21 H3K27me1 (Upstate 07-448) antibodies. concentration of 20 mgml . For mature leaves, nuclei were extracted from rosette leaves 3 and 4 from 4-week-old plants; for young leaves, nuclei were Quantitative PCR assays on genomic DNA. Fifty-thousand 16C nuclei were extracted from rosette leaves 1 and 2 from 9-day-old plants. Flow cytometry prepared from rosette leaves of 28-day-old plants (Col and atxr5 atxr6), as profiles were obtained on a BD FACSCalibur (Becton Dickinson) based upon described above. For sorting, the same 16N gate size was used for both samples. Genomic DNA was extracted from the sorted nuclei using the PicoPure DNA propidium iodide and 90u side angle scatter on a logarithmic scale due to the small size of the nuclei. Analysis was performed with CellQuest Pro software Extraction Kit (Molecular Devices) according to the manufacturer’s recommen- (Becton Dickinson). For nuclei sorting, 2.5 g of mature rosette leaves were col- dations. qPCR was performed using Brilliant II SYBR green QPCR master mix lected from 4-week-old plants, chopped in 5 ml of Galbraith buffer containing (Agilent), also according to the manufacturer’s recommendations. 200 mg of RNase A, filtered and stained with propidium iodide. The procedure Quantitative PCR assays on gel separated genomic DNA. Four 3-week-old for sorting was based on parameters similar to the FACSCalibur on a BD FACS leaves were ground and extracted in 400 ml extraction buffer (sorbitol 350 mM, Aria II, using an 85 mm nozzle with sheath pressure at 35 p.s.i. Analysis was Tris-HCl pH 7.5 100 mM, EDTA 5 mM) followed by lysis in 400 ml of lysis buffer performed with FACSDiva version 6.1.1 (Becton Dickinson). For sequencing, (Tris-HCl pH 7.5 200 mM, EDTA 50 mM, NaCl 2 M, CTAB 2%) plus 27 mlof 400,000 nuclei of each ploidy (2C, 4C, 8C and 16C) were isolated from Col and 10% sarkosyl. After a 30-min incubation at 65 uC, DNA was extracted with 21 m atxr5 atxr6 leaves at a threshold rate of approximately 500 events s , a flow rate chloroform/isoamyl alcohol, precipitated and re-suspended in 100 l of extrac- m of 2.5 and a variable sort rate dependent upon the peak sorted. Genomic DNA tion buffer including RNase A (Qiagen). Around 10 g of this DNA was run in a was extracted from the sorted nuclei using the PicoPure DNA Extraction Kit 1% low melting point agarose gel (Lonza) for both Col0 and atxr5 atxr6 and the (Molecular Devices) according to the manufacturer’s recommendations. band for the genomic DNA was cut out of the gel (BIG) along with the rest of the , For analysis of sorted nuclei by microscopy, 0.25 g of leaves were fixed for lane below that band (SMALL, DNA 12 kb). 20 min in 4% formaldehyde in Tris buffer (10 mM Tris-HCl pH 7.5, 10 mM For the recovery of the BIG fraction of DNA from the gel we diluted it five EDTA, 100 mM NaCl) and washed with Tris buffer 2 3 10 min. The leaves were times with TE (Tris pH 8 plus EDTA) and incubated at 65 uC until the agarose then chopped in Galbraith buffer, filtered and stained with 4 mgml21 DAPI. melted. We then performed serial extractions with phenol, phenol/chloroform m Two-thousand nuclei of each ploidy were sorted directly onto microscope slides. and chloroform followed by isopropanol precipitation and resuspension in 50 l Coverslips were then mounted using Vectashield mounting medium with DAPI of TE. (Vector Laboratories) and sealed with clear nail polish. Thirty nuclei for each For the recovery of the SMALL fraction of DNA from the gel we used Qiagen ploidy type were analysed for three biological samples. Flow cytometry and gel extraction kit and followed the manufacturer’s instructions. DNA was eluted nuclei sorting were performed at the Indiana University Flow Cytometry Core in 50 ml of TE. qPCR was performed using the IQ -SYBR Green Supermix from Facility. Bio-Rad, a Stratagene MX3005P qPCR system. cDNA synthesis, real-time PCR, protein expression and purification, and Illumina library preparation. Illumina libraries for genomic DNA extracted immunofluorescence. These procedures were performed as described prev- from sorted nuclei and ChIP samples were made following the manufacturer’s iously2. Microscopy was performed at the Indiana University-Bloomington instructions. The libraries were sequenced using Illumina Genome Analyser II Light Microscopy Imaging Center. following manufacturer instructions, producing reads of 36 bp in length. Constructs. To make the ATXR6 constructs used to transform A. thaliana, the Illumina read alignment and analysis. Sequenced reads were based-called promoter (293 bp upstream of start codon) and gene (exons and introns) were using the standard Illumina software. We used SeqMap (ref. 21) to align the amplified by PCR and cloned into pENTR/D (Invitrogen). Point mutations in reads to the Arabidopsis thaliana genome, allowing up to two mismatches. the PHD domain, SET domain and the PIP motif were made using pENTR/D- Identical reads were collapsed into single reads. We used two different map- ATXR6 as template by site-directed mutagenesis. For the PHD-domain mutant, ping strategies for all of our data sets: (1) keeping reads that uniquely map to leucine 49 was replaced with tryptophan (L49W). This mutation is based on the the genome and (2) remapping the reads that map to multiple locations in the structure of the PHD domain of the mammalian protein BHC80, which was genome, giving each read a weight. In (2), if a read mapped to x places in the shown to rely on an equivalent residue (M502) for preferentially binding genome, the read was given a score of 1/x, hence uniquely mapping reads got a H3K4me0 (ref. 24). To disrupt the function of the SET domain, we changed score of 1. Mapping strategy (2) was necessary because the majority of re- tyrosine 243 to asparagine (Y243N). This tyrosine is part of the conserved YXG replicating regions were present in heterochromatic regions, and were used to motif of SET-domain proteins, which is responsible for orienting the e-amino define regions of re-replication. Because many heterochromatic regions are group for methyl transfer to occur on lysine25. An equivalent mutation (Y655N) highly repetitive, we found that uniquely mapping the reads results in no in Drosophila Enhancer of zeste (E(z)) was shown to abolish H3K27 methylation signal in those regions. without affecting folding of the protein26. Finally, glutamine 92, isoleucine 95, Because 36-bp reads represented the ends of the DNA fragments in the library, phenylalanine 98 and phenylalanine 99 of the conserved PIP motif (QTKIIDFF) for the analysis, we extended the read so that the data represent the actual DNA of ATXR6 were replaced with alanine. The resulting ATXR6 constructs were fragments of the libraries. The lengths of extensions were determined based on subcloned first into pEG302 (ref. 27) using Gateway technology to acquire a the distribution of the sizes of DNA fragments in the library. Each base pair of the C-terminal Flag-epitope sequence, then into pMDC30 (ref. 28) by restriction read was given a score of 1 (or 1/x). Therefore if a certain nucleotide in the digest of the pEG302 vectors with SpeI and SbfI. genome had x uniquely mapping fragments overlapping, that nucleotide got a pGEX-6P was used to clone the PHD domains (amino acids 25–103, ATXR6; score of x. Data in (1) were normalized to total number of uniquely mapping 57–133, ATXR5) and PHD-SET domains (25–349, ATXR6) for the in vitro reads in wild type, whereas in (2), data were normalized to the number of binding and methyltransferase assays, respectively. The point mutations in the uniquely mapping reads plus the number of reads that map to multiple locations. 14 PHD (L49W) and SET (Y243N) domains were introduced in the pGEX vectors Re-replicating regions were defined by using BLOC . Scores for atxr5 atxr6 by site-directed mutagenesis. log2(16C/2C) were calculated in 60-bp bins, Z-score transformed, and then an For the yeast two-hybrid assay, the coding sequences of ATXR6, ATXR6(pip) average Z-score cutoff of 0.3 was applied for BLOC. and AtPCNA1 (At1g07370) were cloned into pENTR/D, then subcloned into 23. Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. Arabidopsis thaliana pDEST32 or pDEST22 using the Gateway system. DNA methylation mutants. Science 260, 1926–1928 (1993). Histone peptide binding and histone methyltransferase assays. Biotinylated 24. Lan, F. et al. Recognition of unmethylated histone H3 lysine 4 links BHC80 to peptides were obtained from Millipore. The procedure for this assay has been LSD1-mediated gene repression. Nature 448, 718–722 (2007). described previously29. The binding buffer contained 250 mM NaCl. Histone 25. Dillon, S. C., Zhang, X., Trievel, R. C. & Cheng, X. The SET-domain protein methyltransferase assays were preformed as previously described2. superfamily: protein lysine methyltransferases. Genome Biol. 6, 227 (2005).

26. Joshi, P. et al. Dominant alleles identify SET domain residues required for histone 28. Curtis, M. D. & Grossniklaus, U. A Gateway Cloning Vector Set for High- methyltransferase of Polycomb repressive complex 2. J. Biol. Chem. 283, Throughput Functional Analysis of Genes in Planta[w]. Plant Physiol. 133, 462–469 27757–27766 (2008). (2003). 27. Earley, K. W. et al. Gateway-compatible vectors for plant functional genomics and 29. Shi, X. et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene proteomics. Plant J. 45, 616–629 (2006). repression. Nature 442, 96 (2006).

A mechanically stabilized receptor–ligand flex-bond important in the vasculature

Jongseong Kim1, Cheng-Zhong Zhang1, Xiaohui Zhang1{ & Timothy A. Springer1

Haemostasis in the arteriolar circulation mediated by von crystal structures, minus the distance between the N terminus of A1 Willebrand factor (VWF) binding to platelets is an example of an and C terminus of GPIba in the complex structure (7.0 nm). The adhesive interaction that must withstand strong hydrodynamic close correspondence of the calculated distance of 18.2 nm to the forces acting on cells. VWF is a concatenated, multifunctional experimentally determined distance of 18.5 6 0.8 nm provides protein that has binding sites for platelets as well as subendothelial strong support that single A1–GPIba binding and unbinding events collagen1,2. Binding of the A1 domain in VWF to the glycoprotein Ib are being measured. Furthermore, a 26-residue linker yielded a con- a subunit (GPIba) on the surface of platelets mediates crosslinking tour length of 10.6 nm, also in good agreement with calculation of platelets to one another and the formation of a platelet plug for (Supplementary Fig. 4). Moreover, single-molecule tethers were dis- arterioles3,4. The importance of VWF is illustrated by its mutation tinguished by a 67-pN plateau in their force-extension curves at the in von Willebrand disease, a bleeding diathesis1. Here, we describe a B- to S-DNA transition8. novel mechanochemical specialization of the A1–GPIba bond for force-resistance. We have developed a method that enables, for the first time, repeated measurements of the binding and unbinding of acA1 A1 Digoxigenin d Pulling Bead a receptor and ligand in a single molecule (ReaLiSM). We demon- dsDNA Laser strate two states of the receptor–ligand bond, that is, a flex-bond. N N C C N N trap S-S S-S S-S C One state is seen at low force; a second state begins to engage at VWF dimer Relaxation 10 pN with a 20-fold longer lifetime and greater force resistance. VWF multimer GPIbα The lifetimes of the two states, how force exponentiates lifetime, b A1 and the kinetics of switching between the two states are all mea- Bead N sured. For the first time, single-molecule measurements on this N C Pipette system are in agreement with bulk phase measurements. The dsDNA Polypeptide Membrane results have important implications not only for how platelets linker Platelet Biotin bound to VWF are able to resist force to plug arterioles, but also e 15 f how increased flow activates platelet plug formation. 25 30 In the A1–GPIba complex, the VWF A1 domain interacts with the 20 20 10 5,6 Unbinding Count 10 concave face of the leucine-rich repeat (LRR) domain of GPIba 0 0 6 12 18 (Fig. 1). The N terminus of GPIba is within 2.2 nm of the C terminus 15 Extension (nm) of VWF A1, and neither terminus contributes to the complex inter- Force (pN)

Force (pN) 10 5 face. Therefore, we were able to link these termini with a 43-residue Binding polypeptide (Fig. 1c). 5 Unbinding data 20 nm Binding data DNA handles of 802 base pairs were specifically attached to Force clamp data 0 0 cysteine residues added to the C terminus of GPIba and N terminus Tether extension (nm) 0 3 6 9 12 15 18 Receptor-ligand extension (nm) of A1 (Fig. 1c and Supplementary Fig. 2). The handles were in turn attached to beads held by a micropipette and laser trap (Fig. 1d) in a Figure 1 | The A1 and GP1ba single-molecule construct and change in ‘mini laser-tweezers’ instrument suspended from a bungee cord7 extension on unbinding and rebinding. a–d, Schematic diagrams of VWF a a b c d (personal communication, S. B. Smith). By moving the laser trap, ( ), GPIb ( ), the ReaLiSM ( ), and the laser tweezers setup ( ). Ribbon diagrams based on the A1 domain-GP1ba LRR domain complex show the A1–GPIba ReaLiSM construct was subjected to cycles of increas- disulphide side chains as gold spheres. The LRR domain of GP1ba is ing and decreasing force. In each cycle, an abrupt increase in the magenta except LRR repeats 2–4 are grey. The mucin-like region between the length of the tether between the two beads was observed during pull- LRR domain and membrane in GP1ba, and the non-A1 portion of the VWF ing, and an abrupt contraction was observed during relaxation monomer which comprises 90% of its mass, are shown schematically. (Fig. 1e, Supplementary Fig. 3). e, Representative force-extension trace for one cycle of force increase (black) The abrupt extension events during unbinding were fit to the and decrease (red) in force-ramp experiments. f, Fit of receptor–ligand wormlike chain (WLC) model (Fig. 1f), which yielded a persistence extension (unbinding) data to the WLC model. Data were binned by force; one representative bin is shown in the inset. Error bars show 1 s.d. for force length of 0.8 6 0.2 nm and contour length of 18.5 6 0.8 nm. The and extension in each bin (n per bin 5 10 to 85 for unbinding, 26 to 30 for contraction events during rebinding fell on the same curve (Fig. 1f). rebinding, and 428 for force clamp). Fit to the WLC equation was by The expected contour length is calculated as the linker length of 43 occurrence-weighted least squares. Data from receptor–ligand binding and residues times an extension of 3.8 A˚ per residue (16.3 nm), plus the force-clamp experiments were not included in fitting, but fall on the same N- to C-terminal distances in the A1 (1.9 nm) and GPIba (7.0 nm) line.

1Immune Disease Institute, Children’s Hospital Boston and Department of Pathology, Harvard Medical School, 3 Blackfan Circle, Boston, Massachusetts 02115, USA. {Present address: Bioengineering Program and Mechanical Engineering and Mechanics Dept, Lehigh University, 19 Memorial Drive West, Bethlehem, Pennsylvania 18015a, USA. 992 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

30 102 20 adg103 40 nm s–1 5 nm s–1 1 0.19 ± 0.02 pN s–1 10 –1 1.6 ± 0.18 pN 102 10 15 102 –1

Count 1.9 ± 0.12 pN s 1 10–1 10 6 8 10 12 14 k 0 –1 1 off = 0.0027 s 0 0 Lifetime (s) [0.0011 to 0.0066 s–1] 20 20 –1 0 σ = 2.73 ± 0.53 nm be10 nm s–1 Ristocetin, 40 nm s 10 1 k 0 = 0.0015 s–1 –1 –1 2 off 0.32 ± 0.04 pN s 1.8 ± 0.16 pN s [0.0003 to 0.0076 s–1] –1 = 1.72 ± 0.48 nm 2.1 ± 0.14 pN s –1 σ2 10 10 10 h 103 k = k 0 exp (σF/k T) Ristocetin ( ) off off B 0 –1 Count k 1 off = 0.034 s σ1 = 1.15 nm

2 k 0 = 0.0017 s–1 0 0 10 2 off σ = 1.04 nm 30 cf30 2 20 nm s–1 Botrocetin –1 Botrocetin ( ) 0.7 ± 0.18 pN s–1 40 nm s k 0 1 1 off = 0.00041 s-1 –1 10 2.1 ± 0.1 pN s σ1 = 1.02 nm 15 0.9 ± 0.13 pN s–1 15 Lifetime (s)

Count 100

0 0 10–1 0 5 10 15 20 0 10 20 30 40 0 10 20 30 40 Unbinding force (pN) Unbinding force (pN) Force (pN)

Figure 2 | Force spectroscopy and bond lifetime. a–f, Unbinding force 40 nm s21 with a 26-residue linker (filled diamonds, Supplementary Fig. 4). distributions at different pulling rates in absence (a–d) or presence of The grey filled circle shows the lifetime in bulk phase measurements12. The 0.5 mg ml21 ristocetin (e) or 0.1 mgml21 botrocetin (f). Error bars show inset shows additional bond lifetime measurements (filled circles) at Poisson noise. Loading rate averages and s.d. are over rupture events at each constant force (Fig. 3). h, Lifetimes in presence of ristocetin and botrocetin. pulling rate. Curves show the predicted rupture force distributions9 using Errors are Poisson noise. For panels c–e, equation (1) was evaluated the constants from panels g and h. g, h, Bond lifetimes at constant force, separately for bins 1–4 (pathway 1) and bins 4–7/8 (pathway 2). Events in bin estimated from each bin in a–f, using the Dudko–Hummer–Szabo equation. 4 were apportioned between pathway 1 and 2 according to the fits. Fits to the g, Lifetimes from pulling at 5 nm s21 (open circles), 10 nm s21 (open Bell equation, shown in panels g and h, were by occurrence-weighted least squares), 20 nm s21 (open triangles) and 40 nm s21 (open diamonds), and squares (dashed and solid lines).

