Science in Court

www.nature.com/nature Vol 464 | Issue no. 7287 | 18 March 2010 Science in court

Academics are too often at loggerheads with forensic scientists. A new framework for certification, accreditation and research could help to heal the breach.

o the millions of people who watch television dramas such as face more legal challenges to their results as the academic critiques CSI: Crime Scene Investigation, forensic science is an unerring mount. And judges will increasingly find themselves refereeing Tguide to ferreting out the guilty and exonerating the inno- arguments over arcane new technologies and trying to rule on the cent. It is a robust, high-tech methodology that has all the preci- admissibility of the evidence they produce — a struggle that can sion, rigour and, yes, glamour of science at its best. lead to a body of inconsistent and sometimes ill-advised case law, The reality is rather different. Forensics has developed largely which muddies the picture further. in isolation from academic science, and has been shaped more A welcome approach to mend- by the practical needs of the criminal-justice system than by the ing this rift between communities is “National leadership canons of peer-reviewed research. This difference in perspective offered in a report last year from the is particularly has sometimes led to misunderstanding and even rancour. For US National Academy of Sciences important example, many academics look at techniques such as fingerprint (see go.nature.com/WkDBmY). Its given the highly analysis or hair- and fibre-matching and see a disturbing degree central recommendation is that the interdisciplinary of methodological sloppiness. In their view, forensic examiners US Congress create a National Insti- have a poor empirical basis for estimating error rates, and they tute of Forensic Science, which would nature of forensic use protocols that don’t fully take into account the possibility of have strong ties both to academic sci- science.” unconscious investigator bias (see page 344). Many academics are ence and to forensic practice. In the also perturbed to see newer techniques, such as DNA analysis of short term, this institute would help to establish standards and extremely small samples and functional magnetic resonance imag- accreditation procedures and provide independent support for ing, being pressed into service before the results and interpreta- existing forensics entities. In the long term, it would provide fund- tions have been adequately validated for forensic use (see pages ing to develop strong research programmes in forensic sciences. 340 and 347). Such national leadership is particularly important given the highly Forensic scientists, meanwhile, are often resentful of academics interdisciplinary nature of forensic science, which has made it hard who speak high-mindedly of proper procedures now, decades after for the field to coalesce on its own. standard operating procedures have been put in place. They also Congress should follow that recommendation without delay. bristle at being criticized by people who offer little in the way of Strengthening forensic science in the country will be neither cheap support for forensics-relevant research. Perhaps not surprisingly, nor easy. But initial signs point to growing support for this kind of many practitioners have closed themselves off from any open shar- approach (see page 351). In the meantime, more research universi- ing of methods and information with the academic community. ties should establish forensic-science centres and PhD programmes As deep as these differences are, they must be bridged. If they in forensics, so that the next generation of forensic researchers will are not, rapidly evolving techniques will continue to be bandied not feel so distant from their academic counterparts. ■ about in court before they are ready. Forensic practitioners will See online at www.nature.com/scienceincourt

least in the United States, where the 2009 stimulus package desig- Handle with care nated some $19 billion to foster their deployment. The success of such efforts hinges on public acceptance — convincing people that Britain’s Department of Health must respond to they will see real benefits and that their privacy will be rigorously concerns about electronic medical records. safeguarded. But this acceptance is seriously under threat in Brit- ain, as the country begins to realize its plans for digitizing patients’ he worldwide effort to computerize paper medical records is records. driven by the need to cut paperwork costs and minimize the Britain is developing a national database of electronic ‘summary Tchance of medication errors, both of which would benefit care records’ (SCRs). These will contain limited information, such as patients and clinicians. Sufficiently large collections of patient data details of a patient’s prescriptions or allergies, extracted from exist- could also be a boon to researchers, helping them to investigate a host ing paper records. The UK Department of Health says that there are of epidemiological issues such as quickly identifying rare problems currently no plans to repackage these data for use by researchers; caused by new drugs or tracking the spread of a pandemic (see Nature the goal is simply to provide quicker and more accurate records for 458, 278–280; 2009). health-service workers. Other data will be added to the SCRs later, As a result, money is pouring into electronic medical records — not however, and the research potential will grow accordingly.

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Despite government assurances, some patients — and some doc- Governments are right to be enthusiastic about such databases, tors — feel that personal and potentially sensitive information will but wrong to rush ahead before they have laid the groundwork for not be secure in a centralized, national database. And last week, public understanding and trust. This is especially important given concerned that patients were not yet fully aware of the system, the that pharmaceutical companies are increasingly showing an interest British Medical Association called for a halt to the SCR roll-out. It in the potential of electronic records. Although there is nothing in argued that the speed with which the database is being introduced principle wrong with such use of data, it is easy to see how the public means that patients do not necessarily know about it and that even might be suspicious of corporations potentially profiting from highly those who do could not easily exercise their right to opt out of hav- personal information. ing an SCR. The UK government should suspend the SCR roll-out, at least until Those criticisms are well taken. The Department of Health insists UCL’s second evaluation is published next month and it is clear that that it supports the right of any patient to opt out, and denies that it previous concerns have been addressed. It should also ensure that all is rushing implementation of the system. But the roll-out is occur- patients have a clear explanation of the system as well as ready access ring before an evaluation of the SCR scheme by University Col- to the form needed for them to opt out. lege London (UCL) has even been published — and thus before For now, the scant data to be held in the UK database will be of lim- the results of changes made following issues with patient consent ited use to researchers. But those wishing to tap into the potentially raised in a previous UCL evaluation are public. As a result, public rich resources of its successors should work to ensure that the process confidence in the deployment of the system is at risk of being seri- of medical data collection is not only secure, ethical and beneficial to ously undermined. all concerned, but that it is seen to be so. ■

Setting the bar matters. The resulting dearth of scientific input has contributed to a number of ill-considered commission policies, most notably its Europe’s chief science adviser must be given infamously harsh stance against genetically modified organisms. It authority and support to deliver across the board. will be the job of Europe’s chief scientist to turn this around. However, more is needed to ensure that the chief scientist will be urope is well on its way to appointing its first chief scientific able to deliver. Any chief scientist is forced to walk a fine line between adviser. European Commission president José Manuel Barroso remaining independent of political interference and maintaining the Eset the ball rolling last September, when he pledged to create the trust and goodwill of the officials he or she is advising. The difficulties position to fill a gaping lack of rigorous, independent scientific input inherent in so doing were highlighted by the recent sacking of David into European policy-making. Europe’s newly elected commissioners Nutt, an adviser on drugs policy to the kept up the momentum by officially creating the position at a meeting British government, on the grounds “The issues are much on 17 February — remarkably quick action by commission standards, that his academic work seemed to con- too complex and indicating that the issue is being taken seriously. tradict government policy on illegal wide-ranging for And last week, Nature has learned, the as-yet-unfilled position substances. To ensure that European any one individual dodged a bullet. Some commission officials had been pushing to policy receives the independent sci- to master.” bury the science adviser’s office deep within the bureaucracy, mak- ence advice it needs, the commission ing it all but certain that the future incumbent would lack sufficient should draw up principles to which they and the adviser must adhere, power and independence to be successful. But the commission has in a similar vein to the guidelines the British government is develop- now rejected that suggestion, making it clear that the chief scientist ing in the wake of the Nutt affair. will report directly to Barroso, and will have a high enough status in In addition, Europe’s chief scientist cannot be a one-person show. the commission to make decisions without interference. The issues that he or she will have to address are much too complex Further details of the chief scientist’s remit are still under dis- and wide-ranging for any one individual to master. John Holdren, cussion. But the early indications are that the chief scientist will be science adviser to US President Barack Obama, heads an Office of involved mainly in delivering science advice for sound policy-making Science and Technology Policy that comprises more than 70 sci- across the board — in issues such as climate change and energy, for entists, analysts and administrators. A similar team of dedicated example — and will not have only a narrow focus on policies for experts should be created for the European science adviser, because research funding. It also seems that the chief scientist will receive the resources available from the Bureau of European Policy Advis- administrative assistance from the Bureau of European Policy Advis- ers, which currently lacks scientific expertise, are unlikely to be ers, a kind of official government think-tank close to the commis- sufficient. sion president — a move that should further help guarantee the chief Barroso will nominate his chief science adviser in the near future. scientist’s access to Barroso. It is crucial that this important position starts out on the right foot These developments are encouraging. Until now, the Bureau of and is filled on the basis of scientific credibility. Allowing politics to European Policy Advisers has rarely given sufficient weight to sci- interfere, as can so often happen in Europe, will destroy the authority ence in decision-making, concerning itself primarily with economic of the chief scientist and waste a valuable opportunity. ■

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Wind-blown ice Geophys. Res. Lett. doi:10.1029/2009GL042356 (2010) Arctic wind patterns explain half of the annual P. SOUDERS/CORBIS P. variation in the minimum extent of Arctic sea ice, and account for about one-third of the unexpectedly rapid sea-ice decline seen in the past three decades, researchers report. Masayo Ogi of the Japan Agency for Marine- Earth Science and Technology in Yokohama and her colleagues compared published data on summer and winter winds from 1979 to 2009 with data on September sea-ice extent. They found that certain wind anomalies — such as the summer anticyclonic (clockwise) winds over the Beaufort Sea — correlate with low sea ice. The authors say that these winds may enhance the flow of ice out through the Fram Strait east of Greenland, contributing to ice shrinkage. Their analysis identified an increasing trend in these particular wind patterns.

MATERIALS SCIENCE from them have failed. Michael Bunce at NEUROSCIENCE Murdoch University in Perth, Western Ultrathin fibres heat up Australia, and his colleagues took several Rats on the wagon Nature Nanotechnol. doi:10.1038/nnano.2010.27 measures to optimize DNA extraction, Neuron 65, 682–694 (2010) (2010) including warming a DNA-isolation solution Rats that regularly consume alcohol and are The plastic polyethylene is used in a wide containing powdered eggshell to 95 °C. then denied it show increased activity in a range of products, from shopping bags to The authors tackled the eggs of several specific brain region. water pipes. One place it doesn’t appear is in extinct species, including the New Zealand F. Woodward Hopf, at the University of heat exchangers, which dissipate heat and giant moa (Dinornis robustus; microscope California, San Francisco, and his colleagues are essential components of devices such image of eggshell’s inner surface pictured). gave rats access to either a 10% alcohol as refrigerators and air conditioners. That’s They say that their procedure released the solution or sucrose for several weeks and then because, in its bulk form, the plastic can DNA (red arrows) from the eggshell’s calcium cut them off. After a few weeks of abstinence, conduct only a paltry amount of heat. Bulk carbonate crystalline matrix, allowing them the alcohol-exposed rats had increased polymers typically conduct only around to double their yield. They also found that neuronal firing in the core of the nucleus 0.1 watts per metre-kelvin. eggshells are less contaminated with bacterial accumbens — a brain structure associated Gang Chen and his colleagues at the DNA than fossil bones. with motivation and addiction — but those Massachusetts Institute of Technology in For a longer story on this research, that had consumed sucrose did not. Cambridge have markedly improved that see go.nature.com/lx1mzC This elevated activity is linked to reduced figure using thin fibres of polyethylene. They numbers of a certain type of potassium created the fibres by stretching strands of channel that normally depresses neuronal polyethylene gel with a sharp tungsten tip. firing. Alcohol-drinking rats that were given R. SOC. The 50–500-nanometre-wide strands act as a drug to activate these channels — thus conduits, conducting heat at rates as high as dampening firing in this brain region — were 104 watts per metre-kelvin — a rate similar less likely to seek alcohol. Drugs that activate to that of many metals. Chen and his team the channels in humans might prevent relapse believe the work could lead to low-cost plastic to alcoholism, the authors say. replacements for some metal components. VIROLOGY PALAEONTOLOGY Infectious inheritance Egg-stracting DNA Proc. Natl Acad. Sci. USA doi:10.1073/ Proc. R. Soc. B doi:10.1098/rspb.2009.2019 (2010) pnas.0913586107 (2010) Improved techniques have yielded well- The genome of a common herpesvirus may preserved DNA from fossilized eggshells, hitchhike from one generation to the next highlighting a potential new source of ancient on the ends of human chromosomes, and genetic material. reawaken in an infectious form. Eggshells are commonly found at fossil Human herpesvirus-6 infects almost sites, but previous attempts to pull DNA everyone. Peter Medveczky of the University

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JOURNAL CLUB Eran Segal Weizmann Institute of Science, Rehovot, Israel of South Florida in Tampa and his colleagues Scotland. Farming would have supported A computational biologist looks studied four families in which some members higher population densities than hunting at how identical cells come to had unusually high amounts of herpesvirus-6 and gathering. And if the indigenous hunter- differ. DNA in their blood. The researchers found gatherers had adopted farming from the that the viral genome had inserted into caps mainland, they would have taken longer My main interest is in called telomeres at the ends of chromosomes. to learn it, resulting in a slower growth in understanding how complex The viral genomes in parents and their population than is indicated by the data. biological behaviours are encoded children were identical, suggesting that the by DNA. An example of such DNA is heritable. Treating cells from these NEUROSCIENCE behaviour is the ability of genetically families with compounds that stimulate latent identical cells to generate diversity herpesviruses also allowed the viruses to Memory reading in their phenotypes, or observable infect other cells in culture. Curr. Biol. doi:10.1016/j.cub.2010.01.053 (2010) traits, by changing how genes are By decoding patterns of brain activity, expressed from one cell to the next. CONSERVATION researchers can tell which in a list of events a How expression variability occurs person is recalling. over short timescales (for example, Heavy metal history Eleanor Maguire and her colleagues at during a cell cycle) has been well Environ. Sci. Technol. doi:10.1021/es903176w (2010) University College London, UK, showed studied; much less is known about it Endangered California condors (Gymnogyps ten volunteers short films depicting three over longer timescales. californianus; pictured) are heavily affected different actions such as drinking coffee. So I was excited by work by lead poisoning, but current biannual The researchers then asked them to from Narendra Maheshri of testing detects only remember each action the Massachusetts Institute of a fraction of their individually while Technology in Cambridge and his exposure. scanning their brains colleagues. They demonstrate that Myra Finkelstein using functional slow expression fluctuations of a at the University of magnetic resonance yeast gene are regulated locally, California, Santa Cruz, imaging (fMRI). They or in cis, by that gene’s promoter and her colleagues found that unique — a nearby stretch of DNA that

report that analysis of fMRI patterns in PHOTOS/ALAMY ALL CANADA regulates the gene’s expression sequential segments the hippocampus (L. M. Octavio et al. PLoS Genet. 5, of condor feathers can corresponded with e1000673; 2009). provide a history of the recall of specific They studied the yeast protein lead exposure over the memories. FLO11, placing two copies of the 2–4 months of feather These patterns were protein’s promoter in the same growth. stable over 24 hours cell, each in front of an engineered By measuring and allowed the fluorescent ‘reporter’ gene. The lead concentration scientists to predict reporters switched expression and isotope composition in feather and which memory participants chose to recall. slowly and independently, implying blood samples, the researchers identified that the expression fluctuations lead-exposure events that would have been GENOMICS were locally encoded. The authors missed by blood monitoring alone. Their further identified global, or trans, technique, they say, may also be applicable to We are family regulators that affect the fast and other bird species. Science doi:10.1126/science.1186802 (2010) slow expression fluctuations of The human mutation rate is lower than FLO11. The type of expression effect ARCHAEOLOGY previously thought, according to researchers that a regulator exerts seems to who sequenced the entire genomes of four depend on several factors, including Adoption or migration? family members — two siblings with rare the location of the regulator relative J. Archaeol. Sci. 37, 866–870 (2010) genetic disorders, and their parents. to the site at which transcription, The origin of farming in Britain is hotly David Galas and Leroy Hood at the or reading of the DNA, begins, and debated: did the indigenous population Institute for Systems Biology in Seattle, relative to sites for other regulators. adopt farming practices through trade and Washington, and their colleagues estimate Although the mechanistic details exchange with continental Europe or did that roughly 70 new mutations arise per of this encoding are still unclear, migrants bring farming from the mainland? genome between generations. This is lower applying similar approaches to Mark Collard from Canada’s Simon Fraser than earlier estimates based on genomic many more promoters should bring University in Burnaby, British Columbia, comparisons between humans and their us closer to understanding how and his colleagues used radiocarbon dates to closest living relatives, chimpanzees. other complex phenomena are estimate prehistoric population changes in The team also pinpointed four genes encoded by DNA. This will hopefully Britain. They found that about 6,000 years likely to underlie the siblings’ two disorders allow us to one day predict the ago, population density increased sharply and — Miller syndrome and primary ciliary phenotypic effects of human cultivated plants occurred at around this time. dyskinesia. The four genes are a subset of genetic variation. The best explanation for this is that those reported in previous studies. groups of migrant farmers from mainland For a longer story on this research, Discuss this paper at http://blogs. Europe established colonies in England and see go.nature.com/Owe9CD nature.com/nature/journalclub

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● POLICY Review panel: Faced with numerous criticisms and an acknowledged mistake in its last assessment in 2007, the Intergovernmental Panel on Climate Change (IPCC) appointed the InterAcademy Council to conduct an independent review of its SEED VAULT GLOBAL M. TEFRE/SVALBARD procedures on 10 March. The council, an international organization representing national science academies worldwide, will itself appoint a team to conduct the review later this month. The team will target a range of IPCC procedures as well as larger questions of organization, resources and communications. The final report is expected by August. SEED VAULT BANKS HALF A MILLION The Svalbard Global Seed Vault (pictured), which opened two years ago, now holds more than 250 million Stem-cell law: Legislation seeds and 520,000 crop varieties — around a third of the world’s known 1.5 million crop strains. The Global codifying US President Barack Crop Diversity Trust, which oversees the seed bank, announced the passing of the half-a-million landmark on Obama’s policy of allowing 11 March. Installed in the side of a mountain at Longyearbyen, a town in the Norwegian archipelago of Svalbard, federal funding for human the bunker is designed to keep samples of the world’s seeds safe at –18 °C, for backup in case disaster strikes embryonic stem-cell research global food production. was introduced in Congress on 10 March. Representatives Diana DeGette (Democrat, Colorado) Acid ruling: The US would allow US companies and Mike Castle (Republican, Environmental Protection to avoid responsibility for Delaware) sponsored the Stem Agency has agreed to develop accidents. Countries such as Cell Research Advancement guidance to help US states France and Russia have already Act. The aim is to prevent the address the increase in ocean agreed to supply nuclear reactors current presidential policy acidification caused by to India, but an Indo-US nuclear from being changed by future carbon dioxide absorption. deal signed in 2008 has still not administrations. The proposed The decision, spelled out in resulted in trade. law, like Obama’s policy, allows an 11 March federal court federal funding for research on settlement with the Center for Animal market shake-up: On stem-cell lines derived from Biological Diversity, based in SOUND 9 March, drug companies Merck embryos created for reproductive Tucson, Arizona, stops short of and Sanofi-Aventis announced purposes that would otherwise regulation but does not preclude BITES that they would merge their be discarded. such action in the future. “If I could get my animal-health businesses. Merck, hands on the person headquartered in Whitehouse Brain-drain reversal: Israel’s who proposed Station, New Jersey, and Sanofi- cabinet has approved a five- ● BUSINESS Aventis, headquartered in Paris, year plan to bring Israeli- Nuclear stalling: India’s the current will have equal ownership in the born researchers back to coalition-led government has management new venture, which would be the country. As part of the deferred introducing legislation structure I would the largest player in the US$19- scheme, to which the cabinet that would have smoothed the strangle him. billion animal-health market. has allocated US$121 million, way towards Indo-US trade in ” William Brinkman, director of the 30 centres of excellence will nuclear technology. The delayed US energy department’s Office from this September begin to bill would cap the liability of Science, tells a departmental ● RESEARCH be established at the country’s of foreign nuclear reactor committee meeting there will Rocket test-fires: On its second universities, creating positions for suppliers at 5 billion rupees be ‘significant changes’ to the attempt, commercial spaceflight management of ITER, the fusion young scientists, social scientists (US$110 million), a clause that project that has suffered cost company SpaceX has successfully and humanities researchers. The US companies would like as a overruns and delays (see Nature test-fired all nine engines of its universities will have to raise precondition of selling nuclear 463, 721; 2010). Falcon 9 rocket. The firm, based further (unspecified) funds from equipment. Opposition parties in Hawthorne, California, has private sources. in India protested that the cap Source: Fusion Power Associates a contract with NASA to take

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payloads to the International what those charges were, and Space Station once the Shuttle NUMBER added that issues giving rise to retires. The 13 March static test the suspension might “still exist” THE WEEK precedes a full launch, which CRUNCH and needed to be dealt with. could happen within months. 82.4% AHEAD Robot lost at sea: A pioneering Estimated decline 21–25 MARCH underwater robot has been lost off The American Chemical Society the coast of Chile. First launched in bluefin tuna in holds its 2010 spring meeting in 1995, the Autonomous the West Atlantic in San Francisco, California. The J. RAPO/ACAD. FINLAND RAPO/ACAD. J. Benthic Explorer, nicknamed between 1970 and conference’s theme is green and ‘ABE’, was one of the earliest 2007. Delegates sustainable chemistry. completely independent research ➧ go.nature.com/iLMbWg submersibles, neither manned at the Convention nor tethered to its research on International 22–23 MARCH ship. It failed to resurface on its Trade in Endangered London’s Royal Society holds 222nd dive, perhaps because its Species of Wild a meeting to discuss how buoyancy spheres imploded, Geneticist dies: Human scientists deal with uncertainty the Woods Hole Oceanographic Fauna and Flora geneticist Leena Peltonen- in their particular field — and Institution in Massachusetts meeting in Doha, Palotie (pictured above) died on how to communicate it. announced on 9 March. Qatar, are debating 11 March, aged 57. She pioneered ➧ go.nature.com/ZhthnH whether to ban the use of isolated populations to Institute shuffle: The US international trade discover disease genes, analysing 22–26 MARCH National Institute of Standards those in her native Finland to Expect norms and guidelines for and Technology in Gaithersburg, in the fish. illuminate the genetic basis of at geoengineering experiments Maryland, is to consolidate least 20 diseases. Deeply engaged to emerge from a summit in its national laboratories, says in bioethical debates about Pacific Grove, California, where director Patrick Gallagher. He genetics, Peltonen-Palotie was climate scientists are meeting plans to reduce the number head of human genetics at the to talk about the risks of climate of agency labs from ten to six, Wellcome Trust Sanger Institute intervention. putting researchers in larger, near Cambridge, UK, and also ➧ climateresponsefund.org interdisciplinary groups aligned headed research groups at the more with agency missions Institute for Molecular Medicine 24–26 MARCH than individual scientific Finland and the National Institute Both the Global Fund to Fight disciplines. Gallagher also wants for Health and Welfare, both in AIDS, Tuberculosis and Malaria to replace the deputy director Helsinki. She was also an associate and the Global Alliance for position (currently vacant) with member at the Broad Institute in Vaccines and Immunisation meet three associate directors — director of the South African Cambridge, Massachusetts. in The Hague, the Netherlands respectively managing research Astronomical Observatory in to discuss shortfalls in their at the agency’s labs; external Cape Town, has been reinstated Saudi shift: Jean Fréchet, the finances (see Nature 463, 415; industry programmes; and after a disciplinary hearing. high-profile organic polymer 2010, and page 338). information technology, human The country’s main funding chemist at the University of ➧ go.nature.com/HHUHcM resources and facilities. agency, the National Research California, Berkeley, has been Foundation, removed Charles appointed vice-president for from his post in January, citing research at the newly opened oversee education and science ● PEOPLE the leaking of confidential King Abdullah University of at the fledgling university’s Astronomer ‘not guilty’: Phil documents, but says that the Science and Technology in nine research centres, and Charles, a leading astronomer hearing found him “not guilty Saudi Arabia. From June 2010 will develop ‘innovation and who was suspended as on all charges”. It did not explain to June 2011, Fréchet will enterprise’ strategies.

BUSINESS WATCH LITHIUM SURPLUS

The appetite for lithium to supply batteries says that existing suppliers, mainly in South Existing lithiumm supply in mobile phones, laptops and electric cars is America, are fully capable of meeting demand 50 plusp future projectsojectsjt

growing, but supplies are forecast to outstrip (see chart). With the new proposals, the ExistingE lithiumuumm supply TRU GROUP SOURCE: demand over the next decade. That would result is a nearly 50% oversupply in 2012 and LithiumL demandannd 40 keep lithium prices low — and put eager new significant surpluses through to the end of the Dashed lines show projectedjected ddataata suppliers of the element out of business. decade. “Most of the money that’s going into TRU Group, a consultancy based in Toronto, these [new] companies now is going to be lost 30 Canada, says that demand for lithium purified to investors,” Anderson says. sufficiently for use in batteries will more than Chris Hartshorn, an analyst with Lux Research double over the coming decade, thanks in part in Boston, Massachusetts, says that the lithium 20 to electric cars. Mining firms are busily signing supply glut could carry over into the lithium-

deals with car manufacturers to supply lithium, battery market, resulting in an eventual shake- lithium (thousand tonnes) Puriﬁed and a few dozen new entrants are proposing out of battery manufacturers. That is good for 10 2002 2005 2008 2011 2014 2017 2020 other production ventures. the market in the long run, he says, but there will Year But TRU Group president Edward Anderson be some carnage along the way.

3330-33130-331 NewsNews BriefingBriefing MH.inddMH.indd 331331 116/3/106/3/10 115:07:295:07:29 Vol 464|18 March 2010 NEWS Wildlife service plans for a warmer world US interior department seeks ways to save species threatened by climate change.

Akikikis are small birds with limber tongues “For too long, in my view, we have stood idle that are adept at pulling insects from the crev- as the climate-change crisis has grown,” says ices in tree bark. The olive-and-white birds Ken Salazar, secretary of the interior depart- are also headed towards extinction. Earlier ment. Mike Daulton, legislative director of the this month, the US Fish and Wildlife Service National Audubon Society in New York City, (USFWS) listed the akikiki as endangered agrees. “We are getting a late start,” he says. and warned that global warming may hasten The report, a collaboration between the its disappearance by prompting the spread of USFWS, the US Geological Survey, academics from the Cornell Lab of Ornithology in Ithaca, avian malaria. and a collection of environmental and wildlife New York. Every habitat will be disrupted by the The akikiki is just one of more than 200 groups, quantified the vulnerability of each warming climate, but ocean birds are the most birds in the United States threatened by cli- species on the basis of its breeding behaviour, vulnerable to the disruption, with all 67 species mate change, according to a report issued last habitat, migratory pattern and ecological niche. especially at risk because they depend on a rap- week by the US Department of the Interior, George Wallace, vice-president for oceans and idly changing marine ecosystem, according to which oversees the USFWS . Called The State islands at the American Bird Conservancy in the report. And island birds, particularly those of the Birds: 2010 Report on Climate Change, The Plains, Virginia , says the report shows in Hawaii, will face increasing rates of avian S. KAZLOWSKI/NATUREPL.COM it is one of several steps recently taken by the that “we need to consider climate change as we malaria if the warming climate allows mosqui- administration of President Barack Obama to continue conservation work into the future”. toes to spread to higher mountain zones . assess how climate change is affecting wild- Climate change will magnify already exist- The USFWS this month added the akikiki life and to find ways of lessening its impact. ing threats to birds, says Kenneth Rosenberg and another bird from the Hawaiian island of Sex bias blights drug studies The typical patient with chronic severe side effects in women that Research, convened on 8–9 March number of disciplines, researchers pain is a 55-year-old woman — the are not seen in men. Such failures by the US Institute of Medicine’s simply don’t study females, and typical chronic-pain study subject might be spotted earlier if more Forum on Neuroscience and there is so much evidence for sex is an 8-week-old male mouse. To preclinical work was done in Nervous System Disorders. differences at all levels of biological pain researcher Jeffrey Mogil at female model animals, according “This is an issue of enormous organization that to only study McGill University in Montreal, to researchers at the Workshop on importance,” says biologist males, and assume the results apply Canada, that discrepancy is a telling Sex Differences and Implications Irving Zucker at the University to females, is just wrong.” example of the problem that he and for Translational Neuroscience of California, Berkeley. “In a Many diseases other neuroscientists discussed disproportionately affect one last week at a workshop held in San sex: chronic pain, depression Diseases such as Francisco, California: the serious and autoimmune disease, depression can under-representation of females in for instance, more often disproportionately biomedical studies. affect women. affect women, with addiction The lack of female participation, and cardiovascular disease which extends from basic research disproportionately affecting men. in animals to clinical trials in The NIH Revitalization Act of 1993 humans, has obvious consequences attempted to take account of this for women, not least a paucity by requiring clinical trials funded of effective drug treatments for by the National Institutes of Health diseases that predominantly affect to include adequate numbers PHOTOGRAPHY/CORBIS M. CONSTANTINI/ÈS them. But it also affects men , for of women to detect differing example when drug candidates fail treatment effects. to get regulatory approval because The US Government they don’t work in women in Accountability Office reported in late-stage clinical trials, or have 2000 that participation of women

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WHEN IS A SPECIES EXTINCT? SPL Model assesses whether conservation is viable. go.nature.com/hWL6tA

the Chesapeake,” says Paul Schmidt, USFWS assistant director for migratory birds. “We must scale down those big climate models to help the manager on the ground.” Meanwhile, wildlife managers are taking several approaches to counteract the effects of climate change. In Hawaii, for example, one tactic to save rare birds involves keeping large mammals out of sensitive areas. This prevents the animals from trampling the ground, which destroys habitat and also creates water-filled hollows where mosquitoes can breed. Environmental groups have charged that the previous administration of George W. Bush ignored climate change and its effects. But Paul Hoffman, who served as deputy assistant secretary for fish, wildlife and parks under Bush, says it would be wrong to suggest that officials ignored the science. “Quite often, the scientists Climate change threatens some 200 bird species in the United States, especially marine birds. weren’t in agreement about the problem and what the solution ought to be,” he adds. Kauai to its endangered-species list. Also joining eight planned around the nation. Information But the Obama administration’s first budget the list is a picture-wing fly and 45 plant species from those centres about climate’s effects and reflects a change in priorities. For fiscal year from Kauai. Because they are all endemic to the ways to lessen them will feed into a network of 2010, it provided US$25 million for Landscape island, climate change poses a special risk to LandscapePic cap Conservationin heere fhgfhfbhf Cooperatives, bjf which Conservation Cooperatives and $15 million for them, according to the wildlife service. will helpbjfbfbjhbf to coordinate jhbf jhbf jhbfjhfbjhb regional climate-adap- Climate Science Centers. “It went from zero to Last month, Salazar issued an order mar- tation efforts by federal and state governments $40 million in one year,” Schmidt says. Steve shalling the forces of the interior department and private landowners. Holmer of the American Bird Conservancy to study climate impacts on wildlife, and earlier “It’s a daunting task for a refuge manager calls these efforts “a pretty impressive system- this month the department opened a Climate sitting on the eastern shore of Maryland to atic approach across the board”. ■ Science Center in Anchorage, Alaska, the first of understand what climate change means for Janet Fang

in NIH-funded trials had increased their oestrous cycles. But Mogil back to 1909, the proportion of their authors to report the sex of substantially (see www.gao.gov/ has reported that in common tests animal studies using only males animals used in the research. The archive/2000/he00096.pdf). used to measure responses to pain, had actually grown since the early London-based National Centre for The following year, it noted similar data from female mice are no more twentieth century. The authors the Replacement, Refinement and progress in late-stage drug trials variable than those from males speculate that this is because Reduction of Animals in Research overseen by the Food and Drug (J. S. Mogil and M. L. Chanda Pain oestrous cycles in guinea pigs, is developing a set of research Administration (see www.gao.gov/ 117, 1–5; 2005). rats and mice were first clearly reporting guidelines that will call new.items/d01754.pdf). The problem is particularly acute characterized only in the 1920s. for authors to disclose the sex of But data from clinical trials are in neuroscience, in which the ratio of Researchers such as Karen animals. Seán Murphy, chief often not analysed separately on male- to female-only Berkley of Florida editor of the Journal of the basis of sex. Such analyses studies is 5.5 to 1. But “To only study State University Neurochemistry, says that there is could reveal whether a drug has under-representation males, and in Tallahassee “interest” in the guidelines among different adverse effects in men and of females occurs in assume the results have been trying journal editors at its publisher, women, for example. most fields of basic to boost female Wiley-Blackwell. And the accountability office also research , according apply to females, representation Researchers at the workshop said noted in 2001 that although women to an unpublished is just wrong.” for decades, and that funding agencies, too, could made up more than half of full-scale analysis by Zucker Berkley feels that ask grant applicants to specify the safety and efficacy trials, they and postdoctoral researcher some progress has been made, sex of the animals they propose formed just 22% of the participants Annaliese Beery. They investigated citing, for example, the launch studying and justify their decision in initial, small-scale safety studies. the use of female and male animals of an online journal in the field, whenever they chose only male Back at the bench, lab animals in research published during 2009 Biology of Sex Differences, and an animals . Funders could also are still predominantly male, in 10 fields across 42 journals . overall growth in research on sex change the status quo, the even in studies of diseases that They found that studies in eight of differences. researchers claimed , by supporting disproportionately affect women. the fields used only male animals But at the workshop, Berkley and more work on sex differences, Investigators tend to prefer male more often than only females, other scientists agreed that further although the NIH would not animals because it is thought and that the data were often not steps were needed to address comment on whether this was that females might introduce analysed by sex . In two journals the problem. Many endorsed the under consideration. ■ variability through factors such as that the researchers investigated idea that journals should require Erika Check Hayden

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FERTILE HOPES FOR A Hunt for the sterile STERILE NEUTRINO Neutrinos are elusive particles that interact with ordinary matter through the weak neutrino heats up nuclear force, which means they can ﬂy through Earth with little chance of hitting any nuclei along the way. They come in The elusive particles, if they exist, could help solve some of the three types and can switch, or oscillate, from one to the other. most pressing problems in astrophysics. Some experiments have suggested the existence of a fourth type of neutrino. Neutrinos like to keep to themselves. These and astrophysics,” says William Louis of Los Unlike ordinary neutrinos, this 'sterile' ghostly particles are so reluctant to interact Alamos National Laboratory in New Mexico, neutrino would not feel the effects of the with ordinary matter that billions zip harm- who worked on a ground-based experiment in weak nuclear force, making them even lessly through each person every day, and it the mid-1990s that provided one of the first more difficult to detect. takes giant, specialized detectors to capture hints of sterile neutrinos. even a handful of them. Now astronomers The three types of ordinary neutrinos (see n Muon n Electron are finding hints of an even more elusive type graphic) are hard enough to detect. They m neutrino e neutrino of neutrino, one so shy that it could never be interact with matter through the weak nuclear detected directly: the sterile neutrino. force, which means they can pass through huge n Tau Sterile For more than a decade, this subatomic volumes of material yet collide with atomic t neutrino ? neutrino spectre has intrigued theorists and experi- nuclei only rarely. To catch them, physicists menters, but experimental efforts have had build detectors using tanks of mineral oil or trouble catching them. Now, two observations heavy water, and they improve the odds by in space — one in microwaves and the other in focusing on the torrents of neutrinos coming X-rays — are raising hopes again. from nuclear reactors, particle accelerators were released in January1, suggest that the If sterile neutrinos could be identified, and the Sun. most likely number of neutrino families in the they would provide the first glimpse of a early Universe was four — implying that one new realm of physics beyond the tried-and- Unsociable particle more neutrino type awaits discovery. “To say tested standard model. They could also help In 1998, results from an accelerator at CERN, that there’s something else is a major deal,” says to explain a host of astronomical puzzles, and Europe’s particle-physics facility near Geneva, principal investigator Charles Bennett of Johns perhaps even account for the invisible dark Switzerland, indicated that only the three Hopkins University in Baltimore, Maryland. NASA/WMAP SCIENCE TEAM NASA/WMAP matter that is thought to make up 85% of the known families, or flavours, of neutrinos could But he cautions that the error bars are still wide Universe’s mass. be affected by the weak force. Any beyond enough that three might still be the maximum “The question of sterile neutrinos is abso- these three families would have to be ‘sterile’ — number. “I would call this intriguing, but I’m lutely crucial for nuclear particle physics that is, immune to the weak nuclear force. And not losing any sleep right now.” that would mean that they would pass straight A more recent clue has come from the orbit- through existing neutrino detectors. ing Chandra X-ray Observatory. Sterile neutri- Neutrino hunters Why would theorists invoke such an unso- nos, if they exist and if they are heavy enough, ciable particle to begin with? Because of should emit faint pulses of X-rays as they decay another surprising discovery made in 1998: into lighter neutrinos. Alexander Kusenko of that the three ordinary neutrinos have a small the University of California, Los Angeles, has but definite mass. focused on regions of the sky thought to contain The result came as a shock, and not simply lots of dark matter but few stars or other light because the standard model assumes that neu- sources. Sure enough, he claims to have found2 trinos have no mass. The masses of the three the predicted X-ray signature in Willman 1, a neutrinos, determined from the way they oscil- dim dwarf galaxy that orbits the Milky Way. But, FERMILAB VISUAL MEDIA SERVICES FERMILAB VISUAL late, or switch, from one flavour into another, like Bennett, Kusenko says that it is still too early turned out to be incredibly small: the heaviest to be claiming a discovery. “Everything seems to ordinary neutrino is at least seven orders of fit together,” he says, “and it’s tantalizing.” magnitude lighter than the electron. Theorists Kusenko’s neutrinos would be heavy and reasoned that some other particle — perhaps a plentiful enough to be the dark-matter parti- sterile neutrino — should sit in between. cles themselves. But the WMAP data point in The Mini Booster Neutrino Experiment Hints of such a missing link have emerged another direction, towards a sterile neutrino (MiniBooNE) uses 1,280 photomultiplier from the Wilkinson Microwave Anisotropy at the lighter end of the spectrum of possible tubes to capture faint flashes made when a Probe (WMAP), a spacecraft that since 2001 masses. George Fuller, director of the Center neutrino or an antineutrino hits liquid inside has been mapping tiny fluctuations in the radia- for Astrophysics and Space Science at the Uni- a giant tank. New data from that experiment tion left over from the Big Bang. The pattern of versity of California, San Diego, in La Jolla, could be pointing to the existence of four kinds of neutrinos, raising the chances that fluctuations holds clues to the stew of particles would find this lighter mass agreeable, because sterile neutrinos may be real. that existed shortly after the Big Bang. it would help him to solve a long-standing Results from the past seven years, which problem with supernovae. These massive star

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GUIDING LIGHT IN QUANTUM NETWORKS Tiny antennas could direct photons on a chip.

go.nature.com/ruNCbv CN BOON/ALAMY Hobbit origins pushed back When the remains of tiny hominins — nicknamed hobbits — were found on the isolated Indonesian island of Flores in 2003, it sparked an epic hunt to understand the origins of these diminutive cousins of modern humans. Now, discoveries of stone flakes used as primitive tools on the island suggest that the hobbit’s ancestors were there a million years ago, at least 120,000 years earlier than previously thought (A. Brumm et al. Nature doi:10.1038/nature08844; 2010). “Whatever species made it to the island 1 million years ago, it was probably an ancestor of Homo floresiensis,” says William Jungers, an Astronomers have found hints of sterile neutrinos in the faint microwave radiation that ﬁlls the Universe and anthropologist at Stony Brook University in is left over from the Big Bang. The Wilkinson Microwave Anisotropy Probe satellite tracks the temperature variations (shown above) and polarization of this radiation. New York. The metre-high H. floresiensis lived on the island until at least 17,000 years ago, explosions cause neutrons to fuse together, at Los Alamos in which Louis took part. From and its small stature probably evolved in creating elements heavier than iron that end 1993 to 1998, he and his colleagues used the response to the island’s sparse resources. up in other stars and in planets. But they Liquid Scintillator Neutrino Detector experi- The simple stone tools demonstrate the also produce inordinate numbers of neutri- ment to shoot antineutrinos — neutrinos’ skills of its ancestors — people who must nos, which should, in theory, inhibit fusion antimatter counterparts — into 167 tonnes of have hopscotched across islands from because they kill off neutrons by changing mineral oil. Their data on how antineutrinos mainland Asia, traversing deep and swift them into protons and electrons. switch from one species to another suggested ocean channels, before arriving on Flores. Sterile neutrinos could provide a way out. If four distinct flavours. A decade later, data from In 2005, Adam Brumm, an archaeologist some of the neutrinos created in supernovae a follow-up test also at Fermilab — the Mini at the University of Wollongong in morphed into sterile ones, they Booster Neutrino Experiment, Australia, found the first of about 45 stone would fly off without interacting “The question of or MiniBooNE — found no tools while exploring a bowl-shaped gully with neutrons, leaving many of sterile neutrinos is evidence of a fourth neutrino on the island that was like “a hot, steamy them intact. “We’re very inter- absolutely crucial type. But the MiniBooNE had wok”. Three years later, researchers at ested in this, because the stakes used a beam of neutrinos, mak- Roskilde University in Denmark analysed are so high,” says Fuller. for nuclear particle ing it difficult to compare its the ratio of two isotopes of argon trapped physics and results with the antineutrino in volcanic ash overlaying the tools to Mixed messages astrophysics.” data from Los Alamos. Now, determine their age. Sobering news for the sterile- after collecting data for a year Previous tool discoveries showed that neutrino hunters came this month3 from the and a half with antineutrinos, the MiniBooNE hominins had arrived on Flores by 880,000 Main Injector Neutrino Oscillation Search experiment sees a different pattern (see ‘Neu- years ago, suggesting that the hobbit’s (MINOS), an experiment that shoots a beam trino hunters’). When combined with other ancestors might have wiped out some of of neutrinos from an accelerator at the Fermi antineutrino experiments worldwide, says the island’s peculiar indigenous animals, National Accelerator Laboratory (Fermilab) Louis, its data fit “beautifully” to a 3 + 1 model: such as the pygmy elephant-like Stegodon in Batavia, Illinois, underground to a detec- three ordinary neutrinos plus a sterile one4. sondaari and giant tortoises (Geochelone tor 735 kilometres away in Minnesota to Louis, who is also a part of the MiniBooNE spp.), which both disappeared at around the observe neutrino oscillations. The MINOS collaboration, says that the next batch of data, same time. data show that one family of neutrinos does to be released this summer, should give a The new finds imply that the hobbit’s not have a high propensity for turning into better idea of whether this fleeting interloper ancestors coexisted with the creatures sterile ones, although spokesman Robert has finally been caught. ■ for much longer, raising the possibility Plunkett says that the results do leave room Eric Hand that a natural disaster was behind the for sterile neutrinos. “We’re limiting the 1. Komatsu, E. et al. Preprint at http://arxiv.org/ disappearance of the animals. parameter space for them.” abs/1001.4538v1 (2010). The team will return to Flores this Another Fermilab experiment, built to fol- 2. Lowenstein, M. & Kusenko, A. Preprint at http://arxiv. summer, hoping to find older sediments org/abs/0912.0552 (2009). low up on the earliest clues to sterile neutrinos, 3. Adamson, P. et al. Phys. Rev. D 81, 052004 (2010). that could hold earlier evidence of the potentially offers more encouragement. The 4. Karagiorgi, G., Djurcic, Z., Conrad, J. M., Shaevitz, M. H. & island’s first hominins. ■ original evidence came from an experiment Sorel, M. Phys. Rev. D 80, 073001 (2009). Rex Dalton

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HEAD CASE Last year, functional magnetic resonance imaging made its debut in court. Virginia Hughes asks whether the technique is ready to weigh in on the fate of murderers.

rian Dugan, dressed in an orange and rode back to DuPage county jail, about Dugan’s lawyers asked Kiehl to testify and jumpsuit and shackles, shuffled to the 50 kilometres west of Chicago. offered him the chance to scan the brain of a door of Northwestern Memorial Hospi- Kiehl has been amassing data on men such notorious criminal. Kiehl agreed and Dugan’s Btal in downtown Chicago, accompanied as Dugan for 16 years. Their crimes are often case became what is thought to be the first in by four sheriff deputies. It was the first time impulsive, violent, committed in cold blood the world to admit fMRI as evidence. Kiehl’s SOVEREIGN/ISM/SPL that Dugan, 52, had been anywhere near a city and recalled without the slightest twinge of decision has put him at odds with many in his in 20 years. Serving two life sentences for a pair remorse. They are psychopaths, and they are profession, and stirred debate among neuro- of murders he had committed in the 1980s, estimated to make up as much as 1% of the scientists and lawyers. he was now facing the prospect of the death adult male population, and 25% of male pris- “It is a dangerous distortion of science that penalty for an earlier killing. oners. To date, Kiehl has used fMRI to scan sets dangerous precedents for the field,” says Dugan was here on a Saturday this past more than 1,000 inmates, Helen Mayberg, a neurolo- September to meet one of the few people who many from a mobile scanner “It is a dangerous gist at Emory University might help him to avoid that fate: Kent Kiehl, a set up in the courtyard of a distortion of science School of Medicine in neuroscientist at the University of New Mexico New Mexico prison. He says Atlanta, Georgia. Mayberg, in Albuquerque. Dugan, Kiehl and the rest of that the brains of psycho- that sets dangerous who uses brain imaging to the entourage walked the length of the hospi- paths tend to show distinct precedents for the field.” study depression, has testi- tal, crossed a walkway to another building, and defects in the paralimbic fied against the use of sev- took the lift down to a basement-level facility system, a network of brain regions important eral kinds of brain scan in dozens of cases where researchers would scan Dugan’s brain for memory and regulating emotion. since 1992. Although other brain-imaging using functional magnetic resonance imag- techniques have been used in court, it is espe- ing (fMRI). Todd Parrish, the imaging centre’s Mitigating circumstances cially hard to argue that fMRI should be, argue director, offered plastic zip ties to replace the The purpose of the work, Kiehl says, is to critics. The technique reveals changes in blood shackles — no metal is allowed in the same eliminate the stigma against psychopaths flow within the brain, thought to correlate room as the scanner’s powerful magnet — but and find them treatments so they can stop with brain activity, and it has become popular the guards said they weren’t necessary. Dugan committing crimes. But Dugan’s lawyers saw in research. But most fMRI studies are small, entered the machine without restraints, another purpose. During sentencing for capi- unreplicated and compare differences in the and Parrish locked the door — as much to tal crimes, the defence may present just about average brain activity of groups, rather than keep the guards and their weapons out as anything as a mitigating factor, from accounts individuals, making it difficult to interpret to keep Dugan in. of the defendant being abused as a child to for single cases. It is rarely used in diagno- Dugan lay still inside the scanner for evidence of extreme emotional disturbance. sis. Moreover, a recent scan, say some critics, about 90 minutes, performing a series of Kiehl’s research could offer a persuasive argu- wouldn’t necessarily indicate Dugan’s mental cognitive control, attention and moral decision- ment that Dugan is a psychopath and could state when he committed his crimes. making tests. Afterwards he ate a hamburger, not control his killer impulses. After read- In 1983, Dugan kidnapped 10-year-old Jean- sat through an extensive psychiatric interview ing about Kiehl’s work in The New Yorker, ine Nicarico, of Naperville, Illinois. He raped

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her in the back seat of his car and beat her to your brain, there is no way you could have understand, basically all I’m telling them is that death. In 1984, he saw a 27-year-old nurse manufactured it for the purpose of the trial.” this is not someone who’s operating with a full waiting at a stop light on a deserted road. He Brain imaging played into the 1982 trial of deck. And so, they may not be eligible for the rammed into her car, raped her and drowned John Hinckley Jr, who had attempted to assas- harshest punishment possible.” Gur gets so her in a quarry. A year later, he plucked a sinate US President Ronald Reagan. Lawyers many requests to testify that he has a team of 7-year-old girl from her bicycle, raped her, presented a computed tomography X-ray scan psychology residents and interns to vet them. killed her and left her body in a drainage ditch, of his head, arguing that it showed slight brain Still, he doesn’t think that fMRI is reliable weighed down with rocks. shrinkage and abnormally large ventricles, enough for legal settings. “If somebody asked indicating a mental defect. The prosecution’s me to debunk an fMRI testimony, it wouldn’t Plea bargaining expert witnesses said the scans looked normal. be too hard,” Gur says. Police charged Dugan with the third murder Whether imaging influenced the verdict is not That’s mainly because fMRI studies deal three weeks after it happened and listed him known, but Hinckley was found not guilty by in average differences between groups. For as a suspect in the nurse’s death. Through his reason of insanity. example, Kiehl’s work has shown that when lawyers, Dugan offered to confess to all three Over the next decade, lawyers gradually processing abstract words, psychopathic killings, but only if prosecutors took the death switched to positron emission tomography prisoners have lower activity in some brain penalty off the table. Authorities, deeming (PET), which can be used to give a measure of regions than non-psychopathic prisoners and some of Dugan’s statements on the Nicarico metabolic activity in the brain. Gur’s research non-prisoners. But there’s bound to be over- murder to be unreliable, wouldn’t negotiate. team has scanned dozens of patients with mental lap. He has not shown, for example, that any One reason for their reluctance was that two illness and hundreds of healthy volunteers using one person showing a specific brain signature men had already been sent to death row for PET and structural MRI — a technique that looks is guaranteed, with some per cent certainty, to killing Nicarico. at the static structure of the brain and is more be a psychopath or behave like one. But more than ten years later the two established for diagnosis than fMRI. Through his For Kiehl, the scan is just part of the picture convicted men were finally exonerated. With research, he has developed algorithms that can and he conducts extensive interviews to deter- constituents demanding justice for Nicarico, predict whether a person has schizophrenia, for mine a diagnosis of psychopathy. “It’s just one local authorities used DNA evidence to link example, from structural MRI alone with about bit of information that helps us understand Dugan to the crime in 2002. In July last year, 80% accuracy1. Gur has testified in roughly 30 brain function,” he says. he formally pleaded guilty. It was a high-profile criminal cases on behalf of defendants alleged to case for the area, and local media accounts have schizophrenic or brain-damage. Taking the stand depict a community haunted by the girl’s death. “We determine whether the values are On 29 October, Kiehl participated in a ‘Frye The defence lawyers knew that they would have normal or abnormal,” Gur says. “It’s a chal- hearing’ for Dugan’s case. Based on a 1923 a tough time arguing for leniency. They were lenge to explain that to a jury, but when they ruling, the hearing determines whether sci- willing to try anything, including the latest entific evidence is robust enough to be admit- that neuroscience could offer. ted. Joseph Birkett, the lead prosecutor in the Brain imaging has a long history in legal Dugan case, argued that allowing the scans — cases. Lawyers have often used scans as a way the bright colours and statistical parameters of

677–684 (2001) 677–684 to tip the scale in the perpetual battle between which are chosen by the researchers — might 50, opposing expert psychiatric witnesses. You can’t bias the jury. Some studies, prosecutors argued, control your brain waves, the theory goes, and have shown that neuroscientific explanations scans are an objective measure of mental state. can be particularly seductive to the layperson2. GENEVE POLICE DEPT/WBBM TV GENEVE POLICE DEPT/WBBM “The psychiatric diagnosis is still soft data — The judge ultimately “cut the baby in half”, it’s behaviour,” notes Ruben Gur, director says Birkett. He ruled that the jury would not ET AL. BIOL. PSYCHIAT.

of the Brain Behavior Center at the University be allowed to see Dugan’s actual brain scans, AP PHOTO/ of Pennsylvania in Philadelphia. “The brain but that Kiehl could describe them and how he

K. A. KIEHL scan doesn’t lie. If there is tissue missing from interpreted them based on his research. On 5 November, Kiehl took the stand for about six hours. He described the findings of two, three-hour psychiatric interviews with Dugan. Dugan had scored 38 out of a possible 40 on the Hare Psychopathy Checklist, which evaluates 20 aspects of personality and behaviour through a semi-structured interview. (It was developed by Kiehl’s graduate-school mentor, Robert Hare.) That puts him in the 99.5th percentile of all inmates, Kiehl says. Using PowerPoint slides of bar graphs and cartoon brains — but not the scans — Kiehl testified that Dugan’s brain, like those of psychopaths in his other studies3, showed Above: criminal psychopaths show less activity decreased levels of activity in specific areas. than non-criminal control subjects in specific Prosecutors, Kiehl says, went to great lengths emotion-processing areas of the brain, according to Kent Kiehl’s testing. Right: Brian Dugan in 1985. to sow confusion about the data.

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At one point during Kiehl’s testimony, he showed a cartoon brain with Xs marked on various regions, to illustrate where Dugan’s activity was aberrant. The prosecutor, Kiehl recalls, “asked me on cross examination, ‘Are there really Xs in this guy’s brain?’ It’s the adversarial system — they were just out to make it look like nothing made sense.” The next day, the prosecution brought a rebuttal witness: Jonathan Brodie, a psychia- trist at New York University. He refuted the imaging evidence on several grounds. First, there was timing: Kiehl scanned Dugan 26 years after he killed Nicarico. It was impossible to know what was going on in Dugan’s brain while he was committing the act, and it was perhaps not surprising that his brain would look like a murderer’s after committing murder. Kent Kiehl outside the mobile scanner he has used to look at the brains of inmates at a New Mexico prison. Second, Brodie said, there was the issue with average versus individual differences. If you says Steve Greenberg, Dugan’s lawyer. The “Kiehl got a lot of criticism, but I think what look at professional basketball players, most of switched verdict will definitely be brought he did is perfectly reasonable,” says Grafton. them are tall, he told the jury, but not everyone up in the appeal to the state supreme court, “I’ve had judges tell me, ‘Look, everything else over six foot six is a basketball player. says Greenberg, as will the fact that the jurors everybody’s bringing into a mitigation hearing From a technical perspective, Kiehl’s work weren’t allowed to see Dugan’s brain scans. is extremely unreliable, so why should we hold is expertly done, says Brodie. “I have no issue Since November, Kiehl says he has been scans to a different standard?’” with his science. I have an issue with what he contacted by about a dozen lawyers asking for Whatever standards fMRI scans should be did with it. I think it was just a terrible leap.” similar services. In February, he and Parrish held to, neuroscientists and the legal system Even if fMRI could reliably diagnose scanned another man on trial using the same are under pressure to work them out fast. psycho pathy, it wouldn’t necessarily reduce a fMRI scanner at Northwestern Memorial Hos- Although a fledgling technology, fMRI has defendant’s culpability in the eyes pital. For Kiehl the motivations for been knocking at the doors of the courts for a of a judge or jury. Ultimately, the “Kiehl got a lot scanning Dugan were twofold. “It while. Since at least 2007, two US companies, law is based on an individual’s gave me an opportunity to study Cephos in Tyngsboro, Massachusetts, and No rational, intentional action, not of criticism, but one of the classic psychopaths in Lie MRI in San Diego, California, have been brain anatomy or blood flow, says I think what he American history,” he says. That selling ‘deception detection’ services based Stephen Morse, professor of law did is perfectly included privileged access to on fMRI scans. The technology has almost and psychiatry at the University Dugan and his massive case file. certainly been used in plea bargains (like its of Pennsylvania. “Brains don’t kill reasonable.” Kiehl also says he feels a “moral older cousin, the polygraph), and is expected people. People kill people,” says obligation” to help educate the to debut in court within a few years. Use of Morse, who also co-directs the MacArthur jury, and the public, about psychopathy, a fMRI for diagnosing mental disorders, as was Foundation’s Law and Neuroscience Project, disorder that is often maligned in the pop- the case for Dugan, will improve and more which brings together scientists, lawyers and ular press. “You can live in the ivory tower scans will probably show up in court, says Gur. judges to debate how brain technology should for your whole career, and never really have “That’s only a question of time.” be used in legal settings. a practical influence. This was a chance to What matters, though, is how the jury help,” says Kiehl. perceives the evidence and the testimony — and Change of heart Many of his peers are unimpressed. The wit- in Dugan’s case, it’s not clear how much weight Dugan’s sentencing proceedings ended four ness stand, says Mayberg, is “not a soapbox”. it carried. For Michael Euringer, a juror in the days after Brodie’s testimony. The jury delib- “What you might believe to drive your own trial and a retired stockbroker, it didn’t mean erated for less than an hour before coming research is very different from being a spokes- much at all. “I don’t think that they were able to back with a verdict: ten for the death penalty person for science,” she says, adding that people present that idea that he had a brain defect. He and two for life in prison — a death sentence who take the stand should not use the platform was a psychopath, but he was not psychotic,” he requires a unanimous vote. as a way to meet their own scientific agenda. says. “But it all depends on the jury.” ■ But while waiting for the Nicarico family Others are less critical. When DuPage Virginia Hughes is a freelance writer in New to return to the courtroom, one of the jurors county prosecutors discovered that Dugan’s York City. asked for more time and the judge agreed. The defence team would be using brain imaging, 1. Davatzikos, C. et al. Arch. Gen. Psychiatry 62, 1218–1227 jury asked for copies of several transcripts of they contacted Scott Grafton, director of the (2005). testimony, including Kiehl’s, and went back Brain Imaging Center at the University of 2. Weisberg, D. S., Keil, F. C., Goodstein, J., Rawson, E. & Gray, into deliberation. The next day, all 12 jurors California, Santa Barbara. Grafton has testi- J. R. J. Cogn. Neurosci. 20, 470–477 (2008). voted to send Dugan to his death. fied against imaging in many cases, but this 3. Kiehl, K. A. et al. Psychiatry Res. 130, 297–312 (2004). Even with the unfavourable final verdict, time he declined, saying that even potentially See Editorial, page 325; News Features, pages 344 Kiehl’s testimony “turned it from a slam dunk shaky scientific evidence should be allowed and 347; Opinion, page 351; and online at for the prosecution into a much tougher case”, during the sentencing phase of a trial. www.nature.com/scienceincourt.

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THE FINE PRINT A single incriminating fingerprint can land someone in jail. But, Laura Spinney finds, there is little empirical basis for such decisions.

he terrorist explosions that ripped back several decades, that is now being used to programme to classify fingerprints according through Madrid’s crowded commuter challenge fingerprint evidence in US courts. to their visual complexity — including incom- trains on the morning of 11 March Those challenges, in turn, are being fed by plete and unclear prints — and to determine T2004 killed 191 people, wounded some a growing unease among fingerprint examin- how likely examiners are to make errors in KIENZLE/AP T. 2,000 more and prompted an international ers and researchers alike. They are beginning each class. “The vast majority of fingerprints manhunt for the perpetrators. Soon after, to recognize that the century-old fingerprint- are not a problem,” says Itiel Dror, a cognitive Spanish investigators searching the area near identification process rests on assumptions psychologist at University College London one of the blasts discovered an abandoned set that have never been tested empirically, and who is involved in the study. “But even if only of detonator caps inside a plastic bag that bore that it does little to safeguard against uncon- 1% are, that’s thousands of potential errors a single, incomplete fingerprint. They imme- scious biases of the examiners. each year.” diately shared the clue with law-enforcement That unease culminated last year in a sting- colleagues around the world. And on 6 May ing report by the US National Academy of Leaving a mark 2004, the US Federal Bureau of Investigation Sciences (NAS)2, which acknowledged that Even fingerprinting’s harshest critics concede (FBI) arrested Oregon lawyer Brandon May- fingerprints contain valuable information — that the technique is probably more accu- field, proclaiming that his print was a match. but found that long-standing claims of zero rate than identification methods based on Two and a half weeks later, a chagrined FBI error rates were “not scientifically plausible”. hair, blood type, ear prints or anything else was forced to release Mayfield after Spanish Since then, fingerprint examiners have found except DNA. Granted, no one has ever tested police arrested an Algerian national — one themselves in an uncomfortable situation. its underlying premise, which is that every of several terrorists later charged with the “How do you explain to the court that what print on every finger is unique. But no one crime — and found one of his fingerprints to you’ve been saying for 100 years was exagger- seriously doubts it, either. The ridges and be a much better match. The FBI eventually ated, but you still have something meaningful furrows on any given fingertip develop in the admitted that it had made multiple errors in to say?” asks Simon Cole, a science historian at womb, shaped by such a complex combina- its fingerprint analysis1. the University of California, Irvine. tion of genetic and environmental factors that The Mayfield case is a textbook example The only way out of the dilemma is data, not even identical twins share prints. Barring of ‘false positive’ fingerprint identification, says Cole: do the research that will put fin- damage, moreover, the pattern is fixed for life. in which an innocent person is singled out gerprinting on solid ground. And that is what And thanks to the skin’s natural oiliness, it will erroneously. But the case is hardly unique. researchers are starting to do. In January, leave an impression on almost any surface the Psychologist Erin Morris, who works with the for example, the US Department of Justice’s fingertip touches. Los Angeles County Public Defender’s Office, research branch, the National Institute of Jus- The concerns start with what happens after a has compiled a list of 25 false positives, going tice, launched the first large-scale research fingerprint, or ‘mark’, is found at a crime scene

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and sent to the examiners. The problem lies not AFIS technology can scan though the vast officer Shirley McKie, leading her to be falsely so much with the individual examiners, most of fingerprint databases compiled by the FBI and accused of perjury. Such errors may not come whom have undergone several years of special- other agencies and automatically filter out all to light until some other, incontrovertible piece ist training, but more with the ACE-V identifi- but a handful of candidate matches to present of evidence trumps the fingerprint, or until the cation procedure they follow in most countries to the examiner. The examiner will then win- prints are reanalysed in an internal review. But (see graphic). The acronym stands for the four now the candidates down by eye. for examiners and researchers alike, the urgent sequential steps of analysis, comparison, evalu- According to the ACE-V protocol, the question is why they happen at all. ation and verification — the hyphen signifying third step, evaluation, can lead One of the problems with the that the last step is carried out by a different the examiner to one of three con- “The system ACE-V procedure lies in sloppy individual, who repeats the first three. clusions: ‘identification’, mean- execution. For example, the pro- The analysis phase starts at the gross level, ing that mark and exemplar came as it’s designed tocol calls for the analysis and where there are three main patterns — loops, from the same finger; ‘exclusion’, purposely comparison steps to be separated, whorls and arches — that can be used to clas- meaning that they did not, as produces false with a detailed description of the sify prints or to rapidly exclude suspects. Then there is at least one significant dif- mark being made before an exam- comes a second level of analysis, which focuses ference that cannot be explained negatives.” iner ever sees an exemplar. This is on finer details, such as bifurcations and ridge by factors such as smearing; and to prevent circular reasoning, in endings (see graphic), which are highly dis- ‘inconclusive’, meaning that the mark is not which the presence of the exemplar inspires criminating between individuals. If necessary, clear enough for the examiner to be sure. the ‘discovery’ of previously unnoticed fea- the examiner can bore down to a third level of “The system as it’s designed purposely pro- tures in the mark. But this separation doesn’t detail, related to the shape of ridge edges and duces false negatives,” says legal scholar Jen- always happen, says forensic-science consult- the pattern of pores. nifer Mnookin of the University of California, ant Lyn Haber, who together with her hus- Having analysed a mark and noted its dis- Los Angeles. Because the protocol makes it band, psychologist Ralph Haber, co-authored tinctive features, the examiner then goes to possible to have one difference and exclude a the 2009 book Challenges to Fingerprints. To the comparison step: checking for similarities match, but a lot of similarities and still not be save time, she says, many examiners do the or differences with a reference fingerprint, or sure, it builds in a preference for missing the analysis and comparison simultaneously. The ‘exemplar’, retrieved from law-enforcement identification of a criminal rather than risking FBI highlighted this as a factor contributing to files or taken from a suspect. This part of the the conviction of an innocent person. the Mayfield error. process has become increasingly automated, Yet, as the Mayfield case illustrates, false first with the development of automatic fin- positives can slip through the net. In Scot- Misprints gerprint identification systems (AFIS) in the land, for example, an ongoing inquiry is try- Another problem is that the ACE-V protocol 1980s, then with the advent of digital print- ing to understand how a fingerprint found at a itself is sloppy, at least by academic standards. capture technology in the 1990s. Today’s murder scene was wrongly attributed to police For example, it calls for the final verification step to be independent of the initial analysis, FINGERPRINT PATTERNS AND ACE-V but does not lay down strict guidelines for ACE-V protocol what that means. So in practice, the verifier Most fingerprints fall into one of three groups: whorls, loops or arches. But the ridges can contain a multitude of small-scale variations, from 1. Analysis often works in the same department as the bifurcations to hooks, bridges to islands. The precise arrangement of The examiner analyses first examiner and knows whose work he or such features — typically 150 per print — uniquely identifies the print. the features in a print left she is checking — not a form of ‘independ- at a crime scene. ence’ with which many scientists would be Basic patterns Ridge characteristics comfortable. Ridge ending Nor is ACE-V especially strict about what examiners can and cannot know about the Bifurcation case on which they are working. This is espe- 3 Dot cially worrying in light of a study published in 2. Comparison 2006 in which Dror and his colleagues showed The print is then Island (short ridge) compared with those that both experienced and novice fingerprint Whorl held on file. examiners can be swayed by contextual infor- Lake (enclosure) mation. In one experiment, the researchers

Hook (spur) examiner) different (by presented six examiners with marks that, unbeknown to them, they had analysed before. Bridge This time, the examiners were furnished with certain details about the case — that the sus- Double bifurcation

Loop 4. Veriﬁcation pect had confessed to the crime, for example, Trifurcation 3. Evaluation or that the suspect was in police custody at the Does the print come from time the crime was committed. In 17% of their the same ﬁnger? Opposed bifurcation examinations, they changed their decision in Yes the direction suggested by the information. Ridge crossing No This point is emphasized by the conclusion Opposed bifurcation/ in last year’s NAS report that “ACE-V does Unsure Arch ridge ending ? not guard against bias; is too broad to ensure repeatability and transparency; and does not

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guarantee that two analysts following it will in 40% of a given population or in 0.4%? Some obtain the same results.” research has been done on this issue, but not For many critics this is the central issue: fin- on a sufficiently large or systematic scale. Nev- gerprint analysis is fundamentally subjective. ertheless, Champod is optimistic that a prob-

D. RYAN/AP PHOTO RYAN/AP D. Examiners often have to work with incom- abilistic system is within reach. Unlike with plete or distorted prints — where a finger DNA, he says, which has strong subpopulation slid across a surface, for example — and they effects, fingerprint patterns vary little between have to select the relevant features from what populations, simplifying the task. is available. What is judged relevant therefore A probabilistic approach would not changes from case to case and examiner to replace the examiner or address bias, but it examiner. would render the decision-making process Several research groups are now looking less opaque. “Once certainty is quantified, it at this problem, with a view to understand- becomes transparent,” says Champod. Ulti- ing and improving the way that experts make mately, however, it is for the courts to decide a judgement. The FBI has an ongoing study how much weight they accord to fingerprint looking at the quantity and quality of informa- evidence. The fact that courts still routinely tion needed to make a correct decision. Dror’s treat it as infallible — which means a single group is doing a controlled study of the errors incriminating fingerprint can still send some- made by examiners in which they are given one to jail — strikes Mnookin as “distressing marks, told they have been taken from a crime Brandon Mayfield was falsely accused of terrorism jurisprudence”. Champod, too, would like to scene — they were actually made by Dror — on the basis of a fingerprint found at the scene. see its importance downgraded. “Fingerprint and asked to identify them. evidence should be expressed by fingerprint the decisions that are most susceptible to bias examiners only as corroborative evidence,” Expert testimony are those in which the mark is unclear or hard he says. If other strands of evidence limit the Other critics have wondered whether any to interpret, Kershaw introduced blind arbitra- pool of suspects, then a fingerprint is much examiner truly qualifies as an expert. As the tion in cases in which examiners disagree. less likely to be misattributed. Habers point out in their book, examiners Safeguards against bias are relatively easy to To date, judges haven’t shown much incli- rarely find out whether their decision was cor- put in place, but another potential source of nation to alter the status quo. But to be fair, rect, because the truth about a crime is often error might be harder to eliminate. It has to do says Barry Scheck, co-director of the Inno- not known. As a result, they write, “even years with how faithfully the pattern on a finger is cence Project — a group in New York that of experience may not improve [an examin- reproduced when it is inked or scanned to cre- campaigns to overturn wrongful convictions er’s] accuracy”. ate an exemplar. No reproduction is perfect, — they haven’t been given a viable alternative. Some fingerprint examiners have simply notes Christophe Champod, an The probabilistic approach is not rejected these criticisms. In 2007, for example, expert in forensic identification “This is all about yet ready for court. But that may the chairman of Britain’s Fingerprint Society, at the University of Lausanne in be about to change if researchers Martin Leadbetter, wrote in the society’s mag- Switzerland. So a mark recovered adding a culture can come up with ways to help azine4 that examiners who allow themselves to from a crime scene could match of science to the fingerprinting profession re- be swayed by outside information are either exemplars from more than one forensic-science establish itself on a more scien- incompetent or so immature they “should seek person, or vice versa, he says. tific footing. employment at Disneyland”. Exacerbating the problem is the community.” A cultural change will also be But others have taken the criticisms to heart. continued growth in the exem- needed, within both the finger- After hearing about Dror’s research on bias, plar databases that AFIS have to search. print community and the legal system. “This Kevin Kershaw, head of forensic identification Champod thinks that the language of cer- is all about adding a culture of science to the services at Greater Manchester Police, one of tainty that examiners are forced to use hides forensic-science community,” says Harry Britain’s largest police forces, decided to buffer this uncertainty from the court. He proposes Edwards, a senior judge on the District of his examiners from potentially biasing infor- that fingerprint evidence be interpreted in Columbia circuit and co-chair of the NAS mation by preventing investigating officers probabilistic terms — bringing it in line with committee that produced last year’s report. from coming on-site to wait for results, and other forensic domains — and that examiners “From what I have seen, we still have a long potentially talking to the examiners about the should be free to talk about probable or pos- way to go.” ■ case. This is made easier by the fact that in sible matches. In a criminal case, this would Laura Spinney is a freelance writer based in Manchester, as in many British police forces, mean that an examiner could testify that there Lausanne, Switzerland. the forensic division is separated from the was, say, a 95% chance of a match if the defend- others. In the United States, by contrast, most ant left the mark, but a one in a billion chance 1. A Review of the FBI’s Handling of the Brandon Mayfield Case (Office of the Inspector General Oversight and Review of the fingerprint work is done inside police of a match if someone else left it. Division, 2006). departments — a situation that the NAS report To be able to quote such odds, however, 2. Strengthening Forensic Science in the United States: A Path recommended be changed. examiners would need to refer to surveys Forward (National Academies, 2009). 3. Dror, I. E. & Charlton, D. J. Forensic Identification 56, Kershaw also invited Dror to come and teach showing how fingerprint patterns vary across 600–616 (2006). his examiners about the dangers of bias, and populations and how often various compo- 4. Leadbetter, M. Fingerprint Whorld 33, 231 (2007). he changed his service so that the verifier no nents or combinations of components crop up. longer knows whose work he or she is check- For example, is a particular configuration of See Editorial, page 325; Opinion, page 351; and ing. Finally, as Dror’s research indicated that bifurcations, ridge endings and the like found online at www.nature.com/scienceincourt.

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DNA’S IDENTITY CRISIS It may be the gold standard of forensic science, but questions are now being raised about DNA identification from ever-smaller human traces. Natasha Gilbert asks how low can you go?

hen Peter Hoe was found Moreover, the methods and procedures meaning that by measuring several STRs — stabbed to death in his home employed are shrouded in secrecy — leading between 10 and 17 — forensic scientists can in North Yorkshire, UK, in the some forensics researchers to demand that the declare with calculable probability whether Wafternoon of 13 October 2006, interpretation of such profiles, if not the prac- DNA left at a crime scene belongs to a suspect. investigators were able to connect the murder tice itself, be re-evaluated. M. SHIELDS/ALAMY to brothers Terence and David Reed on the Low-copy-number analysis is accepted in A soupçon of cells basis of a small amount of DNA lifted from just a handful of countries including Britain Scientists employ the polymerase chain reaction shards of plastic found near the body. The men and New Zealand, but it is being applied in (PCR) to amplify the STRs to detectable lev- were convicted the next year. many cases. The FSS says that it has used the els. The copies are then separated according But an appeal to the ruling heard in 2009 technique in more than 21,000 serious criminal to size by electrophoresis. The different-sized raised questions about the reliability and inter- cases since its development in the late 1990s. STRs show up as a pattern of peaks on an elec- pretation of DNA profiles drawn from very Recent appeals affirming the validity of low- tropherogram that can be compared against a small amounts of genetic material, a tech- copy-number typing suggest that law enforce- database or an individual. nique known as low-copy-number analysis. ment may increasingly embrace the technique. Standard DNA analysis requires about 200 In the appeal, the Reeds’ lawyers argued that Some suggest that its wider acceptance could picograms of DNA, 33 cells’ worth, or twice that Valerie Tomlinson, an officer involved in the threaten DNA profiling’s 20-year reputation as for haploid sperm cells. Technicians can gener- analysis at the Forensic Science Service (FSS) the gold standard of forensic science. Contrary ally get more than enough DNA from visible based in Birmingham, UK, had overstepped to the public’s perception, “DNA evidence is samples such as blood or semen. But to produce her bounds by speculating how the men’s DNA not flawless”, says Peter Neufeld, co-director profiles from just a few cells, scientists have came to be on the pieces of plastic — thought of the Innocence Project, an advo- developed ways of increasing the to have broken off two knife handles. The cacy group in New York that has "DNA evidence sensitivity of the analysis, includ- appeal failed last December, but a larger ques- campaigned for broader access to ing running more PCR cycles tion looms about how suspects can be fingered DNA evidence to overturn wrong- is not flawless." to copy more DNA or purifying from such a small amount of DNA. ful convictions. the sample after PCR to remove The case is one of the most recent public British geneticist Alec Jeffreys developed unused reagents. In many cases the amount of airings of a highly charged debate in the sci- DNA profiling in the 1980s . Profiles are drawn starting material for low-copy-number analy- ence and law-enforcement communities about from short, repeating sequences of DNA scat- sis is unclear. The quantitation methods, also low-copy-number analysis. Some argue that tered throughout the genome, called short based on PCR, can suggest that no DNA is profiles are not reproducible, are prone to tandem repeats (STRs). Because the number present, but the technicians will still be able to contamination and lack a scientifically vali- of repeats varies widely from person to person, produce at least a partial profile. This sensitivity, dated means for deciding on their accuracy. the length of these STRs is also highly variable, although remarkable, has a downside.

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During analysis of any sample, large or presiding over the case expressed “concern small, random fluctuations occur that distort about the present state of the validation of the the results. STRs present in the original sam- science and methodology”, and its use was sus- ple might not show up — a ‘drop-out’ effect. In pended in British courts. But a UK review of the addition, profiles can show STRs that are not technique published in 2008 concluded that the present in samples. This ‘drop-in’ effect can be method is “robust” and “fit for purpose”, and its caused by contamination. use was reinstated2. In March 2009, a judge in These stochastic effects are easily spotted California ruled that, due to a lack of scientific and ruled out from standard analysis because acceptance of the procedures and statistics there is enough DNA to run multiple controls, used to interpret low-copy-number results, and generally the strong signals from true STRs such evidence was inadmissible. More recently, can be distinguished from the faint peaks of however, a judge in New York’s supreme court drop-ins. But when pushing the signal as far as denied a motion to exclude low-copy-number IMAGES ASSOCIATION ARCHIVE/PRESS BEHAL/PA J. it will go, even faint, artefact peaks are ampli- DNA evidence in a murder trial, ruling that the fied, making them look more acceptable. technique is reliable. Bruce Budowle, a forensic and investigative The courtroom is not the best place for the geneticist at the University of North Texas in science of low-copy-number analysis to be Fort Worth and a former scientist at the Fed- debated, says Allan Jamieson, director of the eral Bureau of Investigation (FBI), gave evi- Forensic Institute in Glasgow, which provides dence criticizing low-copy-number analysis forensic-science services to UK police forces. at the Reeds’ appeal trial. He argues that the Sean Hoey was cleared of all charges relating to Jamieson, who gave evidence in the Reed methods for determining whether a signal the Omagh bombing in Northern Ireland. brothers’ and the Omagh bombing trials, says is true in low-copy-number analysis haven’t that the forensics community must validate been validated. The FSS did not respond to several interview procedures and further investigate the issues In low-copy-number profiling, forensic requests, but Peter Gill, a forensic scientist at that low-copy-number profiling has brought to scientists generally split their limited amount the University of Strathclyde in Glasgow, UK, light, before scientists and courtrooms can have of DNA into two or three samples and run who developed low-copy-number typing in a confidence in the results. “The public does not analyses on two of them. The third, if avail- former post at the FSS1, says that the quality of understand that just because your DNA is on able, is reserved for the defence. The results the science “is not in question”. Although drop- an object it does not mean you have touched it,” of analyses aren’t completely reproducible, in and drop-out effects are more pronounced he says. A point of contention in the Reeds’ case profiles often won’t match and the scientists than in standard analysis because was whether the Reeds had ever generally accept as true those STR signals that of the sensitivity of the technique, “None of the labs come in direct contact with the show up in both runs. The practice is worry- operating in clean conditions and plastic knife handles, or whether ing, says Budowle: “The logic of this approach monitoring negative controls disclose what they might have transferred DNA makes some sense, but the confidence in it has should avoid serious issues. they do. They say indirectly through someone else’s not been assessed.” The effects of drop-in and it is proprietary touch, or, say, by sneezing. drop-out can also be accommo- As Budowle says, “with [low- Inherent bias? dated statistically, reducing the information.” copy-number typing] you can’t Common forensics practices can also lead to reliance on human judgement, say what the tissue source is”. biasing errors, says Dan Krane, a molecular he says. But the probability theory that deals Scientists, he says, “overstep the line” when evolutionist at Wright State University in Day- with these stochastic effects is not yet in use interpreting the results of low-copy-number ton, Ohio. “Forensic scientists tell me that it is in forensic circles. He says that statisticians typing in court cases. easier for them to distinguish between noise have not had the funding to adapt the existing But Gill disagrees. “Scientists are there to and what is really coming from the DNA if theory to forensic use. “It is a disappointment explain what the evidence means,” he says. “It they have a reference sample to work with,” that tools to enable better interpretation have is their responsibility to explain all the pos- he says. Low copy number or not, that refer- not kept pace with developments in forensic sibilities of how the DNA got there and then ence sample is often the suspect’s DNA — and science over the past ten years,” he says. it is up to the court to decide.” The arguments faint peaks in a crime-scene sample might All these concerns have led some countries to are likely to continue. ■ seem more convincing when viewed side by approach the use of low-copy-number analysis Natasha Gilbert writes for Nature from side with a strong peak from the suspect. Only with caution. In 2001, the FBI recommended London. a couple of labs in the United States, he says, that it be used only in certain circumstances in 1. Gill, P., Whitaker, J., Flaxman, C., Brown, N. & Buckleton, J. require blind testing in their protocols. the United States, such as for identifying the Forensic Sci. Int. 112, 17–40 (2000). Critics’ fears are confounded by an unwill- bones of someone suspected of being a missing 2. Caddy, B., Taylor, G. R. & Linacre, A. M. T. A Review of ingness of the labs that use the technique to person. Its use has been controversial in Brit- the Science of Low Template DNA Analysis (Home Office Forensic Science Regulation Unit, 2008); available at reveal their guidelines for interpreting results. ain, too. Sean Hoey was accused of killing 29 http://police.homeoffice.gov.uk/publications/operational- Labs should be forced to disclose details, says people in an explosion that destroyed the town policing/Review_of_Low_Template_DNA_12835. Budowle. Given the technique’s reproducibility centre of Omagh in Northern Ireland in 1998. pdf?view=Binary problems, he argues it is imperative that these Central to the case against him was a low-copy- protocols are robust and reliable. But “none of number analysis of DNA left on bomb parts See Editorial, page 325; News Features, the labs disclose what they do. They say it is connected to the attack. pages 340 and 344; Opinion, page 351; and online proprietary information,” he says. Hoey was freed on appeal in 2007. The judge at www.nature.com/scienceincourt.

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offers the best hope of competing with China and the United States. Behind the scenes, they are pre- What can little Europe do? paring to look far beyond FP8 and set objectives that would transform Europe’s research land- Scientists must engage with the European Union’s redesign of its scape by 2030. This would include the tripling of research programmes to shore up the continent’s competitive position. the share of the EU budget devoted to research (from 4% to 12%) and the adoption of a more he research component of the European daring target for R&D to reach 5% of GDP. (Japan Union (EU) budget — known as the is already touching 4%.) The plan, which is being Tframework programme, now in its sev- prepared for submission at the highest levels, also enth iteration (FP7) — has experienced only sets ambitious objectives for other areas of public incremental change each time the union has policy related to science, from mobility and edu- set its budget. The outcome is a set of activities, cation to patenting and venture capital. under FP7, of limited strategic impact. The hopes of these leaders are raised by the This year will see researchers and policy- early success of the ERC in establishing its makers begin to shape FP8, and it is abundantly awards as the most prestigious in European clear that the steady-as-she-goes approach science, and the high priority being given to won’t do this time around. on 5 March. In a talk to the Lisbon Council, a research in the EU leadership’s public pro- In the coming months, researchers must European think tank, she pledged to “refocus nouncements. Research and innovation take press home the things they need most from research and innovation policy” on five ‘grand pride of place in the economic strategy docu- FP8: more effective ‘joint programming’ challenges’ — energy, climate change, food secu- ment, Europe 2020, released by EU president between member states, less onerous bureau- rity, health and ageing. This focus is supposed to José Barroso on 3 March, and there are plans cratic demands on project participants and excite the public and clarify research outcomes. for the heads of state to discuss research and expansion of the programme’s funding agency, Critics fear, however, that it will give politicians innovation at the Autumn European Council the European Research Council (ERC). more control over what researchers do under meeting in September. The EU has failed to meet, or even approach, the framework programme. At the same time, optimism is challenged its primary research-policy target, set out in Other priorities for FP8, which is expected to by niggling worries. Some doubt that Barroso 2002, to increase investment in research and run from 2014 until 2020, include support for will appoint a really prominent, independently development (R&D) to 3% of gross domestic infrastructure projects and for the expansion of minded chief scientific adviser, as he promised product (GDP). This measure of research inten- the ERC — currently the star in the framework to do last September. They also fear that the sity is still stuck where it was in 2002, at 1.9%. firmament. European Commission will struggle to find a This is unsurprising, perhaps, as it is dominated Another major thrust of FP8 will probably genuine star to accept the new director’s post by the R&D activities of industry, and so sits be the rapid expansion of ‘joint programming’. at the ERC. outside the direct control of politicians. This typically opaque phrase describes the Frustration continues to simmer over the But the extent to which this is a political alignment of research activities in the 27 EU paperwork and strenuous auditing require- failure is underlined by the performance of member states — which between them account ments of the framework programme. Some China. There, R&D investment surged from for 95% of publicly funded research in Europe 7,000 researchers have now signed an online 0.7% of GDP in 1998 to 1.5% by 2008, accord- — with each other, and with the programmes petition (www.trust-researchers.eu) calling ing to the Organisation for Economic Co-oper- of the European Commission. Joint pro- for an auditing regime “based on trust and ation and Development in Paris. The growth gramming gets fiercely independent national responsible partnering”. Their protest articu- comes from strong public investment, backed agencies to work together. A prototype for such lates a serious push to reform the programme’s up by the actions of powerful and globally collaboration is a project on Alzheimer’s dis- financial rules. ambitious industrial companies. The United ease that involves, among others, INSERM in The petition is also a departure from States has now pledged to up its performance, France, the German federal research ministry researchers’ customary forbearance of mal- with President Barack Obama last April copy- and the UK Medical Research Council. But administered EU programmes. European ing Europe’s 3% target, without specifying a fragmentation remains the norm. science would benefit greatly if as many date when it should be reached. researchers as possible make waves about what The battle between the United States and The bigger picture they would like to see in the next EU budget. China for research pre-eminence — and the A key question to be addressed this year and Rival beneficiaries of it, such as farmers and industrial and military strength that is assumed next is how fast new ventures, such as joint business lobby groups, will certainly be doing to follow from it — puts Europe’s puny efforts programming and an expanded ERC, can be so. The ERC should be seen as a beachhead somewhat in the shade. Last month, for exam- built up. Another is whether these will grow onto fresh territory, where EU awards become ple, one prominent European was planning at the expense of the ‘traditional’ part of the hallmarks of excellence. The current economic to call his talk at the annual meeting of the framework programme — applied research crisis gives the scientific community a chance American Association for the Advancement projects with partners in at least three mem- — perhaps its last chance — to build on that, of Science in San Diego, California, ‘What can ber states. This currently consumes two-thirds and to help ensure that little Europe doesn’t little Europe do?’ (A commission official dis- of the programme’s €7-billion (US$10-billion) slide into irrelevance. ■ suaded him.) annual budget. Colin Macilwain is based in the United Europe’s new research commissioner, Ireland’s Research leaders in all member states now Kingdom. Máire Geoghegan-Quinn, took her first stab at agree that a European Research Area, across e-mail: [email protected] answering that rhetorical question in Brussels which scientists can move and collaborate freely, See go.nature.com/ILx8PC for more columns.