The force at which A1 and GPIba dissociated was determined at In force-clamp experiments, feedback was used to maintain a con- different laser trap pulling rates (nm s21) which yielded different stant force on the bead in the laser trap. Hopping between the bound force loading rates (pN s21) (Fig. 2a–d). At low pulling rates, bond and unbound states occurred over a narrow range of forces from 9.55 rupture events had a narrow, unimodal distribution (Fig. 2a, b). At to 10.58 pN (Fig. 3a–d). At the lower end of this force range A1– higher pulling rates, the distribution of dissociation forces was clearly GPIba was predominantly bound (Fig. 3a), and as force increased the bimodal (Fig. 2c, d). The peak at lower force (Fig. 2c, d) corre- proportion of the unbound state increased (Fig. 3b, c) until the sponded to the single peak at lower pulling rates (Fig. 2a, b); the unbound state predominated (Fig. 3d). The receptor–ligand exten- shape of the peak and its shift to higher force with increasing pulling sion measured in force-clamp experiments showed excellent agree- rate fit predictions for dynamic force spectroscopy (curves in Fig. 2)9. ment to the WLC fit to force-ramp data (Fig. 1f). The second peak also occurred at a slightly higher force at 40 nm s21 21 a than at 20 nm s (Fig. 2c, d; note difference in x-axis) and matched 9.55 ± 0.1 pN, ΔX = 9.5 nm 20 predictions (curves in Fig. 2). U 10 Since the lifetime of a bond is a function of the rate at which force is B 9 10 0 applied , with the Dudko–Hummer–Szabo equation , each of the b 10.05 ± 0.1 pN, ΔX = 11.5 nm 20 bins of the rupture force histograms shown in Fig. 2a–d was used to U 10 estimate lifetimes from the four different pulling rates (different B 0 symbols in Fig. 2g). The results demonstrate excellent agreement c ΔX 20 10.27 ± 0.1 pN, = 11.4 nm for the different pulling rates and the robustness of this elegant equa- U Extension (nm) 10 tion. The kinetics show two distinctive force-dependent lifetimes, or B 0 bond dissociation pathways. The first dissociation pathway predo- d 10.58 ± 0.1 pN, ΔX = 11.6 nm 20 minated below 8 pN, whereas above 12 pN a second pathway predo- U 10 minated (Fig. 2g). Indistinguishable results were obtained with a B 0 26-residue linker of distinct sequence (Fig. 2g, Supplementary Fig. 4b, 0 10 20 30 40 50 60 c). Thus, the A1–GPIba bond is a flex-bond, with one state in low Time (s) 100 force and switching to a second state induced by higher force. e 9.55 pN f 10.05 pN g 10.27 pN h 10.58 pN k k k Each pathway was well fit to an exponential decrease of lifetime k 1 off 1 off 1 off 11 –1 1 off k k 10 2 off 2 off k under tensile force (lines, Fig. 2g). The second state has a lower 2 off value of s, the constant that describes how much force exponentiates –2 Survival probability 10 koff (Fig. 2g). Thus, switching at higher force occurs to a bond that is 0 10 20 0 10 20 0 10 20 0 10 20 more force resistant. The estimate of the off-rate in the absence of Dwell time (s) 0 21 force for the pathway at lower force, k1 off , is 0.0027 s (confidence 21 Figure 3 | Force-clamp experiments. a–d, Extension over representative interval of 0.0011 to 0.0066 s ), in excellent agreement with bulk 6 . 5 D 125 60 s periods at the indicated clamped forces (average s.d., n 10 ). X is phase measurements of I-labelled A1 domain dissociation from the average extension between the bound (B) and unbound (U) states. 21 GPIba on agarose beads (koff 5 0.0036 6 0.0002 s ) and intact pla- e–h, Survival fraction of the bound state as a function of force and time in 21 12 telets (koff 5 0.0038 s ) (Fig. 2g) . a–d over periods of 200 to 400 s (n 5 55 to 172). 993 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

To measure kinetics in force-clamp experiments, periods of 200 to In the ReaLiSM construct, force is applied to the N terminus of A1 400 s were segmented into bound and unbound states based on the and C terminus of GPIba (Fig. 1c), similarly to physiologic force in spatial extensions in the trajectory (Methods). The lifetime in the vivo. When VWF is bound to multiple platelets and /or the vessel wall bound state showed double exponential decay (Fig. 3e–h). We fit this at a site of hemostasis, force applied to individual A1 domains will be data to the model shown in Fig. 4 (Methods, equations 2–4). These distributed to both their N and C termini, because of the long-range equations encompass the rates of conversion to state 2 (k12) and back disulphide bond within A1 that connects these termini (Fig. 1a). The conversion to state 1 (k21), as well as k1 off and k2 off (Fig. 4). Fits to all GPIba subunit is linked to the platelet through its C terminus four parameters (Supplementary Information, Supplementary Table (Fig. 1b), where the hydrodynamic force from the platelet will be 1) yielded values for k1 off and k2 off that were in excellent agreement applied. The close physical proximity of the receptor and ligand in with those obtained from the Dudko–Hummer–Szabo equation ReaLiSM is similar to the situation in a multivalent interaction, (inset, Fig. 2g). To obtain values with lower error estimates we also except A1 and GPIba remain in proximity because of the linker rather fixed k2 off at the values determined from the fit to the force-ramp than the presence of interactions between neighbouring A1 domains data, because fewer dissociation events occurred through the second in VWF and GPIb molecules on the platelet surface. 21 pathway at 10 pN (Fig. 3e–h), and fit the other three parameters Above a threshold shear of 5,000 s , VWF transitions from a com- 2–4,20 (Fig. 4, Supplementary Table 2). The estimates show that k12 and pact to an extended conformation that is active in hemostasis .At this shear, the force-loading rate in the largest VWF concatamers is k21 are on the order of 1 s, and both rates trend upward at higher 21 21 force. estimated to be well above the threshold of 0.9 pN s where the In clinical diagnosis of VWD, ristocetin is used to mimic shear- second state of the flex-bond is seen, and thus this second state may enhanced activation of VWF13. Ristocetin is an antibiotic that adven- function early in the initiation of platelet interactions with VWF titiously activates VWF; botrocetin is a snake venom protein that in vivo. activates VWF by a different mechanism and does not mimic shear The effect of the flex-bond is to broaden the distribution of forces (refs 13, 14 and references therein). The force-rupture histogram that A1–GPIba can withstand by extending the working range of the with ristocetin remained bimodal, with a marked shift of the second receptor–ligand bond by about 7 pN (Fig. 2g). This broad force range is peak from 14.8 to 22.4 pN at 40 nm s21 (Fig. 2d, e). In the presence of important, because the force experienced by the A1–GPIba bond will botrocetin a qualitatively different, unimodal distribution was seen, vary widely in vivo, depending on the length of the VWF multimer, and the force-rupture peak was shifted to an even higher level of binding to the vessel wall, the number and distribution of bound 28.4 pN at 40 nm s21 (Fig. 2f). Transformation of the force-rupture platelets, and the shear and elongational forces at the site of bleeding. distributions using the Dudko–Hummer–Szabo equation demon- Our experimental setup offers several advantages over previous strated good agreement with the exponential dependence of lifetime biophysical methods of studying receptor–ligand interactions. The on force embodied by the Bell equation (Fig. 2h). The kinetics further receptor and ligand are held away from bead surfaces by DNA handles, emphasize the selective strengthening by ristocetin of pathway 2 and reducing artefacts from nonspecific interactions between proteins and beads, or between probes and surfaces. Single tethers are unambi- demonstrate that both ristocetin and botrocetin decrease exponen- guously distinguished by overstretching of dsDNA around 67 pN. tiation by force. The ReaLiSM system provides further benefits. Previous studies of We have demonstrated that the bond between the VWF A1 receptor–ligand interactions were on receptors and ligands immo- domain and the platelet glycoprotein Ib a-subunit has two states bilized on separate surfaces. Receptor–ligand complexes were formed (Fig. 4). Only one pathway was seen at lower force-loading rates, 21 when two surfaces approached one another, but repeated measure- whereas at and above loading rates of 0.9 pN s , or at constant force ments on the same receptor and ligand pair were impossible. In , of 10 pN, a second pathway was found, and the force-rupture dis- ReaLiSM, the covalent tether between receptor and ligand not only tribution was bimodal. Agreement between force-ramp and force- enables repeated measurements on a single receptor–ligand complex, clamp experiments was excellent. Moreover, two linkers of different but also provides an intrinsic control that single molecule events are length and sequence showed no effect on off-rate or switching being measured, that is, the tether extension upon receptor–ligand between states. The rate of switching between the states, k12 and dissociation and the contraction upon rebinding. The advantages of k21, was overall increased by force. Thus, tensile force influences ReaLiSM may account for the marked differences in observed off-rates the lifetime of an A1–GPIba bond not only by shifting the equilib- and trends with an earlier AFM study reporting A1–GPIb as a catch- rium between the two states, but also by lowering the barrier for bond22 (Fig. Supplementary Fig. 6), as well as our ability to fit our data conversion between the two states. to equations, and to find an extrapolated off-rate within 1.5-fold, Slip- and catch-bonds are defined as bonds with lifetimes that rather than .1,000-fold, of bulk phase measurements12 (Fig. Sup- decrease and increase, respectively, with applied force. We find plementary Fig. 6). switching between two populations of bonds, each of which is a The finding that A1–GPIba is a flex-bond rather than a catch-bond slip-bond. Since slip- and catch-bonds are defined as opposites of has important conceptual implications. Shear flow above a threshold one another15, we propose a distinct name, the flex-bond. The ana- of 10,000 s21 activates binding of platelets to VWF 2, and this is logy is to muscles and to adhesion molecules such as selectins and thought to be related to the mechanism by which increased shear integrins, that have distinct flexed, low-affinity and extended, high- or elongational flow21 at sites of arteriolar haemorrhage activates affinity states16–19. platelet plug formation in vivo1–4. Conversion from a compact to an extended conformation of VWF, or conformational changes that remove shielding by neighbouring domains, may expose the A1 k 0 = 0.0027 s–1 NC1 off domain to activate binding to platelet GPIba20,23. Our results are B1 σ1 = 2.73 nm Force k , s–1 k , s–1 consistent with these mechanisms, and an increase in the rate of bond 21 12 24 10.05 pN 0.13 k k 0.57 formation triggered by shear flow , rather than an alternative mech- 21 12 N C 10.27 pN 0.28 0.40 anism in which a decrease in the rate of bond dissociation would 1.17 10.58 pN 0.60 U trigger haemostasis22. C N k 0 = 0.0015 s–1 B 2 off Ristocetin is thought to bind near a stretch of C-terminal A1 2 σ = 1.72 nm 2 domain residues in which gain-of-function VWD mutations are Figure 4 | The flex-bond. Schematic model of different states of the found (refs 13, 14 and references therein). Concordance between A1–GP1ba flex-bond, showing direction of tensile force (horizontal arrows) functional effects of antibodies and mutations indicate that ristocetin, and estimated constants. and not botrocetin, induces a conformational state of the A1–GPIba 994 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS bond similar to its shear-induced configuration13. These findings cor- bearing von Willebrand disease mutations. J. Biol. Chem. 279, 23327–23334 relate with our observation that ristocetin selectively stabilizes state 2, (2004). 7. Bustamante, C. & Smith, S. Light-force sensor and method for measuring axial and that flex-bond behaviour is seen in ristocetin and not botrocetin. optical-trap forces from changes in light momentum along an optic axis. US Although only a single A1–GPIba state has been seen in crystal Patent 7,133 132 (2004). structures, mutational data indicate a second, ristocetin- and force- 8. Bustamante, C., Smith, S. B., Liphardt, J. & Smith, D. Single-molecule studies of stabilized state. The N-terminal and C-terminal leucine rich repeats of DNA mechanics. Curr. Opin. Struct. Biol. 10, 279–285 (2000). GPIba (magenta, Fig. 1c), contact A1 in the crystal structure5,6. 9. Evans, E. & Ritchie, K. Strength of a weak bond connecting flexible polymer chains. Biophys. J. 76, 2439–2447 (1999). Although the middle repeats 2–4 (grey, Fig. 1c) make no contacts in 10. Dudko, O. K., Hummer, G. & Szabo, A. Theory, analysis, and interpretation of the crystal structure and are unimportant in botrocetin-stimulated single-molecule force spectroscopy experiments. Proc. Natl Acad. Sci. USA 105, binding of VWF to GPIba, they are required for ristocetin-stimulated 15755–15760 (2008). binding25. Furthermore, the higher the shear, the greater the import- 11. Bell, G. I. Models for the specific adhesion of cells to cells: a theoretical framework ance of repeats 2–4 (ref. 26). Thus, ristocetin and tensile force stabilize for adhesion mediated by reversible bonds between cell surface molecules. Science 200, 618–627 (1978). a second conformational state of the A1–GPIba complex in which 12. Miura, S. et al. Interaction of von Willebrand factor domain A1 with platelet LRR 2–4 of GPIba seem to contact the A1 domain. This evidence for glycoprotein Iba-(1–289). Slow intrinsic binding kinetics mediate rapid platelet two conformational states corresponds closely with our observation adhesion. J. Biol. Chem. 275, 7539–7546 (2000). that the A1–GPIba bond is a flex-bond with two states. Mutations in 13. Dong, J. F. et al. Ristocetin-dependent, but not botrocetin-dependent, binding of the A1 domain in gain-of-function type 2B von Willebrand disease are von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions. Blood 97, 162–168 (2001). also indicative of conformational change. These mutations enhance 14. De Luca, M. et al. Structure and function of the von Willebrand factor A1 domain: binding of A1 to GPIba, yet some map to sites that are buried in the A1 analysis with monoclonal antibodies reveals distinct binding sites involved in domain and may stabilize a conformation that has not yet been seen in recognition of the platelet membrane glycoprotein Ib-IX-V complex and crystal structures27. It will be interesting to examine whether gain of ristocetin-dependent activation. Blood 95, 164–172 (2000). function mutations in VWF and GPIba1 affect switching between the 15. Dembo, M., Torney, D. C., Saxman, K. & Hammer, D. The reaction-limited kinetics of membrane-to-surface adhesion and detachment. Proc. R. Soc. Lond. B 234, two states, and whether structural studies can reveal an extended, as 55–83 (1988). 5,6 well as the currently known flexed state , of the A1–GPIba complex. 16. Phan, U. T., Waldron, T. T. & Springer, T. A. Remodeling of the lectin/EGF-like interface in P- and L-selectin increases adhesiveness and shear resistance under METHODS SUMMARY hydrodynamic force. Nature Immunol. 7, 883–889 (2006). ReaLiSM constructs, with and without cysteines at the N and C termini, were 17. Astrof, N. S., Salas, A., Shimaoka, M., Chen, J. F. & Springer, T. A. Importance of purified from HEK293T cell supernatants. Only the construct with cysteines force linkage in mechanochemistry of adhesion receptors. Biochemistry 45, 15020–15028 (2006). coupled to DNA handles (Supplementary Fig. 2). ReaLiSM DNA handle con- 18. Springer, T. A. Structural basis for selectin mechanochemistry. Proc. Natl Acad. Sci. , jugates at 2 pM were incubated with 4.2 mm anti-digoxigenin Fab-beads. USA 106, 91–96 (2009). Trapped beads were brought close to 2.1 mm streptavidin-beads held in a micro- 19. Thomas, W. E., Vogel, V. & Sokurenko, E. Biophysics of catch bonds. Annu Rev pipette for tether formation. Each tether used for data collection passed a single- Biophys 37, 399–416 (2008). molecule test by showing a plateau in the force-extension curve at the B- to 20. Schneider, S. W. et al. Shear-induced unfolding triggers adhesion of von S-DNA transition. The instrument comprises a dual-beam counter-propagating Willebrand factor fibers. Proc. Natl Acad. Sci. USA 104, 7899–7903 (2007). optical trap, with optics and fluidics in a miniature 25 3 29 3 29 cm head sus- 21. Zhang, X., Halvorsen, K., Zhang, C. Z., Wong, W. P. & Springer, T. A. pended from a bungee cord. Optical force was measured directly from a differ- Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand ence in light momentum. Measurements were at 500 Hertz. In force-ramp Factor. Science 324, 1330–1334 (2009). experiments the trap was pulled away from or relaxed towards the fixed bead 22. Yago, T. et al. Platelet glycoprotein Iba forms catch bonds with human WT vWF at a constant speed. In force-clamp mode, force was auto-adjusted by feedback but not with type 2B von Willebrand disease vWF. J. Clin. Invest. 118, 3195–3207 control, with overall equilibration of the laser tweezer in 5 ms. (2008). 23. Ulrichts, H. et al. Shielding of the A1 domain by the D9D3 domains of von The lifetime t of a bond is a function of the rate at which force is applied9, 10 Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V. J. enabling t to be extracted from force rupture histograms : Biol. Chem. 281, 4699–4707 (2006). P(rupture above F) 24. Chen, S. & Springer, T. A. An automatic braking system that stabilizes leukocyte t(F)~ FF_ :p(rupture at F) rolling by an increase in selectin bond number with shear. J. Cell Biol. 144, 185–200 P ðÞDudkoHummerSzabo equation ð1Þ (1999). z N 25. Shen, Y. et al. Requirement of leucine-rich repeats of glycoprotein (GP) Iba for (hk =2 i~kz1 hi)DF ~ ðÞDudkoHummerSzabo equation shear-dependent and static binding of von Willebrand factor to the platelet h FF_ (F z(k{1=2)DF) k 0 membrane GP Ib-IX-V complex. Blood 95, 903–910 (2000). where P (rupture above F) is the total probability that bond rupture occurs above 26. Shen, Y. et al. Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Iba F, p (rupture at F) is the probability density of rupture events at force F, FF_ is the regulate shear-dependent cell adhesion to von Willebrand factor. J. Biol. Chem. force loading rate, DF is the bin width of the rupture force histogram that starts at 281, 26419–26423 (2006). F , h is the fraction of ruptures in the ith bin, and i and k are bin numbers. 27. Celikel, R., Ruggeri, Z. M. & Varughese, K. I. von Willebrand factor conformation 0 i and adhesive function is modulated by an internalized water molecule. Nature Full Methods and any associated references are available in the online version of Struct. Biol. 7, 881–884 (2000). the paper at www.nature.com/nature. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Received 21 December 2009; accepted 22 June 2010. Acknowledgements Supported by NIH HL-48675 (TAS). The authors are 1. Sadler, J. E. New concepts in von Willebrand disease. Annu. Rev. Med. 56, 173–191 indebted to S. B. Smith and C. Bustamante for help with laser tweezers construction (2005). and insightful discussion. We thank B. Coller, O. K. Dudko and C. Lu for reagents 2. Ruggeri, Z. M. & Mendolicchio, G. L. Adhesion mechanisms in platelet function. and insightful discussions, and J. Dill for software for data analysis. Circ. Res. 100, 1673–1685 (2007). 3. Ruggeri, Z. M., Orje, J. N., Habermann, R., Federici, A. B. & Reininger, A. J. Author Contributions T.A.S. designed and supervised the project. X.Z. cloned the Activation-independent platelet adhesion and aggregation under elevated shear ReaLiSM construct. J.K. designed experiments and collected and analysed data. stress. Blood 108, 1903–1910 (2006). C.-Z.Z. analysed data. T.A.S, J.K. and C.-Z.Z. wrote the paper. 4. Nesbitt, W. S. et al. A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nature Med. 15, 665–673 (2009). Author Information Reprints and permissions information is available at 5. Huizinga, E. G. et al. Structures of glycoprotein Iba and its complex with von www.nature.com/reprints. The authors declare no competing financial interests. Willebrand factor A1 domain. Science 297, 1176–1179 (2002). Readers are welcome to comment on the online version of this article at 6. Dumas, J. J. et al. Crystal structure of the wild-type von Willebrand factor A1- www.nature.com/nature. Correspondence and requests for materials should be glycoprotein Iba complex reveals conformation differences with a complex addressed to T.A.S. ([email protected]).