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Tsunami: unexpected services need more resources for e-mail to some 10,000 GDACS That is promising. We welcome community education. users within 30 minutes; the JRC feedback from the broad blow foils flawless Dale Dominey-Howes, James Goff accurately predicted the height research community. warning system Australian Tsunami Research Centre, of the tsunami near Concepcion. Olivier Küttel Euresearch, University of New South Wales, The risk for the Pacific islands was Effingerstrasse 19, 3008 Bern, The trans-Pacific tsunami High Street, Kensington 2052, considered low because of the Switzerland generated by the magnitude-8.8 New South Wales, Australia wave’s conditions at the source, e-mail: [email protected] Chile earthquake on 27 February e-mail: [email protected] with a sea-level rise of less than a Sabine Herlitschka Austrian Research exposed a problem with metre expected in Hawaii. Promotion Agency, Austria Australia’s tsunami early- The Portuguese Institute of warning system. Tsunami: time for Meteorology is already testing Since the 2004 Indian the reliability of forecasts with S moking out the big Ocean tsunami, the Australian models to be tested an early-warning system that government has spent Aus$70 in warning centres will provide a framework for the tobacco-users — and million (US$64 million) on the IOC’s future northeast Atlantic they’re not in China development and deployment of You highlight, in a News story, the regional centre. the Australian Tsunami Warning controversy over the reliance of It is time for forecasting models The photograph of an Asian System (ATWS). This is jointly tsunami early-warning systems to be tested systematically in person puffing on a cigarette operated by the Australian Bureau on model-based forecasts (Nature operational conditions by the and the map of the top cigarette- of Meteorology and Geoscience 464, 14–15; 2010). Important tsunami-warning centres. These consuming countries, both shown Australia. advances have been made in will complement the arrays of tidal in J. M. Samet and H. L. Wipfli’s On 27–28 February, the near-real-time model-based and deep-sea gauges by filling Opinion article (Nature 463, system worked flawlessly in tsunami forecasting since 2004, information gaps where gauges 1020–1021; 2010), make China detecting the earthquake and by scientists such as those at the are scarce, to minimize false look like a smokers’ paradise. issuing first watch messages, US Pacific Marine Environmental alarms. The challenge is to identify Its large population may then warning messages for Laboratory, the German Research how to formalize the integration make China the top consumer of Australia via numerous channels. Centre for Geosciences and the of model-based forecasts into the cigarettes in the world. But, as is The smooth operation of the European Commission’s scientific process of issuing warnings. shown more clearly in the online physical hardware of the ATWS and technical reference arm, the Delilah H. A. Al Khudhairy, Alessandro version of the Tobacco Atlas map is a credit to all the government Joint Research Centre (JRC). Annunziato Global Security and Crisis (http://go.nature.com/jQNEWc), scientists who have been working These advances make the Management Unit, Institute for the the populations of many other tirelessly since December 2004. models important contributors Protection and Security of the Citizen, countries, especially in Europe, Unfortunately, the final element to tsunami-warning systems. Joint Research Centre, Ispra, Italy smoke more per person — the of the ATWS — the community’s The Intergovernmental e-mail: delilah.al-khudhairy@ heaviest smokers being in Greece response — failed. Oceanographic Commission ec.europa.eu and Ukraine. The Chilean tsunami was (IOC) of the United Nations Jie-Yu Chuang Department of forecast to arrive at Sydney at Educational, Social and Cultural Psychiatry, Tri-Service General about 8.30 in the morning on Organization can help to guide Researchers’ petition Hospital, National Defense Medical 28 February. Despite widespread the optimal use of such forecasts Center, Taipei, Taiwan television and radio broadcasts to improve the effectiveness of aims to simplify e-mail: [email protected] of the tsunami warning, live existing early-warning systems. European funding television coverage showed The JRC has been developing hundreds of people standing on a global tsunami-modelling Your call for simplification of Waterfall is the beaches waiting for the tsunami system, which is integrated European research funding to arrive. Worse, some of them into the joint United Nations/ (Nature 463, 999; 2010) is clue to a case of were deliberately swimming into European Commission Global timely. In February we started mistaken identity the incoming tsunami, despite Disaster Alert and Coordination a pan-European petition for the efforts made by volunteers System (GDACS; www.gdacs. simplification of the funding A photograph (Nature 463, 1007; from Surf Life Saving Australia to org). It is based on a global rules, addressed to the European 2010) captioned to imply that it prevent this. pre-event calculated tsunami Parliament and the Council of shows the coastline of Monterey These people may not have scenario grid and near-real- Ministers. So far, more than 7,300 Bay actually shows the cove at the received the warning messages, time simulated forecasts. This researchers have signed up at mouth of McWay Canyon, about or they may not have understood combined approach gives an www.trust-researchers.eu. 50 km south of Monterey Bay. The the risk, or they may have almost instantaneous estimate The petition aims to give a voice give-away is the ribbon waterfall deliberately ignored the advice of wave height and arrival times, to researchers all over Europe who plunging over the sea cliff from the issued. as well as enabling a warning to want to change the way research mouth of the canyon. What became clear is that we be issued within 20 minutes of an is funded (see page 349). William R. Dickinson Department of need a better understanding of earthquake-inducing tsunami. Right now, many stakeholder Geosciences, University of Arizona, the social dimension of public The system worked effectively discussions are taking place Box 210077, Tucson, Arizona 85718, response to warning messages after the Chilean earthquake, with inside and outside the European USA in Australia, and the emergency alerts transmitted via SMS and Commission to simplify the rules. e-mail: [email protected]

335050 Correspondence.inddCorrespondence.indd 350350 115/3/105/3/10 119:08:489:08:48 Vol 464|18 March 2010 OPINION Making forensic science more scientific The US Congress should create an office to study, standardize and certify those who apply science to crime as well as the techniques they use, urge Peter Neufeld and Barry Scheck.

groundbreaking report by the National (OFSIS) within the US Department of Com- hundreds of millions of dollars in public funds Academy of Sciences (NAS) last year merce to spur independent research, develop are used for forensic science — funds that Arecognized that much of forensic sci- standards and ensure compliance. The direc- would be deployed more wisely and efficiently ence is not rooted in solid science. Many tor of an OFSIS would be appointed by the US through an OFSIS. Additional research and forensic disciplines — such as hair micro- Secretary of Commerce in consultation with the development of forensic assays, devices scopy, bite-mark comparisons, fingerprint the NAS and the National Science Founda- and techniques could also spark job growth analysis, firearm testing and tool-mark analy- tion. The director would work with a foren- and increase revenue in the public, private sis — were developed solely to solve crimes. sic-science commission (including physicists, and academic sectors. An OFSIS would be They evolved mainly in the context of indi- geneticists, criminologists, cognition experts, future-focused rather than directed at past vidual cases, which often had significant vari- chemists and others) and an advisory com- cases, and the US court system is well situated ation in resources and expertise. They have mittee (including experts such as judges, pros- to handle any implications this might have for not been subjected to rigorous experimental ecutors, defence attorneys, law-enforcement existing convictions. scrutiny, and there are no standards or over- officials and forensic scientists). sight in the United States or elsewhere to An OFSIS would develop a research agenda Battling bias ensure that validated, reliable forensic meth- for basic and applied forensic sciences, and The OFSIS proposal also responds to a central ods are used consistently. With the exception would direct a grant programme to encourage concern of the NAS report committee: the of DNA analysis, no forensic method has research partnerships between the private sec- potential for real or perceived bias if this new been proved to reliably and accurately dem- tor and academia. It would also devise stand- function is controlled by law enforcement onstrate a connection between evidence and ards and certification for forensic devices, rather than being a mainly scientific agency. a specific source. assays, techniques, training, practitioners and We anticipate that legislation respond- We see first-hand the consequences of this providers. These would include requirements ing to the NAS report’s recommendations problem. Innocent people are misidentified for written reports and testimony param- will be introduced in the next few months. as suspects and, in some cases, convicted of eters. The Department of Scientists are crucial to crimes they did not commit, while the guilty go Justice would encourage building support for the free. Indeed, unvalidated or improperly used forensic practitioners and “Political and criminal-justice OFSIS proposal. Those forensic science contributed to approximately law-enforcement agencies ends — rather than research who belong to associa- half of the 251 convictions in the United States to comply with standards imperatives — have taken tions should advocate for that have been overturned after DNA testing developed by an OFSIS. organizational position since 1989. In many cases, the real perpetrator Even with a grow- forensic science off course.” statements that support went on to commit further violent crimes. ing consensus in favour our OFSIS proposal. To The NAS made a powerful and persuasive of creating an OFSIS, pockets of resistance learn more and get involved as individuals or case for the gravity of the situation and outlined remain. A small minority of people within groups see www.just-science.org. a road map for meaningful reform. Its central the forensic-science and law-enforcement Political and criminal-justice ends — rather recommendation is for the US Congress to cre- fields defend the status quo because they are than research imperatives — have taken foren- ate a federal entity to ensure consistency, valid- invested in the system as it stands. They do sic science off course. We can bring about sub- ity and reliability in the forensic sciences. not speak for their entire field, but they will stantial improvement only if we trust scientists argue that an OFSIS is not necessary and that to take charge of forensic science, free of poli- Campaign trail laboratory accreditation is sufficient, even tics and independent of outside influence. The To develop legislation based on the report’s though accreditation alone would not resolve US Congress will have the opportunity to take recommendations, we have over the past year many of the underlying problems identified strong action this year. It must: putting foren- met with members of Congress and their staff, in the NAS report. They argue that an OFSIS sic science on a stronger scientific footing in testified at several congressional hearings and would cost too much (particularly as the US the United States will help to solve crimes, worked with key stakeholders in the scien- budget deficit grows) and that it could create promote fair adjudications and strengthen the tific and criminal-justice fields. Members of chaos in the US justice system by reopening integrity of judicial proceedings on our shores the judiciary and science committees in both countless old cases. and beyond. ■ chambers of Congress, plus law-enforcement These concerns do not stand up to scrutiny. Peter Neufeld and Barry Scheck are co-founders officials, a wide range of scientists (including Setting up an OFSIS within the commerce and co-directors of the Innocence Project, statisticians, forensic experts and analysts and department would keep the operation small, which is affiliated with Cardozo School of Law, academic researchers) and others agree that streamlined and efficient, and connect it with New York, New York 10011, USA. the report provides an important opportunity similar functions already undertaken within e-mail: [email protected] to improve our system of justice. the agency (such as the development of per- See Editorial, page 325; News Features, pages We advocate the creation of an office of formance standards for certain technology 340, 344 and 347; and online at www.nature.com/ forensic science improvement and support used by law-enforcement agencies). Already, scienceincourt.

335151 OpinionOpinion - InnocenceInnocence MH.inddMH.indd 351351 112/3/102/3/10 116:29:556:29:55 Vol 464|18 March 2010 BOOKS & ARTS Tales from the climate-change crossroads Four books by prominent global-warming pundits illustrate that exhortation and authority are not enough to solve the climate crisis — it is time for some humility, concludes Roger Pielke Jr.

The public debate about climate change has been human behavior and thinking”. Again, he does 2004 through his public endorsement of Senator transformed in recent months. Last autumn, not say how a new global consciousness is to John Kerry over George W. Bush in a speech many were optimistic about the December be delivered. Instead, he revisits the themes made in the swing state of Iowa. Schneider climate conference in Copenhagen. Climate of his previous book An Inconvenient Truth relates how, at an IPCC meeting, he may have science was seen as the ultimate authority in a (Bloomsbury, 2006), complete with attacks on aided the viability of the Kyoto Protocol after discussion framed as nailing down details on a the policies of former US president George W. explaining climate science to an African national global agreement to support emissions reduc- Bush and nefarious climate deniers. delegate who later changed his position. tion. Today, few observers view the Copenhagen Hansen and Schneider each provide a trou- conference as a step forward. Climate scientists bling inside view of the political battle over cli- Policy pressure are also under fire for uncaught errors and slop- mate change in their respective books, Storms Schneider’s recounting of the IPCC process piness in the latest report of the Intergovern- reveals how few people are able to influence mental Panel on Climate Change (IPCC), and Our Choice: A Plan to Solve the its reports. He recalls holding “private negotia- for questionable behaviour revealed in e-mail Climate Crisis tions” over a bottle of wine with a co-chair to disclosures. by Al Gore settle how to present sea-level rise in his work- In the weeks before Copenhagen, climate Rodale Books: 2009. 416 pp. $26.99 ing group’s report. His comment that IPCC pundits rushed to publish their visions of how Storms of My Grandchildren: The Truth chairman Rajendra Pachauri was keen to the negotiations would and should play out. About the Coming Climate Catastrophe stress “the importance of the Working Group II How did their arguments fare? Al Gore’s rose- and Our Last Chance to Save Humanity report in identifying linkages between climate tinted vision failed to square with Copenhagen’s by James Hansen change and sustainable development around outcome, leaving us with little guidance going Bloomsbury USA: 2009. 320 pp. the world” raises questions as to whether simi- forward. Two first-hand accounts by distin- $25, £18.99 lar pressures might have affected quality control guished climate scientists who advocate action, in that report — particularly on such topics as James Hansen and Stephen Schneider, left me Science as a Contact Sport: Inside the Battle the Himalayan glaciers or the economic costs feeling that their convictions have pushed to Save Earth’s Climate of natural disasters and African agriculture, for them towards simplistic, almost authoritarian by Stephen H. Schneider which the IPCC has recently been criticized. visions of political decision-making. However, National Geographic Society: 2009. Schneider and Hansen relate how they have the technical deliberations of less well-known 304 pp. $28 had considerable access to government deci- names, in a volume edited by Dieter Helm and sion-makers around the world, with whom The Economics and Politics of Cameron Hepburn, remain compelling. they have shared their policy recommenda- Climate Change In Our Choice, Gore speculates on how a tions. Given such opportunities, it is difficult Edited by Dieter Helm and future generation might look back at our deci- to understand Hansen’s frustration that the Cameron Hepburn sions. Writing before Copenhagen, he hedges governments of the United Kingdom, Ger- Oxford University Press: 2009. 400 pp. $55 his bets on the agreement that he believed would many and Japan failed to alter their climate be delivered, calling it historic but insufficient. policies in response to letters that he wrote to He hoped that the meeting would be viewed as of My Grandchildren and Science as a Contact their leaders and meetings he attended with the start of a global shift in attitudes, bolstered by Sport. Hansen invokes religious terms, charac- cabinet ministers. US legislation that was passed on the eve of the terizing himself and Schneider as witness and Hansen’s complaint that leaders of sovereign conference. Clearly, Gore’s assessment was far preacher, respectively. Both are evangelists who countries have not acceded to his demands from the diplomatic meltdown that occurred. hold science as an ascendant authority. implies a criticism of democracy, also present Observers are still trying to make sense of what The tension between the role of scientists as in Schneider’s book. If science leads inexorably happened and what should come next. political advocates and as expert advisers is an to particular political outcomes, then it would Missing from Gore’s account, despite its sub- undercurrent in both books. Schneider explains seem to favour autocratic forms of governance. title, is any “plan to solve the climate crisis”. He that “as scientists, we never recommend which The middle man — the general public — is eas- provides a colourful overview of low-carbon policies should be chosen”; Hansen similarly ily ignored if heads of state need only hear the technologies — wind, solar, nuclear — and sees himself as an “objective scientist”. Yet both expert voice of science. Schneider worries that discusses aspects of the Earth system, such as books largely comprise strong ideological and democracy finds it hard to deal with complex forests and population, that are related to the political commentary based on an unstated issues: if only the public understood the real climate problem. He does not quantify how assumption that science compels action on cli- risks, he explains, they would be “much more those technologies and practices might come mate change. Neither author accepts the label likely to send strong signals to their representa- together to decarbonize the global economy, of advocate, claiming to be speaking for science; tives”. He bemoans a public debate that includes through what policy mechanisms and on nor do they see the paradox in their position. the participation of “special interests” and that what timescale. It is jarring to learn that Gore Both scientists express a desire to influence is filtered through an inept media, a perspective thinks that the solution to the climate crisis will political outcomes. Hansen describes how he echoed by Hansen. come about only through “massive changes in hoped to sway the US presidential election in In The Economics and Politics of Climate

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policy-makers and the public as an important global problem. At the same time, that approach has shown its limitations. Climate science has become deeply politicized and climate politics is in gridlock. Climate change is at risk of becoming an issue of cultural politics, similar to the evolution debate in the United States and elsewhere. If the climate-policy debate is to continue as it has, we should expect more of the same. An alternative way forward would start by admitting the limitations of science in compelling political agreements, and by admitting that we do not know how to complete the challenge of decarbonizing the global economy. There may be greater prospects for political consensus if scientists acknowledge their humility rather than asserting their authority. Incre- mental approaches to climate mitigation that can be modified by experience offer a chance that realistic and democratically grounded actions might rise to a challenge that will be A solar-power plant in Wuhan is part of China’s plans to produce 10 billion watts of solar energy by 2020. with us for decades to come. Each of these contrasting books offers a Change, Helm, an economist at the University with which to embark on the challenge of valuable perspective on how we got to where of Oxford, UK, agrees that the policy outlook responding to climate change, we are nowhere we are, but each gives only limited insight as to for stabilizing atmospheric concentrations of close to a complete solution. where climate policy may be going. The future, carbon dioxide and other greenhouse gases Together, these four books highlight that as Gore says, remains our choice. ■ is “very grim” owing to the myriad complexi- climate policy is at a crossroads. The journey Roger Pielke Jr is professor of environmental ties and contradictions of policies for reducing so far has emphasized science and exhortation: studies at the University of Colorado, Boulder, emissions globally. He argues that far more than that facts, spoken loudly enough, are enough Colorado 80309, USA. His forthcoming book is

CHINA DAILY/REUTERS/CORBIS political will is required to make progress on to win the argument. That path has succeeded The Climate Fix. this issue, the magnitude of which has been in bringing climate change to the attention of e-mail: [email protected] underestimated. A “fundamental rethink” is needed, he says, with a greater focus on international and long-term technological solutions. Other authors who contribute to this impressive volume highlight the challenges A misguided attack on evolution of securing a global agreement. Economic growth, suggest Australian economist Ross What Darwin Got Wrong evolutionary biology has become complacent Garnaut and his colleagues, has entered not by Jerry A. Fodor and Massimo Piattelli- with the nearly 70-year-old Modern Synthesis, just a golden but a “platinum age” that, beyond Palmarini which reconciled the original theory of natu- the current recession, is likely to continue for Farrar, Strauss & Giroux/Profile: 2010. ral selection with Mendelian and population decades. If so, then the IPCC assessments have 288 pp/258 pp. $26/£20 genetics; and that the field needs to extend the underestimated the challenge of emissions conceptual arsenal of evolutionary theory. But reduction, its costs and the mitigation poten- in claiming that there are fundamental flaws tial of existing technologies. Chapters on India On the heels of last year’s anniversary in an edifice that has withstood a century and China reinforce this perspective. celebrations of Charles Darwin’s On the Ori- and a half of critical examination, Fodor and Helm and his co-editor Hepburn, also an gin of Species and C. P. Snow’s ‘Two Cultures’ Piattelli-Palmarini err horribly. economist at the University of Oxford, admit essay, an interdisciplinary view of evolutionary The authors’ argument against “Darwinism” that their book “is not intended to provide theory by philosopher Jerry Fodor and cog- boils down to a two-pronged attack. First, they simple answers”. It presents a range of options nitive scientist Massimo Piattelli-Palmarini claim that biologists’ emphasis on ecological, or that might each contribute to decarboniza- might be anticipated with interest. Unfortu- exogenous, factors is misplaced because endog- tion of the global economy. These include nately, What Darwin Got Wrong fails to bridge enous genetic and developmental constraints cap-and-trade regimes, carbon taxes, behav- these two cultures. play a crucial part in generating organic forms. ioural change, nuclear energy, carbon capture By misusing philosophical distinctions Second, they argue that natural selection can- and storage, renewable-energy technologies, and misinterpreting the literature on natural not be an evolutionary mechanism because sequestration in forests, improving energy effi- selection, Fodor and Piattelli-Palmarini make evolution is a historical process, and history is ciency and geoengineering. Each method has a mess of what could have been an important “just one damned thing after another” with no its merits, flaws and opposition. The bottom contribution. The authors are correct in two overarching logic. line is that although we have plenty of options of their assessments. Namely that: mainstream The first claim represents a distortion of the

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literature. The relative importance of natural selection and internal constraints has always A macromolecular history been contended by biologists: molecular and developmental biologists tend to focus on Giant Molecules: From Nylon to Nanotubes is symptomatic of a general weakness in the internal mechanisms; ecologists and evolu- by Walter Gratzer book when it comes to the physical science of tionary biologists prefer to address external Oxford University Press: 2009. 144 pp. macromolecules. ones. But even Darwin accepted the impor- $24.95, £11.99 Recent developments in the physics and tance of both: in Origin, his ‘laws of variation’ chemistry of macromolecules get short shrift. acknowledge that variation is constrained, and The book’s discussion of ways of measuring his ‘correlation of growth’ implies that organis- The study of large molecules follows two the size of polymer molecules, for example, mal traits are interdependent. strands that have alternately diverged and is many years out of date, and the influential Fodor and Piattelli-Palmarini misappropriate intertwined over the subject’s history. The first work of those such as Nobel laureate Pierre- the famous critique of adaptationism (the idea strand explores the natural macromolecules of Gilles de Gennes, who brought theoretical that natural selection is sufficient to explain biology, including proteins, polysaccharides physics concepts to molecular science, is not every complex biological trait) that Stephen Jay and nucleic acids. The second is concerned mentioned. New methods of polymer chem- Gould and Richard Lewontin presented in their with synthetic macromolecules, the inven- istry, such as living polymerization, ring- ‘spandrels’ paper of 1979. Gould and Lewontin tion of which in the early twentieth century opening metathesis and solid-phase peptide warned about the dangers of invoking natural launched industries based on plastics such synthesis, all of which allow unparalleled con- selection without considering alternatives. But as nylon, polyethylene and Perspex. In Giant trol of the size and architecture of synthetic Fodor and Piattelli-Palmarini grossly overstate Molecules, biophysicist Walter Gratzer weaves macromolecules, are not mentioned, despite that case, concluding that natural selection has together both stories. yielding Nobel prizes. little or no role in the generation of biological Initially the two strands developed together, In recent years, the two strands of macro- complexity, contrary to much evidence. sharing experimental methods and theoretical molecular science have converged again. In their second line of attack, Fodor and Techniques such as laser tweezers and single- Piattelli-Palmarini maintain that biological molecule force spectroscopy have allowed us phenomena are a matter of historical con- to study the behaviour of biological macromol- tingency. They argue that generalizations are ecules as individual physical objects. Aspects impossible because of the interplay of too many of protein behaviour, such as their mechanical local conditions, such as ecology, genetics and unfolding and the structures they form when chance. In their narrow view of what counts as they misfold, re-emphasize the analogies science, only law-like processes allow for the between biological and synthetic macromol- testability of scientific hypotheses. Thus, they ecules. The increasing ability of chemists to A. CAVANAGH/WELLCOME IMAGES A. CAVANAGH/WELLCOME claim, an explanation of adaptations that is control the architecture of synthetic polymers based on natural selection is defensible in only has made new applications possible, especially two cases — if there is intelligent design, or if in nanotechnology. The new forms of carbon there are laws of biology analogous to those — fullerenes, nanotubes and graphene — earn of physics, both of which they reject. Here the their place in the book. authors ignore the entire field of evolutionary Tiny capsules made of synthetic polymers can be Gratzer covers the promise of polymer ecology, countless examples of convergent evo- used to deliver drugs for slow release in the body. nanotechnology in brief. Some applications — lution of similar structures in different lineages such as glues inspired by shellfish; drug-deliv- that show the historical predictability of evolu- approaches. The simpler chemistry of the ery devices based on self-assembled polymer tionary processes, and the literature on experi- synthetic materials offered tractable analogues vesicles; and scaffolds for tissue engineering — mental evolution, in which similar conditions of the natural systems. Then, as the inherent are directly inspired by biology. Others, such consistently yield similar outcomes. There is complexity of biological molecules became as the plastic electronics made possible by clearly a logic to evolution. central to their understanding, the fields split. semi-conducting polymers, use properties of Evolutionary biology is a mix of chance and The flowering of structural biology in the late macromolecules that have not been exploited necessity, as French biologist Jacques Monod 1950s and 1960s was driven by the technique by nature. famously put it, in which endogenous and of X-ray diffraction and owed little to polymer Arguably, DNA is the most important exogenous factors are in constant interplay. science. macromolecule, and I share the author’s par- It is a fertile area for rigorous philosophical Gratzer describes the history engagingly, ticular fascination with its potential uses, for analysis. Fodor and Piattelli-Palmarini offer and includes many anecdotes. He explains example as the basis of synthetic molecular only sterile and wrongheaded criticism. how German chemist Hermann Staudinger’s motors, for information processing and to make Fortunately, other philosophers of science and concept of polymers as giant molecules intricate self-assembled nano-objects. Only theoretical biologists are coming together to became accepted amid controversy, rancour time will tell whether such beautiful laboratory clarify and build on the conceptual foundations and the ugly academic politics of German uni- demonstrations will yield practical technologies of science and explore issues of its practice; this versities in the period up to the Second World that have the impact in the twenty-first century is a better way to bridge the two cultures. ■ War. Not all of the anecdotes he chooses are that plastics had in the twentieth. ■ Massimo Pigliucci is a professor of philosophy reliable: he includes, for example, the widely Richard Jones is professor of physics at the at the City University of New York–Lehman held but incorrect notion that the windows University of Sheffield, Sheffield S3 7RH, UK, and College, Bronx, New York 10468, USA. in medieval cathedrals are thicker at their author of Soft Machines: Nanotechnology and Life. e-mail: [email protected] base because the glass has flowed. This lapse e-mail: [email protected]

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Q&A: Hervé This on flavour and perception French chemist Hervé This is a pioneer of the field of molecular gastronomy, the science of cooking. From perfecting the boiled egg to making custards with meat proteins, he has advised top chefs worldwide. He tells Nature why he is moving on to ‘note-by-note’ cuisine using compounds to build taste and smells, and why turkey is best cooked in the dishwasher.

Why do some meals taste better create intense sensations such as strawberry than others? or coffee water. The home cook could in The principles of cuisine are love, art and principle do the same. technique: perfect technique without love and companionship is lost. A poor Another professional technique is to cook sandwich shared with good friends will taste food for long periods at low temperatures MAÏTEC MAÏTEC/S. D. better than dinner in a three-star Michelin in a vacuum-sealed bag. How might a home restaurant shared by an arguing couple. chef emulate this 'sous-vide’ method? Primates generally avoid bitter tastes such Use the dishwasher! For the next holiday as beer. But the sensory punishment of meal, I recommend that you prepare two drinking bitter beer is more than offset by the turkeys. Cook one in the dishwasher, in a social reward of being in a group. plastic bag, for several cycles of your machine. In this way, you can get low temperatures. Does every ingredient have a unique taste? Butterfly the other turkey and cook it on There is no such thing as unique taste. It is a the grill, creating the maximum expanse of phantasm. The taste of chicken, for example, delicious crispy skin. Then serve the moist, does not exist as a pure concept. Chicken has flavourful meat from the dishwasher turkey many different tastes, depending on the type with the grilled skin. A good accompaniment of chicken and on the way you cook it. them to chefs doing note-by-note cooking, would be foie gras, also cooked in the the farmer could make a profit while dishwasher at low temperature. Should dishes be put together from supporting sustainable agriculture and scratch, molecule by molecule? working within the traditional AOC system. What have you found by studying the Meat, fish, fruits and vegetables are cooking process of meat? organized mixtures of compounds. Cooking The taste of your famous Chantilly Our experiments have clearly shown that traditionally means mixing mixtures, and chocolate mousse is more intense than most meat cannot absorb liquids, be they is not precise. This is why I proposed the the ingredients alone. Why is this? acidic or basic, hot or cold. This finding concept of note-by-note cooking — using The importance of microstructure on flavour has shocked many. But we have recently specific compounds to build consistency, is an area of active and exciting research. In discovered that certain kinds of meat — those taste and odour. It is difficult, but a huge an emulsion of oil in water, such as a dressing, with low amounts of collagenic tissue, such unexplored continent is ahead of us. large oil droplets enhance the perception of as beef fillet — can absorb liquids through oil and small droplets enhance the perception capillary action. This reconciles the results of Are foods from different regions the of odorant molecules that dissolve in the various past scientific studies. same chemically? water phase. The molecular mechanism is Geography can alter the flavour of a food still unclear, but the effect is well established. Are any foods poor candidates for produced with exactly the same methods. My Also, the more surface area you have, the molecular-gastronomy research? friend and colleague Jean-Baptiste Coulon, at more odorant molecules are released. The I try not to work on cheese, wine, bread the French National Institute for Agricultural same holds for foams. So, if your preparation or sausages — the microbiology is too Research in Clermont-Ferrand, has shown contains many small air bubbles, this complicated. Onions and carrots are better that the flavour of reblochon cheese differs increases the flavour of the chocolate. subjects, but progress is slow. After two PhD depending on the cow’s grazing location, and projects studying the creation of carrot stock, that molecular differences are responsible. How might chefs retain the odorant we are only beginning to understand the Here in France, the Appellation d’Origine molecules that are released in cooking molecular diffusion processes involved. ■ Contrôlée (AOC) system [which certifies a vapours? Interview by Michael White, associate editor at food’s geographical origin] is right and there The food industry already recaptures and Nature. is a measurable difference between locations reincorporates ‘essential oils’ that are lost — a real terroir. during cooking processes. As a result, jams Hervé This’s latest book is The Science of the Oven and orange juice, for example, are now (Columbia University Press, 2009). For Q&As with How would local food producers much better. After decades of my promoting molecular chefs Harold McGee and Ferran Adrià, see contribute to note-by-note cooking? it, chefs have recently taken up using go.nature.com/3cs5Yg and go.nature.com/FzrNmn. Take, for example, this bottle of powder rotary evaporators — a device available in Correction made from the phenolic acids from syrah chemistry-supply stores and usually used The brief review of William Langewiesche’s book grapes. A farmer growing grapes for wine for distillation — to extract these often- Fly By Wire (Nature 463, 882; 2010) incorrectly production alone will probably lose money. lost essences. Using a technology that was spelled the first name of Captain Sullenberger as But if you make these phenolics and sell never designed for cooking, chefs can “Chelsey”. It should have read “Chesley”.

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SPECTROSCOPY Expanding versatility Martin Moskovits Gold nanoparticles coated with a thin layer of an oxide allow molecules adsorbed on surfaces as diverse as those of platinum, yeast cells or citrus fruits to be characterized routinely in the laboratory.

Placing molecules on properly nanostructured silver and gold nanoparticles have been used substrates is a crucial part of the working broadly13 and even commercialized. principle of surface-enhanced Raman spec- But Li and colleagues’ insight of making the Silica or troscopy (SERS), a technique that is widely alumina shell silica coat so thin as to increase the plasmonic used to characterize the chemical composition influence of its golden core on the sample of a sample. This approach has been shown Gold core under investigation as much as possible, while to boost the intensity of the molecules’ vibra- avoiding direct contact of the sample with the tional Raman spectrum by factors exceeding Hydrogen core, is new and potentially powerful. And, 1 one million , and has allowed a single molec- layer as the authors have demonstrated, it is espe- ule to be detected under favourable circum- Platinum cially useful in revealing the Raman spectra of stances2,3. One drawback of SERS is that the molecules adsorbed onto metals through the Raman signal, obtained by scattering laser creation of a hot spot — a tiny volume in which light from the sample, is sufficiently strong the optical electromagnetic fields are especially Figure 1 | Spectroscopy with oxide–gold only for molecules residing on gold or silver 4 concentrated — at the junction between the (or hard-to-handle metals such as lithium or nanoparticles. Li and colleagues show that, if silica-coated nanoparticle and the adsorbate/ sodium) substrates that have roughened sur- gold nanoparticles coated with a thin layer of an substrate. oxide (silica or alumina) are arrayed on a sample faces or consist of arrangements of metallic such as a single-crystal platinum surface covered One feature that will have to be clarified in nanoscale features. On page 392 of this issue, by a layer of hydrogen, they act as an amplifier future work is the extent to which molecules 4 Li et al. describe an innovative approach to to the intensity of light scattered by the sample interacting nonspecifically with a substrate — as SERS, termed shell-isolated nanoparticle- when light is shone on it. Such an approach parathion molecules do with orange peel — enhanced Raman spectroscopy (SHINERS), allows the application of surface-enhanced migrate from the substrate to the silica-coated that expands the versatility of SERS. Raman spectroscopy — a form of spectroscopy gold nanoparticles. In this situation, it would The enhancement of the Raman signal that that is usually limited to probing the vibrational be the hot spot between neighbouring nano- distinguishes SERS from other spectroscopic frequencies of substances residing on gold or particles that produces the Raman signal, as techniques arises from collective electron silver substrates — to be expanded to many other opposed to that between the nanoparticles and substrates. excitations called surface plasmons that form the substrate. But such details will be elucidated at the nanostructured substrate’s surface when as SHINERS is put to use in the materials and light strikes the sample. Such a plasmonic res- their technique, they applied it to biological life sciences. ■ onance helps to concentrate the intensity of systems and food. They report seeing new Martin Moskovits is in the Department both the incident light and the Raman radia- vibrational bands in the Raman spectrum of of Chemistry and Biochemistry, tion scattered by the sample in features such yeast cells — presumably due to biomolecules University of California, Santa Barbara, as nanometre-sized clefts and crannies in the that reside in the cells’ surface. They also California 93106, USA. underlying substrate5–8. Since its first demon- detected the potent insecticide parathion by e-mail: [email protected] stration more than 30 years ago1, SERS has spreading the silica-coated nanoparticles on 1. Jeanmaire, D. L. & Van Duyne, R. P. J. Electroanal. Chem. 84, become an active field of research and, along the skin of a parathion-contaminated orange. 1–20 (1977). the way, has spawned the more general field of Essentially, what Li et al. have invented is 2. Nie, S. & Emery, S. R. Science 275, 1102–1106 (1997). plasmonics9,10 and metamaterials11. a ‘powder’ with which one can dust a sample 3. Kneipp, K. et al. Phys. Rev. Lett. 78, 1667–1670 (1997). Li and colleagues’ SHINERS technique4 to detect and identify the molecules at its sur- 4. Li, J. F. et al. Nature 464, 392–395 (2010). 5. Aravind, P. K., Nitzan, A. & Metiu, H. Surf. Sci. 110, 189–204 relies on coating nanoparticles with a very thin face. Researchers have previously attempted (1981). layer of an oxide, such as silica or alumina, and to extend the applicability of SERS to vari- 6. Aravind, P. K. & Metiu, H. J. Phys. Chem. 86, 5076–5084 then spreading these nanoparticles onto a sam- ous materials and substrate morphologies (1982). 7. Inoue, M. & Ohtaka, K. J. Phys. Soc. Jpn 52, 3853–3864 ple. For a layer of hydrogen on a single-crystal by using approaches similar to that used (1983). 4 platinum surface (Fig. 1), the authors were by the authors . The late SERS pioneer 8. Moskovits, M., Tay, L., Yang, J. & Haslett, T. Top. Appl. Phys. able to record intense SHINERS Raman spec- Michael Weaver recorded Raman spectra 82, 215–226 (2002). 9. Maier, S. A. et al. Adv. Mater. 13, 1501–1505 (2001). tra of the vibrational modes of the hydrogen from molecules adsorbed on transition metals 10. Ozbay, E. Science 311, 189–193 (2006). at this interface. Next, and to investigate the by depositing thin layers of those metals on 11. Pendry, J. B., Schurig, D. & Smith, D. R. Science 312, applicability of SHINERS to non-metallic sur- nanostructured gold or silver substrates, while 1780–1782 (2006). faces, they measured the Raman spectrum of a ensuring that the thickness of the layer would 12. Zou, S. Z. & Weaver, M. J. Anal. Chem. 70, 2387–2395 (1998). single-crystal silicon wafer covered with hydro- not damp out the gold’s strong plasmonic 13. Mulvaney, S. P., Musick, M. D., Keating, C. D. & Natan, M. J. gen. Finally, to demonstrate the versatility of resonance too much12. Likewise, glass-coated Langmuir 19, 4784–4790 (2003).

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DRUG DISCOVERY the formation of BRAF–CRAF complexes. Their models suggest that this is because the inhibitor induces a different conformational Inhibitors that activate change in BRAF, which specifically impairs BRAF binding to other RAF kinases. It there- Karen Cichowski and Pasi A. Jänne fore seems that PLX4720 mediates its effects Inhibitors of RAF enzymes can suppress or activate the same signalling by promoting the formation of CRAF–CRAF pathway. The details of how this happens provide a cautionary note for dimers (Fig. 1). Poulikakos and colleagues5 (page 427) also those targeting the pathway for anticancer drug discovery. investigated the biochemical effects of RAF inhibitors. They found that these agents acti- Over the past year, there has been consider- inhibitors in some settings (such as in cells that vate MEK and ERK not only in tumour cells able excitement following the positive thera- lack BRAF-activating mutations). that harbour a mutant version of RAS, but also peutic effects of the compound PLX4032 in Hatzivassiliou and colleagues’ studies6 in cells in which the RAS–RAF–MEK–ERK early clinical trials in patients who have meta- (page 431) are thematically similar, but their pathway is activated by other oncogenes, such static melanoma1. The compound is thought focus is on working out the molecular mecha- as HER2. Consistent with Hatzivassiliou and to selectively inhibit BRAFV600E, a mutant, nisms by which two selective, structurally colleagues’ report6, Poulikakos et al. suggest cancer-associated form of the kinase enzyme unrelated BRAF inhibitors — GDC-0879 and that PLX4720 functions by promoting CRAF– BRAF. The positive clinical effects of PLX4032 PLX4720 — activate the RAF–MEK–ERK CRAF formation (Fig. 1). In addition, they contrast with clinical responses to inhibitors of pathway in RAS-mutant cells. The authors’ make the intriguing observation that, at low MEK, another kinase directly downstream of findings suggest that GDC-0879 promotes concentrations, compounds that are thought to BRAF in the RAS–RAF–MEK–ERK signalling the formation of dimeric complexes of RAF- inhibit all RAFs (pan-RAF inhibitors) stimulate cascade. MEK inhibitors have been less effec- type kinases, specifically BRAF–CRAF, BRAF– the RAF–MEK–ERK pathway. Presumably this tive than PLX4032 in clinical trials2, despite ARAF and CRAF–CRAF complexes. The is because, at low concentrations, the inhibi- the fact that they — like the BRAF inhibitor authors also obtained a crystal structure of a tors bind to only a fraction of the available RAF — selectively killed melanoma cells that carry close analogue of GDC-0879 bound to CRAF. kinases, promoting the formation of complexes BRAFV600E mutations in preclinical studies3. This revealed that drug binding causes a con- of the drug-bound kinases with non-drug- But despite the antitumour effects of formational change in the kinase that promotes bound RAFs. Because only one kinase within PLX4032 on melanomas, about 25% of patients the formation of dimeric RAF complexes. a given complex is drug-bound (inhibited), treated with the drug unexpectedly developed Although PLX4720 also induces activa- such complexation induces the activation of hyperproliferative skin lesions (keratoacantho- tion of the RAF–MEK–ERK pathway, Hatzi- the non-drug-bound kinase, thus promoting mas) and, in some cases, squamous-cell car- vassiliou et al. found that it actually prevents MEK–ERK signalling. cinomas, which disappeared following drug withdrawal1. Adding to this paradox, although most cancer-causing (oncogenic) BRAF muta- a Wild-type cells, b Mutant RAF, c Mutant RAF, d Mutant RAS tions — such as BRAFV600E — enhance the no inhibitors no inhibitors RAF inhibitor kinase’s activity, some patients with melanoma RAS Mutant RAS harbour BRAF-inactivating mutations4. Three 5,6 7 studies, two in this issue and another in Cell , GTP Wild-type BRAF Kinase-dead begin to provide an explanation for these BRAF unexpected findings. CRAF All three studies5–7 started with the same intriguing observation: whereas selective BRAF oro or RAF inhibitors potently suppress RAF–MEK–ERK signalling in BRAF-mutant melanoma cells, BRAFV600E dimer the same inhibitors unexpectedly activate this pathway in a different type of tumour cell — MEK MEK MEK MEK those that carry an oncogenic mutation in the gene KRAS, but which possess a wild-type BRAF gene (Fig. 1). Heidorn et al.7 report that ERK ERK ERK ERK the selective BRAF inhibitor 885-A binds to wild-type BRAF in KRAS-mutant cells and Cell growthTumour growth Cell death, Keratoacanthomas? promotes the formation of BRAF–CRAF com- tumour shrinkage Squamous-cell carcinomas? plexes, in which CRAF is a RAF kinase related to BRAF in this signalling pathway. Formation Figure 1 | The effects of RAF inhibitors in mutant and wild-type cells. a, Activation of the RAS– of the complex activates CRAF, and conse- RAF–MEK–ERK signalling pathway promotes cell growth, but excessive activation is associated quently the MEK–ERK pathway. with cancer. RAS enzymes activate signalling when bound to the nucleotide GTP. b, BRAFV600E is a Heidorn et al. also demonstrate that a mutant, oncogenic version of a RAF kinase enzyme found in the pathway. In tumour cells that harbour ‘kinase-dead’ mutant of BRAF (which behaves BRAFV600E, RAS–GTP activation is low, and ERK signalling is predominantly activated by dimers of like an inhibited BRAF enzyme) similarly binds the mutant kinase. Phosphate groups (blue) are present on the activated kinase. c, In the presence of a V600E to CRAF and cooperates with a mutant form BRAF inhibitor that blocks phosphorylation (green), MEK–ERK signalling in BRAF mutant cells d of the RAS gene to promote MEK–ERK activa- is suppressed, causing cell death and tumour shrinkage. , In RAS mutant cells that contain wild-type BRAF, RAF inhibitors block phosphorylation of RAF monomers (BRAF or CRAF), which go on to tion, thus inducing melanomas in genetically form dimeric complexes5–7 such as BRAF–CRAF or CRAF–CRAF. Similarly, a ‘kinase-dead’ BRAF engineered mice (Fig. 1). The authors therefore that cannot phosphorylate (grey block) forms a complex with CRAF7. The formation of these RAF provide a unifying explanation for the onco- complexes causes excessive signalling, stimulated by the mutant RAS enzyme. This might explain the genic effect of kinase-dead BRAF proteins, formation of skin lesions (keratoacanthomas) and squamous-cell carcinomas observed in melanoma and for the pro-tumorigenic effects of BRAF patients given RAF inhibitors during clinical trials1.