METHODS difference in the light momentum between the incident and output beams, that is, as a difference in the distance-weighted intensity on a position-sensitive Protein expression. The cDNA of human VWF A1 domain (Ile 1262 to Pro 1466 photodetector29. Dual trap positions were changed by deflecting two optical fibres with pre-pro-VWF numbering), and human platelet GPIba (His 1 to Arg 290) with piezo actuators. Trap positions were determined from two position-specific were PCR-amplified, and then used to construct the cDNAs of covalently tethered photodetectors. Here the two traps moved together by force feedback, so the two A1–GPIba with or without additional cysteines flanking the N and C termini position signals were averaged together. All measurements were recorded at (Supplementary Fig. 2a). The sequence of the peptide linker (TGGPTIKPPKP 500 Hz. PKPAPNLLGGPDKTHTKPPKPAPELLGGPGTG) was modified from the hinge Force-rip experiments. Force-rip experiments were carried out by stretching regions of murine IgG2a and human IgG1, with Cys residues either removed or and relaxing the tether between two force values, while moving the trap away substituted with Pro. All Lys residues were followed by Pro; the Lys-Pro sequence from (pulling) or towards (relaxation) the fixed bead held by the micropipette at is resistant to trypsin cleavage28. The engineered cDNAs were cloned into AgeI and a constant speed ranging from 5 to 40 nm s21. The distance was measured XhoI sites of plasmid pHLsec, which encodes a Kozak sequence, an N-terminal between two points on the force-trap position curves, from the point just before secretion signal sequence, a vector-derived ET sequence, and a C-terminal His 6 dissociation to the point when force returned to the same level after dissociation. tag31. A completely different, shorter, 26-residue sequence (GTGENLYFQGG The trap stiffness does not matter for that measurement because at each force the SSSSTTGWRGGHVGT) was used to construct a ReaLiSM to control for the relaxation of the bead in the trap is the same whether the tether is extended or effects of the length and the sequence on ReaLiSM properties. HEK293T cells not. The extra extension in the curves is solely due to unbinding of the receptor were transiently transfected using calcium phosphate or Lipofectamine. Culture and ligand in the ReaLiSM, because the persistence length of the polypeptide supernatants were harvested 3 days after transfection and proteins were purified linker (,1 nm) is significantly shorter than that of the dsDNA handles using Ni-NTA affinity chromatography followed by size-exclusion chromato- (,50 nm). Force loading rates (pN s21) before each rip-event were estimated graphy in 20 mM Tris, pH 8.0, 50 mM NaCl, 0.02% NP-40 and 5 mM EDTA from the curve and further averaged for all rupture events at a given pulling rate (Supplementary Fig. 2b, c). (nm s21). Sample preparation. DNA handles (802 bp) were PCR-amplified using forward The lifetime t of a bond is a function of the rate at which force is applied9, primers with a 59thiol group and reverse primers with either 59biotin or enabling t to be extracted from force rupture histograms10: 59digoxigenin, and activated with 2,29-dithio-dipyridine (DTDP) as described 21 P(rupture above F) previously . For protein–DNA coupling, 1 mM of protein (100 ml) was incubated t(F)~ in 0.1 mM DTT for 30 min under argon at room temperature, followed by FF_ :p(rupture at F) P Dudko Hummer Szabo equation removing DTT with 0.5 ml Zeba desalting columns (Pierce) twice. About N ðÞ ð1Þ (h =2z ~ z h )DF 0.1 mM of protein was allowed to react with 0.1 mM of each DTDP-activated ~ k i k 1 i ðÞDudkoHummerSzabo equation _ z { DNA handle in 20 mM Tris, pH 7.5, 100 mM NaCl, 0.01% NP-40 and 1 mM hk FF(F0 (k 1=2)DF) EDTA under argon for 16 h (typically 50 ml). Protein coupling to DNA handles where P (rupture above F) is the total probability that bond rupture occurs above was assayed by 4–20% native gels (Supplementary Fig. 2d). Material was stored at F, p (rupture at F) is the probability density of rupture events at force F, FF_ is the 280 uC. force loading rate, DF is the bin width of the rupture force histogram that starts at Carboxyl-polystyrene beads of 2.1 and 4.3 mm diameter (Spherotech) F0, hi is the fraction of ruptures in the ith bin, and i and k are bin numbers. We were washed and resuspended in 0.2 ml of 50 mM 2-(N-morpholino) ethane- used this equation to estimate bond lifetime at constant force from the rupture sulphonic acid pH 5.2, 0.05% ProClin 300 (Bangs Laboratories). 1-ethyl-3-(3- force histogram10. dimethylaminopropyl)carbodiimide (2 mg in 10 ml of the same buffer) was The above equation was obtained by inverting the bond rupture force distri- 21 added, followed after 5 min by 50 mg of 5 mg ml streptavidin (Invitrogen) or bution in dynamic force spectroscopy9: 2 1mgml 1 affinity-purified sheep anti-digoxigenin Fab (Roche) in PBS. After Ð { F {1 _ {1 shaking for 1 h at room temperature, beads were washed five times in PBS and exp 0 t(f ) FF(f ) df stored at 4 uC in PBS supplemented with 0.02% Tween 20 and 2 mM sodium p(F)~ ð2Þ t(F)FF_ (F) azide. Crude Bothrops jararaca venom (100 mg, Sigma) in 3 ml 20 mM Tris-HCl, The lifetime as a function of force, t(f), obtained from fittings in Fig. 2g, h, was pH 7.5 and 100 mM NaCl was centrifuged at 15,000g. The supernatant was substituted back into equation (2) with the average loading rate to calculate the applied to 1 ml HiTrap Q fast-flow (GE healthcare) and proteins were eluted rupture force distributions, which are shown as curves in Fig. 2a–f. with a 10 ml gradient of NaCl (0.1–0.46 M) in the buffer over 10 min at a flow Force-clamping experiments. In force-clamping experiments, the position of rate of 1 ml min21. Fractions were assayed for agglutination of platelet-rich the laser trap and the applied force were recorded at 500 Hz for 200–400 s, when plasma. The fractions showing highest activity were loaded on size-exclusion force was auto-adjusted by feedback control to the desired value. The feedback chromatography columns with Superdex 200 (GE Healthcare) in 20 mM Tris- time loop is 5 ms for overall equilibration of the laser tweezers. HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.02% Tween 20. The most active The baseline drift of extension data was corrected by a linear compensation, 21 fractions were pooled and stored at 280 uC. Ristocetin was purchased from x(t) 5 xdata(t)1vt; the slope v was in the range of 0.05 to 0.1 nm s . The average Sigma. spatial extensions for the bound and the unbound states were determined from Single-molecule force experiments. Appropriate dilutions of the receptor and the histogram of spatial extension data. We then assigned the state (U or B) at any ligand single molecule (ReaLiSM) construct (about 2 pM) were incubated with given time based on the spatial extension and generated intervals of bound and polystyrene beads (4.2 mm) functionalized with anti-digoxigenin polyclonal Fab. A unbound states. Intervals shorter than 10 ms (twice the instrument feedback bead trapped in the laser tweezers was brought close to a streptavidin-modified loop of 5 ms) were removed. An example of segmentation of the bound and polystyrene bead (2.1 mm) held by suction in a micropipette. To test for single- unbound states into a hopping diagram is shown in Supplementary Fig. 3. molecule tethers, each tether was first examined for a plateau in the force extension Assuming that the ReaLiSM system has no memory, we can treat each hopping curve (overstretching) at ,67 pN corresponding to the B- to S-DNA transition. event from the bound to unbound state as an independent dissociation of the Force was rapidly increased manually (about 80 nm s21), and as soon as over- bound state. The durations of the bound state were cumulated into lifetime stretching was seen, force was rapidly lowered to 2 pN. Rapidity was desired to distributions (Fig. 3e–h) to obtain dissociation kinetics. For clamping data at decrease the chance of biotin or digoxigenin dissociation from functionalized 10.05, 10.27 and 10.58 pN, exponential fitting of cumulative distributions of beads. Single tethers that passed the overstretching test, and also showed rip events, durations yielded two slopes, indicating two different kinetics. Therefore, data were then subjected to cycles of increasing and decreasing forces (force-rip experi- were fit to the kinetic model of the flex-bond in Fig. 4 and governed by equations ments) or constant force (force-clamp experiments). 3–5, Alternatively, if a multi tether rather than a single tether was obtained, it was d½B 1 ~{(k zk )½B zk ½B ð3Þ readily identified by a force-extension curve extending to 80 pN without any dt 1off 12 1 21 2 overstretching plateau. Such tethers were discarded. To search for further tethers, the bead in the trap was either slightly rotated using flow, or a new bead d½B2 ~{(k zk )½B zk ½B ð4Þ was picked up. dt 2off 21 2 12 1 The instrument (ref. 7, personal communication, S. B. Smith, and http:// tweezerslab.unipr.it) comprises a dual-beam counter-propagating optical trap, d½U ~k1off½B1zk2off½B2ð5Þ with all the optics and fluidics contained in a miniaturized (25 3 29 3 29 cm) dt head that is isolated from vibration by suspending it from a single bungee cord. where [B1] and [B2] are the probabilities of occurrences of the first and second Optical force exerted on the trapped bead was measured in real time as the bound states, and [U] the probability of the unbound state; k1offand k2offare

dissociation rates of the first and the second bound states; k12 is the forward for k2offby interpolation from Bell-model fitting of the force-dependent life- conversion rate (from the first to the second state), and k21 is the backward rate. times in Fig. 2g. This enabled us to estimate the other three rates, as shown in The same equations were used to fit two-state, but not one-state, catch bonds19,30. Supplementary Table 2. Durations in the bound states between 0.1 and 10 s were fitted to the analytical expression for d[U]/dt solved from equations (3–5) by a nonlinear-least-square 28. Kieliszewski, M. J., Leykam, J. F. & Lamport, D. T. Trypsin cleaves lysylproline in a method using MATLAB. Results for all four rate coefficients are summarized in hydroxyproline-rich glycoprotein from Zea mays. Pept. Res. 2, 246–248 (1989). Supplementary Table 1. Because relatively small fractions of long durations 29. Smith, S. B., Cui, Y. & Bustamante, C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 361, 134–162 . ( 3 s) are nonetheless important for determining the two conversion rates (2003). (k12 and k21) as well as the second dissociation rate (k2off), we varied initial guess 30. Thomas, W. et al. Catch-bond model derived from allostery explains force- for k2offin order to obtain best overall fits to data. Estimates were robust to activated bacterial adhesion. Biophys. J. 90, 753–764 (2006). different initial guesses; however, estimations of confidence intervals for the rate 31. Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high- coefficients were inaccurate and showed a broad range. To obtain better esti- level protein production in mammalian cells. Acta Crystallogr. D 62, 1243–1250 mates for the conversion rates and their confidence intervals, we fixed the value (2006).

Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching

Lawrence K. Lee1, Michael A. Ginsburg1, Claudia Crovace2, Mhairi Donohoe1 & Daniela Stock1,3

The flagellar motor drives the rotation of flagellar filaments at 36 and 46 residues of FliG from Salmonella typhimurium disrupt fla- hundreds of revolutions per second1,2, efficiently propelling bacteria gellar assembly and binding to FliF, respectively17.InA. aeolicus,the 3 through viscous media . The motor uses the potentialenergy from an equivalent residues map to the N-terminal three helices (helicesN1–3)of electrochemical gradient of cations4,5 across the cytoplasmic mem- the protein (Fig. 1c), indicating that these promote binding to FliF. braneto generate torque. A rapid switch from anticlockwiseto clock- FliGFL and FliGMC have been captured in different conformations. wise rotation determines whether a bacterium runs smoothly The most obvious conformational difference is in the middle domain. forward or tumbles to change its trajectory6,7. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in Helices the generation of torque8–13 through an interaction with the cation- a C1–6 channel-forming stator subunit MotA12. FliG has been suggested to adopt distinct conformations that induce switching but these struc- ARMC tural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in N-terminal domain Middle domain C-terminal domain rotational switching and uncover the structural basis for the forma- Loop tion of the FliG torque ring. This allows us to propose a model of the C complete ring and switching mechanism in which conformational HelixNM Helix changes in FliG reverse the electrostatic charges involved in torque MC generation. LoopM HelicesN1–5 The structure of the full-length (FL) FliG protein (FliGFL)from Aquifex aeolicus is entirely a-helical and consists of distinct amino- terminal (N), middle (M) and carboxy-terminal (C) globular domains, ARMM which are connected by two long helices (helixNM and helixMC) b c (Fig. 1a). The structure of the middle to C-terminal domain of FliG 14 from Thermotoga maritima (FliGMC) has been previously reported (Supplementary Fig. 1). Like FliGMC, the middle domain of FliGFL HelixC5 HelixN3 contains a single armadillo repeat motif (ARMM)whereasthe FliF-binding C-terminal domain can be further divided into a second ARM helices N terminus (ARMC) and a six-helix bundle (helicesC1–6). An overview of the full-length FliG structure, assembly of the FliG ring and switching mechanism is illustrated in Supplementary Movie 1. In the C-terminal domain, charged residues that are clustered around helixC5 result in a functionally important electrostatic charge C terminus distribution (Fig. 1b) that is involved in torque generation9–12,14. d N-terminal domain Surprisingly, the fold around these charges, comprising helicesC3–6, 10 20 30 40 50 60 70 is repeated in the N-terminal domain (helicesN1–4) (Fig. 1c). These | | | | | | | ALSKAQKAAVLLLSLPEEVSMNIVKELSEEELQKLFALAKDLESVPEEEIENIAEELLDEIKKAGIKI___ helices are moderately conserved and contain four groups of con- ____KNTLMIALLGAPEDIKQKFLSNMSKRAAKLFLEDMEALGPVKKSEIEKAQRQVVNIIRKMIDEGKIE served amino acid triplets (Fig. 1d). Charged residues on helixN3 | | | | | | | seem to invert the charges on the structurally equivalent torque 260 270 280 290 300 310 320 C-terminal domain helixC5 (Fig. 1c, d). However, in the N-terminal domain, this does not result in an obvious polar electrostatic charge distribution, indi- Figure 1 | Structural overview of the full-length FliG monomer. cating that the repeated fold is not echoed with a repeated torque- a–c, Residues are coloured from N to C terminus as a spectrum of colours generating function (Supplementary Fig. 2). from blue to red. Torque helixC5 and helixN3 are labelled with a red and blue asterisk respectively. Helices are shown with charged residues and the FliG is required for assembly of an intact flagellar motor8 where it C3–6 electrostatic potential on helixC5 in b adjacent to helicesN1–4 in c to highlight binds to a structural subunit called FliF, thereby coupling torque to the the conserved fold. d, Sequence alignment of the residues shown in b and 15,16 rest of the flagellar filament (Supplementary Fig. 3). Escherichia coli c. Conserved or similar residues are highlighted, and conserved amino acid cells containing a FliF–FliG fusion mutant can form fully assembled triads are underlined. HelixC5 and helixN3 are encircled and charged residues flagella, and the deletion of groups of 10 residues within the N-terminal on these helices are in red (negative) and blue (positive).

1Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool Street, Darlinghurst, New South Wales 2010, Australia. 2MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. 3Faculty of Medicine, University of New South Wales, Sydney 2052, Australia. 996 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

abc * d* HelixC5 HelixNM HelixMC HelixMC

LoopM

ARMM

A. aeolicus, T. maritima, A. aeolicus T. maritima closed open

Figure 2 | Conformational differences between A. aeolicus FliGFL and T. C-terminal domains of A. aeolicus FliGFL (CterFL)(c) and T. maritima maritima FliGMC.a, b, The middle domains of FliGFL (a; A. aeolicus, closed) FliGMC (CterMC)(d). Torque helixC5 is labelled with a red asterisk. Hinge and FLiGMC (b; T. maritima, open). The equivalent position of the extreme residues M236 and F237 are shown as sticks. clockwise-biased deletion mutant is coloured in magenta. c, d, The

Whereas ARMM is well conserved (Ca root mean squared deviation helixMC and ARMM and alters the register of helixMC, thereby ˚ (r.m.s.d.), 1.0 A), helixMC is packed tightly against ARMM and helixNM changing the orientation of the hydrophobic ridge along helixMC that in FliGFL (‘closed’ conformation, Fig. 2a), but dissociated from ARMM binds to ARMM (Supplementary Fig. 4). In contrast to clockwise- in FliGMC (‘open’ conformation, Fig. 2b). The closed conformation is biased mutants, anticlockwise-biased mutations do not occur at the stabilized by 15 mostly conserved hydrophobic residues, which form a helixMC–middle-domain interface, with the exception of the conser- highly complementary hydrophobic interface between helixMC and vative I122L mutation in E. coli. Combined, these rotationally biased ARMM (Supplementary Fig. 4a, b) that is disrupted in the open con- mutants indicate that the closed conformation represents FliG during formation. A second conformational difference is between the anticlockwise rotation and that switching to clockwise rotation may C-terminal domains of FliGMC (CterMC) and FliGFL (CterFL). Here, involve the dissociation of helixMC from ARMM to an open conforma- ˚ ˚ ARMC and helicesC1–6 are very similar (Ca r.m.s.d., 1.0 A and 0.77 A, tion via a putative hinge loop between ARMM and helixMC (loopM). respectively) and are connected by a highly conserved loop, but their This may explain why mutations at almost any residue in loopM can relative orientations differ between CterFL and CterMC (Fig. 2c, d). bias the rotation direction (Supplementary Movie 3). The middle They are related by rotations of 77.8u around the F237 w angle and domains of FliGFL and FliGMC seem to represent anticlockwise and 25.6u around the M236 w angle (Supplementary Movie 2). This rota- clockwise states respectively, indicating that CterFL and CterMC may tion alters the relative orientation of the torque helixC5, which has follow the same trend. Indeed, a second cluster of mutants occurs 13 been proposed as a mechanism for rotational switching . between the two subdomains (ARMC and helicesC1–6) of the We mapped all known FliG mutations that bias the direction of C-terminal domain. However it is less clear whether these favour rotation of the flagellar motor onto FliGFL (Supplementary Movie 3). either the CterFL or the CterMC conformation (Supplementary Fig. These are clustered in three regions of the protein. Two of these 5). A third cluster of mutations occurs at the loop between helixMC and clusters indicate that the conformational differences between FliGFL the C-terminal domain (loopC), which is another putative hinge loop 14,19 and FliGMC may reflect changes associated with motor switching for that has been implicated in switching . the following reasons. The first cluster is around helixMC, where a In the FliGFL crystal lattice (Fig. 3a), the base of ARMC is packed majority of clockwise-biased mutations occur at the interface between against the base of ARMM of an adjacent monomer (ARMM11) helixMC and the middle domain (Supplementary Fig. 4), and intro- (Supplementary Fig. 6a). The arrangement forms a stack of tandem duce charged or bulky residues that are likely to interfere with the ARM motifs resulting in a right-handed superhelix consisting of closed conformation. Furthermore, deletion of a moderately con- seven a-helices (Fig. 3b). The otherwise exposed hydrophobic patch 14 served 169PAA171 motif (Fig. 2a, b, in magenta) in S. typhimurium on the base of ARMC is completely buried in the large interacting results in an extreme clockwise bias18. This mutation is also likely to surface and forms part of a continuous hydrophobic core that destabilize the closed conformation as it shortens the loop between extends over the entire superhelix. The ARMC–ARMM11 stacking

ab e Conserved tandem ARM right-handed * s 5 *T * superhelix ice C orq el ue ARM h h C ue e lic rq e o s T * C 1 Å r.m.s.d. 5

ARMM+1 34 FliG subunits FliGFL FliGMC A. aeolicus P T. maritima P ( 21) ( 6422) * ** * * F liF –3 b es N1 cd* * Repeated FliG inding helic FliF UNIT **** binding protomers in the 45 nm same orientation curvature

45 nm FliGUNIT FliG monomer

Figure 3 | Structural basis for the formation of FliG multimers. a, The FliG magenta. c, Same as a, but highlighting the orientation of a FliG monomer multimer in the FliGFL crystal lattice. The ARMC–ARMM11 superhelix is and a FliGUNIT in the crystal lattice. Repeated FliGUNIT protomers in the encircled and expanded in b, which shows the ARM superhelix from FliGFL same orientation are shown linearly in d, and with the curvature of a 45-nm (left) adjacent to the ARM superhelix from FliGMC (right). The positions of diameter ring in e. mutations that inhibit FliM binding and flagellar assembly are shown in 997 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010 has marked parallels with eukaryotic ARM motifs20 (Supplemen- The FliG multimer resembles beads on a string, linked by residue tary Movie 4). In all structures containing ARM motifs, these form A193, one of the few amino acids unrestrained by secondary structure tandem repeats that interact extensively, resulting in a right-handed (Supplementary Fig. 6c, e). The repeating ‘beads’ are formed by an 21,22 superhelix that creates a surface for protein–protein interactions . L-shaped protomer (FliGUNIT) that contains two halves of adjacent The stacking is mediated typically by nine hydrophobic residues, FliG monomers (Supplementary Fig. 6a–c). Although loopC probably 20 14 usually consisting of leucine, valine or isoleucine . Similarly, the allows interdomain flexibility in the FliG monomer , the FliGUNIT is a stacking of ARMC–ARMM11 is mediated by eleven leucine, valine more robust arrangement of the three domains in FliG. In a FliGUNIT or isoleucine residues, eight of which are absolutely conserved as the intersubunit b-sheet between loopC–loopM11, in combination hydrophobic residues across all known FliG sequences. This indicates with the superhelix (ARMC–ARMM11), restrains the relative move- that the interaction is not specific to A. aeolicus but extends to all ment of the C-terminal domain (Supplementary Movie 5). The main flagellated bacteria. Indeed, in the crystal packing of the T. maritima body of the FliGUNIT contains a complete ARMC/M11 superhelix that FliGMC structure, the ARMC–ARMM11 interaction forms an identical is tethered to the base of the C-terminal helicesC1–6 through the ˚ (Ca r.m.s.d., 1.0 A) right-handed superhelix (Fig. 3b). Importantly, M236–F237 hinge loop. The torque helixC5 is perpendicular to the this is despite the FliGMC crystal originating from different species, axis of the superhelix at the top of the FliGUNIT (Fig. 3c) and the two constructs and crystal forms with no other conserved crystal contacts. long helices are at the base. HelixMC connects adjacent FliGUNIT Combined, these data indicate that the ARMC–ARMM11 inter- protomers and helixNM places the N-terminal domain next to the action is a real biological interaction and this has several profound FliGUNIT, thereby forming the L-shaped protomer. In the crystal implications. First, FliG forms part of the flagellar motor known as lattice, adjacent FliGUNIT protomers are in opposing orientations the switch complex, which contains two other proteins, FliM and (Fig. 3a and Supplementary Fig. 6a–c). FliN. All three proteins are required for flagellar assembly (Sup- To model the FliG ring, we applied a curvature to the multimer plementary Fig. 3). Mutations at the base of ARMC and ARMM in the crystal lattice, creating a ring with a 45-nm diameter that and on the face of ARMM, which contains a highly conserved corresponds to a region of the flagellar motor known as the C ring, EHPQR motif14, can disrupt flagellar assembly8,10,23 and FliM bind- where the switch complex is thought to reside (Supplementary Fig. 3). 24 ing . On the basis of the structure of a single FliG monomer, it seems The model contains 40 FliGUNIT protomers, where adjacent proto- 14 evident that ARMC and ARMM are separate FliM binding sites . mers are in opposing orientations resulting in a 20-fold symmetry However the ARMC–ARMM11 superhelix indicates that ARMC and (Supplementary Fig. 7a). However, this is inconsistent with the appar- 25 ARMM from adjacent monomers interact to create a surface for a ent 34-fold symmetry of the C ring . Furthermore, the opposing single FliM binding site, which is consistent with all other known arrangement of alternating N- and C-terminal domains is inconsistent ARM superhelices that stack to form a surface for protein–protein with the location of their respective interacting proteins, FliF and interactions. Second, in addition to its interactions with FliF and MotA. These are both on the periplasmic side of the C ring (Sup- FliM, proper assembly of the bacterial flagellum also requires a plementary Fig. 3). Therefore, we generated rings by repeating the FliG–FliG interaction that is independent of other components of FliGUNIT protomers in the same orientation (Fig. 3d). These rings the flagellar motor. This is well supported by mutagenesis studies that satisfy the spatial restraints imposed by the location of the MotA demonstrate the requirement of at least five hydrophobic residues at stators and the FliF ring (Fig. 3e). Furthermore, the 45-nm ring con- 10,23,24 the ARMC–ARMM11 interface for flagellar assembly (Fig. 3b). tains 34 monomers, which is in agreement with the 34-fold symmetry 25 Last, because ARMC forms a right-handed superhelix with ARMM11, of the C ring (Fig. 3e). Attempts to generate rings that match the size it follows that ARMC11 has the same interaction with ARMM12. and symmetry of the MS ring, which is integrated into the cytoplasmic Thus, the ARMC–ARMM11 superhelix is the structural basis for the membrane, were unsuccessful (Supplementary Fig. 7c, d). formation of FliG multimers (Supplementary Fig. 6a, b). When protomers are arranged in the same orientation, the con- As mentioned earlier, a cluster of rotationally biased mutations tinuous FliG polypeptide chain that spans adjacent FliGUNIT proto- 14,19 ˚ highlight the importance of the putative hinge loopC in switching . mers is broken with a distance of around 34 A between A193 and Similarly, such mutations also occur at almost every residue on hinge E192 (Fig. 4b). Remarkably, dissociation of helixMC from ARMM into loopM, which mediates the transition from the closed to open con- an open conformation brings these residues to within a peptide-bond formation. Remarkably, in the FliG multimer, loopC and loopM11 distance (Fig. 4a, b). Similarly, this can be achieved with FliG in the form an intermolecular anti-parallel b-sheet (Supplementary Fig. 6d, closed conformation by rotating the main body of the FliGUNIT f) explaining how mutations in loopC may influence switching. (Fig. 4b, c). These conformational changes may explain why FliG

abc

HelixMC, open (clockwise) HelixMC, closed (anticlockwise)

E192 A193 de

Clockwise Anticlockwise

Reversal of torque helixC5

Figure 4 | Molecular basis of rotational switching. a–c, An expanded view alternative conformational change when helixMC remains in the closed of the encircled region in Fig. 3e is shown with one coloured FliG polypeptide conformation. d, e, The FliG ring with the monomers in the FliGMC and the chain. E192 and A193 are shown as yellow and red spheres respectively. FliGFL crystal structures, respectively. Charged residues on torque helixC5 a, b, Transition from the closed to the open conformation of helixMC. b, c,An are shown in blue (positive) and red (negative). 998 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS alone does not spontaneously form an oligomer without an inter- superhelix (Supplementary Fig. 9b), pointing towards the expected action with the FliF scaffold. location of FliM (Supplementary Fig. 3). Finally, by docking struc- We generated two FliG rings, one with each monomer in the FliGFL tures of FliM and FliN subunits into the remaining density of the C (closed, anticlockwise) conformation and the other with each mono- ring we demonstrate that this density is sufficiently large to accom- mer in the FliGMC (open, clockwise) conformation (Fig. 4d and e, modate rings of both subunits in an arrangement that is consistent respectively). In the context of the complete FliG ring, the conforma- with biochemical studies (Supplementary Fig. 11). tional differences between FliGFL and FliGMC result in a reversal of the electrostatic charges on torque helixC5 of FliG, providing a model for METHODS SUMMARY rotational switching of the flagellar motor. Furthermore, to visualize The full-length FliG protein from A. aeolicus was expressed in E. coli and crystal- the transition from FliGFL to FliGMC, we generated 100 intermediate lized as detailed in Supplementary Information. Crystals belong to space group structures between each state by interpolating the different w and y P21 with one FliG monomer in the asymmetric unit. Native X-ray diffraction torsion angles. This illustrates that the entire conformational change data were collected at the European Synchrotron Radiation Facility (ESRF) in occurs without any significant clashes in the ring (Supplemen- Grenoble, France, and single or multiple wavelength anomalous dispersion (SAD or MAD) data were collected at the Advanced Photon Source (APS) in tary Movie 6). Chicago, USA. The methods used for structure determination are detailed in As the FliG monomer spans two adjacent FliGUNIT protomers, Supplementary Information. X-ray data and refinement statistics are given in neighbouring protomers are restrained to the same open or closed Supplementary Tables 1 and 2, respectively. Still images and movie frames of conformation. This indicates that switching entails a bistable global protein structures were generated with PyMOL (http://www.pymol.org). conformational switch where adjacent FliG monomers are restrained Morphing and modelling software were written in C11. to the same rotational state. However, there is sufficient flexibility in Full Methods and any associated references are available in the online version of the model to allow for opposing conformations in the ring that are the paper at www.nature.com/nature. bridged by protomers in intermediate states (Supplementary Fig. 8). This is compatible with the conformational spread model of co- Received 22 February; accepted 21 June 2010. operativity26, the hallmarks of which were recently observed in the Published online 1 August 2010; corrected 19 August 2010 (see full-text HTML flagellar motor27. version for details). The FliG ring is consistent with three-dimensional electron micro- 1. Lowe, G., Meister, M. & Berg, H. C. Rapid rotation of flagellar bundles in swimming scopy reconstructions. The highest resolution three-dimensional bacteria. Nature 325, 637–640 (1987). electron microscopy reconstruction of the flagellar motor so far 2. Magariyama, Y. et al. Very fast flagellar rotation. Nature 371, 752 (1994). 3. Berg, H. C. & Anderson, R. A. Bacteria swim by rotating their flagellar filaments. has been generated from single-particle averaging of a clockwise- Nature 245, 380–382 (1973). 25 locked 69PAA171-deletion mutant from S. typhimurium .We 4. Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M. & Van der Drift, C. A docked the FliG ring into the C ring, which has 34 distinct lobes on protonmotive force drives bacterial flagella. Proc. Natl Acad. Sci. USA 74, the periplasmic side at the outer periphery. The shape and size of 3060–3064 (1977). 1 these lobes complement that of the ARM superhelix and the C- 5. Hirota, N. & Imae, Y. Na -driven flagellar motors of an alkalophilic Bacillus strain YN-1. J. Biol. Chem. 258, 10577–10581 (1983). terminal domain in the clockwise FliG ring (Supplementary Fig. 9a). 6. Berg, H. C. & Brown, D. A. Chemotaxis in Escherichia coli analysed by three- Notably, the anticlockwise FliG ring is not consistent with the clock- dimensional tracking. Nature 239, 500–504 (1972). wise-locked electron microscopy reconstruction (Supplementary 7. Turner, L., Ryu, W. S. & Berg, H. C. Real-time imaging of fluorescent flagellar Fig. 10). filaments. J. Bacteriol. 182, 2793–2801 (2000). 8. Irikura, V. M., Kihara, M., Yamaguchi, S., Sockett, H. & Macnab, R. M. Salmonella The vertical cross-section of the C ring from various electron micro- typhimurium fliG and fliN mutations causing defects in assembly, rotation, and graphs has two lobes on the periplasmic side of the S. typhimurium C switching of the flagellar motor. J. Bacteriol. 175, 802–810 (1993). ring. The inner lobe is missing in the same micrograph of a flagellar 9. Lloyd, S. A. & Blair, D. F. Charged residues of the rotor protein FliG essential for motor containing a FliG–FliF fusion deletion mutant that does not torque generation in the flagellar motor of Escherichia coli. J. Mol. Biol. 266, contain the first 94 residues of FliG (N-terminal domain and part of 733–744 (1997). 10. Lloyd, S. A., Tang, H., Wang, X., Billings, S. & Blair, D. F. Torque generation in the helixNM) and the position of the C ring is shifted up towards the MS flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM 28 ring compared to the native or full-length FliG–FliF mutants .We or FliN. J. Bacteriol. 178, 223–231 (1996). overlayed the cross-section of the FliG ring onto these micrographs and 11. Yorimitsu, T., Mimaki, A., Yakushi, T. & Homma, M. The conserved charged the C-terminal domain fits well on the outer lobe, the N-terminal residues of the C-terminal region of FliG, a rotor component of the Na1-driven domain on the inner lobe and the ARM superhelix in between flagellar motor. J. Mol. Biol. 334, 567–583 (2003). 12. Zhou, J., Lloyd, S. A. & Blair, D. F. Electrostatic interactions between rotor and (Supplementary Fig. 9b). Furthermore, we deleted the equivalent of stator in the bacterial flagellar motor. Proc. Natl Acad. Sci. USA 95, 6436–6441 the first 94 residues from FliG and overlayed this truncated structure (1998). onto electron micrographs of the vertical cross-section of the FliG–FliF 13. Lloyd, S. A., Whitby, F. G., Blair, D. F. & Hill, C. P. Structure of the C-terminal deletion fusion mutant. The micrographs precisely shadow the struc- domain of FliG, a component of the rotor in the bacterial flagellar motor. Nature 400, 472–475 (1999). ture of the truncated FliG ring, and the shift in the position of the C ring 14. Brown, P. N., Hill, C. P. & Blair, D. F. Crystal structure of the middle and C-terminal relative to the MS ring reflects the shortened link between these rings as domains of the flagellar rotor protein FliG. EMBO J. 21, 3225–3234 (2002). a result of deleting the N-terminal domain (Supplementary Fig. 9c). 15. Francis, N. R., Irikura, V. M., Yamaguchi, S., DeRosier, D. J. & Macnab, R. M. The orientation and location of the FliG rings on the periplasmic Localization of the Salmonella typhimurium flagellar switch protein FliG to the side of the C ring satisfies spatial restraints, which have been deter- cytoplasmic M-ring face of the basal body. Proc. Natl Acad. Sci. USA 89, 6304–6308 (1992). mined from structural, mutagenesis and electron microscopy data. 16. Ueno, T., Oosawa, K. & Aizawa, S. M ring, S ring and proximal rod of the flagellar First, in situ cryo-electron tomograms indicate that the MotA/B sta- basal body of Salmonella typhimurium are composed of subunits of a single tor interacts with the outer circumference of the periplasmic side of protein, FliF. J. Mol. Biol. 227, 672–677 (1992). the C ring29,30 (Supplementary Fig. 3). Supplementary Fig. 9b and 17. Kihara, M., Miller, G. U. & Macnab, R. M. Deletion analysis of the flagellar switch Fig. 4d and e illustrate how helix forms the outer circumference of protein FliG of Salmonella. J. Bacteriol. 182, 3022–3028 (2000). C5 18. Togashi, F., Yamaguchi, S., Kihara, M., Aizawa, S. I. & Macnab, R. M. An extreme the periplasmic side of the C ring and how the electrostatic charges clockwise switch bias mutation in fliG of Salmonella typhimurium and its that are involved in torque generation point towards the periplasm. suppression by slow-motile mutations in motA and motB. J. Bacteriol. 179, Second, all FliF-binding helicesN1–3 are located in the inner circum- 2994–3003 (1997). ference of the ring in an optimal position to interact with the MS ring 19. Van Way, S. M., Millas, S. G., Lee, A. H. & Manson, M. D. Rusty, jammed, and well- oiled hinges: mutations affecting the interdomain region of FliG, a rotor element of (Fig. 3e and Supplementary Figs 3, 9b). Third, in the FliG ring, the Escherichia coli flagellar motor. J. Bacteriol. 186, 3173–3181 (2004). mutations that affect the interaction with FliM segregate to a single 20. Andrade, M. A., Petosa, C., O’Donoghue, S. I., Muller, C. W. & Bork, P. Comparison location on the cytoplasmic side of the ring on the surface of the ARM of ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18 (2001). 999 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

21. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Crystallographic analysis of Acknowledgements We thank the staff at beamline ID14-1 at the ESRF, and at the recognition of a nuclear localization signal by the nuclear import factor beamlines 14-ID (BioCARS) and 23-ID (General Medicine and Cancer Institutes karyopherin a. Cell 94, 193–204 (1998). Collaborative Access Team (GM/CA-CAT)) at the APS for their support. Use of 22. Huber, A. H., Nelson, W. J. & Weis, W. I. Three-dimensional structure of the the APS was supported by the US Department of Energy, Basic Energy Sciences, armadillo repeat region of b-catenin. Cell 90, 871–882 (1997). Office of Science (contract DE-AC02-06CH11357), and use of the BioCARS Sector 23. Brown, P. N., Terrazas, M., Paul, K. & Blair, D. F. Mutational analysis of the flagellar 14 was supported by the National Institutes of Health, National Center for Research protein FliG: sites of interaction with FliM and implications for organization of the Resources (grant RR007707). GM/CA-CAT was funded in whole or in part with switch complex. J. Bacteriol. 189, 305–312 (2007). federal funds from the National Cancer Institute (Y1-CO-1020) and the National 24. Marykwas, D. L. & Berg, H. C. A mutational analysis of the interaction between Institute of General Medical Science (Y1-GM-1104). This work was supported by FliG and FliM, two components of the flagellar motor of Escherichia coli. J. Bacteriol. the Australian Synchrotron Research Program of the Australian Nuclear Science 178, 1289–1294 (1996). Technology Organization. D. Thomas is acknowledged for providing us with the 25. Thomas, D. R., Francis, N. R., Xu, C. & DeRosier, D. J. The three-dimensional electron microscopy density map of the clockwise-locked S. typhimurium mutant structure of the flagellar rotor from a clockwise-locked mutant of Salmonella and O. Perisic and H. Huber are acknowledged for cloning vectors (pOPT-GST) and enterica serovar Typhimurium. J. Bacteriol. 188, 7039–7048 (2006). A. aeolicus genomic DNA, respectively. D.S. was initially funded by an MRC Career 26. Duke, T. A., Le Novere, N. & Bray, D. Conformational spread in a ring of proteins: a development award (UK) and C.C. by an MRC predoctoral fellowship (UK). stochastic approach to allostery. J. Mol. Biol. 308, 541–553 (2001). Author Contributions L.K.L. collected synchrotron data, solved and analysed the 27. Bai, F. et al. Conformational spread as a mechanism for cooperativity in the structure, wrote software to generate FliG rings and switch morphs, and wrote the bacterial flagellar switch. Science 327, 685–689 (2010). manuscript, M.D. collected synchrotron data, M.D. and M.A.G. purified and 28. Thomas, D., Morgan, D. G. & DeRosier, D. J. Structures of bacterial flagellar crystallized selenomethionine-containing protein, C.C. cloned, purified and motors from two FliF–FliG gene fusion mutants. J. Bacteriol. 183, 6404–6412 crystallized the native protein, D.S. conceived and coordinated project, collected (2001). synchrotron data and wrote the manuscript. 29. Liu, J. et al. Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C ring assembly flexion. Author Information The coordinates and structure factors of the A. aeolicus FliG J. Bacteriol. 191, 5026–5036 (2009). monomer have been deposited in the Protein Data Bank under accession code 30. Murphy, G. E., Leadbetter, J. R. & Jensen, G. J. In situ structure of the complete 3HJL. Reprints and permissions information is available at www.nature.com/ Treponema primitia flagellar motor. Nature 442, 1062–1064 (2006). reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/ Supplementary Information is linked to the online version of the paper at nature. Correspondence and requests for materials should be addressed to D.S. www.nature.com/nature. ([email protected]).

METHODS produced a complete model, which was used to phase the native data set to 2.4 A˚ resolution. This was refined with REFMAC and PHENIX37 in several cycles The full-length FliG protein from A. aeolicus was expressed with an N-terminal iterating with model building (see Supplementary Table 2). The refined structure cleavable glutathione S-transferase (GST) tag and purified using a GSTrap HP was assessed with MolProbity38. Partial charges were calculated with PDB2PQR39 column (GE Healthcare). The GST tag was then removed with 63 His-TEV and the Poisson–Boltzmann equation was solved using the APBS module of protease. The protein mixture was passed through HisTrap and GSTrap columns the VMD molecular graphics software40. Morphs and intermediate states were (GE Healthcare) to remove TEV protease and GST, respectively. The flow-through w y containing FliG protein was concentrated and passed over a superdex 200 column generated by interpolating and angles in putative hinge loop regions. (GE Healthcare), equilibrated in 20 mM Tris/Cl, pH 7.4, 100 mM NaCl and eluted as a single peak at the expected size of a FliG monomer. Peak fractions were pooled 31. Leslie, A. G. W. in Joint CCP4 and ESF-EACMB Newsletter on Protein Cystallography No. 26 (Daresbury Laboratory, 1992). and concentrated to 10 mg ml21. The protein was crystallized at 20 uCin 32. Collaborative Computational Project Number 4.. The CCP4 suite: programs for Cryschem sitting drop vapour diffusion plates (Hampton Research) with a pre- protein crystallography. Acta Crystallogr. D 50, 760–763 (1994). cipitant containing 45% 1,4 butanediol and 0.1 M Tris-acetate, pH 8.75. Crystals 33. Leahy, D. J., Hendrickson, W. A., Aukhil, I. & Erickson, H. P. Structure of a were flash-frozen in liquid nitrogen and a native data set was collected at beamline fibronectin type III domain from tenascin phased by MAD analysis of the ID14-1 at the ESRF. Data were processed with MOSFLM31 and SCALA32 (see selenomethionyl protein. Science 258, 987–991 (1992). Supplementary Table 1). Attempts to solve the structure using T. maritima 34. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008). FliGMC coordinates as a template for molecular replacement were unsuccessful. 35. Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter Instead, crystals were grown using selenomethionine-substituted protein and data refinement for the multiple isomorphous replacement and multiwavelength were collected at three different wavelengths at beamline 14-ID-B at the APS. anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997). Selenomethionine-containing protein was overexpressed in E. coli B834 (DE3) 36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta cells (Novagen) with selenomethionine-containing media33 and purified as Crystallogr. D 60, 2126–2132 (2004). described earlier, except that 10 mM b-mercaptoethanol was added to all 37. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002). buffers. Selenomethionine-containing crystals were grown under the same con- 38. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for ditions as native crystals, except that 2 mM b-mercaptoethanol was added and the proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007). drops were micro-seeded with seeds from native crystals. Initial phases were 39. Dolinsky, T. J. et al. PDB2PQR: expanding and upgrading automated preparation obtained by combining SAD data from three isomorphous crystals containing of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, selenomethionine-substituted protein (see Supplementary Table 1). The heavy W522–W525 (2007). atom positions were determined with SHELX34 and refined with SHARP35. 40. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Rounds of model building with Coot36 and refinement with REFMAC532 Graph. 14, 33–38 (1996).