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Although the precise mechanisms by which the therapeutically acceptable doses of these More fundamentally, these reports5–7 suggest different RAF inhibitors activate the RAF– compounds are limited by the side effects that, despite 25 years of intensive study, we MEK–ERK pathway may differ, all three stud- associated with ERK inhibition in normal tis- still do not fully appreciate the complexities ies5–7 demonstrate that inhibitors thought sue, such as intolerable skin rashes2. It is cur- of the RAS–RAF–MEK–ERK pathway. The to be selective for mutant BRAF can activate rently not known whether the greater success unexpected effects (and, in some cases, lack of CRAF through the formation of dimeric RAF of PLX4032 in clinical trials, compared with effects) of cancer therapies that target it under- complexes — a process that is enhanced by MEK inhibitors, is solely the result of a greater score the value of studies aimed at garnering the presence of an oncogenic RAS mutation. therapeutic window, or whether this success detailed mechanistic insight into both normal Consequently, these studies support the same also (or instead) stems from the selectivity of and pathogenic signalling mediated by this fundamental conclusion: mutant-specific PLX4032 for BRAFV600E. The answer to this pathway. Such insight might ultimately lead to BRAF inhibitors should be used for treating question is important for future drug develop- the development of effective new anticancer cancers caused by BRAF mutants (such as ment, as BRAF inhibitors that do not activate therapies. ■ BRAFV600E-associated melanomas), but should CRAF might ultimately cause side effects simi- Karen Cichowski is in the Division of Genetics, be avoided in cancers caused by RAS mutants lar to those caused by MEK inhibitors. Thus, Brigham and Women’s Hospital, Boston, (including other kinds of melanoma). This a compound that inhibits BRAFV600E alone, Massachusetts 02115, USA. Pasi A. Jänne is underscores the need to select patients who if it is possible to develop such an agent, in the Department of Medical Oncology, have BRAF-mutant cancers for current and might ultimately be the best drug for treating Dana-Farber Cancer Institute, Boston, future clinical studies of BRAF inhibitors. BRAFV600E-mutant melanomas. Massachusetts 02115, USA. These findings5–7 may also help to explain Finally, the observation5 that pan-RAF e-mails: [email protected]; the side effects of RAF inhibitors in patients inhibitors might also activate the RAS–RAF– [email protected] with melanoma, and they raise additional ther- MEK–ERK pathway in RAS-mutant cells at 1. Flaherty, K. et al. J. Clin. Oncol. (Meet. Abstr.) 27, 9000 apeutic considerations. Although it remains to low concentrations may suggest that only those (2009). be formally proved, it is tempting to speculate inhibitors that potently inhibit CRAF at clini- 2. Adjei, A. A. et al. J. Clin. Oncol. 26, 2139–2146 that the keratoacanthomas and squamous- cally achievable drug concentrations should (2008). cell carcinomas observed in clinical trials8 of be considered as treatments for RAS-mutant 3. Solit, D. B. et al. Nature 439, 358–362 (2006). 4. Ikenoue, T. et al. Cancer Res. 63, 8132–8137 (2003). RAF inhibitors are attributable to the activa- cancers. Alternatively, MEK inhibitors may 5. Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & tion of ERK signalling by one or more of the be a more appropriate choice of therapeutic Rosen, N. Nature 464, 427–430 (2010). proposed mechanisms5–7 (Fig. 1). Consistent agent. Regardless of the agent chosen, it will 6. Hatzivassiliou, G. et al. Nature 464, 431–435 (2010). with this possibility, Hatzivassiliou et al.6 report probably need to be used in combination with 7. Heidorn, S. J. et al. Cell 140, 209–221 (2010). 8. Arnault, J. P. et al. J. Clin. Oncol. 27, e59–e61 (2009). that GDC-0879 causes hyperactivation of ERK another drug to effectively treat RAS-mutant 9. Spencer, J. M., Kahn, S. M., Jiang, W., DeLeo, V. A. & and hyperproliferation of skin cells in mice. tumours. Weinstein, I. B. Arch. Dermatol. 131, 796–800 (1995). Moreover, RAS mutations are found in up to 20% of squamous-cell carcinomas. RAS mutations are also present7,9 in a subset of actinic keratoses — thick, scaly patches of skin that ASTROPHYSICS sometimes precede keratoacanthomas — supporting the notion that RAS mutations may enhance the pro-tumorigenic side effects of First generation of quasars RAF inhibitors in patients. Although this particular lesion is easily removed, it could be that Giulia Stratta other, less visible pre-malignant lesions associ- The discovery of two quasars in the distant Universe that apparently have ated with RAS mutations might be induced by no hot dust in their environments provides evidence that these systems RAF inhibitors, such as those in the airways of smokers. This suggests that patients treated represent the first generation of their family. with RAF inhibitors should be monitored care- fully for side effects. Nevertheless, the success In seeking to understand how, and when, that accretes matter at high rate from a sur- of BRAF inhibitors in patients with metastatic galaxies such as our own formed, astronomers rounding disk of gas. This accretion process melanoma1, a group of patients who until often turn to a type of galaxy called a quasar. powers the release of intense electromagnetic recently had a poor prognosis, suggests that Because quasars are extremely bright, they radiation in the galactic core that has wave- clinical development of these compounds can be seen at much larger distances from lengths spanning from the near-infrared should continue. Earth than other galaxies, and so allow us to to the X-ray bands of the electromagnetic The trio of studies5–7 might also help us to peer into the early history of the Universe. spectrum. Most of the system’s ultraviolet and understand the clinical differences observed To date, multi-wavelength observations1–3 visible radiation is absorbed by dust close to the when melanomas are treated with MEK or have shown that the most distant quasars accretion disk5,6 and is re-emitted at infrared BRAF inhibitors, and may provide insight known are nearly indistinguishable from their wavelengths. The hottest of this dust is directly into which drugs should be selected to treat closest (lower-redshift) counterparts, sug- heated by the central engine and generates genetically distinct tumours. The reports suggesting that quasars in the early Universe near-infrared radiation7–9. gest that BRAF inhibitors do not inhibit (and were already evolved objects. Now Jiang and Observational studies10,11 have led to the may even potentiate) ERK signalling in normal collaborators4 (page 380) put this view to the discovery of more than 40 quasars at an epoch tissues, such as skin. This effect might actu- test with the Spitzer Space Telescope’s observa- when the Universe was about 7% of its present ally provide a greater therapeutic window for tions of a sample of high-redshift quasars seen age. These studies have shown that such high- anticancer treatment, allowing BRAF inhibi- at an epoch when the Universe was about one redshift (z ≈ 6) quasars have very similar prop- tors to be administered at sufficient doses to billion years old. erties to their lower-redshift analogues in the fully inhibit ERK signalling in BRAF-mutant Quasars are a family within a larger class rest-frame (not redshifted) ultraviolet, visible tumours, while sparing normal tissue. In con- of galaxies called active galactic nuclei. These and X-ray energy bands1–3; light of wavelength trast, MEK inhibitors block ERK signalling galaxies host a supermassive black hole (more λ emitted by a quasar at redshift z is redshifted in both tumour and normal tissue, so that than a million solar masses) in their nucleus on its course to Earth by a factor 1+z owing to

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the expansion of the Universe. However, the probably formed less than half a billion years rest-frame near-infrared properties of distant High-redshift earlier. Therefore, sources of dust other than quasars have until recently remained mostly quasars (z ≈ 6) evolved stars may have been at work at high unexplored12. This is because, at large dis- redshift. The stellar explosions called super- tances, near-infrared light is redshifted to the novae are one possibility. The colours of a red- far infrared, an extremely challenging energy dened quasar at z = 6.2 cannot be accounted range for observational astronomy, as most of for by models of dust extinction at low red- this radiation is blocked by Earth’s atmosphere J0303–0019 shift, but agree well with models of dust cre- and cannot be detected by ground-based tele- ated by supernovae14. A similar theory of scopes. But the advent of dedicated infrared dust originating from supernovae has been log hot-dust abundance log hot-dust space telescopes such as the Infrared Space J0005–0006 proposed to explain the colours of the opti- Observatory, the Infrared Astronomical Sat- cal afterglow of the γ-ray burst GRB 050904 ellite and the Spitzer telescope, as well as the at z = 6.29, for which the ratio of dust content recently launched Herschel Space Observatory log black-hole mass to gas mass in its host galaxy was found to be and Wide-field Infrared Survey Explorer, has much smaller than that of the Milky Way15. made the far-infrared Universe fully accessible Figure 1 | Hot-dust abundance versus black- Together with Jiang and colleagues’ results, 4 to observers. hole mass. Jiang and colleagues find that the these two examples support the idea that the To study the properties of hot dust in the envi- abundance of hot dust in the environment of properties and sources of dust in the early Uni- 4 a quasar as a function of its black-hole mass is ronments of high-redshift quasars, Jiang et al. different for quasars at low and high redshift: verse may be quite different from those of the ■ obtained infrared Spitzer images for a sample at low redshift (not shown), the hot-dust present time. of 21 quasars at z ≈ 6. They found that most of abundance is roughly independent of the system’s Giulia Stratta is at the ASI Science Data Center, these quasars show strong rest-frame near- black-hole mass; at high redshift (dashed line), via Galileo Galilei, 00044 Frascati, Italy. infrared emission, which the authors attribute the abundance approximately scales with black- e-mail: [email protected] to hot dust in the quasars’ surroundings. Two hole mass. In contrast to all quasar systems quasars, however, SDSS J000552.34−000655.8 reported to date, at both low and high redshift, 1. Fan, X. et al. Astron. J. 128, 515–522 (2004). and SDSS J030331.40–001912.9 (hereafter the authors find two high-redshift quasars, 2. Jiang, L. et al. Astron. J. 134, 1150–1161 (2007). J0005−0006 and J0303−0019, that display no 3. Shemmer, O. et al. Astrophys. J. 644, 86–99 (2006). called J0005−0006 and J0303−0019), do not 4. Jiang, L. et al. Nature 464, 380–383 (2010). emission from hot dust. Filled and open circles display any such emission. This is a remark- 5. Antonucci, R. Annu. Rev. Astron. Astrophys. 31, 473–521 denote quasars with black-hole masses estimated able — and puzzling — result, because hot-dust (1993). from MgII and CIV spectral lines, respectively. 6. Elitzur, M. N. Astron. Rev. 52, 274–288 (2008). emission was thought to be a ubiquitous fea- 7. Rieke, G. H. Astrophys. J. 226, 550–558 (1978). ture of quasars. These apparently hot-dust-free 8. Hyland, A. R. & Allen, D. A. Mon. Not. R. Astron. Soc. 199, quasars have no analogues at low redshift. environment is already enriched with dust13. 943–952 (1982). 9. Haas, M. et al. Astron. Astrophys. 402, 87–111 (2003). The second, no less remarkable finding by At low redshift, most dust in the interstellar 10. Fan, X. et al. Astron. J. 132, 117–136 (2006). Jiang and colleagues is that, among all quasars medium is thought to be produced by evolved 11. Willott, C. J. et al. Astron. J. 134, 2435–2450 (2007). in their sample for which black-hole masses stars, which develop it between about half a 12. Jiang, L. et al. Astron. J. 132, 2127–2134 (2006). are known from previous studies, J0005−0006 billion and one billion years after their initial 13. Juarez, Y. et al. Astron. Astrophys. 494, L25–L28 (2009). 14. Maiolino, R. et al. Nature 431, 533–535 (2004). and J0303−0019 have the smallest black-hole starbursts. But at z ≈ 6, the Universe was less 15. Stratta, G., Maiolino, R., Fiore, F. & D’Elia, V. Astrophys. J. masses (about 200 million to 300 million solar than one billion years old, and the first stars Lett. 661, L9–L12 (2007). masses). What’s more, they find that the z ≈ 6 quasars’ abundance of hot dust is approximately proportional to their black-hole mass (Fig. 1), suggesting that these two properties grow at MULTIPLE SCLEROSIS roughly the same rate. The authors suggest, therefore, that J0005−0006 and J0303−0019 are in an early stage of evolution, being too young Closing in on an oral treatment to have formed a detectable amount of hot dust. By contrast, for a sample of low-redshift Roland Martin quasars from the Sloan Digital Sky Survey, the At present, only injectable drugs are available for treating multiple hot-dust abundance is roughly independent sclerosis. So clinical trials indicating that the drug fingolimod might of the black-hole mass. The authors argue that this is the reason for the observed absence of be a step towards an oral treatment for the disease are exciting indeed. hot-dust-free quasars such as J0005−0006 and J0303−0019 at low redshift. At low redshift, Multiple sclerosis affects some 2.5 million people and so destroying them3. The first-line thera- dust has allegedly been recycled between gal- worldwide, with patients often suffering from pies for the most common form of the dis- axies and the intergalactic medium, and the substantial disability at an early age. Several ease, relapsing-remitting multiple sclerosis quasars’ environments are supplied with dust drugs of varying efficacy are available for (RR-MS), have been interferon-β and glati- even before the central black hole starts accret- treating this autoimmune disorder, although ramer acetate4,5. Both of these drugs shift the ing mass rapidly. none can be taken orally. The findings of two immune response from proinflammatory If confirmed, Jiang and colleagues’ discov- clinical trials1,2 published in The New England (capable of causing inflammation) to anti- ery of two hot-dust-free objects out of 21 z ≈ 6 Journal of Medicine indicate that the search for inflammatory (reducing autoimmune activity) quasars (a fraction of 10%) would be an appar- an oral drug for multiple sclerosis might soon and are well tolerated by patients. Nonetheless, ent contradiction of the hypothesis proposed be over. they only moderately reduce the rate of relapse to explain the high content of elements other Multiple sclerosis is characterized by infil- and have local side effects. than hydrogen and helium observed in high- tration of the brain and spinal cord with More severe cases of RR-MS are treated with redshift quasars. This hypothesis is based on autoreactive immune cells; these cause an an antibody-based drug called natalizumab6, the premise that by the time a distant qua- inflammatory response there, damaging which efficiently blocks inflammation in the sar has reached a detectable luminosity, its the myelin sheath that insulates nerve fibres, central nervous system by interfering with the

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N e w ing RR-MS both and early the of phase the heart andheart hascaused cancers known assec- severely restricted it because toxic can be to the progressthe of multiple sclerosis, its is use antrone. Although efficiently blocks drug this rosis, which usually follows RR-MS, ismitox- scle- multiple of form progressive secondary communication). Idec information, drug March 2010,personal sive multifocal leukoencephalitis (Biogen an often fatal brain infection calledprogres- with it, more than 30patients have developed however, isthat, of some 66,000patients treated usually well tolerated. Adownside to drug, this either interferon-β or glatiramer acetate, and is brain.the Natalizumab ismore than effective migration of autoreactive Tlymphocytes into NATURE processes, including formation, blood-vessel ways cell inthe to trigger several downstream signalling path- 5 of transmembrane the S1Preceptor (S1P 2—interactskinase with 1,3, 4and subtypes through action the of enzyme sphingosine the —which isgenerated FTY720-P of FTY720, blood peripheral the to egress lymphocyte and organs such thymus asthe and lymph nodes homing inimmune lymphocyte mote both (S1P). Anessential of function S1P is to pro- natural lipid calledsphingosine-1-phosphate antibiotic myriocin isstructurally similar to a different way. Thissynthetic derivative of the T-lymphocyte migration, albeit inan entirely natalizumab,Like interferes FTY720 with to brain the forblood iscrucial sustaining it. process, and from passage their thedisease T lymphocytes are essential for initiating the patients sis? Studies and animal inboth models human autoimmunethe response inmultiple sclero- patients to disability. develop further on brain imaging data and timeit the took for and interferon-β inits long-term based effects over, placebo wassuperior to both drug this 38–52% compared with interferon-β. More- 50% compared with and placebo the by reduced relapseFTY720 rates by more than (0.5and drug the 1.25milligrams daily), two and trials, for two the tested of doses or interferon-βFTY720 for one year. In the almost 1,300patients, were who given either TheTRANSFORMSdrug. trial or aplacebo with eitheryear FTY720 period randomly for selected treatment over atwo- FREEDOMS trial which can taken orally. be (FTY720), In the immunomodulatory agent calledfingolimod on RR-MS patients desirable. anSo effective oral treatment would behighly injections intra or periodic venous infusions. All the approved treatments require frequent leukaemiasinupondary to 2.8%of patients s a Like S1P,Like active the (phosphorylated) form How promising this does influence drug The two large-scale, phase IIIclinical trials Finally, another approved drug for treat- n d

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1,2 3 6 tested the efficacy of an an of efficacy the tested 1 2 also involved 1,3–5 7 ) . © immunomodulatory not just drug for mul- to its interaction with S1P the mostrelevant relatesthe of FTY720-P effect FTY720-P. ities reported for have been its also analogue cell migration. Not surprisingly, different activ- cell differentiation, proliferation and immune- — much more will be learned from further from further learned be — much more will immune system, beyond its potential asadrug insightsseminal into functioning the of the as investigations have of provided FTY720 remains unknown. Without doubt —just on other cellsof central the system nervous or vessels endothelial cellsliningthe blood wereside effects acting on caused by FTY720 been reported previously called haemorrhagic encephalitis that has tumours, and of atype brain inflammation infections, and benign oedema, cancerous cations, an condition eye known asmacular subtype, and include cardiovascular compli- acting with more than one S1P-receptor trials human reported been two inthe also FTY720 a mystery. reactivatedbeen human inthe remains trials Why or infections have occurred herpes viral infections and may enhance even them. with data from animal studies tions were contrasts noted. Thisobservation infec- ofcases moderately herpesvirus serious be without problems. During both trials combination therapy. Its however, use, willnot for suitable be might it action, of mechanism rejection. Moreover, of because its novel and fordiseases preventing organ-transplant tiple sclerosis but for also other autoimmune a myelin sheath —cellsthatcytes wrap around fibres as nerve S1P tive by effects interacting with S1P brain,Once inthe it potentially posi- exerts brain barrier, which normally brain. shieldsthe can cross system, drug this blood– nervous the cells, remain unaffected, at innumber. least locationas their sites within these differentthe immune compartments, aswell antibody-secreting B-lymphocyte subtypes in number the affects also of specific- FTY720-P ability inthe bloodstream are greatly reduced. in lymph and numbers nodes, their and avail- Consequently, Tlymphocytes remain trapped peripheral and blood from there to brain. the tion from thymus the and lymph to the nodes mediatewhich would migra- their otherwise T lymphocytes cannot respond to S1Psignals, reduced numbers of S1P tor isinternalized for days. several With binding of FTY720-P, recep- cell-surface this cells, monocytes andpolymorphonuclear monocytes cells, immune such asnatural celltypes, killer mise protective immune responses during during responses immune protective mise not does compro-reported that FTY720

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r , which e . Other s e 3 r and and v e d 1,2 , distances, thenseriousthought with modestpayloads over short hovercraft canprove itsworth at aspeedof90knots...Ifthe eighty cars across thechannel eight hundred passengers and car ferry capableofcarrying has beengiven toa400-ton rivers. Seriousconsideration snow, sand,ice andshallow difficult, for example, marsh, which existing vehicles find may well beover surfaces required andearlyapplications flat operational surface is air compressor ...Areasonably and control systems, andthe are mountedthepropulsion is anoval dishontopofwhich version now flying,the‘SR-NI’, development ...The experimental the hard taskofpractical The first hovercraft hasbegun 50 YEARSAGO From of about25miles. duration incovering adistance 1910, being of32minutes’ number offlights,thatMarch 1, the machinehasmadealarge about 48milesperhour, and front elevators ...The speedis ... fittedwithShort’s patent biplaneisshownAnother Short used by theWright brothers ... closely resembles themachines Rolls ...Ingeneral designit and belongingtotheHon.C.S. first ofitskindbuiltinEngland, Wrightis aShort biplane,the Club, oneofthemostinteresting by members oftheRoyal Aëro machines ...Ofshown all ofthesebeingfull-size nine biplanes,andonetriplane, There are twenty monoplanes, across theChannel lastsummer. buyers, isowing toBlériot’s flight popularity, bothwithmakers and design ...there isnodoubtthatits any inherent advantages ofthis in thisexhibition. from Apart the larger numberofmachines Monoplanes comprise by far Motor BoatExhibition’— ‘The InternationalAëro And 100 YEARSAGO From of crossing theAtlantic in24 hr. trans-ocean hovercraft capable could begiven todeveloping a Nature Nature 17March 1910. 19March 1960. NEWS &VIEWS 112/3/10 17:59:48 2 / 3 361 / 1 0

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dissecting this drug’s mechanism of action and 6. Miller, D. H. et al. N. Engl. J. Med. 348, 15–23 (2003). of matter that propagate through the system its side effects. ■ 7. Pascual, A. M. et al. Mult. Scler. 15, 1303–1310 (2009). much like a particle does. 8. Bielekova, B. et al. Nature Med. 6, 1167–1175 (2000). 6 Roland Martin is at the Institute for 9. Yednock, T. A. et al. Nature 356, 63–66 (1992). Neutron-scattering experiments performed Neuroimmunology and Clinical Multiple Sclerosis 10. Chiba, K. et al. J. Immunol. 160, 5037–5044 (1998). on copper benzoate measured the energies and Research, University Medical Center-Eppendorf, 11. Mandala, S. et al. Science 296, 346–349 (2002). momenta of low-energy quasiparticles in this D-20251 Hamburg, Germany. 12. Brinkmann, V. et al. J. Biol. Chem. 277, 21453–21457 (2002). system; the properties of a beam of neutrons e-mail: [email protected] 13. Cinamon, G. et al. Nature Immunol. 5, 713–720 (2004). scattered off the material vary by an amount 14. Miron, V. E. et al. Ann. Neurol. 63, 61–71 (2008). that depends on the energies and momenta of 1. Kappos, L. et al. N. Engl. J. Med. 362, 387–401 (2010). 15. Pinschewer, D. D. et al. J. Immunol. 164, 5761–5770 the quasiparticles. Three quasiparticles were 2. Cohen, J. A. et al. N. Engl. J. Med. 362, 402–415 (2000). (2010). 16. Premenko-Lanier, M. et al. Nature 454, 894–898 found whose energies have the same momen- 3. Sospedra, M. & Martin, R. Annu. Rev. Immunol. 23, 683–747 (2008). tum dependence as the energies of three types (2005). 17. Leypoldt, F. et al. Neurology 72, 1022–1024 (2009) of massive relativistic particle, if the speed of 4. The IFNB Multiple Sclerosis Study Group Neurology 43, 655–661 (1993). The author declares competing financial interests. See online light is replaced by the much lower quasiparti- 5. Johnson, K. P. et al. Neurology 45, 1268–1276 (1995). article for details. cle velocity of approximately 1,100 m s−1. The masses of these quasiparticles were measured from the relativistic energy–momentum relationship. By taking into account some rather SOLID-STATE PHYSICS unusual magnetic interactions in copper benzoate, it was later shown7 that the low-energy behaviour of this material is governed by an Golden ratio seen in a magnet integrable quantum-field theory in one spatial dimension, known as the sine-Gordon model, Ian Affleck for which exact bound-state mass ratios had The golden ratio — an exact ‘magic’ number often claimed to be observed been predicted much earlier4,8–10. This work6 when taking ratios of distances in ancient and modern architecture, thus provided what may have been the first experimental test of exact mass-ratio predic- sculpture and painting — has been spotted in a magnetic compound. tions for integrable quantum-field theories in one spatial dimension, and the agreement According to Albert Einstein’s formula E = mc2, string theory), experimental realizations of was good. when several particles form a collective bound lower-dimensional universes can be found Coldea et al.1 report results of a neutron- state, the mass (m) of the system is reduced in condensed-matter systems. One example scattering experiment on another quasi-one- from the sum of the constituent masses by the is the electrical insulator copper benzoate dimensional magnetic compound, cobalt

binding energy (E) of the ensemble divided (Cu(C6H5CO2)2∙3D2O), which contains chains niobate (CoNb2O6). In this case, the spins of by the square of the speed of light (c). For the of magnetic copper atoms that interact with the Co atoms in a chain order magnetically at strong forces, which hold together protons their neighbours through antiferromagnetic low temperatures according to the one-dimen- and neutrons in nuclei, and quarks in protons Heisenberg interactions. Classically, below a sional Ising model of ferromagnetism: all spins and neutrons, the ratio of the system’s mass critical temperature, these interactions cause point in the same direction, either up or down, to the sum of the constituent masses can be the magnetic moments (spins) of neighbouring along a fixed ‘easy axis’. The authors show that, significantly reduced from 1. For such rela- atoms to point oppositely but along an arbi- at a critical magnetic field of 5.5 tesla applied tivistic bound states of matter, in which the trary axis in three-dimensional space. Because transverse to the easy axis, the system under- constituents rotate around each other at close the chains in copper benzoate are weakly cou- goes a quantum-phase transition beyond which to the speed of light, calculation of these mass pled to one another, each chain can be regarded the Ising magnetic order is destroyed, and each ratios is a challenging problem and requires approximately as a separate one-dimensional spin may be thought of as going into a quantum methods of quantum-field theory (those in universe, one in which the spins act together to superposition of the up and down orientations. which functions defined at all points in space form quasiparticles — collective bound states At field strengths slightly below the critical are treated as quantum variables). Writing in Science, Coldea and colleagues1 describe how they measured such a mass ratio in a magnetic material, and show that it agrees well with an old exact theoretical prediction2. Calculations of mass ratios for bound states of quarks are generally tackled by large-scale computer simulations, which yield large uncer- P. SVARC/PHOTOLIBRARY.COM P. tainties3. But there are integrable models of quantum-field theory — in which particles are restricted to a ‘universe’ of only one spatial dimension — for which such mass ratios can be calculated exactly4. In some of these integrable theories, particle binding is so strong that which particles are fundamental and which are bound states may be a matter of interpretation. Geoffrey Chew argued5, in an idea he dubbed ‘nuclear democracy’, that it is possible that all particles are bound states of matter, and so are Figure 1 | The golden ratio in ancient architecture. The semi-base, height and apothem of the in some sense equal. Great Pyramid of Giza, constructed around 2,550 bc in Egypt, are in the proportions 1:√φ:φ, where Although our Universe has at least three φ = 1.618034 is the golden ratio. Coldea and colleagues1 find that, under a particular magnetic field, spatial dimensions (even more according to the masses of two quasiparticles in cobalt niobate approach the golden ratio.

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value, they1 treat the weak inter-chain Ising approach the golden ratio, indicating that E8 couplings, in a standard approximation, as might underlie the low-temperature physics giving an effective longitudinal magnetic field of this system. Whether such a ratio applies — which acts on the nearly isolated chains — to other solid-state systems remains to be */Ɛɥ(-'( (3(.-Ʀ in addition to the transverse field. seen. More generally, the golden ratio of two Ɛɥ(-'( (3(.- It has previously been shown theoreti- quantities has intrigued mathematicians for cally2 that the one-dimensional Ising model at least 2,400 years, and has been shown to

of ferromagnetism at the critical transverse occur in modern and ancient architecture #5#+ɥ.$ɥ231#22ɥ field, combined with a very weak longitudinal (Fig. 1), sculpture and painting. It has even 4,.41 .1, + Pre-tumoral Pre-malignant +(%- -3 field, is a realization of a particular integrable been claimed to exist in nature, for example in 23 %# 3(224# tissue tumour 34,.41 quantum-field theory. The lightest two parti- the arrangement of branches along the stems cles of such a theory, labelled A and B, obey a of plants and of veins in leaves. ■ #-2(3(9#"ɥ .1, + type of ‘nuclear democracy’: particle B can be Ian Affleck is in the Department of Physics and !1(3(! +ɥ/.(-3ɥ !1(3(! +ɥ/.(-3ɥ regarded as a bound state of two particles of Astronomy, University of British Columbia, $.1ɥ2#-#2!#-!# $.1ɥ2#-#2!#-!# type A and, at the same time, particle A can Vancouver, V6T 1Z1 British Columbia, Canada. be regarded as a bound state of two particles e-mail: [email protected] ƭɥ ,/1.5#"ɥ! -!#1ɥɥɥɥɥɥɥ of type B. This situation was shown2 to require ɥɥɥ/1.3#!3(.- 1. Coldea, R. et al. Science 327, 177–180 (2010). ƭɥ.ɥ2("#ɥ#Ɖ#!32 particle masses in the golden ratio: the unique 2. Zamolodchikov, A. Int. J. Mod. Phys. A 4, 4235–4248 solution of mB/mA = (mA + mB)/mB for positive (1989). masses, which is about 1.618034. Physicists 3. Mawhinney, R. D. Nucl. Phys. B. Proc. Suppl. 83, 57–60 (2000). Figure 1 | Manipulating the threshold for have found this type of quantum-field theory Ann. Phys. 4. Zamolodchikov, A. B. & Zamolodchikov, Al. B. senescence. especially intriguing because of its hidden ‘E8 120, 253–291 (1979). With increasing levels of structure’. E8 is the largest of the ‘exceptional 5. Chew, G. F. The Analytic S-Matrix: A Basis for Nuclear senescence-inducing stress (oncogenes, Democracy (Benjamin, 1966). DNA damage, oxidative damage), tumour symmetry Lie groups’ and occurs in various 6. Dender, D. C., Hammar, P. R., Reich, D. H., Broholm, C. & development goes through three stages. The models of elementary particle physics, includ- Aeppli, G. Phys. Rev. Lett. 79, 1750–1753 (1997). stressors normally reach sufficient intensity to 7. Oshikawa, M. & Affleck, I. Phys. Rev. Lett. 79, 2883–2886 ing both grand unified field theories (in three trigger senescence only at the pre-malignant (1997). 2,3 spatial dimensions) and string theories. 8. Dashen, R. F., Hasslacher, B. & Neveu, A. Phys. Rev. D 11, tumour stage. Two groups show that the critical 1 In their experiment, Coldea and colleagues 3424–3450 (1975). point for triggering senescence can be lowered by show that, in the vicinity of the critical 9. Korepin, V. E., Kulish, P. P. & Faddeev, L. D. JETP Lett. 21, inhibiting either Skp2 or CDK2. Decreasing this 138–139 (1975). critical point results in cellular senescence at the transverse field, the masses of two different 10. Korepin, V. E. & Faddeev, L. D. Theor. Math. Phys. 25, low-energy quasiparticles in cobalt niobate 1039–1049 (1975). pre-tumoral stage, which translates into better protection against cancer.

Moreover, mutations that disable the senescence CANCER response are instrumental in the transition of oncogene-harbouring cells to a malignant stage, again highlighting the importance of A lower bar for senescence senescence in countering malignancy 6. Although the tumour-suppression machinery Manuel Serrano is highly efficient, it is obviously not infallible. Cellular senescence is a physiological mechanism for thwarting the In thinking of ways to improve it, one wonders proliferation of tumour cells. Encouraging cancer-prone cells to senesce why tissues wait until the pre-malignant stage to unleash senescence. Would it not be better might therefore be a way to nip this disease in the bud. to trigger this defensive response earlier, at the pre-tumoral stage? Lin et al.2 (page 374) and Genetic mutations are routine cellular events: In experimental mouse models, senescent cells Campaner and colleagues3 have independently it is estimated that each gene is mutated once can be engulfed by phagocytic cells in a pro- attempted to lower the bar of senescence and in every 20,000 cells1. Considering both the cess that is essentially similar to the removal of look at the effects on cancer. fact that an organism has trillions of cells and apoptotic cells5. The clearance of oncogenically Both teams find that the key to lowering the variety of oncogenic mutations, at any primed cells by induced senescence is gaining the bar for senescence lies in the activity of a given time there are probably millions of cells increasing attention as accumulating evidence6 group of enzymes known as cyclin-dependent primed to become cancerous (oncogenically points to its role in tumour suppression. kinases, or CDKs. Earlier work4 has shown that primed). Fortunately, most of these danger- Tumorigenesis can usually be divided into inhibition of CDK2, CDK4 and CDK6, all of ous cells are disposed of through programmed three stages: the initial proliferation of cells which mediate the initial (G1) phase of the cell- cell death (apoptosis) and cellular senescence. carrying oncogenes (pre-tumoral stage); the division cycle, is crucial for establishing senes- Two papers2,3, including one by Lin et al.2 in formation of a non-invasive tumour (pre- cence. Senescence-inducing stressors inhibit this issue, shed light on the molecular mecha- malignant stage); and the formation of a locally these G1 CDKs by increasing the expression of nisms underlying cancer-associated senes- invasive and metastasizing tumour (malignant CDK inhibitory proteins (CKIs), which include cence, reporting results that could have clinical stage or, simply, cancer) (Fig. 1). The transi- p16, p21 and p27 (refs 4, 7). Because of a large relevance. tion from one stage to the next is generally degree of functional redundancy among the Cellular senescence is a stress response that accompanied by an increase in the levels of three G1 CDKs, elimination of each of these is characterized by robust inhibition of cell the senescence-inducing stressors mentioned enzymes individually or even in pairs has only proliferation; this often becomes irreversible above. Most human and mouse tumour cells minor effects on the normal physiology of a and independent of the initial stress trigger4. stop proliferating and undergo senescence at mammalian organism7. The present studies2,3 Signalling by oncogenic proteins, oxida- the pre-malignant stage6, suggesting that it is build on these previous findings and explore tive damage and persistent DNA damage are at this stage that senescence-inducing signals whether decreasing the activity of G1 CDKs among the best-studied triggers of senescence4. reach sufficient intensity to be effective (Fig. 1). might lower the ‘critical point’ for triggering

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senescence in oncogenically primed cells. These data2,3 lead to an even more excit- normal Myc levels. The challenge ahead is to Lin et al.2 genetically eliminated the Skp2 ing question: if early tumorigenic cells can be test whether these preclinical studies in mice protein, which normally mediates degrada- sensitized to undergo senescence, what about can be translated into more effective cancer tion of a number of proteins, including p21 fully malignant cells? Malignant cells evade therapies. ■ and p27 (ref. 8). Consistent with the viability senescence because they generally lack proper Manuel Serrano is at the Spanish National of mice with reduced G1 CDK activity, mice CKI function, and so retain G1 CDK activity. It Cancer Research Centre (CNIO), 3 Melchor lacking Skp2 are also viable and fertile despite is therefore conceivable that, by inhibiting G1 Fernández Almagro Street, Madrid E-28029, having moderately raised levels of p21 and p27 CDKs, senescence can be induced in malignant Spain. (ref. 8). Lin et al. find that, in the absence of cells. The authors of both papers take a first e-mail: [email protected] Skp2, cancer-prone cells expressing the Ras step in exploring this possibility. oncoprotein or partially lacking the tumour- Lin et al.2 show that human prostate cancer 1. Vijg, J. & Dolle, M. E. Mech. Ageing Dev. 123, 907–915 (2002). suppressor protein Pten become more sensi- cells lacking Pten senesce when treated with 2. Lin, H.-K. et al. Nature 464, 374–379 (2010). tized to senescence. The survival of mice a small-molecule drug that indirectly blocks 3. Campaner, S. et al. Nature Cell Biol. 12, 54–59 (2010). carrying only one copy of the Pten gene the activity of Skp2 (by inhibiting the Nedd8- 4. Collado, M., Blasco, M. A. & Serrano, M. Cell 130, 223–233 (Pten+/– mice) is severely compromised owing activating enzyme9), and the same drug can (2007). 5. Xue, W. et al. Nature 445, 656–660 (2007). to the spontaneous development of several decrease the growth of tumours introduced 6. Collado, M. & Serrano, M. Nature Rev. Cancer 10, 51–57 cancers, most notably in the lymph nodes, into mice. (2010). adrenal glands and prostate. Intriguingly, Lin Campaner et al.3 report that two small- 7. Malumbres, M. & Barbacid, M. Nature Rev. Cancer 9, and colleagues find that mice deficient in both molecule inhibitors of CDK2 can induce 153–166 (2009). +/– –/– 8. Nakayama, K. I. & Nakayama, K. Semin. Cell Dev. Biol. 16, Pten and Skp2 (Pten ; Skp2 ) are strongly senescence in human leukaemia cells that are 323–333 (2005). 2 protected from cancer . overexpressing Myc, but not in those expressing 9. Soucy, T. A. et al. Nature 458, 732–736 (2009). The authors report similar results in mice lacking Pten in the prostate, and those deficient for another tumour suppressor, Arf. In both cases, when deficiency in these tumour EVOLUTIONARY BIOLOGY suppressors was combined with low Skp2 levels, strong resistance to cancer was observed. Importantly, analysis of the tissues protected Pregnant fathers in charge from cancer, particularly the lymph nodes and prostate, showed abundant senescent cells and Anders Berglund a low proliferation rate within the pre-tumoral Pipefish and related species provide rare examples of extreme male tissues. By contrast, the comparable tissues from mice expressing normal Skp2 levels con- parental care. Controlled breeding experiments allow the resulting conflicts tained either no senescent cells (lymph nodes) of interest between female, male and offspring to be explored. or only low levels of them (prostate). Campaner et al.3 focused directly on In sea horses, pipefishes and sea dragons, low-quality eggs from low-quality mothers; he CDK2-deficient mice. In agreement with Lin it is the males that undergo pregnancy. The may even want to use the low-quality eggs as and colleagues’ data, they found that onco- father broods the female’s eggs, and in many food for himself, to gain resources for future genic stress, in this case exerted by the Myc species this takes place in a specialized pouch offspring with better prospects. According to oncoprotein, caused the cells of mice lacking where the babies are protected and can receive Paczolt and Jones6, both eventualities occur: CDK2 to become sensitized to senescence. In oxygen and nutrition1,2. In some pipefish spe- by means of selective abortion and resorption, normal mice, oncogenic expression of Myc cies the father’s commitment is so great that pipefish fathers can manipulate eggs to their in B lymphocytes (white blood cells) led to a males cannot reproduce as fast as females, own ends but to the detriment of low-quality strong apoptotic response, which, despite its and males become a limiting resource for mothers and their eggs. protective potential, was not sufficient to pre- females3,4. Females then compete for males, In their experiments, Paczolt and Jones mated vent the eventual development of lymphoma. and traits that aid females in this competition males of the gulf pipefish, Syngnathus scovelli, By contrast, Myc expression in CDK2-deficient may evolve. This reversal of the usual sex roles to one female each. They allowed the males to mice resulted not only in apoptosis but also in has been well documented in several pipefish complete their pregnancies and give birth, and increased levels of senescent cells in the pre- species3,5. But there remains the crucial ques- then mated each male to a new female. Females tumoral spleen, where B lymphocytes are abun- tion of why the brood pouch evolved to start varied in size, which in this species may reflect dant. This double anti-oncogenic response was with. There are probably many answers, and quality — males were more reluctant to mate accompanied by a lower number of proliferative an intriguing possibility is tackled by Paczolt with smaller females and discriminated against cells and delayed development of lymphoma. and Jones (page 401 of this issue)6 — what if them6. In a related species, the broad-nosed The authors also present conceptually similar the pouch allows a male to resolve a conflict of pipefish Syngnathus typhle (Fig. 1), larger data in the islets of Langerhans in the pancreas, interest for his own benefit? females produce more and larger eggs that give to show that the effect they observed also The conflict is a family one — between par- rise to higher-quality offspring7,8. occurs in other tissues. ents and offspring and between mothers and The idea of mating each male to one female Both studies2,3 demonstrate that it is feasible fathers. All mothers and offspring would of after another was to establish a reproduc- to lower the critical point for senescence in course benefit from getting as much care for tive history for the males, and see whether a oncogenically primed mouse cells. Conse- eggs as possible from the father. But this might previous brood affected what happened to quently, these cells senesce at the pre-tumoral bear on the father’s reproductive prospects: a subsequent one. Indeed it did: Paczolt and stage rather than at the subsequent pre- spending all his valuable resources on one Jones found that if the first brood came from malignant stage (Fig. 1). The earlier and more brood might compromise his future interests. a large female, survival in the second brood sensitive induction of senescence translates Instead, a father could benefit from spending decreased, implying that those males had into better protection from cancer without more on offspring with good prospects (that few resources left to invest. Similarly, if off- interfering with the normal functioning of the is, offspring from a mother that already has spring survival was low in the first clutch, the organism. invested substantially in the eggs), and less on second survived better, implying that males

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confirmed that this strategy can be adaptive. Anders Berglund is in the Department of Males that were reluctant to mate with small Ecology and Evolution, Uppsala University, females and exhibited cryptic (post-copula- Norbyvägen 18 D, SE-752 36 Uppsala, Sweden. A. BERGLUND tory) choice in favour of large females had a e-mail: [email protected] higher fitness than males not adopting cryptic choice — the higher fitness taking the form of 1. Ripley, J. L. & Foran, C. M. J. Comp. Phys. B 179, 325–333 (2009). more offspring being produced. 2. Ripley, J. L. Comp. Biochem. Phys. A 154, 98–104 The pipefish brood pouch may serve sev- (2009). eral functions. It provides safety and nutrition 3. Berglund, A. & Rosenqvist, G. Adv. Study Behav. 32, 131–167 for offspring, and it may serve as an attractant (2003). 4. Scobell, S. K., Fudickar, A. M. & Knapp, R. Anim. Behav. 78, signal for females. But also, as is evident from 747–753 (2009). Paczolt and Jones’s study, it grants fathers bet- 5. Jones, A. G., Walker, D. & Avise, J. C. Proc. R. Soc. Lond. B ter control over reproduction — males may 268, 2531–2535 (2001). use their role as carers not only to nurture the 6. Paczolt, K. A. & Jones, A. G. Nature 464, 401–404 (2010). A video presentation based on this paper can be viewed at eggs but also to favour those from high-quality www.youtube.com/NatureVideoChannel females and disfavour and/or exploit those of 7. Ahnesjö, I. Behav. Ecol. Sociobiol. 38, 167–172 (1996). low-quality females. What at first sight seems 8. Partridge, C. et al. Behav. Ecol. Sociobiol. 63, 345–354 an egalitarian partnership between males and (2009). 9. Sagebakken, G., Ahnesjö, I., Mobley, K. B., Braga Gonçalves, I. females, both investing heavily in their young, & Kvarnemo, C. Proc. R. Soc. Lond. B 277, 971–977 (2010). looks more like brooding sexual conflict. ■ 10. Braga Gonçalves, I. et al. Proc. R. Soc. Lond. B (in the press).