Crystal structure of the a6b6 holoenzyme of propionyl-coenzyme A carboxylase

Christine S. Huang1*, Kianoush Sadre-Bazzaz1*, Yang Shen1{, Binbin Deng2, Z. Hong Zhou2,3 & Liang Tong1

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin- To prepare samples of the PCC holoenzyme for structural studies, dependent enzyme, is essential for the catabolism of the amino the a- and b-subunits were co-expressed in Escherichia coli using a acids Thr, Val, Ile and Met, cholesterol and fatty acids with an bicistronic plasmid. We first obtained crystals of HsPCC but could not odd number of carbon atoms. Deficiencies in PCC activity in improve the diffraction beyond 5.5 A˚ resolution after extensive efforts. humans are linked to the disease propionic acidaemia, an auto- In addition, the crystals exhibited perfect twinning (space group R3). somal recessive disorder that can be fatal in infants1–4. The holo- We then examined a collection of bacterial PCCs, and were able ˚ enzyme of PCC is an a6b6 dodecamer, with a molecular mass of 750 to produce crystals of RpPCC that diffracted to 3.3 A resolution. kDa. The a-subunit contains the biotin carboxylase (BC) and biotin However, these crystals were also perfectly twinned (space group carboxyl carrier protein (BCCP) domains, whereas the b-subunit P3). Finally, we discovered that a PCC chimaera, containing the supplies the carboxyltransferase (CT) activity. Here we report the a-subunit of RpPCC and the b-subunit of Roseobacter denitrificans ˚ crystal structure at 3.2-A resolution of a bacterial PCC a6b6 holo- PCC (RdPCC), produced crystals without twinning (space group P1), enzyme as well as cryo-electron microscopy (cryo-EM) reconstruc- and we determined its structure at 3.2 A˚ resolution (Supplemen- tion at 15-A˚ resolution demonstrating a similar structure for tary Table 1). RdPCC is a close homologue of RpPCC, with their human PCC. The structure defines the overall architecture of b-subunits sharing 88% sequence identity (Supplementary Fig. 2). PCC and reveals unexpectedly that the a-subunits are arranged as The structure of the chimaera is essentially the same as that of the monomers in the holoenzyme, decorating a central b6 hexamer. A native RpPCC dodecamer (Supplementary Text) as well as that of the hitherto unrecognized domain in the a-subunit, formed by resi- HsPCC holoenzyme (see below). dues between the BC and BCCP domains, is crucial for interactions The structure of the a6b6 PCC holoenzyme contains a central b6 with the b-subunit. We have named it the BT domain. The structure hexamer core, in the shape of a short cylinder with a small hole along reveals for the first time the relative positions of the BC and CT its axis (Fig. 1a). This hexameric core can be considered as a trimer of active sites in the holoenzyme. They are separated by approxi- b2 dimers, with each dimer being formed by one subunit from each mately 55 A˚ , indicating that the entire BCCP domain must trans- layer of the structure (Fig. 1b). In contrast, the a-subunits are locate during catalysis. The BCCP domain is located in the active arranged as monomers on both ends of the b6 core, far from the site of the b-subunit in the current structure, providing insight centre of the holoenzyme, with each a-subunit contacting primarily for its involvement in the CT reaction. The structural informa- only one b-subunit (see below). There are no significant conformation establishes a molecular basis for understanding the large tional differences among the six copies of the a- and b-subunits of the collection of disease-causing mutations in PCC and is relevant holoenzyme (Supplementary Information). for the holoenzymes of other biotin-dependent carboxylases, We performed cryo-EM studies on HsPCC and obtained a three- including 3-methylcrotonyl-CoA carboxylase (MCC)5–7 and eukar- dimensional reconstruction at 15-A˚ resolution by single-particle ana- yotic acetyl-CoA carboxylase (ACC)8,9. lysis (Supplementary Figs 3–6). The cryo-EM envelope is remarkably PCCs catalyse the carboxylation of propionyl-CoA to produce similar to the overall shape of the crystal structure (Fig. 1c). In fact, D-methylmalonyl-CoA. These enzymes are found in organisms from the atomic model can be readily fitted into the cryo-EM map, giving a bacteria to humans, with highly conserved amino-acid sequences. cross-correlation value of 0.80, and only the BCCP domain appears For example, the a- and b-subunits of human PCC (HsPCC) and to be in a somewhat different position (Fig. 1d). These studies Ruegeria pomeroyi PCC (RpPCC) share 54% and 65% sequence iden- demonstrate that the structure of HsPCC is highly similar to that tity, respectively (Supplementary Figs 1 and 2). To simplify discus- of the bacterial enzyme. sions, we have numbered residues in bacterial PCCs according to An unexpected discovery from the crystal structure of the holo- their equivalents in HsPCC. The BC and BCCP domains in the enzyme is that there is little direct contact between the BC domain in a-subunit are homologous to their equivalents in ACC and pyruvate the a-subunit and the b-subunit (Fig. 2a). Instead, interactions with carboxylase (PC), whereas the b-subunit is homologous to the CT the b-subunit are primarily mediated by a hitherto unrecognized domain of ACC. The active site of BC is formed by residues from its A domain in the a-subunit, formed by residues 514–653 in the connec- and C (sub-)domains, whereas the B (sub-)domain forms a lid that tion between the BC and BCCP domains (Supplementary Fig. 1). We can assume open and closed conformations9–11. The active site of CT have named it the BT domain, as it mediates BC–CT interactions. is located at the interface of its dimer, and each CT contains two (sub-) The BT domain has well-defined electron density (Supplementary domains, the N and C domains9,12. In contrast to the wealth of Fig. 7), suggesting that it is highly ordered in the holoenzyme. A total information about these domains, little is known about how they of 1,950 A˚ 2 of the surface area of each a-subunit is buried at the are assembled into the holoenzyme of PCC (or ACC). interface with the b-subunits. Only 200 A˚ 2 are contributed by the

1Department of Biological Sciences, Columbia University, New York, New York 10027, USA. 2Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas 77030, USA. 3Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA. {Present address: Department of Antibody Technology, ImClone Systems, 180 Varick Street, 6th Floor, New York, New York 10014, USA. *These authors contributed equally to this work. 1001 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

abα2

β1 α N

BCCP BT β β2 Biotin

B C C β α1 A BC α β3 α3

Figure 1 | Structure of the PCC holoenzyme. a, Structure of the rectangle, the region shown in detail in Fig. 2a. c, Cryo-EM reconstruction of RpPCCa–RdPCCb chimaera, viewed down the threefold symmetry axis. HsPCC at 15-A˚ resolution, viewed in the same orientation as a. The atomic Domains in the a- and b-subunits in the top half of the structure are given model of the chimaera was fit into the cryo-EM envelope. d, Cryo-EM different colours, and those in the first a- and b-subunits are labelled. The a- reconstruction viewed in the same orientation as b. Arrows, a change in the and b-subunits in the bottom half are coloured in magenta and green, BCCP position that is needed to fit the cryo-EM map. All the structure respectively. Red arrow, the viewing direction of b. b, Structure of the figures were produced with PyMOL (www.pymol.org), and the cryo-EM RpPCCa–RdPCCb chimaera, viewed down the twofold symmetry axis. Red figures were produced with Chimera30. BC domain (Fig. 2a). In contrast, the BT domain contributes 1,300 A˚ 2 conserved or show conservative variations among the PCC enzymes to the buried surface area with one b-subunit, and an additional (Supplementary Figs 1 and 2), consistent with our observations that 100 A˚ 2 with an adjacent b-subunit (Fig. 2a). Finally, the BCCP HsPCC has a similar structure. Our mutagenesis data confirm the domain contributes 350 A˚ 2 to the interface (see below). extensive nature of the a–b-subunit interface and suggest that the The BT domain contains a long helix (aV, Supplementary Fig. 1) at holoenzyme can withstand substantial disruptions in it (Supplemen- the amino (N) terminus, followed by an eight-stranded up–down tary Information and Supplementary Table 2). b-barrel (b22-b29) that surrounds the N-terminal two-thirds of Another unexpected discovery from the structure is that the BC the helix (Fig. 2a and Supplementary Fig. 8). The carboxy (C)- domains are arranged as monomers in the PCC holoenzyme (Fig. 1a). terminal one-third of the helix and the long loop connecting to the Studies of the BC subunit of bacterial ACCs have shown a dimeric first b-strand protrude from the b-barrel, and form a ‘hook’ that pro- association9–11, which may be required for its activity15, although vides a major contact with the b-subunit (Fig. 2b and Supplementary monomeric BC mutants are catalytically active16. A conserved Fig. 8). A second area of close contact with the b-subunit is mediated by dimeric association of the BC domain was also observed in PC14,17. a small helix (aW) at the end of the BT domain (Fig. 2c and Sup- However, the BC domains in PCC are monomeric and, in fact, there plementary Fig. 8), which projects away from the b-barrel (Fig. 2a). are no contacts among the a-subunits in the holoenzyme (Fig. 1a). The BT domain does not have any close structural homologues in Our structure defines the molecular basis for the lack of dimerization Protein Data Bank, based on an analysis with the program DaliLite13. of the BC domain in PCC (Supplementary Information). A mono- Remarkably, however, the domain does share some structural similarity meric arrangement of the BC domains also has significant relevance with the PC tetramerization (PT) domain, which helps mediate the for the holoenzyme of eukaryotic ACCs (see below). tetramerization of PC (Supplementary Information and Supplemen- The active site of the BC domain is conserved with that of E. coli tary Fig. 9)14. BC, and all the residues that interact with the substrates of this reac- Many residues are located in the interface between the a-andb- tion have essentially the same conformation in both structures subunits (Supplementary Figs 1 and 2), forming ion-pair, hydrogen- (Supplementary Fig. 10)18. Similarly, residues in the active site of bonding and hydrophobic interactions (Supplementary Information). CT, located at the interface of a b-subunit dimer (Fig. 3a), are also Residues making important contributions to the interface are generally conserved. The structure of this dimer is homologous to those of the 1002 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

a bc α1 P598 α8 E266 T597 αV β29 BC αV R639 BT D328 R540 N263 E326 R272 R539 β22 A269 V265V265V265 α8 P651 β26 β18 β25 F292 αW N domain αW β24 D276 R553 M650 BCCP β23β22 α1 Q643Q643Q643 R416 N115 E318 β1 β3 E76 L64 L649 α2 α8 V543 R117 β4 L72 α2 α0 R289 W111 β1 H551 L646 P287 M547 E68 β3 V288 R642 N S544 β4 H59 Hook Hook S521 α8 508 D71 A520 α2 α1 α8 C β2 C domain 522 C domain Figure 2 | Interactions between the a- and b-subunits in the PCC (a8) of an adjacent b-subunit (labelled b2) is also shown. c, Detailed holoenzyme. a, Interface between the a- and b-subunits in the interactions between helix aW in the BT domain and the b-subunit. The RpPCCa–RdPCCb chimaera. b, Detailed interactions between the hook in view is related to that of a through a 90u rotation around the vertical axis. See the BT domain of the a-subunit and the b-subunits. The C-terminal helix Supplementary Fig. 8 for stereo versions of b and c.

Streptomyces coelicolor and Mycobacterium tuberculosis acyl-CoA car- 75 A˚ 14,17. Our cryo-EM studies on HsPCC have provided direct experi- boxylase b-subunits19,20 and the 12S subunit of Propionibacterium mental evidence that the BCCP domain can be located in different shermanii transcarboxylase (Supplementary Fig. 11)21, which also positions in the holoenzyme (Fig. 1d). Residues 654–660, the linker form similar hexamers. Weaker structural similarity is observed with between the BT and BCCP domains, have weak electron density and the CT domain of yeast ACC12, the CT subunit of bacterial ACC22 and are exposed to the solvent in the current structure (Fig. 1b), suggesting the CT subunit of a bacterial sodium pump23, although these CT that they are flexible and can facilitate the translocation. enzymes only form dimers. A helical sub-domain at the C terminus Further insight into PCC catalysis is obtained from the binding of of the CT domain of yeast ACC is incompatible with the b6 hexamer the BCCP domain and its associated biotin, with well-defined elec- of PCC. tron density (Supplementary Fig. 13), in the CT active site (Fig. 3a). Our structure reveals for the first time the relative positions of the The interface between BCCP and the b-subunit is small, with 350-A˚ 2 BC and CT active sites in the holoenzyme, providing unprecedented surface area burial. Only residues 693–697 around the biotinylation insight into PCC catalysis. The distance between the two active sites in site (Lys 694) interact with the b-subunit, through hydrophobic PCC is about 55 A˚ (Fig. 3a); consequently the entire BCCP domain interactions and one hydrogen bond (Fig. 3b and Supplementary must translocate during catalysis (Supplementary Information and Fig. 14). This weak interaction should also help the BCCP domain Supplementary Fig. 12). A similar situation has been observed in the to leave the CT active site and translocate to the BC active site during structure of PC, where the BC and CT active sites are separated by catalysis.

abdP313 E696 C domain

α N697 K433 β11 N311 M695 β9 ADP α6 L362 A363 K433 1′ Biotin K694 BC P399 M693 α5 * α6 M463 BT Biotin V440 L398 1′ BCCP F397 55 Å R474 c C C domain β11 A201 β1 N β9 Biotin * α6 α6 Y222 CoA CoA * P199

R54 R61

β4 N domain α1 N domain CoA

Figure 3 | The active sites of the PCC holoenzyme. a, Relative positioning BCCP-biotin and the C domain of a b-subunit. Hydrogen-bonding of the BC and CT active sites in the holoenzyme. One a-subunit and a b2 interactions are indicated with the dashed lines in red. The N19 atom of dimer (b1 from one layer and b4 from the other layer) are shown, and the biotin is labelled as 19, hydrogen-bonded to the main-chain carbonyl of viewing direction is the same as Fig. 1b. The two active sites are indicated Phe 397. c, Molecular surface of the CT active site, showing a deep canyon with the stars, separated by a distance of 55 A˚ . The bound positions of ADP where both substrates are bound. d, Schematic drawing of the CT active site. in complex with E. coli BC18 and that of CoA in complex with the 12S subunit See Supplementary Fig. 14 for stereo versions of b and d. of transcarboxylase21 are also shown. b, Detailed interactions between 1003 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 466 | 19 August 2010

The active site of CT is located in a deep canyon at the b-subunit and R165Q mutations may disturb the recognition of the adenine dimer interface (Fig. 3c). The a6 helix from the C domain of one base of CoA (Supplementary Fig. 15). On the other hand, few of the subunit and the a6 helix from the N domain of the other subunit form missense mutations are located in the interface between the a- and the two walls of the canyon. Our structure shows that BCCP-biotin b-subunits of the holoenzyme (Fig. 4 and Supplementary 16). The occupies one half of the canyon, interacting with the C domain of one extensive nature of this interface might make it difficult to disrupt the subunit (Fig. 3d and Supplementary Fig. 14). Propionyl-CoA, the holoenzyme by single-site mutations in this region (Supplementary other substrate of this activity, is expected to occupy the other half Information). of the canyon and interact with the N domain of the second b-subunit The structure of the PCC holoenzyme also has strong implications (Fig. 3d)12,19,21, with the propionyl group located in the centre of the for the structure and function of other biotin-dependent carboxy- canyon (Fig. 3c). Biotin is in a partly folded, unproductive conforma- lases. There are five such enzymes in humans: PCC, MCC, ACC1, tion in the current structure (Fig. 3b), although a conformational ACC2 and PC (Supplementary Fig. 17). MCC is a close homologue of change in the side chain of Lys 694 and the valeryl group of biotin PCC, with the same domain architecture and subunit organization. should readily bring the N19 atom into the proximity of propionyl- Therefore the PCC structure is directly relevant for understanding CoA for catalysis. the MCC enzyme and its disease-causing mutations5–7. Both RpPCC and the RpPCCa–RdPCCb chimaera have a pref- Most importantly, the identification of the BT domain in PCC led us erence for propionyl-CoA over acetyl-CoA as the substrate (Sup- to re-examine the sequences of eukaryotic, multi-domain ACCs. The plementary Table 4). Earlier studies with Streptomyces coelicolor segment containing the BC and BCCP domains in these enzymes is acyl-CoA carboxylase showed that residue Asp 422—equivalent to remarkably similar to the PCC a-subunit, with a linker of about 120 Asp 440 in HsPCC and RpPCC and Val 440 in RdPCC (in helix a5, residues between the two domains (Supplementary Fig. 17). Secondary Fig. 3d)—is important for discriminating between the two substructure prediction shows that this linker contains a helix followed by strates19. However, equivalent mutations in PCCs—D440I for seven or more b-strands, suggesting that it may form a structure similar RpPCC and V440I for the RpPCCa–RdPCCb chimaera—did not to the BT domain in PCC. This putative BT domain of ACC is likely change their substrate preference (Supplementary Table 4). also crucial for mediating interactions between its BC and CT domains. Residue 440 is about 10 A˚ away from the thiol group of CoA In fact, we have observed that purified BC and CT domains of yeast (Fig. 3d) and does not directly contribute to substrate binding. ACC do not interact with each other (unpublished results). Because the Other residues may also be improtant in determining the substrate CT domain dimer of ACC is similar to the b2 dimer of PCC, the a2b2 preference of these enzymes. assembly of PCC might be a plausible model for the organization of the The structure of the holoenzyme establishes a foundation for ACC dimer, the protomer that can also form higher oligomers. This understanding the molecular basis of many disease-causing muta- model implies that the BC domain could be monomeric in the ACC tions in PCC (Fig. 4 and Supplementary Table 3)1–4. Among these, holoenzyme, which is consistent with observations that isolated BC 28,29 only the R399Q mutation in the a-subunit directly disrupts a residue domains of eukaryotic ACCs are monomeric in solution ,incon- in the active site (Supplementary Fig. 15). This side chain stabilizes trast to the dimers for bacterial BC subunits. Therefore, there might be the biotin enolate during BC catalysis (Supplementary Fig. 10)18, and a fundamental difference between the overall architecture of eukaryo- the mutation leads to a large loss in activity18,24. Another mutation, tic, multi-domain ACCs and that of bacterial, multi-subunit ACCs. G668R in the BCCP domain (Fig. 4), abolishes biotinylation. Many of METHODS SUMMARY the other mutations are detrimental to catalysis by destabilizing the Crystallography. The a- and b-subunits of PCC were co-expressed in E. coli, enzyme and/or interfering with holoenzyme assembly1,25–27. Many of with a His-tag on the b-subunit. The PCC holoenzyme was purified by nickel- them, especially those in the b-subunit, are actually located near the affinity and gel-filtration chromatography. Crystals were obtained by the micro- active site (Supplementary Fig. 15), and they may indirectly affect batch method under oil, and the structures were determined by the molecular substrate binding and/or catalysis as well. For example, the R165W replacement method. Cryo-EM. Frozen hydrated human PCC particles at 70 mgml21 concentration were imaged at 350,000 magnification in a 100-kV cryo-electron microscope. A featureless Gaussian oval was used to obtain a low-resolution (40-A˚ ) model from α negative-stain electron microscope images. A 15-A˚ resolution three-dimensional BC reconstruction was obtained from approximately 10,000 cryo-EM particle images, using the structure from the negative stain images as the initial model. Mutagenesis and kinetic studies. Site-specific and deletion mutants were designed based on the structural information, and their effects on the formation BT 399 631 * of the holoenzyme were assessed by nickel-affinity chromatography. The cata- 559 lytic activity of PCC was determined by a coupled enzyme assay, monitoring the hydrolysis of ATP. 551 BCCP Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. N C β1 Received 4 March; accepted 11 June 2010. 668 * * 1. Desviat, L. R. et al. Propionic acidemia: mutation update and functional and structural effects of the variant alleles. Mol. Genet. Metab. 83, 28–37 (2004). 2. Rodriguez-Pombo, P. et al. Towards a model to explain the intragenic complementation in the heteromultimeric protein propionyl-CoA carboxylase. CoA Biochim. Biophys. Acta 1740, 489–498 (2005). 3. Deodato, F., Boenzi, S., Santorelli, F. M. & Dionisi-Vici, C. Methylmalonic and β4 propionic aciduria. Am. J. Med. Genet. C. Semin. Med. Genet. 142, 104–112 (2006). 4. Desviat, L. R. et al. New splicing mutations in propionic acidemia. J. Hum. Genet. 51, 992–997 (2006). Figure 4 | Locations of disease-causing mutations in the PCC holoenzyme. 5. Desviat, L. R. et al. Functional analysis of MCCA and MCCB mutations causing Structure of one a-subunit and one b2-subunit dimer of PCC, in the same methylcrotonylglycinuria. Mol. Genet. Metab. 80, 315–320 (2003). view as Fig. 3a. The locations of the missense mutations associated with 6. Stadler, S. C. et al. Newborn screening for 3-methylcrotonyl-CoA carboxylase propionic acidaemia are indicated with the spheres, coloured by the deficiency: population heterogeneity of MCCA and MCCB mutations and impact domains. The BC and CT active sites are indicated with the stars. on risk assessment. Hum. Mutat. 27, 748–759 (2006). 1004 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 LETTERS

7. Stucki, M., Suormala, T., Fowler, B., Valle, D. & Baumgartner, M. R. Cryptic exon 23. Wendt, K. S., Schall, I., Huber, R., Buckel, W. & Jacob, U. Crystal structure of the activation by disruption of exon splice enhancer. Novel mechanism causing carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl- 3-methylcrotonyl-CoA carboxylase deficiency. J. Biol. Chem. 284, 28953–28957 coenzyme A decarboxylase. EMBO J. 22, 3493–3502 (2003). (2009). 24. Sloane, V. & Waldrop, G. L. Kinetic characterization of mutations found in 8. Wakil, S. J., Stoops, J. K. & Joshi, V. C. Fatty acid synthesis and its regulation. Annu. propionic acidemia and methylcrotonylglycinuria. J. Biol. Chem. 279, 15772–15778 Rev. Biochem. 52, 537–579 (1983). (2004). 9. Tong, L. Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and 25. Jiang, H., Rao, K. S., Yee, V. C. & Kraus, J. P. Characterization of four variant forms attractive target for drug discovery. Cell. Mol. Life Sci. 62, 1784–1803 (2005). of human propionyl-CoA carboxylase expressed in Escherichia coli. J. Biol. Chem. 10. Waldrop, G. L., Rayment, I. & Holden, H. M. Three-dimensional structure of the 280, 27719–27727 (2005). biotin carboxylase subunit of acetyl-CoA carboxylase. Biochem. 33, 10249–10256 26. Muro, S. et al. Effect of PCCB gene mutations on the heteromeric and homomeric (1994). assembly of propionyl-CoA carboxylase. Mol. Genet. Metab. 74, 476–483 (2001). 11. Cronan, J. E. Jr & Waldrop, G. L. Multi-subunit acetyl-CoA carboxylases. Prog. 27. Perez-Cerda, C. et al. Functional analysis of PCCB mutations causing propionic Lipid Res. 41, 407–435 (2002). acidemia based on expression studies in deficient human skin fibroblasts. Biochim. 12. Zhang, H., Yang, Z., Shen, Y. & Tong, L. Crystal structure of the Biophys. Acta 1638, 43–49 (2003). carboxyltransferase domain of acetyl-coenzyme A carboxylase. Science 299, 28. Shen, Y., Volrath, S. L., Weatherly, S. C., Elich, T. D. & Tong, L. A mechanism for the 2064–2067 (2003). potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a 13. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure macrocyclic polyketide natural product. Mol. Cell 16, 881–891 (2004). databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008). 29. Weatherly, S. C., Volrath, S. L. & Elich, T. D. Expression and characterization of 14. Xiang, S. & Tong, L. Crystal structures of human and Staphylococcus aureus recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding pyruvate carboxylase and molecular insights into the carboxyltransfer reaction. domain. Biochem. J. 380, 105–110 (2004). Nature Struct. Mol. Biol. 15, 295–302 (2008). 30. Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory 15. Janiyani, K., Bordelon, T., Waldrop, G. L. & Cronan, J. E. Jr. Function of Escherichia research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). coli biotin carboxylase requires catalytic activity of both subunits of the Supplementary Information is linked to the online version of the paper at homodimer. J. Biol. Chem. 276, 29864–29870 (2001). www.nature.com/nature. 16. Shen, Y., Chou, C.-Y., Chang, G.-G. & Tong, L. Is dimerization required for the catalytic activity of bacterial biotin carboxylase? Mol. Cell 22, 807–818 (2006). Acknowledgements We thank N. Whalen and H. Robinson for access to the X29A 17. St. Maurice, M. et al. Domain architecture of pyruvate carboxylase, a biotin- beamline at the National Synchrotron Light Source; J. Schwanof and R. Abramowitz dependent multifunctional enzyme. Science 317, 1076–1079 (2007). for access to the X4A beamline; M. Sampat for help during the initial stages of the 18. Chou, C.-Y., Yu, L. P. C. & Tong, L. Crystal structure of biotin carboxylase in project; and W.W. Cleland for discussions. This research was supported in part by complex with substrates and implications for its catalytic mechanism. J. Biol. National Institutes of Health grants DK067238 (to L.T.), GM071940 and Chem. 284, 11690–11697 (2009). AI069015 (to Z.H.Z.). C.S.H. was also supported by a National Institutes of Health 19. Diacovich, L. et al. Crystal structure of the b-subunit of acyl-CoA carboxylase: training program in molecular biophysics (GM08281). structure-based engineering of substrate specificity. Biochem. 43, 14027–14036 Author Contributions C.S.H., K.S.-B., Y.S. and B.D. performed the experiments, (2004). analysed the data and commented on the manuscript. L.T. and Z.H.Z. designed and 20. Lin, T. W. et al. Structure-based inhibitor design of AccD5, an essential acyl-CoA performed the experiments, analysed the data and wrote the manuscript. carboxylase carboxyltransferase domain of Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 3072–3077 (2006). Author Information The atomic coordinates are deposited in Protein Data Bank 21. Hall, P. R. et al. Transcarboxylase 12S crystal structure: hexamer assembly and under accession number 3N6R. Reprints and permissions information is available substrate binding to a multienzyme core. EMBO J. 22, 2334–2347 (2003). at www.nature.com/reprints. The authors declare no competing financial 22. Bilder, P. et al. The structure of the carboxyltransferase component of acetyl-CoA interests. Readers are welcome to comment on the online version of this article at carboxylase reveals a zinc-binding motif unique to the bacterial enzyme. Biochem. www.nature.com/nature. Correspondence and requests for materials should be 45, 1712–1722 (2006). addressed to L.T. ([email protected]).

METHODS holey carbon grids by plunge-freezing immediately after dilution following Protein expression and purification. The a-subunit of PCC was amplified by standard procedures. Cryo-EM was performed using a JEOL 1200EX electron PCR from genomic DNA and inserted into the pET-26b vector (Novagen) using microscope. Electron micrographs were recorded on Kodak SO163 films at the restriction enzymes NdeI and NotI (New England Biolabs). The b-subunit 350,000 magnification and digitized using a Zeiss SCAI scanner with a step size ˚ was cloned into pET-28a using NdeI and EcoRI, introducing an N-terminal of 14 mm per pixel, corresponding to 2.8 A per pixel on the sample (Sup- hexahistidine tag. The PCCa insert in pET-26b, together with the upstream plementary Fig. 4). 40 ribosomal binding site, was then placed to the 39-end of the PCCb insert to make Single particle analysis was performed using the EMAN software package .To a bicistronic expression plasmid. The plasmid was transformed into E. coli eliminate possible model bias, a featureless elliptical Gaussian ball was used as a BL21Star (DE3) cells (Invitrogen). Biotin at 20 mg l21 concentration was sup- starting model to process the high-contrast negative-stain images to obtain a plemented into the growth media. After induction with 1 mM IPTG, the cells low-resolution structure of HsPCC (Supplementary Fig. 5). This low-resolution were allowed to grow overnight at 25 C. Cells were lysed by sonication in a buffer reconstruction exhibited features consistent with D3 symmetry, in agreement u 34 containing 20 mM Tris (pH 7.4), 250 mM NaCl, 5% (v/v) glycerol, 0.1% (v/v) with the expected symmetry of the b6 core of PCC . Subsequently, D3 symmetry Triton X-100 and 10 mM b-mercaptoethanol. Soluble PCC was purified by Ni- was imposed on the three-dimensional model obtained from the negative stain NTA (Qiagen) and gel filtration (Sephacryl S-300, GE Healthcare) chromato- images. graphy. Purified protein was concentrated to 15 mg ml21 in a buffer containing For the cryo-EM reconstruction, approximately 15,000 particles were picked 25 mM Tris (pH 7.4), 250 mM NaCl, 2 mM DTT and 5% (v/v) glycerol, flash- from digitized micrographs semi-automatically using the boxer program in frozen in liquid nitrogen and stored at 280 uC. The N-terminal His-tag on PCC- EMAN. The three-dimensional reconstruction from the negative-stain images ˚ b was not removed for crystallization. Complete biotinylation of the a-subunit was low-pass filtered to 40-A resolution and used as the starting model for was confirmed by an avidin gel-shift assay. processing the cryo-EM images. Particle images were classified and class averages Protein crystallization. Crystals of the RpPCCa–RdPCCb chimaera were generated (Supplementary Fig. 6). D3 symmetry was imposed during refinement obtained at 4 uC using the microbatch method under paraffin oil. The protein and three-dimensional reconstruction. The resolution of the three-dimensional was at 15 mg ml21 concentration, and the precipitant solution contained 0.1 M reconstruction was assessed by monitoring the Fourier shell correlation between HEPES (pH 8.0), 22% (w/v) PEG3350, 0.2 M NaCl and 16% (v/v) glycerol. Most three-dimensional reconstructions from the two half sets of the whole data set of these crystals diffracted X-rays poorly and were highly mosaic. A few of good (Supplementary Fig. 7). The structure was refined iteratively until no further quality were identified after screening through many of them. improvement in the resolution of the reconstruction could be obtained. Crystals of RpPCC were obtained at 20 uC using the microbatch method under Approximately 10,000 particles were used in the final three-dimensional recon- paraffin oil. The protein was at 15 mg ml21 concentration, and the precipitant struction (Supplementary Fig. 7). The University of California, San Francisco solution contained 0.2 M succinic acid (pH 6.5), 22% (w/v) benzamidine and (UCSF) Chimera program was used to create three-dimensional graphical repre- 41 22% (w/v) PEG3000. The diffraction quality of most of these crystals was also sentations . The atomic model of PCC was first manually fitted into the cryo- very poor. EM density map, and the fitting was refined using the fit-model-to-map module Crystals of HsPCC were obtained at 4 uC using the sitting-drop vapour- of Chimera. diffusion method. The protein was at 15 mg ml21 concentration, and the pre- Mutagenesis and kinetic studies. Site-specific mutations were introduced in cipitant solution contained 0.1 M Tris (pH 8.5), 5% (w/v) PEG8000, 13% (v/v) RpPCC with the QuikChange Kit (Stratagene) and sequenced for confirmation. PEG300 and 8% (v/v) glycerol. Deletion mutations were created by introducing a stop codon through mutagenesis Data collection and structure determination. An X-ray diffraction data set to at the desired position in RpPCC. The mutant plasmids were transformed into 3.2-A˚ resolution on the RpPCC-a–RdPCC-b chimaera was collected at the X29A E. coli, and the formation of the holoenzyme was assessed by nickel-affinity beamline of the National Synchrotron Light Source. The crystal belonged to chromatography. space group P1, with cell parameters of a 5 133.9 A˚ , b 5 159.2 A˚ , c 5 153.7 A˚ , The catalytic activity of PCC was determined using a coupled enzyme assay, 34,42 a 5 113.9u, b 5 101.0u and c 5 109.0u. There is one a6b6 dodecamer in the converting the hydrolysis of ATP to the disappearance of NADH . The reac- asymmetric unit/unit cell. The diffraction data were processed and scaled with tion mixture contained 100 mM HEPES (pH 8.0), 0.5 mM ATP, 8 mM MgCl2, the HKL package31. The structure was solved by the molecular replacement 40 mM KHCO3, 0.5 mM propionyl-CoA or acetyl-CoA, 0.2 mM NADH, 0.5 mM method with the program Phaser32. The structures of the BC subunit of E. coli phosphoenolpyruvate, 7 units of lactate dehydrogenase, 4.2 units of pyruvate ACC33, the b-subunit of S. coelicolor acyl-CoA carboxylase34 and the BCCP kinase and 200 mM KCl. The absorbance at 340 nm was monitored for 5 min. domain of Staphylococcus aureus PC35 were used as search models. Sixfold 31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in non-crystallographic symmetry averaging was performed with the program 36 oscillation mode. Methods Enzymol. 276, 307–326 (1997). DM in the CCP4 package . The atomic model was built into the electron density 32. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 37 map with the program O . The structure refinement was performed with the (2007). 38 program CNS . Non-crystallographic symmetry restraints were used during the 33. Thoden, J. B., Blanchard, C. Z., Holden, H. M. & Waldrop, G. L. Movement of the refinement. The data processing and refinement statistics are summarized in biotin carboxylase B-domain as a result of ATP binding. J. Biol. Chem. 275, Supplementary Table 1. 16183–16190 (2000). An X-ray diffraction data set to 3.2-A˚ resolution on RpPCC was collected at 34. Diacovich, L. et al. Crystal structure of the b-subunit of acyl-CoA carboxylase: the X29A beamline of the National Synchrotron Light Source. The crystal structure-based engineering of substrate specificity. Biochemistry 43, belonged to space group P3, with cell parameters of a 5 b 5 246.3 A˚ and 14027–14036 (2004). c 5 133.5 A˚ . There are three a b assemblies in the asymmetric unit, situated 35. Xiang, S. & Tong, L. Crystal structures of human and Staphylococcus aureus 2 2 pyruvate carboxylase and molecular insights into the carboxyltransfer reaction. at the crystallographic threefold axis. The crystal exhibited perfect merohedral Nature Struct. Mol. Biol. 15, 295–302 (2008). twinning, with a twinning fraction of 0.49. The structure was solved by the 36. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 39 molecular replacement method with the program COMO , using the structure 50, 760–763 (1994). of the PCC chimaera as the search model. Twinned structure refinement was 37. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for performed with the program CNS. building protein models in electron density maps and the location of errors in An X-ray diffraction data set to 5.5-A˚ resolution on HsPCC was collected at these models. Acta Crystallogr. A 47, 110–119 (1991). the X4A beamline of the National Synchrotron Light Source. The crystal 38. Brunger, A. T. et al. Crystallography & NMR System: a new software suite for belonged to space group R3, with cell parameters of a 5 b 5 196.1 A˚ and macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998). c 5 979.5 A˚ . The crystal also exhibited perfect merohedral twinning, and 39. Jogl, G., Tao, X., Xu, Y. & Tong, L. COMO: a program for combined molecular attempts to solve this structure have been unsuccessful. replacement. Acta Crystallogr. D 57, 1127–1134 (2001). 40. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high- Electron microscopy. Highly purified HsPCC sample was diluted to 70 mgml21 resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999). with 1 3 PBS buffer (pH 7.4). For negative stain electron microscopy, an aliquot 41. Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory of 3-ml sample was placed onto a carbon-film-coated, glow-discharged, 300- research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). mesh copper grid. Excess sample was blotted away after 1 minute. The sample 42. Blanchard, C. Z., Lee, Y. M., Frantom, P. A. & Waldrop, G. L. Mutations at four was stained twice with 2.5% uranyl acetate solution and air dried. For cryo-EM, active site residues of biotin carboxylase abolish substrate-induced synergism by frozen hydrated HsPCC particles were suspended across holes of Quantifoil biotin. Biochemistry 38, 3393–3400 (1999).

CORRIGENDUM doi:10.1038/nature09360 An allosteric mechanism of Rho-dependent transcription termination Vitaly Epshtein, Dipak Dutta, Joseph Wade & Evgeny Nudler

Nature 463, 245–249 (2010) In this Letter, the statement ‘‘Mutation of R933A in the trigger loop (TL) domain, which prevents tagetitoxin (Tgt) from binding to RNAP’’ should have been accompanied by the citation: ‘‘I. Artsimovitch, personal communication’’.

CORRIGENDUM doi:10.1038/nature09361 Grassroots initiatives Amy Maxmen

Nature 466 (suppl.), S20 (2010) This Outlook article contained some errors: the UN World Food Programme is a partner with, not a donor to, the Global Health Committee. The Zahara Children’s Center is not open yet. Sok Thim has not had tuberculosis.

CORRIGENDUM doi:10.1038/nature09362 Quantum oscillations in a molecular magnet S. Bertaina, S. Gambarelli, T. Mitra, B. Tsukerblat, A. Mu¨ller & B. Barbara

Nature 453, 203–206 (2008) The authors report that in a recent extension of this Letter, considering different concentrations, they were not able to reproduce the very weak signal of the ground-state EPR transition (the red curves termed ‘1/2’ in Figs 3 and 4). The authors report that the signal is hardly observable (with intensity lower than that of the cavity background); but the more important S 5 3/2 transitions (blue curves in Figs 3 and 4), which give rise to well-defined oscillations with several periods and high signal intensity, are reproducible. The extension of the work — about the concentration dependence of decoherence — will be published1 but leads to the same major conclusion as in the original manuscript. The authors thank J. Du and colleagues for independently bringing the mentioned problem to their attention.

1. Shim, J. H. et al. Driven spin-bath decoherence in the molecular magnet V15. Preprint at Æhttp://arxiv.org/abs/1006.4960æ (2010).

CORRIGENDUM doi:10.1038/nature09363 Above-room-temperature ferroelectricity in a single-component molecular crystal Sachio Horiuchi, Yusuke Tokunaga, Gianluca Giovannetti, Silvia Picozzi, Hirotake Itoh, Ryo Shimano, Reiji Kumai & Yoshinori Tokura

Nature 463, 789–792 (2010) In this Letter, the crystallographic a and b axes had been labelled incorrectly in Figs 3a, 4b and in Supplementary Fig. 1. The a- and b-axes labels need to be exchanged to correct these figures. Namely, the yellow plates of croconic acid crystal are elongated along the crystallographic b axis in the corrected Supplementary Fig. 1. The correct Fig. 3 depicts the surface image of crystal (010) plane. The errors do not involve the polar (crystallographic c) axis focused, and these corrections do not alter the essence or conclusion of the paper.

ProSPectS Search tool for career data launched A new online facility allows users to delve into Naturejobs’s career and salary survey data on their own terms, explains Gene russo.