Figure 1 | Courting couple. A male (front) and female of the species Syngnathus typhle engage in CELL BIOLOGY a nuptial dance. Like other pipefish, including the species (S. scovelli) studied by Paczolt and Jones6, it is the father that broods the resulting eggs. Actin filaments up against a wall

can save resources for future broods. Cécile Sykes and Julie Plastino It could be that larger females are better at The front of motile cells is thought to be pushed out by branched filaments of manipulating males to invest more in their actin protein abutting the cell membrane. New work challenges this textbook eggs, but this explanation seems less likely than one invoking male control. The eggs of view, showing that actin branches grow away from, or obliquely to, a surface. small females never did very well, not even when those females were preceded in mating Monomers of the protein actin assemble into fixed cells suggests that branches are oriented by another small female, which suggests that a filamentous scaffold that propels the cell towards the cell membrane9. Together, these the pattern of brood survival is due to a male forward, and that can also power the move- observations led to the interpretation that, at an adaptive strategy of cryptic choice (choice after ment of intracellular pathogens and transport NPF-coated surface, forward-facing branches mating). So pipefish fathers invest more in off- vesicles, as well as biomimetic objects such as form, elongating by actin-monomer addition spring from preferred mates. Can they also beads and liposomes1,2. The complex formed to the barbed ends abutting the surface and so exploit non-preferred females by using their by actin-related proteins 2 and 3 (the Arp2/3 pushing the surface forward. eggs as a source of nutrition? Possibly they can: complex) is a ‘nucleator’ of actin polymeri- Achard et al.3 call this view into question. in related Syngnathus species, nutrients move zation, which causes branching in this scaf- The authors coated glass rods with an NPF, and not only from father to offspring1 but also from fold. The existing model for actin-mediated incubated them with the ingredients for actin- offspring to father9. movement proposes that the fast-growing filament formation: the Arp2/3 complex and Evidently, cryptic male support for eggs (barbed) ends of actin filaments are directed a mixture of actin and profilin (a protein that from preferred females for mating would ben- towards a surface, such as the cell membrane, binds to actin monomers), to ensure that fila- efit these females’ offspring; that is, one might where polymerization is catalysed. Writing in ment elongation occurred only at barbed ends. expect post-copulatory sexual selection to Current Biology, Achard et al.3 turn this model Their trick was to use a fluorescently labelled reinforce pre-copulatory selection. A differ- on its head by providing a real-time readout form of actin that bleaches quickly; the newly ent possibility is that fathers might compensate of single-filament dynamics in a network polymerized filament-ends fluoresce, whereas for the shortcomings of low-quality mothers growing on a surface. They show that when older parts of the network are less visible. The by investing more in their eggs. Something the growing filament-ends encounter a wall (in authors then observed this biomimetic system similar has, for instance, been shown in the this case, a rod or a bead, but by analogy a cell using TIRF (total internal reflection fluores- broad-nosed pipefish, where mothers made membrane), they orient away from it. cence) microscopy, which limits background up for the shortcomings of low-quality fathers The previous view of actin-based motility fluorescence. by providing them with eggs with extra pro- came from three main observations. First, in Surprisingly, they saw that all of the fila- tein10. Perhaps the sex that makes the choice of solution, the Arp2/3 complex is activated by ments’ barbed ends were directed away from partner is more likely to invest differentially in nucleation-promoting factors (NPFs) of the the rod surface. On adding a capping protein, the offspring of high-quality partners, whereas WASp/Scar family; consequently, it nucleates which stops filament elongation, barbed ends the chosen sex is more likely to adopt the com- new actin filaments as branches at a 70° angle were still oriented away from the surface, pensatory strategy of providing extra support on the sides of the ‘mother’ filaments, and near although the region containing the growing for those of low-quality partners. the growing barbed end4,5. Second, live imaging filaments was closer to the surface. The authors From Paczolt and Jones’s observations6 it shows that new actin material is incorporated also found that explosive Arp2/3-dependent seems that male gulf pipefish opt for differ- near the leading-edge membrane of motile actin polymerization was initiated by actin- ential allocation of support, and a simulation cells and near the surface of moving biomi- filament seeds, or ‘primers’. This is consistent model using realistic reproductive parameters metic objects6–8. Third, electron microscopy of with the results of experiments performed in

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INORGANIC CHEMISTRY copper(ii) nitrate. None of the manganese ions in Pores off the peg the porous material was displaced by Cu2+ — even Porous materials provide a pictured here as red structures) though both types of ion nanometre-scale playground for self-assemble in the presence of bear the same charge — chemists to investigate unusual manganese ions (small yellow leaving the framework reactions that occur in confined spheres) to form a regularly shaped architecture intact. spaces. But in the most widely used pore (multicoloured faceted The authors could easily of these materials — zeolites and structure) of about 7 nm3 in volume. alter the oxidation state metal–organic frameworks — it These roughly cubic units extend in of the linking manganese is difficult to control the size and three dimensions throughout the ions while they were in the geometry both of pore openings and material to make a remarkably open framework. When they of the pores themselves. Writing in framework. increased the oxidation Nature Chemistry, Scott Mitchell and The material also incorporates state from +2 to +3, they by Cu2+ ions. But larger organic his colleagues report a solution to potassium and lithium ions (not observed a reduction in the number molecules that are too large to fit this problem in which the molecular shown) to balance the negative of charge-balancing lithium and inside the pore framework instead building blocks used to create a charge carried by the oxide ions potassium ions. These charge- interact with pore entrances, porous framework are themselves in the wheel. These metal ions fit balancing ions could still be replaced blocking the entry of Cu2+ ions. ready-made pore openings into the channels and cavities of with Cu2+ ions, but only by about half Because a wide range of (S. G. Mitchell et al. Nature Chem. the material loosely enough that as many as before. polyoxometalate wheels are doi:10.1038/nchem.581; 2010). they can be replaced with other The system’s ion-exchange available, Mitchell and colleagues’ The authors’ building block is a positive ions if the compound is properties could be further tuned work opens the door to a variety of polyoxometalate — a ‘wheel’ of soaked in a suitable solution. For by blocking the pore openings porous materials that have tunable metal, oxygen and phosphorus example, Mitchell et al. found that with organic molecules. The team properties. The authors are currently atoms that has an opening of about 35 copper(ii) ions (Cu2+) could found that small organic molecules investigating the material’s catalytic 1 nanometre in diameter. Six of be incorporated into each pore by introduced into the material’s pores and ion-sensing capabilities. these wheels (three of which are soaking the material in a solution of can subsequently be displaced Neil Withers

solution4, but that had never been incorporated produced on a surface by an actively polymer- What remains unclear is why most filaments in into biomimetic or cell-motility models. izing zone that is constrained to grow between motile cells are oriented with their barbed ends Achard et al. combine these observations to the surface and a ‘dead zone’ of capped fila- towards the membrane. Achard and colleagues’ propose the following model (Fig. 1). On acti- ments (Fig. 1). In the case of spherical beads, data suggest that this orientation cannot be vation by an NPF on the polymerizing surface, stress builds up on the surface because actin explained solely by the molecular mechanism the Arp2/3 complex captures a floating primer, filaments are organized in a connected net- of the Arp2/3-complex, which would give becoming attached to its side to generate a work, covering the entire bead surface. Rupture rise to actin-filament barbed ends oriented branch point that nucleates a daughter filament; of the dead zone relaxes stress, giving space for predominantly away from the membrane. Gen- the new filament can grow away from, tangen- the formation of a freshly polymerizing zone eral reorganization due to stresses in the actin tial to or towards the surface. Filaments ori- that propels the object forward. This zone, in network may cause filament reorientation. ented away from or tangential to the surface will turn, becomes capped, leading to a new cycle Moreover, proteins that bind to actin barbed elongate until stopped by the capping protein. of stress-and-rupture. ends at or near the leading edge — for example, Those growing towards the surface, however, In the context of the cell membrane, one formins11 — may contribute to the forward ori- will be stalled by the surface until they escape can imagine that the newly polymerizing zone entation of the barbed ends. Nonetheless, one and restart growth tangentially to it. All tan- at the surface braces against the dead zone to point is certain: Achard and colleagues’ results3 gentially oriented filaments act as primers for push out the membrane. The wide distribution fundamentally change our view of how, at the further branching events at the surface. of filament orientations previously observed molecular level, actin polymerization catalysed In such a scenario, a propulsive force is by electron microscopy9,10 fits with this model. by the Arp2/3 complex powers movement. ■ Cécile Sykes and Julie Plastino are in the Laboratoire Physico-chimie, Institut Curie, NPF (WASp/Scar) Surface Stalled ﬁlament Movement Centre de Recherche, CNRS UMR 168, Université Paris VI, Paris F-75248, France. Escape e-mails: [email protected]; [email protected]

Arp2/3 Forward- Stress and Outward- 1. Pollard, T. D. & Borisy, G. G. Cell 112, 453–465 (2003). complex facing branch network facing branch 2. Plastino, J. & Sykes, C. Curr. Opin. Cell Biol. 17, 62–66 (2005). reorganization et al. Curr. Biol. 20, Capping protein 3. Achard, V. 423–428 (2010). 4. Machesky, L. M. et al. Proc. Natl Acad. Sci. USA 96, Pointed end 3739–3744 (1999). 5. Amann, K. J. & Pollard, T. D. Proc. Natl Acad. Sci. USA 98, Barbed end 15009–15013 (2001). Actin primer Dead zone6. zone Active Wang, Y. L. J. Cell Biol. 101, 597–602 (1985). 7. Cameron, L. A. et al. Curr. Biol. 11, 130–135 (2001). 8. Paluch, E., van der Gucht, J., Joanny, J.-F. & Sykes, C. Figure 1 | Actin-network assembly gets a fresh look. Achard et al.3 propose that, after a primer has Biophys. J. 91, 3113–3122 (2006). been captured at a surface, branches form in all directions. Outward-facing branches grow until 9. Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M. & Borisy, G. G. J. Cell Biol. 139, 397–415 (1997). capped. Forward-facing branches stall, escape and eventually grow tangentially. Tangential filaments 10. Koestler, S. A. et al. Nature Cell Biol. 10, 306–313 (2008). act as primers for explosive actin-network growth. Capped parts of the network constitute the dead 11. Gupton, S. L., Eisenmann, K., Alberts, A. S. & zone, whereas the active polymerizing zone is constrained to the surface. Waterman-Storer, C. M. J. Cell Sci. 120, 3475–3487 (2007).

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Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Li-Jun Ma1*, H. Charlotte van der Does2*, Katherine A. Borkovich3, Jeffrey J. Coleman4, Marie-Jose´e Daboussi5, Antonio Di Pietro6, Marie Dufresne5, Michael Freitag7, Manfred Grabherr1, Bernard Henrissat8, Petra M. Houterman2, Seogchan Kang9, Won-Bo Shim10, Charles Woloshuk11, Xiaohui Xie12, Jin-Rong Xu11, John Antoniw13, Scott E. Baker14, Burton H. Bluhm11, Andrew Breakspear15, Daren W. Brown16, Robert A. E. Butchko16, Sinead Chapman1, Richard Coulson17, Pedro M. Coutinho8, Etienne G. J. Danchin8{, Andrew Diener18, Liane R. Gale15, Donald M. Gardiner19, Stephen Goff20, Kim E. Hammond-Kosack13, Karen Hilburn15, Aure´lie Hua-Van5, Wilfried Jonkers2, Kemal Kazan19, Chinnappa D. Kodira1{, Michael Koehrsen1, Lokesh Kumar1, Yong-Hwan Lee21, Liande Li3, John M. Manners19, Diego Miranda-Saavedra22, Mala Mukherjee10, Gyungsoon Park3, Jongsun Park21, Sook-Young Park9{, Robert H. Proctor16, Aviv Regev1, M. Carmen Ruiz-Roldan6, Divya Sain3, Sharadha Sakthikumar1, Sean Sykes1, David C. Schwartz23, B. Gillian Turgeon24, Ilan Wapinski1, Olen Yoder25, Sarah Young1, Qiandong Zeng1, Shiguo Zhou23, James Galagan1, Christina A. Cuomo1, H. Corby Kistler15 & Martijn Rep2

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Fusarium species are among the most diverse and widely dispersed transmembrane transporters5. We sequenced two additional plant-pathogenic fungi, causing economically important blights, Fusarium species, Fv, a maize pathogen that produces fumonisin root rots or wilts1. Some species, such as F. graminearum (Fg) and mycotoxins that can contaminate grain, and F. oxysporum f.sp. lyco- F. verticillioides (Fv), have a narrow host range, infecting predomi- persici (Fol), a tomato pathogen. Here we present the comparative nantly the cereals (Fig. 1a). By contrast, F. oxysporum (Fo), has a analysis of the genomes of these three species. remarkably broad host range, infecting both monocotyledonous and dicotyledonous plants2 and is an emerging pathogen of immu- Results nocompromised humans3 and other mammals4. Aside from their Genome organization and gene clusters. We sequenced Fv strain differences in host adaptation and specificity, Fusarium species also 7600 and Fol strain 4287 (Methods, Supplementary Table 1) using a vary in reproductive strategy. Some, such as Fo, are asexual, whereas whole-genome shotgun approach and assembled the sequence using others are both asexual and sexual with either self-fertility (homo- Arachne (Table 1, ref. 6). Chromosome level ordering of the scaffolds thallism) or obligate out-crossing (heterothallism) (Fig. 1b). was achieved by anchoring the assemblies either to a genetic map for Previously, the genome of the cereal pathogen Fg was sequenced Fv (ref. 7), or an optical map for Fol (Supplementary Information A and shown to encode a larger number of proteins in pathogeni- and Supplementary Table 2). We predicted Fol and Fv genes and re- city related protein families compared to non-pathogenic fungi, annotated a new assembly of the Fg genome using a combination of including predicted transcription factors, hydrolytic enzymes, and manual and automated annotation (Supplementary Information B).

1The Broad Institute, Cambridge, Massachusetts 02141, USA. 2University of Amsterdam, Amsterdam 1098XH, The Netherlands. 3University of California Riverside, California 92521, USA. 4University of Arizona, Tucson, Arizona 85721, USA. 5Universite´ Paris-Sud, 91405 Paris, France. 6Universidad de Cordoba, Cordoba 14071, Spain. 7Oregon State University, Corvallis, Oregon 97331, USA. 8CNRS, Universite´s Aix-Marseille, 13628 Aix-en-Provence, France. 9Penn State University, University Park, Pennsylvania 16802, USA. 10Texas A&M University, College Station, Texas 77843, USA. 11Purdue University, West Lafayette, Indiana 47907, USA. 12University of California, Irvine, California 92697, USA. 13Centre for Sustainable Pest and Disease Management, Rothamsted Research, Harpenden AL5 2JQ, UK. 14Pacific Northwest National Laboratory, Richland, Washington 99352, USA. 15USDA ARS, University of Minnesota, St. Paul, Minnesota 55108, USA. 16USDA-ARS-NCAUR, Peoria, Illinois 61604, USA. 17European Bioinformatics Institute, Cambridge CB10 1SD, UK. 18University of California, Los Angeles, California 90095, USA. 19CSIRO Plant Industry, Queensland Bioscience Precinct, St Lucia, Brisbane, Queensland, 4067 Australia. 20BIO5 Institute, University of Arizona, Tucson, Arizona 85721, USA. 21Seoul National University, Seoul 151-742, Korea. 22Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. 23University of Wisconsin-Madison, Madison, Wisconsin 53706 USA. 24Cornell University, Ithaca, New York 14853, USA. 2517885 Camino Del Roca, Ramona, California 92065, USA. {Present addresses: 454 Life Sciences, Branford, Connecticut 06405, USA (C.D.K.); University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA (L.L.); INRA, Institut National de la Recherche Agronomique, 06903 Sophia-Antipolis, France (E.G.J.D.); Seoul National University, Seoul 151-742, Korea (S.-Y.P.). *These authors contributed equally to this work. 367 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 464 | 18 March 2010

a F. verticillioides b c d

F. oxysporum

Asexual F. graminearum

F. solani

M. grisea

N. crassa

A. nidulans 0.1 substitution per site Figure 1 | Phylogenetic relationship of four Fusarium species in relation to Branches are labelled with the percentage of 10,000 bootstrap replicates. other ascomycete fungi and phenotypic variation among the four Fusarium b–d, Phenotypic variation within the genus Fusarium: b, disease symptoms species. a, Maximum-likelihood tree using concatenated protein sequences of (top to bottom) kernel rot of maize (Fv), wilt of tomato (Fol), head blight of 100 genes randomly selected from 4,694 Fusarium orthologous genes that of wheat (Fg) and root rot of pea (Fs); c, the perithecial states of Fv have clear 1:1:1:1 correlation among the Fusarium genomes and have unique (Gibberella moniliformis), Fol (no sexual state), Fg (G. zeae) and Fs (Nectria matches in Magnaporthe grisea, Neurospora crassa and Aspergillus nidulans. haematococca); and d, micro- and macroconidia of Fv, Fol, Fg and Fs. Scale The tree was constructed with PHYML35 (WAG model of evolution36). bars, 10 mm. Fg produces only macroconidia.

The Fol genome (60 megabases) is about 44% larger than that of its biosynthesis (SMB) gene clusters. Microarray analyses confirmed the most closely related species, Fv (42 Mb), and 65% larger than that of co-expression of genes in 14 of 18 Fg and 10 of 16 Fv SMB gene Fg (36 Mb), resulting in a greater number of protein-encoding genes clusters. Ten out of the 14 Fg and eight out of the 10 Fv co-expressed in Fol (Table 1). SMB gene clusters are novel (Supplementary Information D, Sup- The relatedness of the three Fusarium genomes enabled the genera- plementary Fig. 5 and Supplementary Table 9, and online materials), tion of large-scale unambiguous alignments (Supplementary Figs 1–3) emphasizing the potential impact of uncharacterized secondary and the determination of orthologous gene sets with high confidence metabolites on fungal biology. (Methods, Supplementary Information C). On average, Fol and Fv Lineage-specific chromosomes and pathogenicity. The genome orthologues display 91% nucleotide sequence identity, and both assembly of Fol has 15 chromosomes, the Fv assembly 11 and the have 85% identity with Fg counterparts (Supplementary Fig. 4). Fg assembly only four (Table 1). The smaller number of chromo- Over 9,000 conserved syntenic orthologues were identified among somes in Fg is the result of chromosome fusion relative to Fv and Fo, the three genomes. Compared to other ascomycete genomes, these and fusion sites in Fg match previously described high diversity three-species orthologues are enriched for predicted transcription fac- regions (Supplementary Fig. 3, ref. 5). Global comparison among tors (P 5 2.6 3 1026), lytic enzymes (P 5 0.001), and transmembrane the three Fusarium genomes shows that the increased genomic transporters (P 5 7 3 1029) (Supplementary Information C and territory in Fol is due to additional, unique sequences that reside Supplementary Tables 3–8), in agreement with results reported for mostly in extra chromosomes. Syntenic regions in Fol cover approxi- the Fg genome5. mately 80% of the Fg and more than 90% of the Fv genome (Sup- Fusarium species produce diverse secondary metabolites, includ- plementary Information E and Supplementary Table 10), referred to ing mycotoxins that exhibit toxicity to humans and other mammals8. as the ‘core’ of the genomes. Except for telomere-proximal regions, In the three genomes, we identified a total of 46 secondary metabolite all 11 mapped chromosomes in the Fv assembly (41.1 Mb) correspond to 11 of the 15 chromosomes in Fol (41.8 Mb). The co-linear order of genes between Fol and Fv has been maintained within these Table 1 | Genome statistics. chromosomes, except for one chromosomal translocation event and Species F. oxysporum F. verticillioides F. graminearum a few local rearrangements (Fig. 2a). Strain 4287 7600 PH-1 The unique sequences of Fol are a substantial fraction (40%) of the Sequence coverage (fold) 6810 Genome size (Mb) 59.941.736.2 Fol assembly, designated as Fol lineage-specific (Fol LS) regions, to Number of chromosomes 15 11* 4 distinguish them from the conserved core genome. The Fol LS Total scaffolds 114 31 36 regions include four entire chromosomes (chromosomes 3, 6, 14 N50 scaffold length (Mb) 1.98 1.96 5.35 and 15), parts of chromosome 1 and 2 (scaffold 27 and scaffold 31, Coding genes 17,735 14,179 13,332 Median gene length (bp) 1,292 1,397 1,355 respectively), and most of the small scaffolds not anchored to the Repetitive sequence (Mb) 16.83 0.36 0.24 optical map (Fig. 2b). In total, the Fol LS regions encompass 19 Mb, Transposable elements (%) 3.98 0.14 0.03 accounting for nearly all of the larger genome size of Fol. NCBI accession AAXH01000000 AAIM02000000 AACM00000000 Notably, the LS regions contain more than 74% of the identifiable * Fv was reported to contain 12 chromosomes7, 11 chromosomes were mapped to the assembled transposable elements (TEs) in the Fol genome, including 95% of all genome, and no genetic markers from the smallest chromosome (600 kb or less) were found in DNA transposons (Fig. 2b, Supplementary Fig. 6 and Supplementary the sequence data. N50 represents the size N such that 50% of the nucleotides is contained in scaffolds of size N or greater. Table 11). In contrast to the low content of repetitive sequence and 368 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES

a b i 14 1 27 Chr 1 ii kb iii

15 6 10 31 i 14 Chr 2 ii 13

Chromosome iii 12 i 47 18 32 7 25 11 50366 Chr 3 ii iii i 8 4 Chr 4 ii iii i 26 2

37112 Chr 5 ii iii i 9 33 41 21 4253 Chr 6 ii iii F. oxysporum i 5 13 Chr 7 ii 23857 iii i 3 29 Chr 8 ii iii i 11 17 Chr 9 ii 10603 iii

i 20 15 45 24681 Un 12345678910 Chr 10 ii iii 7453 16770 26087 35403 kb i 35 12 1 2 3 4 5 6 7 8 9 10 11 Un Chr 11 ii F. verticillioides Chromosome iii Syntenic alignments of i 19 23 F. verticillioides chromosomes Chr 12 ii c iii Chr 1 Chr 7 Chr 8 i 16 39 Chr 2 47 18 32 7 25 Chr 13 ii iii Chr 3 Chr 9 22 51 36 Chr 3 i Chr 4 Chr 10 Chr 14 ii iii Chr 5 Chr 11 i 37 38 24 28 Chr 6 Unmapped Chr 15 ii 9 33 41 21 4253 iii i 30 34 40 43 44 48 4946 52 5450 Un ii

Chr 6 iii

123456 Length of linkage groups (Mb) Figure 2 | Whole genome comparison between Fv and Fol.a, Argo37 dotplot blocks; row ii displays the syntenic mapping of Fv chromosomes, with one of pair-wise MEGABLAST alignment (1 3 10210) between Fv and Fol major translocation between chr 4/chr 12 in Fol and chr 4/chr 8 in Fv; row iii showing chromosome correspondences between the two genomes in the represents the density of transposable elements calculated with a 10 kb black dashed boxes. The vertical blue lines illustrate the chromosomal window. LS chromosomes include four entire chromosomes (chr 3, chr 6, translocations, and the red dashed horizontal boxes highlight the Fol LS chr 14 and chr 15) and parts of chromosome 1 and 2 (scaffold 27, scaffold chromosomes. b, Global view of syntenic alignments between Fol and Fv and 31), which lack similarity to syntenic chromosomes in Fv but are enriched the distribution of transposable elements. Fol linkage groups are shown as for TEs. c, Two of the four Fol LS chromosomes showing the inter- (green) the reference, and the length of the light grey background for each linkage and intra- (yellow) chromosomal segmental duplications. The three traces group is defined by the Fol optical map. For each chromosome, row i below are density distribution of TEs (blue lines), secreted protein genes represents the genomic scaffolds positioned on the optical linkage groups (green lines) and lipid metabolism related genes (red line). Chr, separated by scaffold breaks. Scaffold numbers for Fol are given above the chromosome; Un, unmapped. minimal amount of TEs in the Fv and Fg genomes (Table 1 and Information E and Supplementary Tables 12–18). Among the genes Supplementary Table 11), about 28% of the entire Fol genome was with a predicted function related to pathogenicity were known identified as repetitive sequence (Methods), including many retro- effector proteins (see below) as well as necrosis and ethylene-inducing elements (copia-like and gypsy-like LTR retrotransposons, LINEs peptides9 and a variety of secreted enzymes predicted to degrade or (long interspersed nuclear elements) and SINEs (short interspersed modify plant or fungal cell walls (Supplementary information E and nuclear elements) and DNA transposons (Tc1-mariner, hAT-like, Supplementary Tables 14, 15). Notably, many of these enzymes are Mutator-like, and MITEs) (Supplementary Information E.3), as well expressed during early stages of tomato root infection (Supplemen- as several large segmental duplications. Many of the TEs are full- tary Tables 15, 16 and Supplementary Fig. 8). The expansion of genes length and present as highly similar copies. Particularly well repre- for lipid metabolism and lipid-derived secondary messengers in Fol sented DNA transposon classes in Fol are pogo, hAT-like elements LS regions indicates an important role for lipid signalling in fungal and MITEs (in total approximately 550, 200 and 350 copies, respec- pathogenicity (Supplementary Fig. 9 and Supplementary Tables 13, tively). In addition, there are one intra-chromosomal and two inter- 17). A family of transcription factor sequences related to FTF1,a chromosomal segmental duplications, totalling approximately 7 Mb gene transcribed specifically during early stages of infection of and resulting in three- or even fourfold duplications of some regions F. oxysporum f. sp. phaseoli (Supplementary Information E and Sup- (Fig. 2c). Overall, these regions share 99% sequence identity (Sup- plementary Table 4; ref. 10) is also expanded. plementary Fig. 7), indicating recent duplication events. The recently published genome of F. solani11, a more diverged Only 20% of the predicted genes in the Fol LS regions could be species, enabled us to extend comparative analysis to a larger evolu- functionally classified on the basis of homology to known proteins. tionary framework (Fig. 1). Whereas the ‘core’ genomes are well These genes are significantly enriched (P , 0.0001) for the functional conserved among all four sequenced Fusarium species, the Fol LS categories ‘secreted effectors and virulence factors’, ‘transcription regions are also absent in Fs (Supplementary Fig. 2). Additionally, factors’, and ‘proteins involved in signal transduction’, but are defi- Fs has three LS chromosomes distinct from the genome core11 and the cient in genes for house-keeping functions (Supplementary Fol LS regions. In conclusion, each of the four Fusarium species 369 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 464 | 18 March 2010

carries a core genome with a high level of synteny whereas Fol and Fs a 1.0

each have LS chromosomes that are distinct with regard to repetitive 0.9 3% sequences and genes related to host–pathogen interactions. 0.8 Origin of LS regions. Three possible explanations for the origin of LS 0.7 regions in the Fol genome were considered: (1) Fol LS regions were present in the last common ancestor of the four Fusarium species but 0.6 were then selectively and independently lost in Fv, Fg and Fs lineages 0.5 0.4 during vertical transmission; (2) LS regions arose from the core BSR_Fol/Fg genome by duplication and divergence within the Fol lineage; and 0.3 (3) LS regions were acquired by horizontal transfer. To distinguish 0.2 among these hypotheses, we compared the sequence characteristics 0.1 9.5% 9% of the genes in the Fol LS regions to those of genes in Fusarium core 0.0 regions and genes in other filamentous fungi. If Fol LS genes have 1.00.90.80.70.60.50.40.30.20.10.0 clear orthologues in the other Fusarium species, or paralogues in the BSR_Fol/Fv core region of Fol, this would favour the vertical transmission or b 1.0

duplication with divergence hypotheses, respectively. We found that, 0.9 4.2% whereas 90% of the Fol genes in the core regions have homologues in 0.8 the other two Fusarium genomes, about 50% of the genes on Fol LS 0.7 regions lack homologues in either Fv or Fg (1 3 10220). Furthermore, there is less sequence divergence between Fol and Fv orthologues in 0.6 core regions compared to Fol and Fg orthologues (Fig. 3a), consistent 0.5 0.4 with the species phylogeny. In contrast, the LS genes that have homo- BSR_Fol/Fg logues in the other Fusarium species are roughly equally distant from 0.3 both Fv and Fg genes (Fig. 3b), indicating that the phylogenetic 0.2 history of the LS genes differs from genes in the core region of the 0.1 48.5% 4.3% genome. 0.0 Both codon usage tables and codon adaptation index (CAI) ana- 0.0 1.00.90.80.70.60.50.40.30.20.1 lysis indicate that the LS-encoding genes exhibit distinct codon usage BSR_Fol/Fv (Supplementary Information E.5, Supplementary Fig. 10 and c Supplementary Table 19) compared to the conserved genes and the F. graminearum 100 genes in the Fv genome, further supporting their distinct evolutionary F. oxysporum (conserved) origins. The most significant differences were observed for amino 98 100 acids Gln, Cys, Ala, Gly, Val, Glu and Thr, with a preference for G F. verticillioides 100 and C over A and T among the Fol LS genes (Supplementary Table 20). F. oxysporum (LS) 100 Such GC bias is also reflected in the slightly higher GC-content in their F. solani third codon positions (Supplementary Fig. 11). M. grisea Of the 1,285 LS-encoded proteins that have homologues in the NCBI protein set, nearly all (93%) have their best BLAST hit to other asco- N. crassa mycete fungi (Supplementary Fig. 12), indicating that Fol LS regions A. nidulans are of fungal origin. Phylogenetic analysis based on concatenated sampling of the 362 proteins that share homologues in seven selected 0.1 substitution per site ascomycete genomes — including the four sequenced Fusarium Figure 3 | Evolutionary origin of genes on the Fol LS chromosomes. The genomes, Magnaporthe grisea12, Neurospora crassa13 and Aspergillus scatter plots of BLAST score ratio (BSR)30 based on three-way comparisons of nidulans14 — places their origin within the genus Fusarium but basal proteins encoded in core regions (a) and the Fol LS chromosomes (b). The to the three most closely related Fusarium species Fg, Fv and Fol (Fig. 3c, numbers indicate the percentage of genes that lack homologous sequences in Supplementary Table 21). Taken together, we conclude that horizontal Fv and Fg (lower left corner), present in Fv but not Fg (x-axis) and present in Fg but not in Fv (y-axis). c, Discordant phylogenetic relationship of proteins acquisition from another Fusarium species is the most parsimonious encoded in the LS regions. The maximum-likelihood tree was constructed explanation for the origin of Fol LS regions. using the concatenated protein sequences of 100 genes randomly selected LS regions and host specificity. F. oxysporum is considered a species from 362 genes that share homologues in seven selected ascomycetes complex, composed of many different asexual lineages that can be genomes including the four Fusarium genomes, M. grisea, N. crassa and A. pathogenic towards different hosts or non-pathogenic. The Fol LS nidulans. The trees were constructed with PHYML35 (WAG model of regions differ considerably in sequence among Fo strains with different evolution36). The percentages for the branches represent the value based on a host specificities, as determined by Illumina sequencing of Fo strain 10,000 bootstrapping data set. Fo5176, a pathogen of Arabidopsis15 and EST (expressed sequence tag) sequences from Fo f. sp. vasinfectum16, a pathogen of cotton (Sup- non-pathogenic F. oxysporum isolates (Supplementary Fig. 14), indi- plementary Information E.2). Despite less than 2% overall sequence cating that Fol LS chromosomes may also be specifically involved in divergence between shared sequences of Fol and Fo5176 (Supplemen- pathogenic adaptation. tary Fig. 13A), for most of the sequences in the Fol LS regions there is no Transfer of Fo pathogenicity chromosomes. It is well documented counterpart in Fo5176. (Supplementary Fig. 13B). Also Fov EST that small proteins are secreted during Fol colonizing the tomato sequences16 have very high nucleotide sequence identity to the Fol xylem system20,21 and at least two of these, Six1 (Avr3) and Six3 genome (average 99%), but only match the core regions of Fol (Sup- (Avr2), are involved in virulence functions22,23. Interestingly, the plementary Information E.2). Large-scale genome polymorphism genes for these proteins, as well as a gene for an in planta-secreted within Fo is also evident by differences in karyotype between strains oxidoreductase (ORX1)20, are located on chromosome 14, one of the (Supplementary Fig. 14)17. Previously, small, polymorphic and con- Fol LS chromosomes. These genes are all conserved in strains causing ditionally dispensable chromosomes conferring host-specific virulence tomato wilt, but are generally not present in other strains24. The have been reported in the fungi Nectria haematococca18 and Alternaria genome data enabled the identification of the genes for three addi- alternata19. Small (,2.3 Mb) and variable chromosomes are absent in tional small in planta-secreted proteins on chromosome 14, named 370 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES

SIX5, SIX6 and SIX7 (Supplementary Table 22) based on mass spec- markers (Fig. 4c, Supplementary Fig. 15 and Supplementary trometry data obtained previously20. Together these seven genes can Information F). The parental strains, as well as the sequenced strain be used as markers to identify each of the three supercontigs (SC 22, Fol4287, each have distinct karyotypes. This enabled us to determine 36 and 51) localized to chromosome 14 (Supplementary Table 23 and with chromosome electrophoresis whether the entire chromosome Supplementary Fig. 15). 14 of Fol007 was transferred into Fo-471 strains. All Fo-471 strains In view of the combined experimental findings and computational had the same karyotype as Fo-47, except for the presence of one or evidence, we proposed that LS chromosome 14 could be responsible two additional small chromosomes (Fig. 4d). The chromosome pre- for pathogenicity of Fol towards tomato, and that its mobility sent in all Fo-471 strains (Fig. 4d, arrow number 1) was confirmed to between strains could explain its presence in tomato wilt pathogens, be chromosome 14 from Fol007 based on its size and a Southern comprising several clonal lineages polyphyletic within the Fo species hybridization using a SIX6 probe (Fig. 4e). Interestingly, two double complex, but absence in other lineages24. To test these hypotheses, we drug-resistant strains (Fo-471 1C and Fo-471 2A in Fig. 4a), which investigated whether chromosome 14 could be transferred and caused the highest level of disease (Fig. 4a, b), have a second extra whether the transfer would shift pathogenicity between different chromosome, corresponding in size to the smallest chromosome in strains of Fo, using the genes for in planta-secreted proteins on chro- the donor strain Fol007 (Fig. 4d, arrow number 2). mosome 14 as markers. Fol007, a strain that is able to cause tomato To rigorously assess whether additional genetic material other wilt, was co-incubated with a non-pathogenic isolate (Fo-47) and than chromosome 14 may have been transferred from Fol007 into two other strains that are pathogenic towards melon (Fom) or banana Fo-471 strains, we developed PCR primers for amplification of 29 (Foc), respectively. A gene conferring resistance against zeocin (BLE) chromosome-specific markers from Fol007 but not Fo-47. These was inserted close to SIX1 as a marker to select for transfer of chro- markers (on average two for each chromosome) were used to screen mosome 14 from the donor strain into Fo-47, Fom or Foc. The Fo-471 strains for the presence of Fol007-derived genomic regions receiving strains were transformed with a hygromycin resistance gene (Supplementary information F.4 and Supplementary Fig. 16). All (HYG), inserted randomly into the genome; three independent Fo-471 strains were shown to have the chromosome 14 markers hygromycin resistant transformants per recipient strain were (Supplementary Fig. 17), but not Fol007 markers located on any core selected. Microconidia of the different strains were isolated and chromosome, confirming that core chromosomes were not trans- mixed in a 1:1 ratio on agar plates. Spores emerging on these plates ferred. Interestingly, the two Fo-471 strains (1C and 2A) that have after 6–8 days of incubation were selected for resistance to both zeocin the second small chromosome and caused more disease symptoms and hygromycin. Double drug-resistant colonies were recovered with were also positive for an additional Fol007 marker (Supplementary Fom and Fo-47, but not using Foc as the recipient, at a frequency of Fig. 17), associated with a large duplicated LS region in Fol4287: roughly 0.1 to 10 per million spores (Supplementary Table 24). scaffold 18 (1.3 Mb on chromosome 3) and scaffold 21 (1.0 Mb on Pathogenicity assays demonstrated that double drug-resistant chromosome 6) (Fig. 2c). The presence of most or all of the sequence strains derived from co-incubating Fol007 with Fo-47, referred to of scaffold 18/21 in strains 1C and 2A was confirmed with an addi- as Fo-471, had gained the ability to infect tomato to various degrees tional nine primer pairs for genetic markers scattered over this region (Fig. 4a, b). In contrast, none of the double drug-resistant strains (data not shown, see Supplementary Tables 25a, b for primer derived from co-incubating Fol007 with Fom were able to infect sequences) (Fig. 4d). tomato. All Fo-471 strains contained large portions of Fol chro- Taken together, we conclude that pathogenicity of Fo-471 strains mosome 14 as demonstrated by PCR amplification of the seven gene towards tomato can be specifically attributed to the acquisition of Fol

a b d Marker Fol 4287 Fol 007 1A 1B 1C 2A 2B 2C 3A 3B 3C Fo -47 H1 Fo -47 H2 Fo -47 H3 3 2 5.7 4.6 1 3.5 0 (Mb) O

1A 2A 3A Disease index (a.u.) 2 1 H Fom

Fo -47 2 Fol 007 Fom 1A Fom 2A Fom 3A Fom 1B Fom 2B Fom 3B Fom 1C Fom 2C Fom 3C Fo -47 1A Fo -47 2A Fo -47 3A Fo -47 1B Fo -47 2B Fo -47 3B Fol007 Fo -47 1C Fo -47 2C Fo -47 3C Fo isolate c Fom Fo-47 e

1B 2B 3B Marker Fol 4287 Fol 007 1A 1B 1C 2A 2B 2C 3A 3B 3C Fo -47 H1 Fo -47 H2 Fo -47 H3

Fo-47 Fom H1 1A 1B 1C Fom H2 2A 2B 2C Fom H3 3A 3B 3C Fo-47 H1 1A 1B 1C Fo-47 H2 2A 2B 2C Fo-47 H3 3A 3B 3C Fol B Chr 14 SIX1 SIX2 1C 2C 3C SIX3 SIX5 SIX7 SIX6 ORX1 SIX6 probe Figure 4 | Transfer of a pathogenicity chromosome. a, Tomato plants bold. Three independent transformants of Fom and Fo-47 with a randomly infected with Fol007, Fo-47 or double drug resistant Fo-471 strains (1A inserted hygromycin resistance gene (H1, H2, H3) were investigated. d, Fo- through 3C) derived from this parental combination, two weeks after 471 strains derived from a Fol007/Fo-47 co-incubation have the same inoculation as described for b. b, Eight of nine Fo-471 strains derived from karyotype as Fo-47, plus one or two chromosomes from Fol007. Protoplasts pairing Fol007 and Fo-47 show pathogenicity towards tomato. Average from Fol4287, Fol007 (with BLE on chromosome 14), three independent disease severity in tomato seedlings was measured 3 weeks after inoculation HYG transformants of Fo-47 (lane Fo-47 H1, H2 and H3) and nine Fo- in arbitrary units (a.u.). The overall phenotype and the extent of browning of 471strains (lane 1A to 3C, the number 1, 2 or 3 referring to the HYG resistant vessels was scored on a scale of 0–4: 0, no symptoms; 1, slightly swollen and/ transformant from which they were derived) were loaded on a CHEF or bent hypocotyl; 2, one or two brown vascular bundles in hypocotyl; 3, at (contour-clamped homogeneous electric field) gel. Chromosomes of S. least two brown vascular bundles and growth distortion (strong bending of pombe were used as a molecular size marker. Arrows 1 and 2 point to the stem and asymmetric development); 4, all vascular bundles are brown, additional chromosomes in the Fo-471 strains relative to Fo-47. e, Southern plant either dead or stunted and wilted. c, The presence of SIX genes and blot of the CHEF gel shown in d, hybridized with a SIX6 probe, showing that ORX1 in Fom, Fo-47 and Fol isolates and in double drug-resistant strains chromosome 14 (arrow 1 in d) is present in all strains except Fo-47 (H1, H2 derived from co-incubation of Fol/Fom and Fol/Fo-47, assessed by PCR on and H3). genomic DNA. Co-incubations were performed with the isolates shown in 371 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 464 | 18 March 2010 chromosome 14, which contains all known genes for small in planta- identified using SignalP (http://www.cbs.dtu.dk/services/SignalP/) after remov- secreted proteins. In addition, genes on other LS chromosomes may ing trans-membrane/mitochondrial proteins based with TMHMM (http:// further enhance virulence as demonstrated by the two strains con- www.cbs.dtu.dk/services/TMHMM/), Phobius (except in the first 50 amino taining the additional LS chromosome from Fol007. We did not find acids), and TargetP (RC score 1 or 2) predictions. Small cysteine-rich secreted a double drug-resistant strain with a tagged chromosome of Fo-47 in proteins were defined as secreted proteins that are less than 200 amino acids in length and contain at least 4% cysteine residues. GPI (glycosyl phosphatidyl the Fol007 background. Also, a randomly tagged transformant of inositol)-anchor proteins were identified by the GPI-anchor attachment signal Fol007 did not render any double drug-resistant colonies when co- among the predicted secreted proteins using a custom PERL script. incubated with Fo-47 (data not shown). This indicates that transfer between strains may be restricted to certain chromosomes, perhaps Received 26 June 2009; accepted 20 January 2010. determined by various factors, including size and TE content of the 1. Agrios, G. N. Plant Pathology 5th edn (Academic Press, 2005). chromosome. Their propensity for transfer is supported by the fact 2. Armstrong, G. M. & Armstrong, J. K. in Fusarium: Diseases, Biology and Taxonomy that the smallest LS chromosome in Fol007 moved to Fo-47 without (eds Nelson, P. E., Toussoun, T. A. & Cook R.) 391–399 (Penn State University being selected for drug resistance in two out of nine cases. Press, 1981). 3. O’Donnell, K. et al. 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The genome sequence of the rice blast fungus Magnaporthe of new pathogenic lineages in otherwise distinct and incompatible grisea. Nature 434, 980–986 (2005). genetic backgrounds28. Fol LS regions are enriched for genes related 13. Galagan, J. E. et al. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868 (2003). to host–pathogen interactions. The mobilization of these chromo- 14. Galagan, J. E. et al. Sequencing of Aspergillus nidulans and comparative analysis somes could, in a single event, transfer an entire suite of genes required with A. fumigatus and A. oryzae. Nature 438, 1105–1115 (2005). for host compatibility to a new genetic lineage. If the recipient lineage 15. Thatcher, L. F., Manners, J. M. & Kazan, K. Fusarium oxysporum hijacks COI1- had an environmental adaptation different from the donor, transfer mediated jasmonate signaling to promote disease development in Arabidopsis. could increase the overall incidence of disease in the host by intro- Plant J. 58, 927–939 (2009). 16. Dowd, C., Wilson, I. W. & McFadden, H. Gene expression profile changes in ducing pathogenicity in a genetic background pre-adapted to a local cotton root and hypocotyl tissues in response to infection with Fusarium environment. Such knowledge of the mechanisms underpinning oxysporum f. sp. vasinfectum. Mol. Plant Microbe Interact. 17, 654–667 (2004). rapid pathogen adaptation will affect the development of strategies 17. Teunissen, H. A. et al. Construction of a mitotic linkage map of Fusarium oxysporum for disease management in agricultural settings. based on Foxy-AFLPs. Mol. Genet. Genomics 269, 215–226 (2003). 18. Miao, V. P., Covert, S. F. & VanEtten, H. D. A fungal gene for antibiotic resistance on a dispensable (‘‘B’’) chromosome. Science 254, 1773–1776 (1991). METHODS SUMMARY 19. Harimoto, Y. et al. Expression profiles of genes encoded by the supernumerary Generation of genome sequencing and assembly. The whole genome shotgun chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple (WGS) assemblies of Fv (83 coverage) and Fol (6.83 coverage) were generated pathotype of Alternaria alternata. Mol. Plant Microbe Interact. 20, 1463–1476 using Sanger sequencing technology and assembled using Arachne6. Physical (2007). maps were created by anchoring the assemblies to the Fv genetic linkage map7 20. Houterman, P. M. et al. The mixed xylem sap proteome of Fusarium oxysporum- and to the Fol optical map, respectively. infected tomato plants. Mol. Plant Pathol. 8, 215–221 (2007). Defining hierarchical synteny. Local-alignment anchors were detected using 21. van der Does, H. C. et al. Expression of effector gene SIX1 of Fusarium oxysporum PatternHunter (1 3 1010) (ref. 29). Contiguous sets of anchors with conserved requires living plant cells. Fungal Genet. Biol. 45, 1257–1264 (2008). 22. Houterman, P. M. et al. The effector protein Avr2 of the xylem colonizing fungus order and orientation were chained together within 10 kb distance and filtered to Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly. ensure that no block overlaps another block by more than 90% of its length. Plant J. 58, 970–978 (2009). Identification of repetitive sequences. Repeats were detected by searching the 23. Rep, M. et al. A small, cysteine-rich protein secreted by Fusarium oxysporum during genome sequence against itself using CrossMatch ($ 200 bp and $ 60% colonization of xylem vessels is required for I-3-mediated resistance in tomato. sequence similarity). Full-length TEs were annotated using a combination of Mol. Microbiol. 53, 1373–1383 (2004). computational predictions and manual inspection. Large segmental duplica- 24. van der Does, H. C. et al. The presence of a virulence locus discriminates Fusarium tions were identified using Map Aligner30. oxysporum isolates causing tomato wilt from other isolates. Environ. Microbiol. 10, Characterization of proteomes. Orthologous genes were determined based on 1475–1485 (2008). BLASTP and pair-wise syntenic alignments (SI). The blast score ratio tests31 were 25. Gladyshev, E. A., Meselson, M. & Arkhipova, I. R. Massive horizontal gene transfer in bdelloid rotifers. Science 320, 1210–1213 (2008). used to compare relatedness of proteins among three genomes. The EMBOSS 26. O’Donnell, K., Kistler, H. C., Cigelnik, E. & Ploetz, R. C. Multiple evolutionary origins tool ‘cusp’ (http://emboss.sourceforge.net/) was used to calculate codon usage of the fungus causing Panama disease of banana: concordant evidence from 32 frequencies. Gene Ontology terms were assigned using Blast2GO software nuclear and mitochondrial gene genealogies. Proc. Natl Acad. Sci. USA 95, 20 (BLASTP 1 3 10 ) and tested for enrichment using Fisher’s exact test, corrected 2044–2049 (1998). for multiple testing33. A combination of homology search and manual inspection 27. Gale, L. R., Katan, T. & Kistler, H. C. The probable center of origin of Fusarium was used to characterize gene families34,35. Potentially secreted proteins were oxysporum f. sp. lycopersici VCG 0033. Plant Dis. 87, 1433–1438 (2003). 372 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES

28. Li, M., Ma, B., Kisman, D. & Tromp, J. Patternhunter II: highly sensitive and fast Initiative of USDA’s National Institute of Food and Agriculture through the Microbial homology search. J. Bioinform. Comput. Biol. 2, 417–439 (2004). Genome Sequencing Program (2005-35600-16405) and conducted by the Broad 29. Zhou, S. et al. Single-molecule approach to bacterial genomic comparisons via Institute Sequencing Platform. Wayne Xu and the Minnesota Supercomputing optical mapping. J. Bacteriol. 186, 7773–7782 (2004). Institute for Advanced Computational Research are also acknowledged for their 30. Rasko, D. A., Myers, G. S. & Ravel, J. Visualization of comparative genomic support. The authors thank Leslie Gaffney at the Broad Institute for graphic design analyses by BLAST score ratio. BMC Bioinformatics 6, 2 (2005). and editing and Tracy E. Anderson of the University of Minnesota, College of 31. Conesa, A. et al. Blast2GO: a universal tool for annotation, visualization and Biological Sciences Imaging Center for spore micrographs. analysis in functional genomics research. Bioinformatics 21, 3674 (2005). 32. Blu¨thgen, N. et al. Biological profiling of gene groups utilizing Gene Ontology. Author Contributions L.-J.M., H.C.D., M.R. and H.C.K. coordinated genome Genome Inform 16, 106–115 (2005). annotation, data analyses, experimental validation and manuscript preparation. 33. Cantarel, B. L. et al. The Carbohydrate-Active EnZymes database (CAZy): an L.-J.M. and H.C.D. made equivalent contributions and should be considered joint expert resource for Glycogenomics. Nucleic Acids Res. 37 (Database issue), first authors. H.C.K. and M.R. contributed equally as corresponding authors. K.A.B., D233–D238 (2009). C.A.C., J.J.C., M.-J.D., A.D.P., M.D., M.F., J.G., M.G., B.H., P.M.H., S.K., W.-B.S., C.W., 34. Miranda-Saavedra, D. & Barton, G. J. Classification and functional annotation of X.X. and J.-R.X. made major contributions to genome sequencing, assembly, eukaryotic protein kinases. Proteins 68, 893–914 (2007). analyses and production of complementary data and resources. All other authors 35. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large are members of the genome sequencing consortium and contributed annotation, phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003). analyses or data throughout the project. 36. Whelan, S. & Goldman, N. A general empirical model of protein evolution derived Author Information All sequence reads can be downloaded from the NCBI trace from multiple protein families using a maximum-likelihood approach. Mol. Biol. repository. The assemblies of Fv and Fol have been deposited at GenBank under the Evol. 18, 691–699 (2001). project accessions AAIM02000000 and AAXH01000000. Detailed information 37. Engels, R. et al. Combo: a whole genome comparative browser. Bioinformatics 22, can be accessed through the Broad Fusarium comparative website: http:// 1782–1783 (2006). www.broad.mit.edu/annotation/genome/fusarium_group.3/MultiHome.html. Supplementary Information is linked to the online version of the paper at Reprints and permissions information is available at www.nature.com/reprints. www.nature.com/nature. This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence, and is freely available to all Acknowledgements The 43 sequence of F. verticillioides was provided by Syngenta readers at www.nature.com/nature. The authors declare no competing financial Biotechnology Inc. Generation of the other 43 sequence of F. verticillioides and 6.83 interests. Correspondence and requests for materials should be addressed to sequence of F. oxysporum f. sp. lycopersici was funded by the National Research H.C.K. ([email protected]) or M.R. ([email protected]).

Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence

Hui-Kuan Lin1,2,3, Zhenbang Chen1,2,4{, Guocan Wang1,2,4*, Caterina Nardella1,2,4*, Szu-Wei Lee3*, Chan-Hsin Chan3, Wei-Lei Yang3, Jing Wang3, Ainara Egia4, Keiichi I. Nakayama5, Carlos Cordon-Cardo2{, Julie Teruya-Feldstein2 & Pier Paolo Pandolfi1,2,4

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19Arf–p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19Arf–p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19Arf–p53 response is impaired, whereas a Skp2–SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

Cellular senescence represents an irreversible form of cell-cycle arrest senescence only in oncogenic conditions. Remarkably, this sen- that can be triggered by a variety of insults. Induction of cellular escence response is triggered in a p19Arf–p53-independent manner. senescence (for example, by oncogenic Ras) results in p19Arf Skp2 pharmacological inactivation may therefore represent a general (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) and approach towards a ‘pro-senescence’ therapy for cancer prevention p53 accumulation, which is critical for this senescence response. and treatment. Recent studies suggest that cellular senescence can act as an important tumour-suppressive mechanism to restrict tumour develop- Skp2 loss restores cellular senescence by Ras and E1A ment in vivo1–7. Skp2 deficiency delays cell cycle progression11,12. We therefore asked Inactivation of Pten functions is frequently observed in human whether Skp2 deficiency would trigger cellular senescence. We isolated cancers8–10. Although Pten negatively regulates cell proliferation mouse embryonic fibroblasts (MEFs) from wild-type and Skp22/2 and survival, we surprisingly discovered that acute Pten inactivation mice and determined cellular senescence in these cells by senescence- triggers the accumulation of p19Arf–p53 and cellular senescence2. associated b-galactosidase (SA-b-gal) staining. Although Skp22/2 MEFs Concomitant inactivation of p53 (also known as Trp53 in mice, proliferated less than wild-type MEFs (Supplementary Fig. 1e)11,12, cel- and TP53 in humans) and Pten abrogates this senescence response, lular senescence in Skp22/2 MEFs was comparable to that in the wild- in turn promoting invasive and lethal prostate cancer2. Although type MEFs (Supplementary Fig. 1a). In contrast, acute inactivation of these findings further underscore the critical importance of the cel- Pten in MEFs markedly increased cellular senescence as previously lular senescence Arf-p53 failsafe pathway, the frequent loss or muta- reported (Supplementary Fig. 1a)2.Thus,Skp2 deficiency by itself does tion of ARF or P53 in human cancers would compromise the not elicit cellular senescence. tumour-suppressive efficacy of this response, thereby limiting thera- Ectopic overexpression of proto-oncogenic Ras (Ras(G12V)) in peutic potential. MEFs elicits cellular senescence through the p19Arf–p53 pathway19,20. Skp2 is a critical component of the Skp2–SCF complex, which acts Simultaneous co-expression of E1A and Ras in MEFs overcomes as an E3 ligase to target p27 and other substrates for ubiquitylation Ras-induced senescence by preventing activation of the p19Arf–p53 and degradation11,12. Recent studies suggest that Skp2 may have pathway and resulting in oncogenic transformation20.Thus,E1A oncogenic activity13–16. Notably, SKP2 overexpression is frequently enables Ras to overcome the cellular senescence response. As Skp2 also observed in human cancer11,12,17, strongly suggesting that SKP2 over- cooperates with oncogenic Ras to induce cell transformation13,itis expression may contribute to tumorigenesis. Skp2-knockout mice conceivable that Skp2 might also display its oncogenic activity by are viable and fertile18. Hence, specific inactivation of Skp2 may rep- antagonizing Ras-induced cellular senescence. On this basis, we tested resent an appealing therapeutic modality. Here we show that Skp2 whether endogenous Skp2 activity is required for cellular transforma- inactivation profoundly restricts tumorigenesis by eliciting cellular tion induced by Ras and E1A. Although cellular senescence was not

1Cancer Biology and Genetics Program, 2Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. 3Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. 4Cancer Genetics Program, Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA. 5Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka 812-8582, Japan. {Present addresses: Department of Biochemistry and Cancer Biology, Meharry Medical College, 1005 Dr D. B. Todd Jr Boulevard, Nashville, Tennessee 37208-3599, USA (Z.C.); Irving Cancer Research Center, Room 309, 1130 St. Nicholas Avenue, New York, New York 10032, USA (C.C.-C.). *These authors contribute equally to this work. 374 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES observed in wild-type MEFs after Ras and E1A overexpression, Skp2 a WT Pten+/– *** deficiency triggered cellular senescence (Supplementary Fig. 1b, c). We 40 *** *** 35 also found that the ability of Ras to induce cellular senescence was 30 far greater in Skp22/2 MEFs than in wild-type MEFs (Supplemen- 25 Arf 20 tary Fig. 1b, c). It should be noted that the induction of p19 and 15 p53 protein levels in Skp22/2 MEFs by Ras was comparable if not lower 10 +/– –/– –/– Senescence (%) β -gal Pten Skp2 Skp2 5 than that of wild-type MEFs (Supplementary Fig. 1d). Moreover, Skp2 0 +/– –/– –/– inactivation profoundly restricted cell proliferation and transforma- WT tion after Ras and E1A overexpression (Supplementary Fig. 1e, f). Pten Skp2 Skp2 +/– Thus, Skp2 inactivation triggers cellular senescence in the presence of powerful oncogenic signals, even when the p19Arf–p53 response is Pten evaded. b c shRNA-ctrlshp53 p53-DN Skp2 loss causes senescence in Pten1/2 and Arf2/2 mutants –/– p53 Skp2 We assessed whether Skp2 inactivation would trigger cellular senescence +/– +/– –/– p21 even when cells experience loss of major tumour-suppressive networks WT Pten Pten Skp2 WT-IR β-actin such as those controlled by Pten and p19Arf–p53. Tothis end, we crossed p53 50 2/2 1/2 2/2 40 Skp2 mutants with Pten and Arf mutants. The resulting α -tubulin 30 compound mice were further intercrossed to generate MEFs of different 20 genotypes for cell proliferation and senescence assays. As aforemen- 2/2 10 tioned, Skp2 MEFs grew much slower than wild-type MEFs, Senescence (%) 0 whereas wild-type and Pten1/2 MEFs grew comparably (Supplemen- shp53 tary Fig. 2a). No obvious cellular senescence was observed in wild-type, d p53-DN –/– Pten1/2 and Skp22/2 MEFs (Fig. 1a). Surprisingly, Pten1/2 Skp22/2 WT Arf shRNA-ctrl 2/2 MEFs had a slower growth rate than Skp2 MEFs and exhibited 50 *** full-blown characteristics of cellular senescence such as flattened large *** *** 40 cells and positive SA-b-gal staining (Fig. 1a and Supplementary Fig. 2a). We also detected cellular senescence in Pten1/2 Skp22/2 MEFs under 30 –/– –/– –/– hypoxic conditions (Supplementary Fig. 2b). We did not see coopera- β -gal Arf Skp2 Skp2 20 tion between Pten inactivation and Skp2 deficiency in triggering apop- 10 tosis, although Skp22/2 MEFs had a higher rate of apoptosis than Senescence (%) 0 18 wild-type MEFs (Supplementary Fig. 3a) . However, the apoptosis rate –/– –/– –/– WT 1/2 2/2 Arf intheprostateofPten Skp2 micewashigherthaninwild-type, Skp2 Skp2 1/2 2/2 –/– Pten and Skp2 mice (see later and Supplementary Fig. 3b). Arf As cellular senescence is largely dependent on activation of the Arf 2,21,22 Figure 1 | Skp2 loss triggers a new senescence response in MEFs in the p19 –p53 pathway in MEFs , we determined whether this pathway context of Pten inactivation and Arf deficiency by a p19Arf–p53-independent is activated in Pten1/2 Skp22/2 MEFs. Notably, we found that p19Arf 1/2 2/2 pathway. a, Primary MEFs at passage 5 from various mouse embryos were and p53 protein levels in Pten Skp2 MEFs were comparable to plated for senescence assay. WT, wild type. b, Cell lysates were collected from levels in wild-type MEFs (Fig. 1b and Supplementary Fig. 4), suggesting primary MEFs of various genotypes of mouse embryos for western blot that the p19Arf–p53 pathway may not be involved in the senescence analysis. The lysates from wild-type MEFs treated with c-irradiation for response in Pten1/2 Skp22/2 MEFs. To test this hypothesis further, 60 min served as a positive control for p53. c, Primary Pten1/2 Skp22/2 MEFs we exposed MEFs of various genotypes to two well-established p53- infected with retroviruses expressing various control shRNA (shRNA-ctrl), inactivating tools: a short hairpin RNA (shRNA) against p53 (ref. 23) or p53 shRNA (shp53), or dominant-negative p53 (p53-DN) were plated for d a dominant-negative p53 mutant (p53-DN)24. Notably, in both condi- senescence assay andwestern blot analysis. , Primary MEFs at passage 5 from 2/2 various genotypes of mouse embryos were plated for senescence assay. tions cell growth was promoted in wild-type and Pten MEFs Results are presented as mean 6 s.d. from a representative experiment (Supplementary Fig. 5a, b), but neither of them overcame the cellular performed in triplicate. ***P , 0.001 using two-tailed Student’s t-test, n 5 3. senescence nor the growth arrest in Pten1/2 Skp22/2 MEFs (Fig. 1c and Supplementary Fig. 5c), suggesting that Skp2-deficiency cooperates cellular senescence that does not involve DNA damage and can sup- with Pten inactivation to trigger a new senescence response by a press transformation even when the p19Arf–p53 response is impaired. p19Arf–p53-independent pathway. p19Arf induction is required for cellular senescence in MEFs in the p27, p21 and Atf4 induction contribute to senescence Arf context of acute Pten inactivation25, whereas loss of p19 leads to cell We next examined the molecular mechanism by which Skp2 defi- immortalization2,21,22. We investigated whether Skp2 inactivation ciency synergizes with oncogenic insults to trigger cellular sene- could elicit cellular senescence in an Arf-deficient genetic background. scence. Although p53 and p19Arf levels remained unchanged, we 2 2 2 2 Notably, Arf / Skp2 / MEFs showed massive cellular senescence found that Skp2 deficiency cooperated with Pten inactivation or similar to Pten1/2 Skp22/2 MEFs (Fig. 1d). Moreover, p53 expression Arf loss to induce p27 expression (Fig. 2a, b). p21 expression was was not induced in Arf2/2 Skp22/2 MEFs (Supplementary Fig. 6a). also increased in Pten1/2 Skp22/2 and Arf2/2 Skp22/2 MEFs (Fig. 2a, This cellular senescence profoundly suppressed the growth of Arf2/2 b). E2F1, cyclin D1 and Cdt1, involved in cell cycle progression and MEFs (Supplementary Fig. 6b). Skp2 deficiency also induced cellular DNA replication, are also targets for Skp2 (refs 12, 29). We found that senescence after p53 inactivation (Supplementary Fig. 6c, d). cyclin D1, but not E2F1 and Cdt1, were induced in Pten1/2 Skp22/2 DNA damage has been recently associated with cellular sene- MEFs (Supplementary Fig. 7a). Because cyclin D1 promotes cell cycle scence26–28. However, we found no evidence of DNA-damage- progression, its upregulation is unlikely to be involved in mediating response activation in Pten1/2 Skp22/2 MEFs, as determined by senescence in Pten1/2 Skp22/2 MEFs. the levels of phosphorylated-(S15)-p53 and -c-H2ax (also known Endoplasmic reticulum (ER) stress proteins such as BiP (also as c-H2afx) (Supplementary Fig. 4). Collectively, these results sup- known as Hspa5 or GRP78), phospho-Perk (p-Perk), and Atf4 are port the notion that Skp2 inactivation can trigger a new type of induced after oncogenic insults and have an important role in cellular 375 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 464 | 18 March 2010

–/– strongly indicate that the concomitant upregulation of p27, –/– Skp2 p21and Atf4 is a required and powerful engine for the induction of +/– +/– –/– –/– /– /– Skp2 – – cellular senescence upon Skp2 inactivation. abWT Pten Pten Skp2 WT Arf Arf Skp2 c shGFP shp27-1 sh27-2 p27 p27 p27 Skp2 loss restricts tumorigenesis independently of Arf-p53 p21 p21 p21 We found that inactivation of Skp2 in the presence of an oncogenic Atf4 Atf4 α-tubulin stress results in the induction of cellular senescence that opposes trans- Arf p-eIF2α p-eIF2α formation in vitro even when the p19 –p53 response is impaired. We β-actin Hsp90 next determined whether Skp2 loss restricts tumorigenesis in vivo through similar mechanisms, and first analysed tumorigenesis in 2/2 1/2 d e shGFP shAtf4-1shAtf4-2 Skp2 Pten compound mutants (Supplementary Fig. 11a). Atf4 Although Pten heterozygous inactivation reduced lifespan in mice, compound Skp2 deficiency prolonged overall survival (Fig. 3a). β -gal β-actin 1/2 50 Pten mice develop lymphadenopathy and adrenal tumours (pheo- 32,33 1/2 40 chromacytoma) at complete penetrance . As expected, Pten mice 30 g 20 shGFP shp21-1 sh21-2 developed adrenal tumours with 100% penetrance by 1 year of age, 10 p21 whereas Skp2 loss remarkably abrogated adrenal tumour formation in Senescence (%) 0 shGFP shp27-1 shp27-2 compound mutants (P , 0.0001; Fig. 3b, top, c and Supplementary α-tubulin Fig. 11b). Pten protein expression in adrenal tissues was comparable 1/2 1/2 2/2 f h between wild-type, Pten and Pten Skp2 mice, before or after tumour occurrence, suggesting that there is no loss of heterozygosity at the Pten locus in the adrenal tissues in any of these mutants and con- β -gal β -gal

50 ditions (Supplementary Fig. 11c). Lymphadenopathy after Pten inac-

40 40 tivation was also profoundly inhibited by Skp2 loss (P , 0.01; Fig. 3b, 30 30 20 20 a WT n = 31 b P < 0.0001 10 10 Senescence (%) 110 Skp2–/– 100

0 Senescence (%) 0 10/10 shGFP shAtf4-1 shAtf4-2 shGFP shp21-1 shp21-2 100 n = 34 80 90 Pten+/– 60 Adrenal 80 n = 42 40 (10–12 months) Figure 2 | Upregulation of p27, p21 and Atf4 drives cellular senescence in +/– –/– 70 Pten Skp2 20 1/2 2/2 2/2 2/2 n = 36 Pten Skp2 and Arf Skp2 MEFs. a, b, Cell lysates were 60 0 0/10 0/10 50 collected from primary MEFs of various genotypes of mouse embryos for incidence (%) Tumour WT Pten+/– Pten+/– 1/2 2/2 40 P = 0.0013 1.6 Skp2–/– western blot analysis. c, d, Pten Skp2 MEFs at passage 2 infected with 30 1.4 20 1.2 lentiviruses expressing green fluorescent protein (GFP) shRNA (shGFP) or 1.0 Lymphoid Cumulative survival (%) 10 ** c 0.8 (5 months) p27 shRNA (shp27-1 and -2) were plated for western blot analysis ( ) and 0 0.6 senescence assay (d). Results are mean 6 s.d. from a representative 0.0 2.5 5.0 7.5 10.0 12.5 15.0 (g) Weight 0.4 1/2 2/2 Months 0.2 e f 0 experiment performed in triplicate. , , Pten Skp2 MEFs at passage 2 Pten+/– Pten+/– –/– infected with lentiviruses expressing GFP shRNA or Atf4 shRNA were plated c Pten+/– Pten+/–Skp2–/– Skp2 for western blot analysis (e) and senescence assay (f). g, h, Pten1/2 Skp22/2 MEFs at passage 2 infected with lentiviruses expressing GFP shRNA or p21 d Pten+/– Pten+/–Skp2–/– shRNA were plated for western blot analysis (g) and senescence assay (h). ×10

30 senescence . We did not find a significant increase in BiP or p-Perk β -gal 1 2 2 2 (Supplementary Fig. 7b and data not shown) in Pten / Skp2 / ×40 MEFs. In contrast, Atf4 was markedly induced in Pten1/2 Skp22/2 MEFs (Fig. 2a). Likewise, we also observed a marked increase in Atf4 e +/– +/– –/– protein levels, but not p-Perk, in Arf2/2 Skp22/2 MEFs (Fig. 2b and Pten Pten Skp2 f Supplementary Fig. 7c). The induction of Atf4 protein levels in Pten1/2 Skp22/2 MEFs was not accompanied by messenger RNA Ki67 upregulation, nor by the enhanced Atf4 protein stability (Sup- 40 ** ** plementary Fig. 8 and data not shown). Instead, we observed an 30 increase in phosphorylated eIF2a (p-eIF2a; also known as p-Eif2s1) 20 1/2 2/2 2/2 2/2 in Pten Skp2 and Arf Skp2 MEFs compared to wild-type cells (%) 10 Ki67-positive 0 cells (Fig. 2a, b). Because p-eIF2a positively regulates Atf4 trans- WT Pten+/– Pten+/– lation31, our results indicate that Atf4 upregulation is probably trig- p27 (×40) p27 (×10) Skp2–/– gered by the enhancement of p-eIF2a levels. 1/2 2/2 Figure 3 | Skp2 deficiency restricts tumorigenesis after Pten inactivation As p27, p21 and Atf4 were induced in both Pten Skp2 and by inducing cellular senescence in vivo.a, Kaplan–Meier plot analysis of 2/2 2/2 Arf Skp2 MEFs, we next determined whether their upregula- cumulative survival of indicated mouse genotypes. b, Top, adrenal tissues tion contributes to senescence. p27 (also known as Cdkn1b) shRNA from various mouse genotypes (10–12 months) were analysed for efficiently abrogated p27 expression and partially rescued growth tumorigenesis; bottom, lymphoid tissues within the neck were obtained and arrest and cellular senescence in Pten1/2 Skp22/2 MEFs (Fig. 2c, d weighed from female mice of various genotypes (around 5 months). and Supplementary Figs 9a, b and 10a). Similarly, knockdown of Atf4 **P , 0.01 using two-tailed Student’s t-test, n 5 6. c, Histological analysis of adrenal tissues from 12-month-old Pten1/2 and Pten1/2 Skp22/2 mice. or p21 (also known as Cdkn1a) in these cells also partially reversed 1/2 cellular senescence and cell arrest (Fig. 2e–h and Supplementary Pten mice developed adrenal tumour (pheochromocytoma), which was profoundly inhibited in Pten1/2 Skp22/2 mice. d, Senescence analysis of Figs 9c and 10b, c). Concomitant knockdown of Atf4, p21 and p27 lymphoid tissue from 5-month-old Pten1/2 and Pten1/2 Skp22/2 mice. in these cells reversed cellular senescence more efficiently than their e, Lymphoid tissues from 5-month-old mice of the indicated genotypes were individual knockdown (Supplementary Fig. 10d). In contrast, in obtained for p27 immunohistochemistry. f, Quantification of Ki67 staining 2/2 Skp2 MEFs, p27 knockdown accelerated growth whereas Atf4 of lymphoid tissues from 5-month-old mice. Results are mean 6 s.d. knockdown did not (Supplementary Fig. 10e, f). These results **P , 0.01 using two-tailed Student’s t-test, n 5 3. 376 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES bottom and Supplementary Fig. 11d). Tumorigenesis was also markedly mouse prostates leads to invasive prostate cancers, it does not affect suppressed in other organs (for example, in the prostate, where the overall survival2. We did not detect a difference in overall survival prostatic intraepithelial neoplasia (PIN) incidence was profoundly between Ptenpc2/2 and Ptenpc2/2 Skp22/2 mice (Supplementary restricted by Skp2 inactivation; data not shown). Fig. 14b). To determine whether Skp2 inactivation along with Pten inactiva- Prostate cancer development in these mice was monitored by tion would trigger cellular senescence in vivo, we performed SA-b-gal magnetic resonance imaging (MRI) and histopathological analysis. staining in the few hyperplastic lymphoid lesions still identified in Consistent with our previous findings2, MRI analysis showed pro- 1/2 2/2 Pten Skp2 mice (see, for example, Supplementary Fig. 10d). state tumour masses in Ptenpc2/2 mice at 6 months of age, which We observed both cellular senescence and p27 induction in the were significantly reduced in Ptenpc2/2 Skp22/2 mice (Supplemen- 1/2 2/2 lymphoid tissues from Pten Skp2 mice (Fig. 3d, e and Sup- tary Fig. 14c). The average size of the prostate in Ptenpc2/2 mice was plementary Fig. 12), which inversely correlated with cell proliferation tenfold larger than in wild-type mice, whereas complete Skp2 loss (Fig. 3f). markedly reduced tumour weight after complete Pten inactivation We then examined whether Skp2 inactivation would also restrict (Fig. 5a). Histological analysis showed that Skp2 loss inhibited invas- 2/2 2/2 tumorigenesis after Arf loss by crossing Skp2 with Arf mice ive prostate cancer after Pten inactivation, albeit PIN lesions were still (Supplementary Fig. 13). Skp2 inactivation markedly prolonged the pc2/2 2/2 2/2 2/2 observed in Pten Skp2 mice (Supplementary Fig. 14d, e). overall survival of Arf mice (Fig. 4a). Around 33% of Arf mice Furthermore, this suppressive effect by Skp2 loss was persistent, as developed sarcoma and/or lymphoma within 1 year (Fig. 4b–d)34,35. 2/2 2/2 we also observed a profound reduction in tumour weight and inva- In contrast, none of the Arf Skp2 compound mutant mice sive prostate cancer in Ptenpc2/2 Skp22/2 mice at 15 months of age showed signs of tumour formation (P , 0.02; Fig. 4b–d). (Supplementary Fig. 14f, g). We next investigated, in vivo, the molecular basis for tumour sup- Senescence after Pten and Skp2 inactivation in the prostate pression elicited by Skp2 inactivation. We found that p27 protein Complete Pten inactivation in the prostate triggers a tumour- expression was synergistically induced in prostates from compound 2 suppressive cellular senescence response . We therefore examined mutants, as determined by immunohistochemistry and western blot whether this response could be further potentiated by Skp2 loss and analysis (Supplementary Fig. 15a, b), whereas p53 expression was affect tumorigenesis after complete Pten inactivation in the prostate. comparably induced in Ptenpc2/2 and Ptenpc2/2 Skp22/2 mice For prostate-specific inactivation, we made use of Cre-loxP-mediated recombination and probasin (Pbsn, also known as PB)-Cre4 transgenic mice expressing the Cre recombinase after puberty in the a 6 months of age d prostatic epithelium2. We obtained PtenloxP/loxP;PB-Cre4 and Vehicle MLN4924 PtenloxP/loxPSkp22/2;PB-Cre4 compound mutant mice, hereafter pc2/2 pc2/2 2/2 β -gal referred to as Pten and Pten Skp2 mice, respectively 900 800 (Supplementary Fig. 14a). Although complete Pten inactivation in 700 * 600 531.8 mg * 500 e 400 900 800 n = 5 a b P < 0.02 300 112 mg 200 41.6 mg 700 P = 0.002 AP weight (mg) 100 600 105 Arf–/– n = 21 0 100 35 9/27 pc–/– pc–/– 500 –/– –/– WT Pten Pten 95 Arf Skp2 n = 13 30 Skp2–/– 400 90 300 ** 85 25 12 months b 200 80 20 100 Tumour weight (mg) 75 0 70 P = 0.036 15 Vehicle MLN4924 65 10 60 55 5 0/10 0/12 β -gal 50 Tumour incidence (%) 0 Cumulative survival (%) pc–/– 45 –/– –/– WT Pten f Oncogenic insults Skp2 loss 40 WT Arf (e.g. one allele of (or Skp2 inhibitors) 0 5 10 15 20 Skp2 Months –/– Pten loss or Arf Arf inactivation)

c Arf–/– Arf–/– Skp2–/– d Arf–/– Arf–/– Skp2–/– β -gal

Ptenpc–/–Skp2–/– Skp2–/– c

Proliferation Senescence (p27, p21 and Atf4 ) β -gal

WT Ptenpc–/– β -gal Tumorigenesis Ptenpc–/–Skp2–/– Skp2–/– Skin Lymphoid Figure 5 | Skp2 deficiency restricts prostate cancer development by Figure 4 | Skp2 inactivation restricts tumorigenesis upon Arf deficiency. triggering cellular senescence in vivo. a, Biopsy of anterior prostate (AP) a, Kaplan–Meier plot analysis of cumulative survival of Arf2/2 and tumours at 24 weeks from various genotypes of mice and their actual sizes Arf2/2 Skp22/2 mice. b, A cohort of wild-type, Arf2/2 and Arf2/2 Skp22/2 and weights. Results are mean 6 s.d. *P , 0.05 using two-tailed Student’s mice were analysed for tumorigenesis within a 1-year period. Arf 2/2 t-test, n 5 5. b, c, Senescence analysis of anterior prostate from Ptenpc2/2 Skp22/2 mice did not develop any tumour up to 1 year observation. Nine out and Ptenpc2/2 Skp22/2 mice aged 3 months (b) or 15 months (c). A of twenty-seven Arf 2/2 mice developed either sarcoma or lymphomas, representative section from three mice is presented for each genotype. whereas none of 12 Arf 2/2 Skp22/2 mice developed tumours. The statistic Original magnifications, 340 (b) and 320 (c). d, PC3 cells were treated with was analysed by chi-squared test, x2. c, Histopathological analysis of skin vehicle or 0.1 mM MLN4924 for 4 days and collected for cellular senescence tissues from Arf2/2 and Arf2/2 Skp22/2 mice at 1 year old. Arrow indicates assay. e, Nude mice bearing PC3 xenograft tumours (around 300 mm3) were sarcoma. d, Histopathological analysis of lymphoid tissues lymphoid tissues treated with vehicle or MLN492, and tumour weight was measured. from Arf2/2 and Arf2/2 Skp22/2 mice at 1 year of age. The arrow indicates f, Working model for tumour-suppressive cellular senescence driven by lymphoma. Original magnification, 340 (c, d). oncogenic insults and Skp2 deficiency. 377 ©2010 Macmillan Publishers Limited. All rights reserved ARTICLES NATURE | Vol 464 | 18 March 2010

(Supplementary Fig. 15c). Skp2 deficiency profoundly enhanced cel- effective in view of the fact that complete Skp2 inactivation in the mouse lular senescence upon Pten inactivation (Fig. 5b). This was observed is compatible with life, whereas cellular senescence is only triggered by at earlier time points and inversely correlated with cell proliferation Skp2 inactivation in conjunction with oncogenic conditions. (Supplementary Fig. 16a, b). Notably, this response was also sustained over time. We could detect massive b-gal positivity in pros- METHODS SUMMARY tates from Ptenpc2/2 Skp22/2 mice even at 15 months of age, whereas PtenloxP/loxP, Arf2/2 and Skp22/2 mice were generated as described previ- b-gal positivity was barely detected at that age in prostates from ously2,18,35. Female PtenloxP/loxP mice were crossed with male PB-Cre4 transgenic Ptenpc2/2 mice (Fig. 5c). Thus, Skp2 inactivation potentiates and mice for the prostate-specific deletion of Pten. MEFs from wild-type and 2/2 40,41 sustains over time the senescence response elicited by an oncogenic Skp2 mice were prepared as previously described and cultured in stimulus, suggesting that pharmacological inhibition of Skp2 may be DMEM containing 10% FBS. Cellular senescence was determined by assessing SA-b-gal activity, and the in vivo cell proliferation assay was performed by Ki67 used as a powerful pro-senescence approach for cancer therapy and staining on the paraffin tissue sections. The cell transformation assay was deter- chemoprevention. mined by the soft agar assay. p53 shRNA is from S. W. Lowe and the pBabe-p53 dominant-negative construct is a gift from M. Oren. MLN4924 was obtained Skp2–SCF complex inactivation triggers senescence from Millennium Pharmaceuticals. To corroborate the potential use of such an approach for cancer Full Methods and any associated references are available in the online version of therapy, we determined whether pharmacological inactivation of the paper at www.nature.com/nature. the Skp2–SCF complex induces cellular senescence in p53-deficient cells and, importantly, suppresses the growth of the pre-formed Received 9 October 2009; accepted 11 January 2010. tumours. To this end, we took advantage of MLN4924 (ref. 36)— 1. Sharpless, N. E. & DePinho, R. A. Cancer: crime and punishment. Nature 436, an inhibitor for the neddylation of cullin 1, which is a component of 636–637 (2005). Skp2–SCF complex. We used PC3 prostate cancer cells for this pre- 2. Chen, Z. et al. Crucial role of p53-dependent cellular senescence in suppression of clinical analysis because these cells are both p53-null and Pten-null, Pten-deficient tumorigenesis. Nature 436, 725–730 (2005). 3. Xue, W. et al. 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Shim, E. H. et al. Expression of the F-box protein SKP2 induces hyperplasia, dysplasia, and low-grade carcinoma in the mouse prostate. Cancer Res. 63, induction. Our results are supported by recent reports showing that 1583–1588 (2003). acute inactivation of the von Hippel–Lindau (VHL) tumour sup- 16. Lin, H. K. et al. Phosphorylation-dependent regulation of cytosolic localization and pressor in vitro or overexpression of the human T-lymphotropic virus oncogenic function of Skp2 by Akt/PKB. Nature Cell Biol. 11, 420–432 (2009). type 1 (HTLV-1) Tax triggers Skp2 downregulation and cellular sen- 17. Chiarle, R. et al. S-phase kinase-associated protein 2 expression in non-Hodgkin’s 37,38 lymphoma inversely correlates with p27 expression and defines cells in S phase. escence . Second, we show that cellular senescence driven by Skp2 Am. J. Pathol. 160, 1457–1466 (2002). inactivation along with oncogenic insults takes place without the 18. Nakayama, K. et al. Targeted disruption of Skp2 results in accumulation of cyclin E Arf activation of the p19 –p53 failsafe pathway. Although senescence and p27Kip1, polyploidy and centrosome overduplication. EMBO J. 19, 2069–2081 is also observed in p53/Pten-null cells such as PC3, it will be important (2000). to determine the specific genetic states that favour evasion of this 19. Lin, A. W. et al. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev. 12, failsafe mechanism, also in a cell-type-specific manner. For instance, 3008–3019 (1998). loss or constitutively low expression of p27, p21 and Atf4 could impair 20. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras this response. This knowledge will in turn identify new pharmaco- provokes premature cell senescence associated with accumulation of p53 and INK4a logical nodes of tumour-type-specific intervention. Third, we show p16 . 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Down-regulation tive syndrome39. of wild-type p53 activity interferes with apoptosis of IL-3-dependent As Skp2 can in principle be subjected to specific pharmacological hematopoietic cells following IL-3 withdrawal. EMBO J. 13, 1368–1374 (1994). 25. Chen, Z. et al. Differential p53-independent outcomes of p19Arf loss in inhibition because of its enzymatic activity, our results call for the oncogenesis. Sci. Signal. 2, ra44 (2009). development and optimization of Skp2 small molecule inhibitors. 26. Bartkova, J. et al. Oncogene-induced senescence is part of the tumorigenesis Skp2 pharmacological inhibition could be particularly appealing and barrier imposed by DNA damage checkpoints. Nature 444, 633–637 (2006). 378 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 ARTICLES

27. Di Micco, R. et al. Oncogene-induced senescence is a DNA damage response 39. Agarwal, A. et al. Absence of SKP2 expression attenuates BCR-ABL-induced triggered by DNA hyper-replication. Nature 444, 638–642 (2006). myeloproliferative disease. Blood 112, 1960–1970 (2008). 28. Mallette, F. A., Gaumont-Leclerc, M. F. & Ferbeyre, G. The DNA damage signaling 40. Lin, H. K., Bergmann, S. & Pandolfi, P. P. Cytoplasmic PML function in TGF-b pathway is a critical mediator of oncogene-induced senescence. Genes Dev. 21, signalling. Nature 431, 205–211 (2004). 43–48 (2007). 41. Yang, W. L. et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. 29. Yu, Z. K., Gervais, J. L. & Zhang, H. Human CUL-1 associates with the SKP1/SKP2 Science 325, 1134–1138 (2009). complex and regulates p21CIP1/WAF1 and cyclin D proteins. Proc. Natl Acad. Sci. USA 95, 11324–11329 (1998). Supplementary Information is linked to the online version of the paper at 30. Denoyelle, C. et al. Anti-oncogenic role of the endoplasmic reticulum differentially www.nature.com/nature. activatedby mutationsin theMAPK pathway. Nature CellBiol. 8, 1053–1063 (2006). Acknowledgements We are grateful to C. J. Sherr, S. W. Lowe and M. Oren for mice 31. Kim, I., Xu, W. & Reed, J. C. Cell death and endoplasmic reticulum stress: disease and reagents. We would also like to thank B. Carver, L. DiSantis, J. Clossey and relevance and therapeutic opportunities. Nature Rev. Drug Discov. 7, 1013–1030 S. Megan for editing and critical reading of the manuscript, J. A. Koutcher, C. Le, (2008). C. Matei and M. Lupa for MRI analysis, as well all the members of the Pandolfi 32. Di Cristofano, A. et al. Impaired Fas response and autoimmunity in Pten1/2 mice. laboratory for comments and discussion. We extend our thanks to M. Rolfe, Science 285, 2122–2125 (1999). P. G. Smith, and Millennium Pharmaceuticals for discussion and for providing the 33. Di Cristofano, A., Pesce, B., Cordon-Cardo, C. & Pandolfi, P. P. Pten is essential for MLN4924 compound. This work was supported by NIH grants to P.P.P. and M.D. embryonic development and tumour suppression. Nature Genet. 19, 348–355 (1998). Anderson Trust Scholar Award and DOD Prostate Cancer New Investigator Award 34. Kamijo, T., Bodner, S., van de Kamp, E., Randle, D. H. & Sherr, C. J. Tumor spectrum to H.K.L. in ARF-deficient mice. Cancer Res. 59, 2217–2222 (1999). Author Contributions H.K.L. and P.P.P. designed the experiments and wrote the 35. Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the manuscript; H.-K.L., Z.C., G.W., S.-W.L., C.N., C.-H.C., W.-L.Y., J.W. and A.E. ARF alternative reading frame product p19 . Cell 91, 649–659 (1997). performed the experiments; C.C.-C. and J.T-F. performed the histopathological 36. Soucy, T. A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to analysis of the mice; K.I.N. provided the Skp22/2 mice. treat cancer. Nature 458, 732–736 (2009). 37. Young, A. P. et al. VHL loss actuates a HIF-independent senescence programme Author Information Reprints and permissions information is available at mediated by Rb and p400. Nature Cell Biol. 10, 361–369 (2008). www.nature.com/reprints. The authors declare no competing financial interests. 38. Kuo, Y. L. & Giam, C. Z. Activation of the anaphase promoting complex by HTLV-1 Correspondence and requests for materials should be addressed to P.P.P. tax leads to senescence. EMBO J. 25, 1741–1752 (2006). ([email protected]).

METHODS laboratories), anti-Ki67 (Novocastra), anti-Pten (Neomarkers) and anti-p53 shRNA-mediated silencing. For the retrovirus-infection system, p27 shRNA (Novocastra) antibodies. (59-GTGGAATTTCGACTTTCAG-39), Atf4 shRNA (59-GAGCATTCCTTTAG Cell proliferation, transformation and senescence. Primary MEFs were iso- TTTAG-39), and GFP shRNA (59-GCAAGCTGACCCTGAAGTTC-39) were sub- lated from individual embryos of various genotype at passage 2, infected with cloned into the pSUPER-puro vector (Oligoengine) according to standard proce- retroviruses or lentiviruses expressing GFP shRNA, p27 shRNA or Atf4 shRNA 21 dures and transfected into Phoenix packaging cells. For lentiviral shRNA infection, for 2 days, selected with 2 mgml puromycin for 4 days, and plated for the cell 4 293T cells were co-transfected with p27, Atf4, p21, Skp2 or GFP control shRNA proliferation and senescence assay. For cell proliferation assay, 2 3 10 MEFs along with packing plasmids (DVPR8.9) and envelope plasmid (VSV-G) using were seeded in 12 wells in triplicate, collected, and stained with trypan blue at Lipofectamine 2000 reagents according to the manufacturer’s instructions. Skp2- different days. Numbers of viable cells were directly counted under the micro- 4 lentivivral shRNA-1 (59-GATAGTGTCATGCTAAAGAAT-39), p27-lentivivral scope. To determine cellular senescence, MEFs were plated at 10 cells per well of shRNA-1 (59-CGCAAGTGGAATTTCGACTTT-39), p27-lentivivral shRNA-2 a 6-well plate in triplicate, and after 4 days SA-b-gal activity was measured using (59-CCCGGTCAATCATGAAGAACT-39), Atf4-lentivivral shRNA-1 (59-GCGAG the senescence detection kit (Calbiochem) and quantified (around 100–200 cells TGTAAGGAGCTAGAAA-39), Atf4-lentivivral shRNA-2 (59-CGGACAAAGATA per well). For in vivo cellular senescence, frozen sections 6-mm thick were stained CCTTCGAGT-39), p21-lentivivral shRNA-1 (59 CTGGTGTCTGAGCGGCCT for b-gal as described earlier. For in vivo cell proliferation, the paraffin section GAA-39), p21-lentivivral shRNA-2 (59-GACAGATTTCTATCACTCCAA-39), was used for Ki67 staining, and the percentages of Ki67-positive cells (around and GFP shRNA (59-GCAAGCTGACCCTGAAGTTC-39) were transfected with 500 cells) from each sample were counted. For transformation assay, wild-type packing plasmids into 293T cells for 2 days, and virus particles containing p27, p21, and Skp22/2 MEFs (3 3 104) infected with Ras(G12V) and E1A were suspended Atf4, Skp2 or GFP shRNA were used to infect mammalian cells. All the infected cells in a medium containing 0.3% agar onto solidified 0.6% agar per well of a 6-well were cultured in a medium containing the appropriate antibiotics. plate, and the number of colonies was counted after 21 days. Western blot analysis and immunohistochemistry. Cell lysates were prepared Apoptosis assay. Primary MEFs of various genotypes of mouse embryos were with RIPA buffer (PBS, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS cultured in 10% FBS for 2 days; cells were collected and labelled with Annexin- and protease inhibitor cocktail (Roche)). The following antibodies were used for V–FITC, followed by a flow cytometry analysis. Arf western blot analysis: anti-p19 (NeoMarkers), anti-p53 (Novocastra), anti-p21 MRI. Individual mice were subjected to MRI assessment for the detection of (Santa Cruz), anti-b-actin (Sigma), anti-Hsp90 (BD transduction laboratories), prostate tumours as described42. anti-p27 (BD transduction laboratories), anti-a-tubulin (Sigma), anti-phospho- In vivo drug treatment in the preclinical tumour model. Nude mice bearing p53 (Ser15) (Cell Signaling), anti-phospho-H2ax (Ser139) (Cell Signaling), PC3 xenograft tumours (around 300 mm3) were treated with vehicle or anti-phospho-eIF2a (Ser51) (Cell Signaling), anti-eIF2a (Cell Signaling), anti- 90 mg kg21 MLN492. Tumour weight was measured at the time of collection phospho-Perk (Thr980) (Cell Signaling), anti-cyclin D1 (Santa Cruz), anti-E2F1 after 15 days of treatment with a scheduling regimen of 3 days of treatment (Santa Cruz), anti-Cdt1 (Proteintech Group), anti-Ras (Oncogene), anti-E1A followed by 3 days without treatment for a total of three courses. (Neomarkers), and anti-Atf4 (Santa Cruz). For immunohistochemistry, tissues were fixed in 10% formalin and embedded in paraffin in accordance with 42. Chen, Z. et al. Crucial role of p53-dependent cellular senescence in suppression of standard procedures. Sections were stained with anti-p27 (BD transduction Pten-deficient tumorigenesis. Nature 436, 725–730 (2005).

Dust-free quasars in the early Universe

Linhua Jiang1, Xiaohui Fan1,2, W. N. Brandt3, Chris L. Carilli4, Eiichi Egami1, Dean C. Hines5, Jaron D. Kurk2,6, Gordon T. Richards7, Yue Shen8, Michael A. Strauss9, Marianne Vestergaard1,10 & Fabian Walter2

The most distant quasars known, at redshifts z < 6, generally have However, J000520006 was not detected at 15.6 and 24 mm, and properties indistinguishable from those of lower-redshift quasars J030320019 was not detected at 24 mm. Note that J000520006 was in the rest-frame ultraviolet/optical and X-ray bands1–3. This puzz- not detected in our earlier, much shallower Spitzer 24 mm imaging4. ling result suggests that these distant quasars are evolved objects Figure 1 shows the Spitzer spectral energy distributions (SEDs) of even though the Universe was only seven per cent of its current age six representative objects in our sample. Their continuum shapes at at these redshifts. Recently one z < 6 quasar was shown not to have the wavelengths of the IRAC bands (0.5 # l0 # 1 mm) are consistent any detectable emission from hot dust4, but it was unclear whether that indicated different hot-dust properties at high redshift or if it is simply an outlier. Here we report the discovery of a second quasar without hot-dust emission in a sample of 21 z < 6 quasars. 10–13 Such apparently hot-dust-free quasars have no counterparts at low redshift. Moreover, we demonstrate that the hot-dust abundance 10–14 in the 21 quasars builds up in tandem with the growth of the central black hole, whereas at low redshift it is almost independent 10–15 of the black hole mass. Thus z < 6 quasars are indeed at an early J0005–0006 J0303–0019 evolutionary stage, with rapid mass accretion and dust formation. ) –13

The two hot-dust-free quasars are likely to be first-generation –2 10

quasars born in dust-free environments and are too young to have cm –1 formed a detectable amount of hot dust around them. 10–14 More than 40 quasars have been discovered at redshifts z < 6 (refs (erg s (erg λ

5, 6); at this epoch, the Universe was less than one billion years old. λ f 10–15 They harbour black holes with masses higher than 108 solar masses J0842+1218 J1137+3549 8 2,7 (10 M[) and emit radiation at about the Eddington limit . According to unification models of active galactic nuclei, the black 10–13 hole accretion disk is surrounded by a dusty structure8,9. A significant fraction of the quasar’s ultraviolet and optical radiation is absorbed 10–14 by the dust and is re-emitted at infrared wavelengths. In particular, the hottest dust is directly heated by the central engine and produces 10–15 near-infrared (NIR) emission10–12. Radiation from hot dust is J1250+3130 J1411+1217 observed to be a ubiquitous feature among quasars, and is strong 0.4 140.4 1 4 λ μ evidence for these unification models. 0 ( m) In order to study the properties of hot dust at high redshift, we Figure 1 | Spitzer SEDs of z < 6 quasars. < Filled circles, rest-frame SEDs of obtained deep infrared photometry of 21 z 6 quasars using the six representative quasars from our sample in the four IRAC channels, the Spitzer Space Telescope (referred to here as Spitzer). The quasar IRS PUI blue band (15.6 mm) and the MIPS 24 mm band. Typical on-source sample spans a redshift range of 5.8 , z , 6.4 and a luminosity range integration time of the Spitzer observations was 10230 min per source per of 227.9 , M1450 ,225.4, where M1450 is the absolute AB mag- band. The integration time for J000520006 at 15.6 and 24 mm was about nitude of the continuum at rest-frame 1,450 A˚ . A cosmology with four hours and for J030320019 at 24 mm was 50 min. The IRAC 4.5 mm 21 21 ˚ H0 5 70 km s Mpc , Vm 5 0.3 and VL 5 0.7 is adopted. The bandpass includes the strong Ha emission line (6,563 A) at this redshift, observations were carried out in the four IRAC channels (3.6, 4.5, enhancing the flux. Open circle (in J030320019), problematic photometry 5.8 and 8.0 mm), the IRS Peak-Up Imaging (PUI) blue band due to contamination from heavily blended neighbours. Circles with (15.6 mm) and the MIPS 24 mm band. These bands correspond to a downward arrows, 2s upper limits; error bars, measurement uncertainties s rest-frame wavelength range of 0.5 # l # 3.5 mm and trace the disk (1 ). Grey lines, template formed from the average of a large number of low- 0 redshift type 1 quasar SEDs13, and normalized to each object at l 5 3.6 mm. and hot dust radiation for z < 6 quasars. Here we use l and l0 to Blue and green lines, respectively disk and hot-dust components from the denote observed and rest-frame wavelengths, respectively. All the best model fits (the IRAC data are used to fit the disk emission, and the IRS quasars except SDSS J000552.342000655.8 (hereafter J000520006; PUI and MIPS data are then used to constrain the hot dust emission); red we use JHHMM 6 DDMM for brevity) and J030320019 were lines, sum of the two. In J000520006 and J030320019, a power law is not a detected with high signal-to-noise ratios in all the Spitzer bands used. good description for the disk emission at l0 . 1 mm.

1Steward Observatory, University of Arizona, 933 North Cherry Avenue, Tucson, Arizona 85721, USA. 2Max-Planck-Institut fu¨r Astronomie, Ko¨nigstuhl 17, D-69117 Heidelberg, Germany. 3Department of Astronomy and Astrophysics, Pennsylvania State University, 525 Davey Laboratory, University Park, Pennsylvania 16802, USA. 4National Radio Astronomy Observatory, PO Box 0, Socorro, New Mexico 87801, USA. 5Space Science Institute, 4750 Walnut Street, Suite 205, Boulder, Colorado 80301, USA. 6Max-Planck-Institut fu¨r Extraterrestrische Physik, Giessenbachstrasse, D-85748 Garching, Germany. 7Department of Physics, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, USA. 8Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, MS-51, Cambridge, Massachusetts 02138, USA. 9Department of Astrophysical Sciences, Peyton Hall, Princeton, New Jersey 08544, USA. 10Dark Cosmology Centre, the Niels Bohr Institute, Juliane Maries Vej 30, DK-2100, Copenhagen O, Denmark. 380 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

abz 01372 465 2 2

1 1 Slope = –0.04 ± 0.02 (5,100 Å)] λ 0 0

Slope = –0.03 ± 0.04 (3.5 μ m)/ λ L λ –1 J0303–0019 –1 J0303–0019 z≈6 quasars log[ λ L PG quasars12, 20, 21 J0005–0006 J0005–0006 Ref. 19 Ref. 13 –2 Ref. 22 –2

44 45 46 47 0.0 0.2 0.4 0.6 0.8 –1 log[λLλ(5,100 Å) (erg s )] log(1 + z)

Figure 2 | Luminosity and redshift dependence of the hot-dust abundance derived from the IRAC 3.5 mm and MIPS 24 mm fluxes, respectively. The for type 1 quasars. a, Hot-dust abundance (that is, rest-frame NIR-to- other circles represent 362 low-redshift (0 , z , 5.5) quasars from the optical flux ratio) as a function of optical luminosity at rest-frame 5,100 A˚ . literature12,13,19–22.Typical errors (1s) for z < 6 quasars are given in the upper b, Hot dust abundance as a function of redshift. Red circles, the 21 z < 6 left corner of a.Ina and b, dashed lines show the best linear fit to the sample quasars in our sample. Their rest-frame 5,100 A˚ and 3.5 mm luminosities are and its 3s range. with the average SED of low-redshift type 1 (unobscured) qua- narrow-line Seyfert 1 galaxies, because such galaxies tend to have 13 sars . At 15.6 and 24 mm(l0 < 2 and 3.5 mm), however, the quasar NIR emission similar to or stronger than that of ordinary Seyfert SEDs show wide diversity. For instance, J125013130 has strong galaxies16. NIR excess, whereas J000520006 and J030320019 are undetected NIR spectroscopy7,15 and our IRAC photometry show that in the MIPS 24 mm band. In a type 1 quasar, the radiation at J000520006 and J030320019 have normal rest-frame ultraviolet/ l0 , 1 mm is mostly from the accretion disk; at longer wavelengths optical SEDs and broad emission lines (although among the narrow- of a few micrometres, hot-dust radiation dominates over the disk est of the sample). It is very unlikely that emission from hot dust is radiation. We use a simple model to fit the Spitzer SEDs, consisting obscured by optically thick cooler dust at larger scales, as the broad- of a power-law disk component and a hot-dust blackbody with a line region is seen along the line of sight. Furthermore, these objects temperature of 1,200 K (ref. 14; Fig. 1). Most of the quasars in our are undetected at 250 GHz (r.m.s. noise of 0.5 mJy), ruling out cool sample show strong NIR emission excess that we interpret as due dust emission17,18. J000520006 was also observed by the Chandra to hot dust, but J000520006 and J030320019 do not show any X-ray satellite. Its X-ray emission is consistent with a typical type 1 detectable emission from hot dust. These two quasars have the quasar, indicating no unusual obscuration3. Although hot-dust emis- narrowest emission lines (line widths ,2,000 km s21) of the objects sion was thought to be a ubiquitous feature in quasars, it apparently in the sample7,15. They are not obviously luminous counterparts of does not exist in these two z < 6 quasars.

a 1 1 z < 0.7 z ≈ 6 Slope = 1.1 ± 0.2 0

Slope = 0.04 ± 0.05 (Hβ) –1 0 1

(5,100 Å)] 0.7 < z < 1.9 λ

Slope = –0.05 ± 0.04 (Mg II)

(3.5 μ m)/ λ L –1 λ –1 J0303–0019 1 z > 1.9 log[ λ L 0 J0005–0006 (Mg II and C IV ) Slope = 0.12 ± 0.08 (C IV) –1 –2 7 8 9 10 8 9 10 log[MBH (M . )] Figure 3 | Correlation between the hot-dust abundance and black hole same quasars measured with the two emission lines. All the mass values have mass for type 1 quasars. a, Sample of 223 low-redshift SDSS quasars13 been re-calibrated using the latest mass scaling relations23,29. The probability whose black hole masses (MBH) were derived from one of the three emission based on the generalized Kendall’s t test that a correlation is present between lines Hb,MgII and C IV (top to bottom: ref. 27). Typical errors (1s) are given the hot-dust abundance and black hole mass at z < 6 is 0.9987. Dashed line, in the top panel. Dashed lines, the best linear fits. b,11z < 6 quasars with best linear fit using the Buckley–James regression method and the EM black hole mass measurements from Mg II (filled circles) and/or C IV (open regression algorithm (they agree within the errors). The statistics were circles) in the literature2,7,15,28. Typical errors (1s) are given in the upper left calculated within the Astronomy Survival Analysis software package30.Ifa corner of the panel. The circle pairs connected by short lines represent the quasar has two measurements from Mg II and C IV, we take the average. 381 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

Indeed, hot-dust-free quasars like J000520006 and J030320019 and J030320019 are living in extremely young environments; they do not have counterparts at low redshift. Figure 2 illustrates the hot- are likely to be first-generation quasars, which have been born in a dust abundance for our z < 6 sample, as well as for an unbiased dust-free medium and are in their earliest evolution stage. Dust is sample of 362 low-redshift quasars with rest-frame NIR flux mea- being produced for the first time in those environments. surements in the literature12,13,19–22. Here we use the ratio of the rest- Received 23 November 2009; accepted 26 January 2010. frame NIR-to-optical (3.5 mm to 5,100 A˚ ) emission as an indicator of the hot-dust abundance. The hot-dust abundances for most objects 1. Fan, X. et al. A survey of z . 5.7 quasars in the Sloan Digital Sky Survey. III. are close to 1, with little dependence on luminosity over four orders Discovery of five additional quasars. Astron. J. 128, 515–522 (2004). , , 2. Jiang, L. et al. Gemini near-infrared spectroscopy of luminous z , 6 quasars: of magnitude or on redshift over a range of 0 z 6. The tight chemical abundances, black hole masses, and Mg II absorption. Astron. J. 134, correlation between the two luminosities suggests that hot dust is 1150–1161 (2007). directly heated by quasar activity. However, J000520006 and 3. Shemmer, O. et al. Chandra observations of the highest redshift quasars from the J030320019 do not show any hot-dust emission and thus signifi- Sloan Digital Sky Survey. Astrophys. J. 644, 86–99 (2006). cantly deviate from the correlation. No known quasars at low redshift 4. Jiang, L. et al. Probing the evolution of infrared properties of z , 6 quasars: Spitzer observations. Astron. J. 132, 2127–2134 (2006). have hot-dust abundances as low as these z < 6 quasars. This is the 5. Fan, X. et al. Constraining the evolution of the ionizing background and the epoch first concrete evidence that SEDs from the quasar central engine have of reionization with z , 6 quasars. II. A sample of 19 quasars. Astron. J. 132, evolved significantly at high redshift: accretion disks in most z < 6 117–136 (2006). quasars may have reached maturity, but dust structures are still evol- 6. Willott, C. J. et al. Four quasars above redshift 6 discovered by the Canada-France High-z Quasar Survey. Astron. J. 134, 2435–2450 (2007). ving rapidly. 7. Kurk, J. D. et al. Black hole masses and enrichment of z , 6 SDSS quasars. Quasars are powered by mass accretion onto central black holes Astrophys. J. 669, 32–44 (2007). and hot dust is directly heated by quasar activity, so the black hole 8. Antonucci, R. Unified models for active galactic nuclei and quasars. Annu. Rev. masses may provide useful clues to the puzzle as to why J000520006 Astron. Astrophys. 31, 473–521 (2003). and J030320019 do not have hot dust. Black hole masses in distant 9. Elitzur, M. The toroidal obscuration of active galactic nuclei. N. Astron. Rev. 52, 274–288 (2008). quasars can be estimated using mass scaling relations based on broad 10. Rieke, G. H. The infrared emission of Seyfert galaxies. Astrophys. J. 226, 550–558 emission line widths and continuum luminosities23. Figure 3 presents (1978). the hot-dust abundance as a function of the estimated black hole 11. Hyland, A. R. & Allen, D. A. An infrared study of quasars. Mon. Not. R. Astron. Soc. mass for quasars at redshifts from z , 0.7 to z < 6. At low redshift, 199, 943–952 (1982). 12. Haas, M. et al. The ISO view of Palomar-Green quasars. Astron. Astrophys. 402, the hot-dust abundance is almost independent of the black hole mass. 87–111 (2003). This explains why we do not find low-redshift quasars like 13. Richards, G. T. et al. Spectral energy distributions and multiwavelength selection J000520006 and J030320019, because at low redshift dust has sup- of type 1 quasars. Astrophys. J. 166 (Suppl.), 470–497 (2006). posedly been recycled between galaxies and the intergalactic medium, 14. Glikman, E., Helfand, D. J. & White, R. L. A near-infrared spectral template for and quasar host galaxies are rich in dust even before the central black quasars. Astrophys. J. 640, 579–591 (2006). 15. Kurk, J. D. et al. Near-infrared spectroscopy of SDSS J0303–0019: a low- hole first undergoes rapid accretion. At z < 6, however, the hot-dust luminosity, high-Eddington-ratio quasar at z , 6. Astrophys. J. 702, 833–837 abundance is roughly proportional to the black hole mass, indicating (2009). that the two grow at about the same rate. The two hot-dust-free 16. Ryan, C. J., De Robertis, M. M., Virani, S., Laor, A. & Dawson, P. C. The central quasars with the lowest hot-dust abundances have the smallest black engines of narrow-line Seyfert 1 galaxies. Astrophys. J. 654, 799–813 (2007). 8 17. Wang, R. et al. Millimeter and radio observations of z , 6 quasars. Astron. J. 134, hole masses (2–3 3 10 M[ ) and highest Eddington luminosity 617–627 (2007). ratios (,2) in the z < 6 sample. This suggests that they are in an early 18. Wang, R. et al. Thermal emission from warm dust in the most distant quasars. stage of quasar evolution with rapid mass accretion, but are too Astrophys. J. 687, 848–858 (2008). young to have formed a detectable amount of hot dust around them. 19. Hines, D. C. et al. Spitzer observations of high-redshift QSOs. Astrophys. J. 641, 85–88 (2006). In the local Universe, most dust in the interstellar medium is 20. Neugebauer, G. et al. Continuum energy distributions of quasars in the Palomar- produced by low- and intermediate-mass AGB stars, which develop Green Survey. Astrophys. J. 63 (Suppl.), 615–644 (1987). 0.5–1 billion years after the initial starburst. At z < 6, the age of the 21. Haas, M. et al. Dust in PG quasars as seen by ISO. Astron. Astrophys. 354, Universe was less than one billion years, and the first star formation 453–466 (2000). probably occurred less than half a billion years earlier. Therefore, 22. Herna´n-Caballero, A. et al. Mid-infrared spectroscopy of infrared-luminous galaxies at z , 0.5–3. Mon. Not. R. Astron. Soc. 395, 1695–1722 (2009). other dust production mechanisms may play an important role at 23. Vestergaard, M. & Peterson, B. M. Determining central black hole masses in high redshift. For example, quasar activity itself can efficiently pro- distant active galaxies and quasars. II. Improved optical and UV scaling duce dust through outflowing winds24. The strong correlation relationships. Astrophys. J. 641, 689–709 (2006). between the hot-dust abundance and black hole mass in z < 6 quasars 24. Elvis, M., Marengo, M. & Karovska, M. Smoking quasars: a new source for cosmic dust. Astrophys. J. 567, 107–110 (2002). suggests that hot dust in these quasars may be produced in this way. 25. Maiolino, R. et al. A supernova origin for dust in a high-redshift quasar. Nature 431, Supernovae are another alternative source of dust at high redshift. 533–535 (2004). For example, the colours of a reddened quasar at z 5 6.2 and the host 26. Stratta, G., Maiolino, R., Fiore, F. & D’Elia, V. Dust properties at z 5 6.3 in the host galaxy of GRB 050904 at z 5 6.29 cannot be explained by low-redshift galaxy of GRB 050904. Astrophys. J. 661, 9–12 (2007). extinction curves, but are well fitted by dust originating from super- 27. Shen, Y., Greene, J. E., Strauss, M. A., Richards, G. T. & Schneider, D. P. Biases in 25,26 virial black hole masses: an SDSS perspective. Astrophys. J. 680, 169–190 (2008). novae . Although the reddening dust in these two cases would be 28. Barth, A. J., Martini, P., Nelson, C. H. & Ho, L. C. Iron emission in the z 5 6.4 quasar on larger scales than the hot dust considered in our study, it does SDSS J114816.641525150.3. Astrophys. J. 594, 95–98 (2003). support the notion that dust properties can show strong evolution at 29. Vestergaard, M. & Osmer, P. S. Mass functions of the active black holes in distant high redshift. quasars from the Large Bright Quasar Survey, the Bright Quasar Survey, and the color-selected sample of the SDSS fall equatorial stripe. Astrophys. J. 699, Until now, multi-band observations have shown that z < 6 quasars 800–816 (2009). are well-evolved objects in the early Universe as they are indistin- 30. Lavalley, M., Isobe, T. & Feigelson, E. in Astronomical Data Analysis Software and guishable from lower-redshift quasars in the wavebands observed. Systems I (eds Worrall, D. M., Biemesderfer, C. & Barnes, J.) 245–247 (ASP Conf. However, our Spitzer observations, especially the discovery of two Ser. Vol. 25, Astronomical Society of the Pacific, 1992). hot-dust-free quasars, indicate that z < 6 quasars are indeed young Acknowledgements This work is based on observations made with the Spitzer and their hot dust evolves rapidly. The strong relation between the Space Telescope, which is operated by the Jet Propulsion Laboratory, hot-dust abundance and black hole mass shows an evolutionary rela- California Institute of Technology, under a contract with NASA. Support for this work was provided by NASA through an award issued by JPL/Caltech. tion at z $ 6 (Fig. 3): small black holes are growing rapidly, with hot X.F. acknowledges support by NSF AST, a Packard Fellowship for Science and dust building up at similar rates, towards a mature structure with a Engineering, and a John Simon Guggenheim Memorial Fellowship. W.N.B. was hot-dust abundance close to one. We thus suggest that J000520006 supported by the NASA ADP program. C.L.C. thanks the 382 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

Max-Planck-Gesellschaft and the Humboldt-Stiftung for support through the J.D.K. analysed NIR spectra of two hot-dust-free quasars; Y.S. and M.V. measured Max-Planck-Forschungspreis. J.D.K. thanks the DFG for support via black hole masses. All authors helped with the scientific interpretations and German-Israeli Project Cooperation. The Dark Cosmology Centre is funded by commented on the manuscript. the Danish National Research Foundation. Author Information Reprints and permissions information is available at Author Contributions L.J. and X.F. designed the project, reduced and analysed the www.nature.com/reprints. The authors declare no competing financial interests. data, and prepared the manuscript; W.N.B., M.A.S. and F.W. performed statistics Correspondence and requests for materials should be addressed to L.J. and edited the manuscript; C.L.C., E.E., D.C.H. and G.T.R. prepared observations; ([email protected]).

A transiting giant planet with a temperature between 250 K and 430 K

H. J. Deeg1,2, C. Moutou3, A. Erikson4, Sz. Csizmadia4, B. Tingley1,2, P. Barge3, H. Bruntt5, M. Havel6, S. Aigrain7,8, J. M. Almenara1,2, R. Alonso9, M. Auvergne5, A. Baglin5, M. Barbieri3,10, W. Benz11, A. S. Bonomo3, P. Borde´12, F. Bouchy13,14, J. Cabrera4,15, L. Carone16, S. Carpano17, D. Ciardi18, M. Deleuil3, R. Dvorak19, S. Ferraz-Mello20, M. Fridlund17, D. Gandolfi21, J.-C. Gazzano3, M. Gillon22, P. Gondoin17, E. Guenther21, T. Guillot6, R. den Hartog17, A. Hatzes21, M. Hidas23,24,G.He´brard13, L. Jorda3, P. Kabath4{, H. Lammer25,A.Le´ger12, T. Lister23, A. Llebaria3, C. Lovis9, M. Mayor9, T. Mazeh26, M. Ollivier12,M.Pa¨tzold16, F. Pepe9, F. Pont7, D. Queloz9, M. Rabus1,2{, H. Rauer4,27, D. Rouan5, B. Samuel12, J. Schneider15, A. Shporer26, B. Stecklum21, R. Street23, S. Udry9, J. Weingrill25 & G. Wuchterl21

Of the over 400 known1 exoplanets, there are about 70 planets that that the event originated on the target star itself. A false alarm from transit their central star, a situation that permits the derivation of possible nearby eclipsing binary stars, the light of which might have their basic parameters and facilitates investigations of their atmo- spread into CoRoT’s large (1699 3 2199) aperture mask, could therefore spheres. Some short-period planets2, including the first terrestrial be excluded8. Radial velocity observations (Fig. 2 and Supplemen- exoplanet3,4 (CoRoT-7b), have been discovered using a space mission5 tary Table 1) with the HARPS spectrograph9 spanning from 21 designed to find smaller and more distant planets than can be seen September 2008 to 20 September 2009 verified a Jupiter-mass planet from the ground. Here we report transit observations of CoRoT-9b, on a moderately eccentric orbit. which orbits with a period of 95.274 days on a low eccentricity of To assign reliable absolute values to the size and mass of the CoRoT- 0.11 6 0.04 around a solar-like star. Its periastron distance of 0.36 9b planet, a precise characterization of the planet host star is required. astronomical units is by far the largest of all transiting planets, yield- Spectra taken at the Thu¨ringer Landessternwarte and with the UVES ing a ‘temperate’ photospheric temperature estimated to be between spectrograph on the Very Large Telescope Unit 2 (ESO) indicated a 250 and 430 K. Unlike previously known transiting planets, the G3V star of nearly solar metallicity. Its evolutionary age is not strongly present size of CoRoT-9b should not have been affected by tidal heat constrained, ranging from 0.15 to 8 Gyr. We favour the higher end of dissipation processes. Indeed, the planet is found to be well described the age range, given the quiescent light curve and the absence of by standard evolution models6 with an inferred interior composition chromospheric activity in the spectra. Its rotational velocity of v sin 21 consistent with that of Jupiter and Saturn. irot , 3.5 km s implies (for a rotational inclination irot < 90u) a slow CoRoT observed the CoRoT-9 host star for 145 days starting on 15 rotation period of .14 days. Depletion of Li has been noted as a April 2008, in the target window E1_0651 of the survey-field LRc02 in feature of planet-hosting stars10 and indeed, its doublet at 6,707 A˚ the constellation Serpens Cauda. Two transit events (Fig. 1) were was not found in our spectra. detected in CoRoT’s three-colour photometry, the first one on 16 The combination of the light curve analysis, the radial velocity data May 2008. A model fit to the first transit event indicated a Jupiter- and the determined stellar parameters allows the derivation of absolute sized planet on an equatorial transit (for values, see Table 1). Alerted values for the planet’s mass, radius and density (Table 1). The planet by this event, two spectra of CoRoT-9 were obtained with the SOPHIE has a radius quite similar to Jupiter, with Rp 5 (1.05 6 0.04)RJupiter,but 7 spectrograph on 5 and 25 August 2008, showing a velocity difference a lower mass of Mp 5 (0.84 6 0.07)MJupiter,leadingtoadensityof of 35 6 21 m s21 between both epochs, which is compatible with a 0.90 6 0.13 g cm23, or about 68% that of Jupiter. giant planet. Tidal heating is expected to play a negligible role in the planet’s Observations of a transit on 1 June 2009 with the Wise Observatory evolution. The ratio of the rate of energy from tidal circularization 1 m telescope (Israel)and the IAC 80 cm telescope (Tenerife) confirmed (assuming an initial eccentricity similar to that of Jupiter) to that

1Instituto de Astrofı´sica de Canarias, C. Via Lactea S/N, E-38205 La Laguna, Tenerife, Spain. 2Department de Astrofı´sica, Universidad de La Laguna, E-38200 La Laguna, Tenerife, Spain. 3Laboratoire d’Astrophysique de Marseille, CNRS and Universite´ de Provence, 38 rue Fre´de´ric Joliot-Curie, F-13388 Marseille cedex 13, France. 4Institute of Planetary Research, German Aerospace Center, Rutherfordstrasse 2, D-12489 Berlin, Germany. 5LESIA, Observatoire de Paris, CNRS, Place J. Janssen, 92195 Meudon cedex, France. 6Universite´ de Nice- Sophia Antipolis, Observatoire de la Coˆte d’Azur, CNRS UMR 6202, 06304 Nice cedex 4, France. 7School of Physics, University of Exeter, Exeter, EX4 4QL, UK. 8Oxford Astrophysics, University of Oxford, Keble Road, Oxford OX1 3RH, UK. 9Observatoire de l’Universite´ de Gene`ve, 51 chemin des Maillettes, CH 1290 Sauverny, Switzerland. 10Dipartimento di Astronomia, Universita` di Padova, 35122 Padova, Italy. 11Universita¨t Bern, Physics Institute, Sidlerstrasse 5, CH 3012 Bern, Switzerland. 12Institut d’Astrophysique Spatiale, Universite´ Paris XI, F-91405 Orsay, France. 13IAP, 98bis boulevard Arago, F-75014 Paris, France. 14Observatoire de Haute-Provence, CNRS/OAMP, 04870 St Michel l’Observatoire, France. 15LUTH, Observatoire de Paris, CNRS and Universite´ Paris Diderot, 5 place Jules Janssen, F-92195 Meudon, France. 16Rheinisches Institut fu¨r Umweltforschung an der Universita¨tzu Ko¨ln, Aachener Strasse 209, D-50931, Ko¨ln, Germany. 17Research and Scientific Support Department, ESTEC/ESA, PO Box 299, 2200 AG Noordwijk, The Netherlands. 18NASA Exoplanet Science Institute/Caltech, South Wilson Avenue, Mail Code 100-22, Pasadena, California 91125, USA. 19University of Vienna, Institute of Astronomy, Tu¨rkenschanzstrasse 17, A-1180 Vienna, Austria. 20Institute of Astronomy, Geophysics and Atmospheric Sciences, Universidade de Saõ Paulo, Brasil. 21Thu¨ringer Landessternwarte, Sternwarte 5, D-07778 Tautenburg, Germany. 22University of Lie`ge, Alleé du 6 aouˆt 17, Sart Tilman, Lie`ge 1, Belgium. 23Las Cumbres Observatory Global Telescope Network, Inc., 6740 Cortona Drive, Suite 102, Santa Barbara, California 93117, USA. 24Sydney Institute for Astronomy, School of Physics, The University of Sydney, New South Wales 2006, Australia. 25Space Research Institute, Austrian Academy of Science, Schmiedlstrasse 6, A-8042 Graz, Austria. 26School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel. 27Center for Astronomy and Astrophysics, TU Berlin, Hardenbergstrasse 36, 10623 Berlin, Germany. {Present addresses: European Southern Observatory Chile, Alonso de Co´rdova 3107, Vitacura, Casilla 19001, Santiago de Chile, Chile (P.K.); Departamento de Astronomıá y Astrofı´sica, Pontificia Universidad Cato´lica de Chile, Casilla 306, Santiago 22, Chile (M.R.). 384 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

a Table 1 | Star and planet parameters of the CoRoT-9/9b system Parameter Value 1.005 ID (CoRoT-Window-Id) LRc02_E1_0651 ID (CoRoT/Exo-Dat) 0105891283 1.000 ID (GSC 2.3)N1RO059308 Position (J2000) 18:43:08.81 1 06:12:15.19 0.995 Magnitudes (ref. 17) U, B, V, r9,i9 14.74, 14.55, 13.69, 13.33, 12.86 Results from light-curve analysis 0.990 Planet period, P 95.2738 6 0.0014 days

Normalized flux Transit epoch, T0 HJD 24 54603.3447 6 0.0001 Transit duration, T14 8.08 6 0.10 h 0.985 Relative transit depth, DF/F0 0.0155 6 0.0005 Radius ratio, Rp/Rs 0.115 6 0.001 0.980 Impact parameter, b z0:06 b 0:01{0:01 Limb-darkening coefficient, u 0.57 6 0.06 0.004 Scaled semimajor axis*, a/R 93 6 3 0.000 s Orbital inclination*, i z0:01 0 89:99{0:04 –0.004 23 Residuals Stellar density*, rs 1.68 6 0.20 gcm 1/3 –0.004 –0.002 0.000 0.002 0.004 Ms /Rs* 1.06 6 0.04 (solar units) Orbital phase Results from radial velocity observations Radial velocity semi-amplitude, K 38 6 3 ms21 Figure 1 | Light curve and model fit of the CoRoT-9b transit. a, Phase- Orbital eccentricity, e 0.11 6 0.04 folded light curve from CoRoT around the first observed transit of CoRoT- Argument of periastron, z9 v 37{37u 9b, with the phase set to zero at mid-transit. The data from the three colour- Systemic radial velocity 19.791 6 0.002 km s21 210 bands of CoRoT have been summed to a ‘white’ band and the points have Mass function, f (5.3 6 1.3) 3 10 Msun been binned from an original cadence of 32 s to a duration of 96 s; the error Results from spectral typing of CoRoT-9 bars indicate the dispersion of the points within the bins. The second transit Stellar spectral type G3V 4 54 0 09 observed by CoRoT is not shown because it was only partially detected; its Stellar surface gravity{, log g . 6 . c.g.s. 5625 80 first 2 h were lost in an instrument interruption. The solid line represents the Stellar effective temperature{, Teff 6 K Metallicity{, [M/H] 20.01 6 0.06 model fit from which the primary fit-parameters in Table 1 have been Stellar mass, M (0.99 6 0.04)M derived. They indicate a transit close to the equator, and a stellar limb star Sun Stellar radius, Rstar (0.94 6 0.04)RSun 21 darkening that is in good agreement with predictions for the CoRoT pass Stellar rotational velocity, vsinirot #3.5 km s 21 band . This fit was based on the white-light data of the first transit event and Stellar rotational period, Prot $14 days included (1) the removal of a 2.5% contaminating fraction of light not Stellar distance 460 pc originating from CoRoT-9, (2) the generation of a model for the planetary Absolute physical parameters from combined analysis 0 84 0 07 transit22 assuming a linear relation for the stellar limb-darkening, and (3) the Planet mass, Mp ( . 6 . )MJupiter 23 Planet radius, Rp (1.05 6 0.04)RJupiter fitting of the model using a heuristic optimization algorithm . The 3 Planet density, r 0.90 6 0.13 gcm2 contaminating fraction of 2.5% was determined from pre-launch imagery24, p Planet orbit semi-major axis, a 0.407 6 0.005 AU taken with the INT/WFC on La Palma, after convolving these images with z0:025 Planet–star distance at transit, at 0:377 AU CoRoT’s point-spread function, and measuring the contribution of nearby {0:015 stars within CoRoT’s aperture mask. b, Residuals after subtraction of the * These values take into account the eccentricity as found by the radial-velocity observations. { These values are based on high-resolution (R < 65,000) spectra taken on 21 and 22 model fit. September 2008 with the ESO VLT/UVES in Dic1 mode (390 1 580), a slit width of 0.7 arcsec and analysis with the VWA software18,19. The planet’s quoted period was determined from the first transit observed by CoRoT and from 11 ground-based photometry of a transit on 5 September 2009, taken with the Euler 1.2 m from insolation is 30 times lower for CoRoT-9b than for telescope from La Silla, Chile, and the 2 m LCOGT Faulkes Telescope North at Haleakala HD80606b, and at least 1,000 times lower relative to all other transi- Observatory. For the transit duration, the time from first to fourth contact is given. The mass 12 e 3 2 2 3/2 3 ting planets. Similarly, we estimate a mass loss rate of about function indicates the ratio 5 (Mpsini) /(Mp 1 Ms) and is given by f 5 (1 2 e ) K P/(2pc), 6 21 where c is the gravitational constant. The stellar spectral type is based on data taken with the 8 3 10 gs , which is the lowest one among the known transiting spectrograph (R < 2,100) at the Thu¨ringer Landessternwarte (Tautenburg, Germany) on 27 July gas giants; escape processes have therefore not affected the planet 2008. The value for the stellar surface gravity is determined directly from spectroscopy. An alternative, logg 5 4.49 6 0.04, can be derived from the stellar density given by the light-curve significantly since its origin. That CoRoT-9b has a much larger 1/3 analysis and a weakly influencing component of Mstar . The stellar mass is derived from periastron distance than any other known transiting exoplanet also StarEvol20 evolutionary tracks. The stellar radius is derived from the quoted stellar mass and the 30 11,13 stellar density from the transit-fit. RJupiter 5 71492 km; MJupiter 5 1.8986 3 10 g. AU, strongly constrains its composition, independently of hypotheses astronomical units. on possible missing energy sources and associated radius inflation: An evolutionary model of CoRoT-9b (Fig. 3) shows a good match to the observed radius between 0 and 20 Earth masses of heavy elements, variations are expected to be very small for two reasons. First, from 6 comparable to the composition of giant planets in our Solar System . its low tidal dissipation we estimate a timescale for rotational slow- Effective temperatures of CoRoT-9b have been calculated using a down of the order of 100 Gyr; so its rotation should still be close to the black-body approximation for the host-star emission and a uniform unknown, but probably rapid, primordial rate. Second, the planet’s redistribution of absorbed heat energy between the planet’s day and radiative timescale at the photosphere, which is inversely proportional night side. Following the planet classification based on temperature to the cube of the photospheric temperature15, should be around 50 regimes in ref. 14, there are two possible outcomes for CoRoT-9b: It times longer than for a standard hot-giant planet with a 1,500 K irra- may be among class III planets with temperatures .350 K, which have diation temperature. clear atmospheres without cloud cover and low Bond albedos of Figure 4 shows a diagram of the eccentricity versus the period for all 0.09–0.12. With such an albedo, CoRoT-9b’s temperatures would known planets and for all transiting planets. As can be seen, CoRoT-9b range from 380 K at apoastron to 430 K at periastron. CoRoT-9b is the first transiting planet among those with longer periods that does may also be among Class II planets, with much lower temperatures not represent a case of extreme eccentricity with associated extreme (250–290 K) owing to a significantly higher Bond albedo of ,0.8 from temperature changes (for example, HD80806b’s temperature is the condensation of H2O in the upper troposphere. The strong inverse estimated to rise from ,800 K to ,1,500 K over a six-hour period dependency of the albedo on temperatures14 near 350 K will make it near periastron16). On the contrary, it is the first transiting planet possible to lock the planet into either class II or class III. Transits occur whose general properties coincide with the largest-known population not far from periastron, so temperatures during transit would be only of planets, those of longer periods and low-to-moderate eccentricities, slightly (,30 K) lower than at periastron. Day/night temperature but which were previously known only from radial velocity surveys. 385 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

60 5,000

40 ) –1 20 500 0

–20 CoRoT-9b HD 80606b Radial velocity (m s –40 50 Period, P (day) –60 HD 17156b

–0.4 –0.2 0 0.2 0.4 Orbital phase 0 Figure 2 | CoRoT-9 radial velocities. Radial velocity data from the HARPS spectrograph after subtraction of the systemic velocity, versus the orbital phase, set to zero at periapsis. The error bars on individual points are 1- 0.5 sigma measurement errors, derived from the fit of a cross correlation 0 0.2 0.4 0.6 0.8 1 function to the observed spectra. The solid line is the best-fit elliptical orbital Eccentricity, e solution; the root-mean-square of its residuals is 6.3 m s21. This solution was also constrained by the known ephemeris of the planetary transits. An Figure 4 | The orbital parameters of CoRoT-9b among extrasolar planets. F-test comparing a best-fit circular orbit to the adopted elliptic orbit gives The eccentricity and period of all 339 exoplanets for which both values are 95% confidence for the presence of an ellipticity that is significantly different known as of 1 November 2009. Solid dots are the 58 transiting planets among from zero. them—most of them have short periods of less than about 5 days and zero or low eccentricities. Only two further transiting planets have orbital periods Our results on CoRoT-9b show that these planets may be expected to longer than ten days; these are HD1715627 with 21.2 days and HD8080628–30 with be rather similar to the giants of our Solar System. 111.4 days. However, both of them also have the highest eccentricities among CoRoT-9b’s period is about ten times longer than of any other planets of similar periods. Open dots represent the remaining exoplanets, known only from radial velocity observations. Data are sourced from ref. 1. planet discovered through the transit method, which demonstrates the method’s potential to find longer-periodic planets. Further Received 30 November 2009; accepted 19 January 2010. observations of such planets will, however, present new challenges. CoRoT-9b’s distance from its host-star of ,0.9 milliarcseconds is too 1. Schneider, J. The Extrasolar Planets Encyclopedia Æhttp://exoplanet.eu/index.phpæ close for imaging, and regardless of its actual Bond albedo, its relative (1999–2010). 2. Dvorak, R. et al. CoRoT’s first seven planets: An overview. In Proc. ‘‘New secondary eclipse depths will be undetectable, being at the parts per Technologies for Probing the Diversity of Brown Dwarfs and Exoplanets’’ (EDP million level in both the visible and the near-infrared regime. The publications, 2009); preprint at Æhttp://arxiv.org/abs/0912.4655æ. most promising aspect of CoRoT-9b is that it will allow for spectro- 3. Le´ger, A. et al. Transiting exoplanets from the CoRoT space mission. VIII. CoRoT- scopy during primary transits, which may lead to the detection of 7b: the first super-Earth with measured radius. Astron. Astrophys. 506, 287–302 (2009). species at moderate temperatures in the visible and infrared spectra, 4. 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21. Sing, D. K. Stellar limb-darkening coefficients for CoRoT and Kepler. Astron. and Spain. CoRoT data are available to the public from the CoRoT archive: http:// Astrophys. 510, 21–27 (2010). idoc-corot.ias.u-psud.fr. The team at IAC acknowledges support by grant 22. Mandel, K. & Agol, E. Analytic light curves for planetary transit searches. ESP2007-65480-C02-02 of the Spanish Ministerio de Ciencia e Innovacio´n. The Astrophys. J. 580, L171–L175 (2002). German CoRoT Team (TLS and Univ. Cologne) acknowledges DLR grants 23. Geem, Z. W., Kim, J. H. & Loganathan, G. V. A new heuristic optimization 50OW0204, 50OW0603 and 50QP07011. Observations with the HARPS algorithm: Harmony Search. Simulation 76, 60–68 (2001). spectrograph were performed under the ESO programme ID 082.C-0120, and 24. Deleuil, M. et al. Exo-Dat: an information system in support of the CoRoT/ observations with the VLT/UVES under ID 081.C-0413(C). exoplanet science. Astron. J. 138, 649–663 (2009). 25. Morel, P. & Lebreton, Y. CESAM: a free code for stellar evolution calculations. Author Contributions H.J.D. coordinated the analysis and its interpretation. P. Astrophys. Space Sci. 316, 61–73 (2008). Barge., S.A., J.M.A., R.A., J.C., L.C., T.M., M.O., M.P. and B. Samuel contributed to 26. Guillot, T. The composition of transiting giant extrasolar planets. Phys. Scr. 130, the treatment of the light curve and the detection of the transits in the CoRoT data. 014023 (2008). F.B., D.Q., C.M., G.H., M.M., C.L., F. Pepe, A.H., W.B., S.A., S.U. and F. Pont prepared, 27. Barbieri, M. et al. HD 17156b: a transiting planet with a 21.2 day period and an performed and analysed radial velocity observations; A. Baglin, M.A., J.S., L.J., P. eccentric orbit. Astron. Astrophys. 476, L13–L16 (2007). Borde´.A.Le´ger, A. Llebaria and P.B. contributed fundamentally to the definition, 28. Fossey, S. J., Waldmann, I. P. & Kipping, D. M. Detection of a transit by the design and operation of the CoRoT instrument. A.E., B.T., S.C., R.D., M.F., M.G., M. planetary companion of HD 80606. Mon. Not. R. Astron. Soc. 396, L16–L20 Hidas, T.L., H.R., D.R., R.S., A.S., H.J.D., R.d.H., R.A., M.R., P.K., B. Stecklum and D.C. (2009). performed ground-based photometry; Sz.C., R.A., M.B. and A. S. Bonomo worked 29. Moutou, C. et al. Photometric and spectroscopic detection of the primary transit on light curve modelling and parameter fitting. M.D., H.B., D.G., J.-C.G., E.G. and of the 111-day-period planet HD 80606 b. Astron. Astrophys. 498, L5–L8 (2009). M.F. constitute the team that performed the stellar typing and related 30. Garcia-Melendo, E. & McCullough, P. R. Photometric detection of a transit of HD observations. T.G., M. Havel, J.S., H.L., G.W. and S.F.-M. performed the modelling 80606b. Astrophys. J. 698, 558–561 (2009). of the planet and the interpretation of its characteristics. All authors discussed the results and commented on the manuscript. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Acknowledgements The CoRoT space mission has been developed and is Correspondence and requests for materials should be addressed to H.J.D. operated by CNES, with the contributions of Austria, Belgium, Brazil, ESA, Germany ([email protected]).