In late June, we released an analysis of many select a topic from the drop-down menu, suggest that among those who reported being of the results from Nature and Naturejobs’s such as salary, then a filter — such as career very satisfied with their current position, first international salary survey, which stage or satisfaction. The results are instantly ‘degree of independence’ and ‘guidance received more than 10,500 responses from available as a graph and a table. received from co-workers’ were among the scientists worldwide (see go.nature.com/ Interested in how much money our biologist leading factors contributing to contentment. RVvqWe). It detailed trends related not only respondents make? Choose ‘Current salary We have chosen to display and make to scientists’ incomes, but also to their job of primary job’, then filter with ‘Primary available for selection only categories and satisfaction. Accompanying the analysis subject/research area’ and ‘Biology’. The large subcategories with sample sizes of at least 50. were expert commentaries (see go.nature. number of scientists on US$40,000–50,000 This means that data from many countries — com/8Z6tnr) and a podcast sampling some reflects in part the large number of postdoc those for which we had small sample sizes — of the survey’s more interesting outcomes respondents (which, in turn, reflects the large will not be available. And, as explained in our (see go.nature.com/VwUzzp). number of postdocs out there). frequently-asked-questions section, there are This week, we are offering readers an Want to know how biologists rate the biases in the data that we have done our best additional online-only resource, free with different sources of job satisfaction? Choose to address (see go.nature.com/MP91Ge). Naturejobs registration. Our search tool ‘Sources of job satisfaction’ and filter with We hope this new tool helps inform, will allow users to probe the data set in ‘Primary subject/research area’ and ‘Biology’. enlighten and even entertain, whether users even more detail — on their own terms Users can generate quite subtle results. Try are browsing the results, using them for a and according to their own interests (see choosing ‘Sources of job satisfaction’, then presentation or simply sharing them with go.nature.com/xV3dnZ). filtering ‘Overall job satisfaction’ with the colleagues. ■ Using the tool is intuitive. Readers first subcategory ‘Very satisfied’. The results Gene Russo is the editor of Naturejobs.

Stefania Mondello, a postdoc at the University of Florida, received the 2010 Premio Award, presented to outstanding female Italian scientists working in North America, at the annual Q&A conference of the Italian Association for Women Inventors and Innovators in Bari, Italy, in June.

What attracted you to biology? recovery depends got it without the a new treatment for traumatic As a child I read about Louis on accurate, early strong team effort brain injury in the past 30 years. Pasteur, creator of the first treatment that takes needed to do this vaccine for rabies, and Alexander into account the type of research. My What opportunities come with Fleming, discoverer of penicillin. person’s genetics. mentors helped me this award? Their research contributed to develop a unique set The award is given by Bridges a better world and I decided I How is your award- of skills — merging to Italy, which supports wanted to be a physician-scientist winning work my clinical focus women scientists and fosters to do the same. innovative? on the brain with a collaborations between Italy and We aim to use biomarkers to knowledge of biomarkers and the United States. As biomedicine Why did you focus on assess the magnitude of the genetics — and this award just in Italy continues to move traumatic brain injury? brain injury as well as to help makes me want to work harder to towards conducting science with While at the University of determine the most effective use those skills to find treatments. more clinical utility, I hope to Messina Medical School in Italy, treatment. People can seem to advocate for more investment in I began to focus my PhD training have a mild-to-moderate brain What are your long-term young investigators. on neural intensive care. I found injury, yet it is impossible to goals? it fascinating that you need to accurately measure the damage. Now that we’ve found diagnostic What inspires you? know how so many organs are We hope our work will one biomarkers, we are working Two quotes illustrate my related to treat these patients, the day change medical practice to on their clinical validation belief that practical people are brain being the most complex. prevent secondary complications — confirming the different important, but so are dreamers. I did my thesis on how best to and improve the survival of the biomarkers in people with My mentor, Ronald Hayes, said: use biomarkers — molecules 1.4 million people in the United traumatic brain injury compared “We seek the truth as scientists, that can serve as indicators of States alone who experience a with controls, assessing their we heal the sick as clinicians, disease progression — as targets traumatic brain injury each year. diagnostic accuracy, and and we teach others to do the for therapy. Yet none existed demonstrating that they provide same as mentors.” And Marie for traumatic brain injury, so I You’re only 31 — were you information that aids medical Curie said: “A scientist is not decided to focus on developing surprised to get the award? decision-making. Once we have only a technician; he is also these biomarkers. People Yes, I was. I left Italy only two clinical validation, our goal is a child placed before natural with traumatic brain injuries years ago, and these types of to secure US Food and Drug phenomena which impress him are often young people hurt award are rarely given to someone Administration approval by 2012. like a fairy tale.” ■ in dramatic situations. Their so young. But I would not have The agency has not approved Interview by Virginia Gewin.

Weighing the options Comparative-effectiveness research answers questions that could transform medical policy and practice. Tamar Nordenberg examines the opportunities for researchers to find both funding and fulfilment.

eremy Rassen wasn’t exactly unhappy patient-centred outcomes studies, are government, including the National with his marketing job, applying designed to answer the practical questions Institutes of Health (NIH), the Agency for his computer-science skills to that have long confounded health-care Healthcare Research and Quality (AHRQ) help companies analyse their sales providers. Which medication is the best and the Department of Veterans Affairs, has Japproaches and increase their profits. choice for a particular patient? And how been supporting and conducting these types But he didn’t feel fulfilled either. “It was do other available care and treatment of study for decades. But the field has been interesting day to day, but not what I wanted strategies — such as surgery, medical the subject of renewed focus in the past two to do with my career,” says Rassen, whose devices, diagnostic tests, behavioural change years. In the United States, US$1.1 billion job involved shedding light on clients’ and methods of delivering health care — of federal money was tagged for CER under business strategies, including helping a stack up in real terms? The President Barack Obama’s large magazine publisher to understand its answers would help people “Clinicians 2009 stimulus package — customers’ subscription-purchasing and with conditions such as hunger for reliable $400 million to the NIH, cancellation trends. “I wanted to put my osteoporosis, for which there $300 million to the AHRQ, analytical skills to a use that would benefit are few data on how the information for and $400 million to the my community.” available treatment options treating patients. Office of the Secretary of So, after some ten years in marketing, work in the long term. They want to know, Health and Human Services. Rassen entered a doctor of sciences “Clinicians I have talked By comparison, in 2008, programme at the Harvard School of Public to hunger for reliable ‘Does drug A work the AHRQ programme Health in Boston, Massachusetts. He had information for treating better than drug B?’” responsible for CER had a decided to transfer his long-standing skills their patients,” Rassen says. budget of $30 million. in computer science and commerce to “They really want to know, ‘Does drug A Rassen, who completed his doctoral the public-health arena — specifically to work better than drug B in routine care?’” programme in 2008, now receives stimulus comparative-effectiveness research (CER) Comparative studies often focus exclusively funds in the form of an AHRQ Mentored and pharmacoepidemiology. Luring him on the health benefits and risks of alternative Research Scientist Development Award to CER, and to drug-comparison studies in medical strategies, but researchers in some (K01) to study comparative-effectiveness particular, were the immediate effects that it cases follow up with a consideration of cost- methods for evaluating the treatments that promised on patient health. effectiveness as well. patients receive in hospital. The award pays Comparative-effectiveness studies, or CER is not new: the US federal 75% of what he calls his “standard junior-

1010 © 2010 Macmillan Publishers Limited. All rights reserved NATURE|Vol 466|19 August 2010 CAREERS faculty salary”, along with $25,000 a year in factors.” An increased potential for bias “Researchers who wish to perform research support, for up to five years while is introduced, for example, when studies high-quality systematic reviews need a he conducts the research. must rely for data on databases of health- host of skills that enable them to criticize, In the United Kingdom, the National insurance claims — with little knowledge categorize and synthesize voluminous and Institute for Health Research, the largest of outside factors that might influence complex contributions to the literature in I

single programme in the National Health the treatment — rather a sophisticated way,” says lb H

Service’s research and development than randomly assigning Michael Lauer, director of the n portfolio, has steadily increased support treatments to comparable US National Heart, Lung and for comparative-effectiveness studies, patients. “That is the magic of Blood Institute’s division of says Tom Walley, director of the institute’s it,” says Rassen. “How do you cardiovascular science, and health technology assessment programme. measure which drug treats an co-chairman of the NIH’s His programme, for example, which has a indication better, when you’re CER committee. “They need big CER component, has seen a significant not going out and designing to be well versed in clinical budget increase, from £13 million (US$20 a randomized trial protocol, medicine and assessment million) in 2005 to an expected £88 million with patients coming back for of scientific hypotheses and by 2011. Walley describes the research as visits every six months?” medical statistics.” “systematic reviews and clinical trials based To account for the many Investigators in on identified patient needs”, and says that variables involved — in his comparative effectiveness health-service-funded comparative studies recent study of the drug commonly have medical or focus on patient outcomes. “We’ll almost bivalirudin during coronary Michael Lauer says that CER other clinical degrees, but never fund a study based on a change in interventions, for example — requires a host of skills. alternatives include master’s- a biochemical marker unless it is very Rassen relies heavily on his level training, doctoral well linked with real patient outcomes, pharmacoepidemiology background. But degrees or postdoctoral training in public- such as viral load with HIV.” He adds that because realistic health studies can present health areas, or specific training in clinical comparative studies funded by the National challenges far beyond those of controlled trials and systematic reviews. Regardless Health Service are “pragmatic” and driven clinical trials, CER benefits from broad of academic background, outcomes by patient needs, “not by publishing an multidisciplinary input. investigator Leslee Shaw of Emory University article in a journal”. In addition to clinical medicine and School of Medicine in Atlanta, Georgia, France is among the other countries epidemiology, useful experience for counsels aspiring comparative-effectiveness investing in CER; its National Authority comparative-effectiveness researchers researchers to pursue diverse areas such as for Health (HAS) looks at the comparative includes biomedical informatics, biostatistics, economic analysis, quality-of-life analysis, effectiveness of health-care options — health-services research, health-system risk modelling and systematic reviews in the assessing drugs, medical devices and economics, research into methods of five or ten years after earning their degrees. procedures and making recommendations decision-making, genomics, proteomics, “A diversity of experience early on puts you to the Ministry of Health. HAS is library science and communications. in a better position to provide input on trial an independent organization with a For prospective studies, Walley says design,” she says. For Shaw herself, the call government-appointed board, and had an that the National Health Service looks for was to cardiology, for the field’s preventive operating budget of €71.7 million (US$92.6 investigators with a thorough knowledge health-care potential in particular. As a non- million) in 2008. of the design and conduct of clinical trials. clinician scientist conducting CER — in her Funded systematic reviews tend to involve case, focusing on imaging research, including A marriage of diverse disciplines clinical experts, as well as those from a studies of patients with stable angina — Shaw Comparative-effectiveness investigators wide range of further disciplines such as says that her partnerships with cardiology such as Rassen have set their sights on information science, research designs and clinicians have brought the patient-care measuring medical strategies’ benefits methods, and data management and meta- perspective to her studies. and harms in realistic settings, as well as analysis (see ‘Practical application’). Scientists such as Rassen and Shaw can in wider populations than are possible look forward to ongoing opportunities for in efficacy trials conducted under tightly PractIcal aPPlIcatIon comparative studies. In the United States, the controlled conditions. Comparative How to win support for a comparative- flow of funding for this research will continue, studies — including clinical trials, but effectiveness study. although the bulk of stimulus-act money has more commonly observational studies, ● Form an idea that promises to have a already been allocated, says Slutsky. modelling, and secondary data analyses positive effect on health outcomes, making Lauer says that there has been “a great using registries and linked databases — sure that it meshes with the funding agency’s deal of interest in CER in the past five years”. attempt to fill knowledge gaps by providing articulated priorities. although decision- And he expects interest to grow as the US trustworthy information about options for makers must consider many of the same health-care reform act is implemented. With diagnosis, treatment and monitoring. things as for randomized, placebo-controlled health care a high priority worldwide, other Such studies involve myriad complexities, clinical trials, funding applications for countries will probably also continue to says Jean Slutsky, director of AHRQ’s comparative-effectiveness research tend invest, creating opportunities for researchers Center for Outcomes and Evidence. also to be judged on their direct relevance to who enjoy work that could have a direct, “Because these studies take place in real- clinical practice or public policy. near-term impact on patients. world circumstances and represent diverse ● Present an application that is clear and well Since his career switch, Rassen has found patients from the community, they involve written. reviews by people within and outside CER to be a major challenge, but well worth challenges unlike those of studies conducted your department and discipline are strongly it. “It is a lot of hours,” he says, “and also in highly controlled settings,” she says. encouraged. difficult work that requires deep analysis to ● Rassen notes that trial design must “address address issues that are relevant to all determine if your ‘answer’ represents a true the important stakeholders in the research, the question being studied in a realistic causal relationship or a mere association.” ■ including patients, health-care providers and population and provide enough information Tamar Nordenberg is a freelance health policy-makers. T.N. to adjust for these serious confounding writer based in Washington DC.

1011 © 2010 Macmillan Publishers Limited. All rights reserved FUTURES 1014 FUTURES again under her breath: and murmured the same word again and him outside, Lena running along behind, Quickly she picked him up and marched darkening. face her around, turned thumb in her mouth. darling, always leaned over and whispered: “Stay special, lar routine. “See She was guiding his sister though a simi he saw his mother over in the far corner. opaque glass jars, some crying. Eventually, skins with lavender-smelling creams in looked into long mirrors, patting their aisles,women alongthese all tables; stood line after line of dressing-room walls. To his left and right, in long aisles, came out of holes placed high up in the main room, where jets of fragrant fog through a long, tiled vestibule into the search of her. He remembers passing Women’s Municipal Powder Room in and Merlin wandered into the nearest dayone disappeared she alive, still Three years ago, when his mother was his father always says. What’s the Deterioration? tions.Where babiesdo came from? not like to be asked the wrong ques for asking. Alison, like his mother, does phone conversations.” when she hangs up. on the phone. “Stay special.” for differences in hair and skin colour. centres. All the girls look like her, allowing that flash and reload in the city’s shopping girl in the huge infomercialswall-to-wall Merlin’s stepmother Alison looks like the Susan Lanigan With age comes …? Stay special before. It looks barely human. It looks like nothing Merlin has ever seen body towards the café near the entrance. and palm trees, a hunched figure drags its the mall’s atrium. Alongside the fountain off!” “Merlin!”shouts.“Stopshe wandering shoe-shopping.him taken has Alison But Merlin has seen something odd in motherHis out. cried “Mama!”he “Stayspecial,” her echoed, sister his “Don’t ask Alison questions like that,” He’s known he would get into trouble “You shouldn’t listen to other people’s “When you say ‘stay special’?” She glares at him. “What?” “What that does Merlin mean?” asks “Love you too,” she says to her girlfriend

…

stay special.”

2 0 - 10

M any left.” ders out loud. “I didn’t think there were it down the highway. to the car. him to pee in his pants as she drags him him and pulls him away, almost causing lin’s heart racing even before she grabs she lets out a sudden shriek that sets Mer window. When she sees the bent figure, with Alison behind him. knock. He opens itthe door: is his father Later, in his room, Merlin hears a gentle of the world, remember! Stay special. 3-pentanoic acid idebenone, dibenzoylmethane, dithiolane- Then it chants the chemical compounds: But today’s woman can stay safe for longer. says. ing a new face cream. announcvoicemellifluouscrooning, asking any more questions. On comes a .” at h t stage when — a stage when you look like isn’t caught in time and it progresses to a a c Alison switches on the radio to stop him “But what’s the —” “Degraded is when the Deterioration “What’s ‘Degraded’?” “A real-life Degraded woman,” she won He shrinks into the seat. “Shut up, Merlin.” “What’s going on?” he says, as she floors Alison leaps to her feet and races to the “There’s a funny person in the mall!” “Can’t it wait? I’m kinda busy here.” “Alison,” he calls. m i l l We all know that changes can occur. a n

P u b l i s h e r s

. Then the wrap: L i m i t e d Life can be tough .

A l l r i g h t s r e s Women Women e r v e d , it it , - - - growing older.” to the condition of senescence. Simply put, says heavily, “is a name given by women son’s shoulders. Deterioration,”“The he is tearful. don’t know why she’s mad at me.” Merlin about it and I don’t know what it means. I ues. “No, really, what is it? Everyone talks blurts out. There is a silence, but he contin Merlin’s father says. Dublin, and blogs at www.joyofwriting.net. She lives on the east coast of Ireland near Susan Lanigan is a programmer and writer. Love you both, stay special.” then at the little girl who never started. who has stopped Deteriorating forever, fingers and looks again: first at the woman ture. The glass is gathering dust. at him. Merlin puts his hand on the pic of Merlin’s mother and sister looks down On his bedroom windowsill, a photograph rictus grin, her eyes like glass. at her stepson, trying to smile — but it is a murs, moving away. Alison turns to look pump in through those little holes.” notjust nice, sweet-smelling stuff they permission?” Alison is harsh as a saw. “It’s sneaked off to that Powder Room without His father tightens his arm around Ali “What’sMerlinDeterioration?” the “I hear you had a little trouble earlier,” “Stay special, Mama. Stay special, Lena. Slowly, reverently, he wipes it off with his “Come with me, Alison,” his father mur “Weren’t you paying attention when you “Suitable?” Merlin asks. of his drink. I men is seen as normal. As you can see, eyes water. Room — on a suitable day.” His father’s both to the Women’s Municipal Powder early with Lena and presented them it. One day she snuck out of the house ten, of course. But she wouldn’t hear of Sisters of her group. I told her not to lis mother was severely pressured by the his hand is on Alison’s shoulder. the first time, Merlin sees how wrinkled glass clatter with his indignation. For Obscene.” The ice cubes in his father’s that’s an obscene way of looking at it. think NowI early-to-mid-thirties. lines around the female mouth. About rule that it starts at the first sign of deep

…” His voice trails off. He takes a gulp His father sighs. “Merlin, older age in “So does that mean I’ll get it?” “In the last few years of her life, your “They — women — have this rough NATURE | Vol 466 | 19 August 2010

■ - - - - -

Jacey NATURE | Vol 466 | 19 August 2010 BRIEF COMMUNICATIONS ARISING

Laryngeally echolocating bats Arising from: N. Veselka et al. Nature 463, 939–942 (2010)

Echolocation of bats is a fascinating topic with an ongoing contro- the difference frequency of both signals. The beat note arises from versy regarding the signal processing that bats perform on the echo. alternating constructive and destructive interference between the Veselka et al.1 found that bats that use the larynx for producing the signal and the local oscillator. This technique is widely used, for echolocating ultrasound have a stylohyal bone that connects the example in commercial frequency-modulated (FM) radio receivers. larynx to the auditory bulla. I propose that the stylohyal bone is used It allows for very precise and phase-sensitive frequency measure- for heterodyne detection of Doppler-shifted echoes. This would ments. Such precise and separate measurements of the Doppler- allow very precise frequency resolution and phase-sensitive analysis shifted return signals at both ears of the bat could yield two compo- of the returning echoes for determining the velocity of echolocated nents of the velocity vector of the object that scatters the bat’s ultra- objects like insects. sound signal, for example an insect. This would enable the bat to The stylohyal bone connects the larynx, the area where the ultra- predict the location of moving objects with much higher accuracy. If sound is generated, to the auditory bulla that is in turn connected to the stylohyal bones or the auditory bulla resonate at the frequency of the cochlea. Veselka et al.1 found by micro-computed tomography the outgoing signal with very little damping, they could preserve the that this connection is present in all of the 26 species of laryngeally outgoing signal to bridge the time lag between the end of the out- echolocating bats that they analysed. The function of this bone is not going signal and the beginning of the return signal. yet entirely clear. Veselka et al.1 list three possible functions: the Ulrich Wittrock1 stylohyal bone could transmit the outgoing signal in order to ensure 1Mu¨nster University of Applied Sciences, Stegerwaldstr. 39, 48565 its accurate registration by the brain, the bone could provide a mech- Steinfurt, Germany. anical reafference, or it could dampen vibrations of the middle ear in e-mail: [email protected] order to prevent self-deafening. Received 18 February; accepted 14 April 2010. I would like to suggest another purpose. The stylohyal bone could transmit the outgoing signal to both ears in order to provide what is 1. Veselka, N. et al. A bony connection signals laryngeal echolocation in bats. Nature 463, 939–942 (2010). called a ‘local oscillator’ in electronic signal processing. In heterodyne detection schemes the reference signal from the local oscillator is Competing interest: declared none. mixed with the signal to be detected. The result is a beat note at doi:10.1038/nature09156

Veselka et al. reply Replying to: U. Wittrock Nature 466, doi:10.1038/nature09156 (2010)

Wittrock suggests that a stylohyal–tympanic connection in larynge- the delay necessary for possibility (1) but could also introduce sig- ally echolocating bats as the one described in our study1could trans- nificant damping of bone-conducted vibrations. Possibilities (1) and mit laryngeal vibrations to both ears2. This could represent a ‘local (2) remain to be investigated experimentally, but clearly represent oscillator’, forming part of a heterodyne-like detection system for productive areas for future research. precise target detection and localization. The essence of this exciting Nina Veselka1, David D. McErlain2,3, David W. Holdsworth2,4, idea is that the externally transmitted echo (signal of interest) Judith L. Eger5, Rethy K. Chhem6,7, Matthew J. Mason8, Kirsty L. Brain8, received by the ears would be mixed (multiplied) with an internally Paul A. Faure9 & M. Brock Fenton1 transmitted copy of the outgoing biosonar sound (reference signal) 1Department of Biology, Schulich School of Medicine and Dentistry, via vibrations of the stylohyal. The multiplicative mixing would gen- University of Western Ontario, London, Ontario N6A 5B7, Canada. erate two new signals—one at the sum and the other at the difference e-mail: [email protected] of the original inputs—and after low-pass filtering the remaining 2Imaging Research Laboratories, Robarts Research Institute, Schulich components would include a difference frequency signal that varied School of Medicine and Dentistry, University of Western Ontario, in its rate of amplitude modulation (AM). London, Ontario N6A 5B7, Canada. Wittrock’s proposal is a logical extension of the first function that we 3Department of Medical Biophysics, Schulich School of Medicine and 1 suggested for the stylohyal–tympanic connection in bats . A hetero- Dentistry, University of Western Ontario, London, Ontario N6A 5B7, dyne-like mechanism may be possible because AM rates up to a few Canada. kilohertz could be encoded in the responses of primary auditory affer- 4Department of Surgery, Schulich School of Medicine and Dentistry, ent neurons.This mechanism applies most obviously to high duty cycle University of Western Ontario, London, Ontario N6A 5B7, Canada. echolocating bats—species that regularly exploit Doppler-shifted 5Department of Natural History, Royal Ontario Museum, 100 Queen’s echoes (for example Rhinolophidae, Hipposideridae, Pteronotus par- Park, Toronto, Ontario M5S 2C6, Canada. nellii)—but its use is not precluded in low duty cycle bats that emit calls 6Department of Radiology, Medical University of Vienna, Division of containing more than a single (narrowband) frequency. Indeed, a Human Health, 1090 Vienna, Austria. heterodyning mechanism may help to explain why so many bats emit 7Division of Human Health, International Atomic Energy Agency, downward hyperbolic frequency-modulated (FM) sweeps. In these Wagramer Strasse 5, PO Box 200, 1400 Vienna, Austria. signals, such linear period modulated chirps emphasize (elongate) 8 the duration of the lowest frequencies in the FM sweep. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. One difficulty with the heterodyning hypothesis lies in identifying 9 the nonlinear biological mechanism for multiplying bone-conducted Department of Psychology, Neuroscience & Behaviour, McMaster vibrations with airborne-conducted vibrations. Another potential University, Hamilton, Ontario L8S 4K1, Canada. difficulty is the difference in velocity between the speed of sound in bone (,3,500 m s21) and the speed of sound in air (,344 m s21), 1. Veselka, N. et al. A bony connection signals laryngeal echolocation in bats. Nature 463, 939–942 (2010). which could result in a latency difference at the cochlea. For the 2. Wittrock, U. Laryngeally echolocating bats. Nature 466, doi:10.1038/nature09156 stylohyal–tympanic bony connection to function as part of a hetero- (2010). dyning system, either (1) there is a mechanism to delay vibrations Author Contributions P.A.F., D.D.M. and M.B.F. wrote the response in consultation with the transmitted along the hyoid apparatus, and/or (2) the tympanic must other authors (N.V., D.W.H., J.L.E., R.K.C., M.J.M. and K.L.B.). continue to ‘ring’ after the outgoing biosonar vocalization has ended so that the signal can be mixed with later returning echoes. Competing interest: declared none. Intervening elastic elements within the hyoid chain may generate doi:10.1038/nature09157

Inferring echolocation in ancient bats Arising from: N. Veselka et al. Nature 463, 939–942 (2010)

Laryngeal echolocation, used by most living bats to form images of O. finneyi falls outside the size range seen in living echolocating bats their surroundings and to detect and capture flying prey1,2, is con- and is similar to the proportionally smaller cochleae of bats that lack sidered to be a key innovation for the evolutionary success of bats2,3, laryngeal echolocation4,8, suggesting that it did not echolocate. and palaeontologists have long sought osteological correlates of echolocation that can be used to infer the behaviour of fossil bats4–7. Veselka et al.8 argued that the most reliable trait indicating echolocation capabilities in bats is an articulation between the stylohyal bone (part of the hyoid apparatus that supports the throat and larynx) and a the tympanic bone, which forms the floor of the middle ear. They examined the oldest and most primitive known bat, Onychonycteris finneyi (early Eocene, USA4), and argued that it showed evidence of this stylohyal–tympanic articulation, from which they concluded that O. finneyi may have been capable of echolocation. We disagree with their interpretation of key fossil data and instead argue that O. finneyi was probably not an echolocating bat. The holotype of O. finneyi shows the cranial end of the left stylohyal resting on the tympanic bone (Fig. 1c–e). However, the stylohyal on the right side is in a different position, the tip of the stylohyal extends beyond the tympanic on both sides of the skull, and both tympanics are crushed. In our opinion, the skull is too deformed to provide evidence of the spatial relationships of these bones in life. Micro-computed tomography (MCT) images of the skull make clear 8 the extent of crushing and fragmentation (Fig. 1b, d). Veselka et al. b noted that the stylohyal–tympanic contact on the left side might be a taphonomic artefact, but nevertheless favoured the interpretation that O. finneyi was an echolocating bat. Available evidence indicates otherwise. Four osteological traits have been postulated as indicators of laryngeal echolocation in bats: (1) an enlarged orbicular apophysis on the 3,4 3–7 malleus ; (2) an enlarged cochlea ; (3) an enlarged paddle-like or c bifurcated cranial tip on the stylohyal3,4; and (4) an articulation between the stylohyal and the tympanic8. Studies in other groups (for example, talpid moles9) indicate that large orbicular apophyses may occur in non-echolocating lineages, hence this trait cannot be considered a definitive indicator of echolocation8. However, the hypothesis that relative cochlear size is a good indicator of the echolocation abilities of bats3–8 has not been refuted. The cochlea of

Figure 1 | Location and orientation of the stylohyal in an extant echolocating bat and in O. finneyi, as revealed by MCT scans. a, Lateral view of the skull of Myzopoda aurita (United States National Museum (USNM) 449282), an extant echolocating bat; note the depth of the braincase and ring-like tympanic (indicated in orange) where the stylohyal (red) articulates with the base of the skull. The cranial end of the stylohyal is de expanded to form a bifurcated tip, and the stylohyal is fused to the tympanic along the length of its course across that bone. b, Lateral view of the skull of 1 the holotype of O. finneyi (Royal Ontario Museum (ROM) 55351A). The cranium is crushed flat and lies directly under a thin, dense sediment layer 2 (shown in blue). This layer is free of any sculptured bone fragments; the 3 layers below it include multiple bone fragments that are all that remain of the braincase and rostrum roof. c, Ventral view of the same specimen (ROM 4 55351A). Both ear regions are preserved but crushed flat. The right and left stylohyals lie at different angles relative to the tympanic ring, indicating that neither was fused to the tympanic. d, e, Individual MCT slices through the 4 basicranial region of the same specimen, with d through a plane slightly 3 dorsal to e. The stylohyals are marked with numerals indicating thirds from 1 2 the cranial end (1) to the distal end (4). The right stylohyal runs perpendicular to the tympanic and is fractured between points 2 and 3. The left stylohyal runs parallel to the edge of the tympanic ring (90u offset from the right stylohyal) and shows evidence of fractures between points 1 and 2, 2 and 3, and 3 and 4. Neither element shows unambiguous articulation with the tympanic or any other bone. Scale bars, 10 mm. E8 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 BRIEF COMMUNICATIONS ARISING

Data presented by Veselka et al.8 indicate that cranial expansion of Nancy B. Simmons1, Kevin L. Seymour2,Jo¨rg Habersetzer3 & the stylohyal and an articulation between this structure and the Gregg F. Gunnell4 tympanic are 100% correlated in extant bats. Previous reports that 1American Museum of Natural History, Central Park West at 79th Street, two families of echolocating bats (Nycteridae and Megadermatidae) New York, New York 10024, USA. lack stylohyal modifications3,4,10,11 overlooked expansions of the e-mail: [email protected] stylohyal where it articulates with the tympanic. We found uniform 2Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario M5S 2C6, presence of expansion and flattening of the stylohyal in both families. Canada. Observed correlations across all extant bat families indicate that this 3Forschungsinstitut Senckenberg, Senckenberganlage 25, Frankfurt am is a definitive marker of laryngeal echolocation, and that expansion Main D-60325, Germany. 4 and flattening of the cranial stylohyal should be considered a Museum of Paleontology, University of Michigan, Ann Arbor, Michigan fundamental part of the stylohyal–tympanic articulation rather than 48109-1079, USA. an independent feature. In O. finneyi, the stylohyal is rod-like and Received 31 March; accepted 26 May 2010. has no cranial expansion or flattening other than a tiny knob at 1. Fenton, M. B. Echolocation: implications for ecology and evolution of bats. Q. Rev. Biol. the proximal end. We hypothesize that this knob might be an ossi- 59, 33–53 (1984). fied, fused typanohyal, which in some non-echolocating bats (for 2. Moss, C. F. & Surlykke, A. Auditory scene analysis by echolocation in bats. J. Acoust. 12 Soc. Am. 110, 2207–2226 (2001). example, Rousettus, Eonycteris ) and insectivores (Echinosorex, 3. Simmons, N. B. & Geisler, J. H. Phylogenetic relationships of Icaronycteris, Erinaceus13) is connected to the stylohyal by a thin ligament or cartil- Archaeonycteris, Hassianycteris,andPalaeochiropteryx to extant bat lineages, with age; regardless, it is not comparable to the condition seen in any extant comments on the evolution of echolocation and foraging strategies in 8 Microchiroptera. Bull. Am. Mus. Nat. Hist. 235, 1–182 (1998). echolocating bat. In contrast with Veselka et al. , we conclude that 4. Simmons, N. B., Seymour, K. L., Habersetzer, J. & Gunnell, G. F. Primitive early Eocene O. finneyi did not have a stylohyal–tympanic articulation as it clearly bat from Wyoming and the evolution of flight and echolocation. Nature 451, 818–821 lacks one of the definitive components of this feature: a modified (2008). 5. Novacek, M. J. Evidence for echolocation in the oldest known bats. Nature 315, stylohyal with an expanded and flattened cranial end. 140–141 (1985). Reconstructions of behaviours of extinct animals require careful 6. Novacek, M. J. Auditory features and affinities of the Eocene bats Icaronycteris and consideration of preservation artefacts in fossils as well as patterns of Palaeochiropteryx (Microchiroptera, incertae sedis). Am. Mus. Novit. 2877, 1–18 (1987). 7. Habersetzer, J. & Storch, G. Cochlea size in extant Chiroptera and middle Eocene form and function among extant animals. Our analyses show that Microchiroptera from Messel. Naturwissenschaften 79, 462–466 (1992). the only two unambiguous pieces of evidence available at this time 8. Veselka, N. et al. A bony connection signals laryngeal echolocation in bats. Nature (cochlear size and stylohyal morphology) support the hypothesis 463, 939–942 (2010). 9. Mason, M. J. Evolution of the middle ear apparatus in talpid moles. J. Morphol. 267, that O. finneyi was not an echolocating bat. Because postcranial 678–695 (2006). morphology indicates that O. finneyi could fly and phylogenetic 10. Griffiths, T. A., Truckenbrod, A. & Sponholtz, P. J. Systematics of megadermatid bats analyses place it on the most basal branch within Chiroptera4, the (Chiroptera, Megadermatidae), based on hyoid morphology. Am. Mus. Novit. 3041, 1–21 (1992). ‘flight first’ hypothesis for the origin of flight and echolocation in 11. Griffiths, T. A. Phylogenetic systematics of slit-faced bats (Chiroptera, Nycteridae), bats3,4 remains the best-supported hypothesis for the origins of these based on hyoid and other morphology. Am. Mus. Novit. 3090, 1–17 (1994). key features. 12. Sprague, J. M. The hyoid region of placental mammals with especial reference to the bats. Am. J. Anat. 72, 385–472 (1943). 13. Sprague, J. M. The hyoid region in the insectivora. Am. J. Anat. 74, 175–216 (1944). METHODS Author Contributions Comparative study of fossil and living bats was carried out by N.B.S. Micro-computed tomography (MCT) images of O. finneyi (Fig. 1b–e) were and G.F.G. MCT scanning was coordinated by J.H. and interpreted by J.H. and K.L.S. N.B.S. obtained with an MCT apparatus using a special ‘region of interest’ algorithm wrote the manuscript with contributions from J.H., K.L.S. and G.F.G. (RayScan 200 XE, RayScan Technologies). CT data for Myzopoda aurita (Fig. 1a) were provided by the University of Texas CT laboratory. Image processing was Competing financial interests: declared none. done with VGStudio MAX 2.0.1 (Volume Graphics). doi:10.1038/nature09219

Veselka et al. reply Replying to: N. B. Simmons, K. L. Seymour, J. Habersetzer & G. F. Gunnell Nature 466, doi:10.1038/nature09219 (2010).

We appreciate the comments of Simmons et al.1 and welcome the provide data about the form of the stylohyal and the size of the new information they have provided about the oldest fossil bat, cochlea, leading them to conclude that the bat did not echolocate. Onychonycteris finneyi, as well as their confirmation of contact Simmons et al. propose that the elongated stylohyal in bats is an between the stylohyal and tympanic bones in Myzopoda aurita,an ossified combination of stylohyal, tympanohyal and the ligament/ extant laryngeal echolocator. Two skeletal features—relatively large cartilage between these two elements. The predictions arising from cochleae and contact between the stylohyal and tympanic bones— this hypothesis can be tested by studying patterns of development identify extant bats with the capacity for laryngeal echolocation. and ossification in living bats. Although the size of the cochlea can be measured in O. finneyi, the We join Simmons et al.1 in awaiting the discovery and description stylohyals may or may not have contacted the tympanics. Simmons et of further fossil bats that can help to resolve the question of the origin al.1 disagree with our interpretation2 of the possible contact between and timing of the evolution of flight and echolocation in bats. the stylohyal and the tympanic bone in O. finneyi, which indicated Nina Veselka1, David D. McErlain2,3, David W. Holdsworth2,4, that this Eocene bat may have had the capacity for laryngeal echolo- Judith L. Eger5, Rethy K. Chhem6,7, Matthew J. Mason8, Kirsty L. Brain8, cation, and have a different interpretation of our results. Paul A. Faure9 & M. Brock Fenton1 We agree with Simmons et al.1 that the known specimens of 1Department of Biology, University of Western Ontario, London, Ontario O. finneyi do not provide clear morphological evidence about contact N6A 5B7, Canada. between the stylohyal and tympanic bones and, by extension, about e-mail: [email protected] the applicability of this character for identifying this bats’ capacity for 2Imaging Research Laboratories, Robarts Research Institute, University laryngeal echolocation. As they note, the holotype of O. finneyi does of Western Ontario, London, Ontario N6A 5B7, Canada. E9 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 466 | 19 August 2010 BRIEF COMMUNICATIONS ARISING

Veselka et al. reply Replying to: N. B. Simmons, K. L. Seymour, J. Habersetzer & G. F. Gunnell Nature 466, doi:10.1038/nature09219 (2010).

We appreciate the comments of Simmons et al.1 and welcome the provide data about the form of the stylohyal and the size of the new information they have provided about the oldest fossil bat, cochlea, leading them to conclude that the bat did not echolocate. Onychonycteris finneyi, as well as their confirmation of contact Simmons et al. propose that the elongated stylohyal in bats is an between the stylohyal and tympanic bones in Myzopoda aurita,an ossified combination of stylohyal, tympanohyal and the ligament/ extant laryngeal echolocator. Two skeletal features—relatively large cartilage between these two elements. The predictions arising from cochleae and contact between the stylohyal and tympanic bones— this hypothesis can be tested by studying patterns of development identify extant bats with the capacity for laryngeal echolocation. and ossification in living bats. Although the size of the cochlea can be measured in O. finneyi, the We join Simmons et al.1 in awaiting the discovery and description stylohyals may or may not have contacted the tympanics. Simmons et of further fossil bats that can help to resolve the question of the origin al.1 disagree with our interpretation2 of the possible contact between and timing of the evolution of flight and echolocation in bats. the stylohyal and the tympanic bone in O. finneyi, which indicated Nina Veselka1, David D. McErlain2,3, David W. Holdsworth2,4, that this Eocene bat may have had the capacity for laryngeal echolo- Judith L. Eger5, Rethy K. Chhem6,7, Matthew J. Mason8, Kirsty L. Brain8, cation, and have a different interpretation of our results. Paul A. Faure9 & M. Brock Fenton1 We agree with Simmons et al.1 that the known specimens of 1Department of Biology, University of Western Ontario, London, Ontario O. finneyi do not provide clear morphological evidence about contact N6A 5B7, Canada. between the stylohyal and tympanic bones and, by extension, about e-mail: [email protected] the applicability of this character for identifying this bats’ capacity for 2Imaging Research Laboratories, Robarts Research Institute, University laryngeal echolocation. As they note, the holotype of O. finneyi does of Western Ontario, London, Ontario N6A 5B7, Canada. E9 ©2010 Macmillan Publishers Limited. All rights reserved BRIEF COMMUNICATIONS ARISING NATURE | Vol 466 | 19 August 2010

3Department of Medical Biophysics, University of Western Ontario, 8Department of Physiology, Development and Neuroscience, University London, Ontario N6A 5B7, Canada. of Cambridge, Downing Street, Cambridge CB2 3EG, UK. 4Department of Surgery, Schulich School of Medicine & Dentistry, 9Department of Psychology, Neuroscience & Behaviour, McMaster University of Western Ontario, London, Ontario N6A 5K8, Canada. University, Hamilton, Ontario L8S 4K1, Canada. 5Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario M5S 2C6, Canada. 1. Simmons, N. B., Seymour, K. L., Habersetzer, J. & Gunnell, G. F. Inferring echolocation 6Department of Radiology, Medical University of Vienna, Division of in ancient bats. Nature 466, doi:10.1038/nature09219 (2010). Human Health, 1090 Vienna, Austria. 2. Veselka, N. et al. A bony connection signals laryngeal echolocation in bats. Nature 463, 939–942 (2010). 7Division of Human Health, International Atomic Energy Agency, Wagramer Strasse 5, PO Box 200, 1400 Vienna, Austria. doi:10.1038/nature09246