A trapped single ion inside a Bose–Einstein condensate

Christoph Zipkes1, Stefan Palzer1, Carlo Sias1 & Michael Ko¨hl1

Improved control of the motional and internal quantum states of and therefore decays more slowly than the van der Waals interaction ultracold neutral atoms and ions has opened intriguing possibilities between two neutral atoms. Here r is the internuclear separation, q for quantum simulation and quantum computation. Many-body denotes the charge of the ion, a is the d.c. polarizability of the neutral effects have been explored with hundreds of thousands of quantum- atom and e0 is the vacuum permittivity.qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi The characteristic radius of the 1 degenerate neutral atoms , and coherent light–matter interfaces have interaction potential is r* 5 maq2=(4pBe )2, m denotes the reduced been built2,3. Systems of single or a few trapped ions have been used to 0 demonstrate universal quantum computing algorithms4 and to search mass of the two collision partners and B is Planck’s constant divided by p 87 174 1 * 5 for variations of fundamental constants in precision atomic clocks5. 2 . For example, for neutral Rb atoms and Yb ions, r 307 nm. Until now, atomic quantum gases and single trapped ions have been In the s-wave scattering regime, we expect the elastic scattering cross- section to be ,4pr*2. Owing to the slow decay of the polarization treated separately in experiments. Here we investigate whether they potential, pure s-wave scattering can be reached only at collision energies can be advantageously combined into one hybrid system,by exploring in the nanoelectronvolt energy range20,21. At higher collision energies, the immersion of a single trapped ion into a Bose–Einstein condensate E ?B2/mr*2, when several partial waves contribute to scattering, the of neutral atoms. We demonstrate independent control over the two 20 components of the hybrid system, study the fundamental interaction cross-section is still determined by r* and takes the following form : 1=3 processes and observe sympathetic cooling of the single ion by the p2 B2 condensate. Our experiment calls for further research into the s(E)~p 1z r2 16 mr2E possibility of using this technique for the continuous cooling of 6 quantum computers .Wealsoanticipatethatitwillleadtoexplora- One particularly valuable application of the strong atom–ion tions of entanglement in hybrid quantum systems and to fundamental interaction is sympathetic cooling of the ion using ultracold atoms. studies of the decoherence of a single, locally controlled impurity The possibility of cooling objects by immersing them in an ultracold 7,8 particle coupled to a quantum environment . atomic gas has been discussed theoretically for ions13, polar mole- Hybrid systems allow the exploration of physics much more cules22 and nanomechanical resonators23. The atomic gas provides a advanced than that which can be studied using the component reservoir that is much colder than the motional energy scales of the systems alone. In particular, the immersion of distinguishable trapped ion, and if elastic collisions dominate, sympathetic cooling24 particles into a quantum liquid has contributed significantly to our can be achieved. understanding of many-body systems. In liquid helium, for example, We prepare a single trapped ion inside a Bose–Einstein condensate vortex lattices have been observed using charged particles as markers for by combining an ion trap and magnetic and optical traps for the the vortex lines9. Moreover, in conventional10,11 and high-transition- neutral atoms into one set-up (Fig. 1 and Methods). We use a linear temperature12 superconductors, single impurity atoms lead to quasi- Paul trap to trap a single 174Yb1 ion. The neutral atoms are pre- particle excitations that profoundly affect the superconducting pared25, using standard techniques, as an ultracold but initially 87 properties. In future investigations of distinct single particles in com- non-degenerate gas of neutral Rb atoms in the jF 5 2, mF 5 2æ bination with quantum matter, it will be important to have a high hyperfine ground state in a magnetic trap, located 8 mm away from degree of control over these particles. In this regard, the excellent the centre of the ion trap. Subsequently, we magnetically transport control possible over the external and internal degrees of freedom of the neutral atoms into the ion-trapping region and prepare a Bose– a single ion trapped in a Paul trap is highly promising. Immersed into a Einstein condensate of approximately 3 3 104 atoms in an optical quantum gas, a single trapped ion not only acts as a probe but could also dipole trap several tens of micrometres away from the trapped ion. be used for local manipulation. Numerous applications of this hybrid During the preparation cycle of the neutral atoms (magneto-optical system have been foreseen, including sympathetic cooling13, the nuc- trapping, magnetic transport and evaporation in the magnetic trap), leation of localized density fluctuations in a Bose gas14–16, scanning which lasts for ,60 s, and before interaction with the ultracold neutral probe microscopy with previously unattainable spatial resolution17,18 cloud, the ion is not laser-cooled. Owing to resonant heating from the and hybrid atom–ion quantum processors19. The majority of these radio-frequency radiation used in evaporative cooling of the neutral proposals are based on there being a strong collisional interaction atoms, the ion reaches a temperature of 27.5 6 0.6 K, as measured by between the ion and the neutral atoms. laser fluorescence (Methods). During preparation of the Bose– The interaction between a single ion and a neutral atom is dominated Einstein condensate in the optical dipole trap, the ion initially partly by the polarization potential. Asymptotically, this potential has the overlaps with the large thermal cloud of neutral atoms at 200 nK, for form20 2 s. After this interaction, the temperature of the ion is measured by laser fluorescence to be T , 0.5 K, a reading whose precision is limited a 2 ~{ 1 q only by the resolution of the fluorescence measurement technique. V(r) 2 4 (4pe0) 2r This signals sympathetic cooling of the ion by the neutral atom cloud.

is the amplitude of the micromotion in the Paul trap. Comparison of the theoretical estimate with our data suggests a micromotion amplitude of approximately 2 nm, which is compatible with our trap parameters. Extrapolating to the limit in which d R 0 leads to kBT < Bv, which could allow ground-state cooling. We time-resolve the dynamics of the immersion cooling process by immersing a hot ion directly into the Bose–Einstein condensate. For this measurement we partly suppress pre-cooling of the ion in the thermal cloud of neutral atoms by displacing the ion diagonally in the x–y plane. As a result, the initial temperature of the ion is approxi- z mately 4 K. Then we quickly move the ion into the Bose–Einstein y x condensate, wait for a variable interaction time, release the neutral atoms and measure the ion’s temperature using the fluorescence method (Fig. 3a). The fluorescence method, although limited in its temperature resolution, is a reliable and independent method in this Figure 1 | Experimental apparatus. The linear Paul trap (grey) is positioned temperature range. We observe sympathetic cooling of the ion in the between the solenoids (copper) used to magnetically trap the neutral atoms. The neutral atoms are first prepared outside the ion trap and are condensate on a timescale of a few tens of milliseconds. After 60 ms, subsequently moved into the trap through a bore in the end-cap electrode. In the ion has reached a temperature of T 5 0.6 6 0.7 K, corresponding the final position, the atoms are loaded into a crossed-beam optical dipole to a temperature as low as our resolution limit in this measurement. trap (red) in which the final evaporative cooling to Bose–Einstein We simultaneously monitor the atom loss rate (Fig. 3b). While the condensation is performed (inset). The drawing is to scale and the distance ion is hot, atom losses are very small and we estimate a cooling between the square magnetic field coils is 36 mm. efficiency of approximately 1,000 vibrational quanta per collision. This is very efficient in comparison with laser-cooling, in which To observe interactions between the ion and the Bose–Einstein typically only a few quanta are removed per collision. Furthermore, condensate, we quickly move the pre-cooled ion over a distance of while the ion is hot the radius of its trajectory is comparable to the size Dx 5 140 mm, into the centre of the condensate. The potential depth of the condensate. As the ion cools, it becomes more strongly loca- of the optical dipole trap is 1 mK, which is less than the vibrational lized at the centre of the condensate and the loss rate of atoms level spacing of the ion-trap potential. Therefore, every collision will increases, possibly as a result also of the increase in the collisional result in the loss of a neutral atom. We monitor this loss as a function cross-section, s(E). of the interaction time. The data are displayed in Fig. 2. From the Apart from elastic scattering, there are other possible interaction decay of the atom number, we estimate the cross-section for atom processes between an ion tightly confined in a Paul trap and ultracold 213 2 loss to be sal 5 (2.2 6 0.2) 3 10 m (Methods). The measured neutral atoms. First, atoms and ions can undergo charge-exchange atom-loss cross-section allows us to estimate more precisely the col- collisions13,20,21,27 of the type Yb1 1 Rb R Yb 1 Rb1 or Yb1 1 Rb R lision energy between the ion and the neutral atoms and, thus, the ion YbRb1, the second of which creates a charged molecule. This process 1 temperature. An ion of mass mion trapped in a harmonic oscillator of is signalled by a loss of the Yb ion from the ion trap. Our choice of frequency vpionffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiat a temperature T has a root mean squared spread in elements is made with the aim of suppressing charge exchange by 2 position of kBT=mionvion. Convolving the position spread with the making the process off-resonant. Second, there are inelastic collisions theoretical expression for the elastic-scattering cross-section, due to micromotion of the ion. Collisions in the presence of this s(E 5 kBT), and equating the convolution with the measured result driven oscillation can result in energy transfer from the driving elec- for sal yields T 5 2.1 6 0.5 mK. This temperature is comparable to tric field to the colliding partners. the Doppler temperature limit of laser-cooling, TD 5 0.5 mK. Buffer- A small charge-exchange collision rate is indicated by a low probability gas cooling in the simplified case of a classical gas of hard spheres has for loss of the ion in collisions with the neutral atoms. The ion loss 26 2 2 been predicted to reach temperatures of T < mionV d /2kB, where d probability is determined using the same experimental sequence as for the loss of neutral atoms (Fig. 2) and is shown as a function of the interaction time in Fig. 4a. We observe a linear increase in the ion loss

30,000 ab5 40,000

4 30,000 20,000 3 20,000 2

Temperature (K) Temperature 10,000 1 10,000 Condensate atom number Condensate atom number 0 0 0102030405060 0102030405060 Interaction time (ms) Interaction time (ms) 0 0 102030405060Figure 3 | Time-resolved sympathetic cooling of a single ion. a Interaction time (ms) , Temperature decrease as a function of interaction time. The temperature is measured by laser fluorescence and the standard deviation of the Figure 2 | Atom loss from a Bose–Einstein condensate due to collisions maximum-likelihood estimate is shown. b, Atom loss during sympathetic with a single ion. The solid line is a fit using a theoretical model (Methods) cooling. While the ion is hot, atom losses are small and the cooling rate and is used to determine sal, the cross-section for neutral-atom loss. Each approaches 1,000 vibrational quanta per collision. When the ion cools, it data point is averaged over approximately 40 repetitions of the experiment localizes inside the region of higher density and the atom loss rate increases. and the standard error is given. The bare atom loss rate, that is, the loss rate The standard errors are shown. Each data point is averaged over without the ion present, has been subtracted. approximately 200 repetitions of the experiment. 389 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

ab resonances21. This will allow fundamental studies of systems compris- 15 ing a single impurity in a bath, for example the spin-boson model7 or ) 8,000 29 –1 the bosonic version of Anderson’s orthogonality catastrophe . 10 6,000 METHODS SUMMARY We store a single 174Yb1 ion in a radio-frequency Paul trap that creates a three- 5 4,000 dimensional harmonic oscillator potential of characteristic frequencies 5 4 vH,ion 5 2p 3 2 3 10 Hz and vx,ion 5 2p 3 5 3 10 Hz. We cool and probe the ion using near-resonant laser light. The laser light is detuned by D 52C/2 Atom number loss (s Ion loss probability (%) Ion loss probability 0 2,000 from the S1/2 R P1/2 transition at a wavelength of 370 nm, where C 5 2p 3 20 MHz is the linewidth of the atomic transition. The intensity of 0204060 –10–20–30 01020 30 the light scattered from the ion is sensitive to the ion’s velocity, which is used 87 Interaction time (ms) Offset electric field (V –1m ) to determine the temperature. We move an ultracold cloud of neutral Rb atoms in the jF 5 2, mF 5 2æ hyperfine ground state into the ion-trapping Figure 4 | Inelastic atom–ion collisions. a, Loss probability for a single ion volume using a time-varying inhomogeneous magnetic field. There, Bose– in a Bose–Einstein condensate. The time-dependent part of the loss is Einstein condensates of up to 3 3 104 atoms are prepared by transferring the attributed to interaction processes between atoms and ions. We determine neutral atoms into a far-detuned optical dipole trap formed by two crossed laser an upper bound on the charge-exchange collision rate from a linear fit to the beams of wavelength 935 nm, and then performing forced evaporation by con- data. Every data point is averaged over approximately 170 repetitions of the tinuously lowering the trap depth. The final trap frequencies of the optical trap experiment. b, Micromotion-induced atom losses for a single ion in a are vx,opt 5 2p 3 51 Hz, vy,opt 5 2p 3 144 Hz and vz,opt 5 2p 3 135 Hz. magnetically trapped thermal cloud. The data are averaged over 40 repetitions of the experiment for each data point and for two different Full Methods and any associated references are available in the online version of orientations of the offset electric field. The standard error is given. the paper at www.nature.com/nature. Received 20 December 2009; accepted 22 January 2010. probability with interaction time andfindthatchargeexchangeissup- pressed with respect to elastic collisions by a factor of 6 3 1026.From 1. Bloch, I., Dalibard, J. & Zwerger, W. Many-body physics with ultracold gases. Rev. 5 6 3 220 3 21 Mod. Phys. 80, 885–964 (2008). this, we estimate a loss rate constant of Kce (1.4 0.5) 10 m s , 2. Brennecke, F. et al. Cavity QED with a Bose–Einstein condensate. Nature 450, 27 which is five orders of magnitude smaller than in the homonuclear case 268–271 (2007). owing to the large energy gap, of 2 eV, between the collision channels. 3. Colombe, Y. et al. Strong atom–field coupling for Bose–Einstein condensates in an The bare ion loss probability in the measurement interval is 1024,which optical cavity on a chip. Nature 450, 272–276 (2007). we derive from our ion-trap lifetime of 8 min. The results clearly signal 4. Blatt, R. & Wineland, D. J. Entangled states of trapped atomic ions. Nature 453, 1008–1015 (2008). the suppression of charge-exchange processes for our choice of elements. 5. Rosenband, T. et al. Frequency ratio of Al1 and Hg1 single-ion optical clocks; The effect of micromotion on ultracold atom–ion collisions has not metrology at the 17th decimal place. Science 319, 1808–1812 (2008). been deeply explored so far. For the classical collision regime, theor- 6. Daley, A. J., Fedichev, P. O. & Zoller, P. Single-atom cooling by superfluid etical considerations of micromotion-induced collisions exist26,28 and immersion: a nondestructive method for qubits. Phys. Rev. A 69, 022306 (2004). 7. Leggett, A. J. et al. Dynamics of the dissipative two-state system. Rev. Mod. Phys. the influence of such collisions on charge-exchange processes has been 59, 1–85 (1987). 27 seen . We investigate micromotion effects experimentally by apply- 8. Recati, A., Fedichev, P. O., Zwerger, W., von Delft, J. & Zoller, P. Atomic quantum ing a d.c. electric field, Eoffset, to the ion. The electric field slightly dots coupled to a reservoir of a superfluid Bose-Einstein condensate. Phys. Rev. displaces the ion from the geometric centre of the trap and introduces Lett. 94, 040404 (2005). an excess micromotion of amplitude d < 2.3 nm 3 E V21 m. The 9. Yarmchuk, E. J., Gordon, M. J. V. & Packard, R. E. Observation of stationary vortex offset arrays in rotating superfluid helium. Phys. Rev. Lett. 43, 214–217 (1979). ultracold atomic cloud is then overlapped with the ion and we study 10. Abrikosov, A. & Gorkov, L. Contribution to the theory of superconducting alloys atom loss from the trap. In our measurements, we use a thermal atom with paramagnetic impurities. Sov. Phys. JETP 12, 1243–1253 (1961). cloud of approximately 2 3 105 atoms at 200 nK, trapped in a mag- 11. Yazdani, A., Jones, B. A., Lutz, C. P., Crommie, M. F. & Eigler, D. M. Probing the local effects of magnetic impurities on superconductivity. Science 275, 1767–1770 (1997). netic trap with radial and axial frequencies vH 5 2p 3 28 Hz and ,mag 12. Pan, S. et al. Imaging the effects of individual zinc impurity atoms on vx,mag 5 2p 3 8 Hz, respectively. We centre the magnetic trap on the superconductivity in Bi2Sr2CaCu2O81d. Nature 403, 746–750 (2000). position of the displaced ion by applying homogeneous magnetic 13. Makarov, O. P., Coˆte´, R., Michels, H. & Smith, W. W. Radiative charge-transfer 1 offset fields for each value of Eoffset. For the actual measurement, we lifetime of the excited state of NaCa . Phys. Rev. A 67, 042705 (2003). quickly move the ion to the centre of the atomic cloud, hold it there for 14. Coˆte´, R., Kharchenko, V. & Lukin, M. D. Mesoscopic molecular ions in Bose- Einstein condensates. Phys. Rev. Lett. 89, 093001 (2002). 8 s and measure the rate of loss of neutral atoms from the cloud. 15. Massignan, P., Pethick, C. J. & Smith, H. Static properties of positive ions in atomic Figure 4b shows our data for different values of the Eoffset. For Bose-Einstein condensates. Phys. Rev. A 71, 023606 (2005). 21 increased micromotion with jEoffsetj . 5Vm , we find a clear sig- 16. Goold, J., Doerk-Bending, H., Calarco, T. & Busch, T. Ion induced density bubble in nature of the effects of inelastic collisions due to micromotion leading a strongly correlated one dimensional gas. Preprint at Æhttp:// to larger atom loss rates. We attribute the shape of the curve to the arxiv1.library.cornell.edu/abs/0908.3179æ (2009). 17. Kollath, C., Ko¨hl, M. & Giamarchi, T. Scanning tunneling microscopy for ultracold energy dependence of atom–ion scattering rate, which scales in pro- atoms. Phys. Rev. A 76, 063602 (2007). 1/3 portion to jEoffsetj . 18. Sherkunov, Y., Muzykantskii, B., d’Ambrumenil, N. & Simons, B. D. Probing Our results highlight the possibilities for sympathetic cooling of a ultracold Fermi atoms with a single ion. Phys. Rev. A 79, 023604 (2009). single ion using an ultracold atomic bath to temperatures that are 19. Idziaszek, Z., Calarco, T. & Zoller, P. Controlled collisions of a single atom and an ion guided by movable trapping potentials. Phys. Rev. A 76, 033409 (2007). relevant for quantum information processing. Cooling qubits by 20. Coˆte´,R.&Dalgarno,A.Ultracoldatom-ioncollisions.Phys.Rev.A62, 012709 (2000). immersion into a superfluid has been predicted to maintain their 21. Idziaszek, Z., Calarco, T., Julienne, P. S. & Simoni, A. Quantum theory of ultracold quantum coherence and entanglement6. Therefore, atomic Bose– atom-ion collisions. Phys. Rev. A 79, 010702 (2009). Einstein condensates may serve as ideal refrigerators for ion-trap 22. Solda´n, P. & Hutson, J. M. Interaction of NH(X3S2) molecules with rubidium quantum computers. The successful immersion of a single trapped atoms: implications for sympathetic cooling and the formation of extremely polar molecules. Phys. Rev. Lett. 92, 163202 (2004). ion in a Bose–Einstein condensate makes several new experiments 23. Treutlein, P., Hunger, D., Camerer, S., Ha¨nsch, T. W. & Reichel, J. Bose-Einstein possible. The high degree of spatial control of a single ion facilitates condensate coupled to a nanomechanical resonator on an atom chip. Phys. Rev. the manipulation and study of quantum-degenerate samples with Lett. 99, 140403 (2007). nanometre precision. Resolving single sites in a three-dimensional 24. Larson, D. J., Bergquist, J. C., Bollinger, J. J., Itano, W. M. & Wineland, D. J. 17,18 Sympathetic cooling of trapped ions: a laser-cooled two-species nonneutral ion optical lattice is within reach , and even the creation of entangle- plasma. Phys. Rev. Lett. 57, 70–73 (1986). ment between distant lattice sites seems feasible. The atom–ion inter- 25. Palzer, S., Zipkes, C., Sias, C. & Ko¨hl, M. Quantum transport through a Tonks- action is predicted to be tunable by means of atom–ion Feshbach Girardeau gas. Phys. Rev. Lett. 103, 150601 (2009). 390 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

26. Major, F. G. & Dehmelt, H. G. Exchange-collision technique for the rf spectroscopy from the Engineering and Physical Sciences Research Council, the European of stored ions. Phys. Rev. 170, 91–107 (1968). Research Council (grant number 240335) and the Herchel Smith Fund (C.S.). 27. Grier, A. T., Cetina, M., Orucˇevic´, F. & Vuletic´, V. Observation of cold collisions between trapped ions and trapped atoms. Phys. Rev. Lett. 102, 223201 (2009). Author Contributions All authors contributed to the design, to data acquisition and 28. DeVoe, R. G. Power-law distributions for a trapped ion interacting with a classical to the interpretation of the presented work. C.Z. and S.P. contributed equally to the buffer gas. Phys. Rev. Lett. 102, 063001 (2009). construction of the apparatus and to the acquisition of the data. 29. Sun, J., Rambow, O. & Si, Q. Orthogonality catastrophe in Bose-Einstein Author Information Reprints and permissions information is available at condensates. Preprint at Æhttp://arxiv.org/abs/cond-mat/0404590æ (2004). www.nature.com/reprints. The authors declare no competing financial interests. Acknowledgements We are grateful to N. Cooper, C. Kollath, D. Lucas, Correspondence and requests for materials should be addressed to M.K. D. Moehring, E. Peik and C. Wunderlich for discussions. We acknowledge support ([email protected]).

METHODS exhibit less fluorescence owing to the large Doppler shift. During fluorescence Ion trapping. The ion is confined in the ponderomotive potential (pseudopo- detection, the temperature of the ion will change as a result of Doppler laser tential) of a linear Paul trap that is created from a very rapidly oscillating electric cooling and the fluorescence will increase to its saturation value over a time span quadrupole field. The trap operates at a radio frequency of V 5 2p 3 42.7 MHz, of several hundred microseconds. We detect the time-resolved fluorescence of which is well above the typical frequencies used in evaporative cooling of the the ion on a photomultiplier tube using a binning time of 25 ms. The frequency of neutral atoms. The distance between the ion and the radio-frequency electrodes the detection laser is red-detuned by D 52C/2 from the S1/2 R P1/2 transition of 174Yb1 (C 5 2p 3 20 MHz is the linewidth of the atomic transition). We analyse is R 5 0.5 mm and the applied voltage of VRF < 250 V gives rise to a radial 5 the temporal increase of the fluorescence by numerically calculating the solution confinement frequency of vH,ion 5 2p 3 2 3 10 Hz. The axial confinement fre- 4 174 1 of the time-dependent optical Bloch equations and performing a thermal quency is vx,ion 5 2p 3 5 3 10 Hz. A single Yb ion is loaded into the ion 33 2 trap by isotope-selective two-photon ionization30 from a pulsed neutral ytter- ensemble average . The resulting temperature is deduced by a x fit to the bium atomic beam. Subsequently, the ion is laser cooled on the S1/2 R P1/2 time-resolved data. For an ion laser cooled to the Doppler temperature of transition at 370 nm. Repumping light at 935 nm is used to clear population TD 5 0.5 mK, this measurement results in a temperature of T 5 0.2 6 0.3 K, out of the metastable D3/2 state. We continually cancel residual d.c. electric fields, indistinguishable from the temperature of an ion sympathetically cooled in a which would result in excess micromotion of the ion, to better than 0.5 V m21 thermal bath, which by the fluorescence method is T 5 0.3 6 0.5 K. The relatively using a photon-correlation technique31. Using the same technique, we establish coarse temperature resolution of this method, of a few hundred millikelvins, is an upper bound of 7 nm on uncompensated micromotion of the ion along the determined by photon shot noise and the number of averages. symmetry axis of the linear trap. Determination of the scattering cross-section. We model the Bose–Einstein Preparation of Bose–Einstein condensates. We transport the cold, but non- condensate with a density distribution n(r) in the Thomas–Fermi approxi- degenerate, neutral atomic cloud from its initial position into the ion trap by mation and with the ion pinned at the centre of the Bose–Einstein condensate displacing the potential minimum of the magnetic Ioffe trap using suitably timed (r 5 0). The cross-section for atom loss, sal, has contributions from the scatter- changes of the electrical currents in the solenoids. The neutral atoms enter the ing cross-section as well as from the spatial extent of the ion. The cold ion locally ion trap through a 700-mm-wide bore in the end-cap electrode, moving along the depletes the condensate and the characteristic speedq atffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi which the condensate symmetry axis of the linear ion trap. Shortly before the neutral atoms arrive at 2 2 density re-adjusts is the speed of sound, vs 5 4pB a0n(0)=m . Here their final position, we displace the ion by 140 mm from its ideal position. We Rb a0 5 5.45 nm is the s-wave scattering length of the neutral rubidium atoms then load the neutral atoms into an optical dipole trap formed by two crossed and mRb is their mass. We assume that in every collision an atom is lost from laser beams operating at a wavelength of 935 nm and centred on the ideal posi- the trap, because the vibrational level spacing of the ion trap is larger than the tion of the ion. Each of the beams is focused to a waist of approximately 70 mm potential depth of the optical dipole trap. In this model, the atom loss obeys the and has a power of up to a few hundred milliwatts. Using the dipole trap, we rate equation perform evaporative cooling by lowering the laser intensity to form Bose– Einstein condensates of 3 3 104 atoms. The optical-trap frequencies are dN 153=5vv 9=5m4=5 ~{ ffiffiffi Rb s N(t)3=5 ð1Þ v 5 2p 3 51 Hz, v 5 2p 3 144 Hz and v 5 2p 3 135 Hz. We apply p 2=5 4=5 al x,opt y,opt z,opt dt 8pa B a homogeneous bias magnetic field of less than 1 G to prevent depolarization of 0 1/3 the neutral atomic sample. The cross-talk between the trapping potentials for where vv 5 (vx,optvy,optvz,opt) . We fit the solution of equation (1) to our data atoms and ions is measurable but very small. The electric quadrupole field of the to extract the scattering cross-section sal. ion trap provides a potential for the neutral atoms via the d.c. Stark effect. This 1 gives rise to a weak anti-trapping harmonic potential of frequency ,10 Hz that 30. Balzer, C. et al. Electrodynamically trapped Yb ions for quantum information processing. Phys. Rev. A 73, 041407 (2006). does not significantly interfere with atom trapping. In the time-of-flight images 31. Berkeland, D. J., Miller, J. D., Bergquist, J. C., Itano, W. M. & Wineland, D. J. of the neutral atoms, we observe this anti-trapping potential as an additional Minimization of ion micromotion in a Paul trap. J. Appl. Phys. 83, 5025–5033 acceleration during expansion of the cloud. The effect of the optical trapping (1998). potential on the trapped ion is considerably weaker and can be neglected. 32. Epstein, R. J. et al. Simplified motional heating rate measurements of trapped ions. Temperature measurements of the ion. We measure the temperature of the ion Phys. Rev. A 76, 033411 (2007). using a laser fluorescence technique32,33. When illuminated with a near-resonant 33. Wesenberg, J. H. et al. Fluorescence during Doppler cooling of a single trapped laser beam, a cold ion displays a high level of fluorescence whereas a hot ion will atom. Phys. Rev. A 76, 053416 (2007).

Shell-isolated nanoparticle-enhanced Raman spectroscopy

Jian Feng Li1, Yi Fan Huang1, Yong Ding2, Zhi Lin Yang1, Song Bo Li1, Xiao Shun Zhou1, Feng Ru Fan1,2, Wei Zhang1, Zhi You Zhou1, De Yin Wu1, Bin Ren1, Zhong Lin Wang2 & Zhong Qun Tian1

Surface-enhanced Raman scattering (SERS) is a powerful spectro- nanoparticle acts as an Au tip in the TERS system, and thus this scopy technique that can provide non-destructive and ultra- technique simultaneously brings thousands of TERS tips to the sub- sensitive characterization down to single molecular level, compar- strate surface to be probed (Supplementary Fig. 3). We can obtain the able to single-molecule fluorescence spectroscopy1–15. However, jointly enhanced Raman signal contributed by all of these nano- generally substrates based on metals such as Ag, Au and Cu, either particles, which is two to three orders of magnitude higher than that with roughened surfaces or in the form of nanoparticles, are for a single TERS tip. Moreover, the use of a chemically inert shell required to realise a substantial SERS effect, and this has severely coating around the Au nanoparticle protects the SERS-active nano- limited the breadth of practical applications of SERS. A number of structure from contact with whatever is being probed. The main approaches have extended the technique to non-traditional sub- virtue of such a shell-isolated mode is its much higher detection strates14,16,17, most notably tip-enhanced Raman spectroscopy sensitivity and vast practical applications to various materials with (TERS)18–20 where the probed substance (molecule or material diverse morphologies. We call this new technique shell-isolated surface) can be on a generic substrate and where a nanoscale gold nanoparticle-enhanced Raman spectroscopy (SHINERS). tip above the substrate acts as the Raman signal amplifier. The drawback is that the total Raman scattering signal from the tip ab area is rather weak, thus limiting TERS studies to molecules with Transition metal shell large Raman cross-sections. Here, we report an approach, which we name shell-isolated nanoparticle-enhanced Raman spectroscopy, in which the Raman signal amplification is provided by gold nanoparticles with an ultrathin silica or alumina shell. A Support Support monolayer of such nanoparticles is spread as ‘smart dust’ over the surface that is to be probed. The ultrathin coating keeps the nanoparticles from agglomerating, separates them from direct c d contact with the probed material and allows the nanoparticles to Silica or alumina shell Au tip Probed conform to different contours of substrates. High-quality Raman molecules spectra were obtained on various molecules adsorbed at Pt and Au single-crystal surfaces and from Si surfaces with hydrogen mono- Any substrate and Any subst layers. These measurements and our studies on yeast cells and rate and morpholo morphology citrus fruits with pesticide residues illustrate that our method gy Au core significantly expands the flexibility of SERS for useful applications ef in the materials and life sciences, as well as for the inspection of food safety, drugs, explosives and environment pollutants. The invention of TERS led to a breakthrough in the substrate and surface generalities of SERS18,19. It uses a working mode that separates 20 nm 20 nm 20 nm the probed substance from a Au tip that acts as the Raman signal g Au/SiO Au/Al O amplifier (Fig. 1a–c). The largely enhanced electromagnetic field 2 2 3 generated on the probe tip under the excitation of a suitable laser can extend to the sample that is in close proximity to the tip apex. In 0.5 µm 5 nm 5 nm principle, the Raman signals from any substrate regardless material and surface smoothness can be enhanced by the tip apex with high Figure 1 | The working principles of SHINERS compared to other modes. spatial resolution19. However, the total Raman scattering signal from Schematic of the contact mode. a, Bare Au nanoparticles: contact mode. the tip area (about 20–50 nm in diameter) is rather weak, so TERS b, Au core–transition metal shell nanoparticles adsorbed by probed studies have been largely limited to molecules having large Raman molecules: contact mode. c, Tip-enhanced Raman spectroscopy: non- d e cross-sections. Moreover, its instrument is sophisticated and expen- contact mode. , SHINERS: shell-isolated mode. , Scanning electron microscope image of a monolayer of Au/SiO nanoparticles on a smooth Au sive, making it impractical for many applications. 2 surface. f, HRTEM images of Au/SiO2 core–shell nanoparticles with Our approach is to replace the Au tip by a monolayer of Au nano- different shell thicknesses. g, HRTEM images of Au/SiO2 nanoparticle and particles (Fig. 1d), each coated with an ultrathin shell of silica or Au/Al2O3 nanoparticle with a continuous and completely packed shell about alumina (here denoted Au/SiO2 or Au/Al2O3 nanoparticles). Each 2 nm thick.

1State Key Laboratory of Physical Chemistry of Solid Surfaces and College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. 2School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332–0245, USA. 392 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

We rationally designed gold nanoparticles coated with a thin, region can be observed (curve IV). Then, when we changed the shell uniform, fully enclosed and optically transparent shell of silica or thickness from 2 to 20 nm, the surface Raman signal also disappeared alumina so that the gold core generates a large surface enhancement, (curve V), illustrating that the ultrathin shell for the Au/SiO2 nano- and so that the shell controls the distance of the core from the surface particles is essential to extend the strong electromagnetic field from to be studied. The physical property (strong electromagnetic field) of the Au core to the probed surface. Taken together, our data suggest the Au nanoparticles is thus transferred to enhance the chemical that the Au/SiO2 nanoparticles with the ultrathin configuration are signal, for example, Raman vibrational bands, of the molecules on necessary to obtain the Pt–H signal with two additional lines of the material surfaces we are examining. Moreover, the chemically evidence. inert shell ensures that there is no direct contact between the probed To examine the capability of SHINERS to distinguish two different substance and the gold core. To prepare the shell-isolated nano- single-crystal facets, we have analysed the potential-dependent particles, Au nanoparticles with a diameter of ,55 nm were synthe- Raman spectra for different single-crystal surfaces of the same metal sized as cores21, onto which ultrathin silica shells were coated to form (Au). As far as we know, it is the first time high-quality surface 22–25 2 Au/SiO2 nanoparticles , which were then spread onto sampled Raman signals have been obtained for ions such as SCN adsorbed surfaces as a monolayer for measurement (Fig. 1e). at Au(111) and Au (100) in an electrochemical environment. One To elaborate the principle of SHINERS, we carried out a systematic advantage of SHINERS over surface infrared spectroscopy is that it study on the Raman scattering intensity as a function of the silica shell provides direct vibrational information about the metal–molecule thickness from 2 to 20 nm. The experimental data are in good agree- bonds (in the low-frequency region of Supplementary Fig. 6). ment with the three-dimensional finite-difference time-domain cal- To illustrate that the shell can easily be changed from silica to other culated result26 (Supplementary Figs 4 and 5). The calculated results materials, we coated the Au nanoparticles with an ultrathin, uniform 5 reveal that the magnitude of the maximally enhanced electric field is Al2O3 layer, using the atomic layer deposition method (see about 85 times greater for the 4-nm shell and about 142 times greater Supplementary Methods for details), and performed the same experi- for the 2-nm shell than that of the incident light; the highest enhancement. The SHINERS spectra obtained from 55-nm Au/2-nm Al2O3 ment of Raman scattering appears at the junction between the nanoparticles spread over Pt(111) (see Fig. 2b) have spectral features particle and the substrate as hot spots with magnitudes of about almost identical to those from the silica-coated nanoparticles, indi- 5 3 107 and 4 3 108 fold, respectively. We now present the applica- cating that changing the shell material is feasible and the quality of the tions of SHINERS to a few unusual cases in which the TERS tech- data are preserved. nique shows either undetectable or extremely low signals. To examine the capability of SHINERS for non-metallic materials, We first applied SHINERS on the atomically flat single-crystal we used an atomically flat silicon (Si) wafer, used widely in the surfaces of different materials in the aqueous solution or electro- semiconductor industry (Fig. 2c). Following the basic procedure of chemical environment, which hardly produces a detectable Raman surface treatment, the Si sample was first treated with H2SO4 (98%), signal using existing techniques. Hydrogen adsorption is a very then decorated with Au/SiO2 nanoparticles for the SHINERS mea- important system in surface science, chemistry and industry, includ- surement; no Si–H band could be detected (curve IX). The strong ing fuel cell applications. To our knowledge, no Raman spectrum of SHINERS signal of hydrogen binding on the Si surface can be iden- any hydrogen adsorbed at single-crystal surfaces has been reported tified after being treated with HF solution (curve X). The broad band 21 because of its extremely low Raman cross-section. We spread the Au/ at about 2,149 cm is attributed to the Si–H stretching vibration 27 SiO2 nanoparticles over a Pt(111) single-crystal electrode in 0.1 M mode . When the sample was further cleaned using O2 plasma, the NaClO4 and applied the electrode potential in the region of the Si–H band vanished (curve XI). This confirms that the Si–H bond is hydrogen evolution reaction. As can be seen in Fig. 2a from curves formed only after treatment of HF solution. There have been no SERS I, II and III, a broad band at about 2,023 cm21 was observed, being or TERS studies on the Si–H system on smooth Si surfaces, although attributed to the Pt–H stretching vibration mode according to the there have been a few Raman studies of hydrogen bonded to highly previous reports on highly roughened Pt surfaces17. In addition, we porous Si27. This result shows that SHINERS has a potential applica- collected Raman signals on Pt(111) under the same conditions but tion in semiconductor industrial processes. without Au/SiO2 nanoparticles. No Raman peak in this frequency Unlike SERS using bare Au nanoparticles or TERS using a bare Au tip, by using Au/SiO2 nanoparticles we can obtain more meaningful ν surface Raman signals because the chemically inert shell prevents abPt–H cdirect interaction between the probed adsorbates (or surface com- ν 2,034 Pt–H ponents) and bare Au nanoparticles or the tip, which often distorts 2,034 IX the true vibrational information (Supplementary Figs 7, 8, 9 and 10). 2,028 SiO or Al O ν 2 2 3 Si–H SHINERS has extraordinary potential for characterizing biological I VI 2,149 2,023 Au 2,029 structures such as living cells. Yeast cells were selected because they are

II Pt(111) Au one of the most extensively studied model eukaryotic organisms from genetics to biochemistry. In yeast the cell walls are of considerable 2,024 VII Si(111) III interest, owing to their sensitivity towards the different biological Pt(111) X functions of the cell. Figure 3 shows the SHINERS spectra (curves IV I–III) from different locations of the same sample. They are very VIII V XI different from the normal Raman spectra (curve V) of yeast cells, Au but similar to the SERS spectra of mannoproteins28, which are con- 2,000 2,100 2,000 2,100 2,000 2,200 Raman shift (cm–1) Raman shift (cm–1) sidered to be the main components of the yeast cell wall. There are Raman shift (cm–1) Pt(111) some other peaks attributed to amide, protein backbone and amino Figure 2 | Detection of hydrogen adsorption on single-crystal flat surfaces acid vibrations, related to the bioactivity of living cells such as protein of Pt and Si by SHINERS. a, Potential-dependent SHINERS spectra of secretion, movement, and so on28. These results demonstrate the hydrogen adsorbed on a Pt(111) surface. Curve I, at 21.2 V; curve II, potential of SHINERS as a safe and convenient technique for in-situ 21.6 V; curve III, 21.9 V; curve IV, without Au/SiO2 nanoparticles; curve V, with the thicker shell nanoparticles at 21.9 V. b, Potential-dependent detection of cell wall proteins and as a possible probe of the dynamics SHINERS spectra of hydrogen adsorbed on a Pt(111) surface using Au/ and mechanisms of living systems. Al2O3 nanoparticles. Curve VI, 21.2 V; curve VII, 21.6 V; curve VIII, SHINERS can also be used for inspecting food safety, drug security 21.9 V. c, SHINERS spectra on Si(111) wafer treated with 98% sulphuric and environment protection accurately and rapidly. The shell- acid (curve IX), 30% HF solution (curve X) and O2 plasma (curve XI). isolated nanoparticles could simply be spread as ‘smart dust’ over 393 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

ab1,414 long-standing limitations (inaccessibility or difficulty) of SERS for 1,587 1,166 * 1,488 the precise characterization of various materials, surfaces and bio- 1,208 1,337 * * * Microscope logical samples. The use of a thin-shell coating around the SERS- active metal nanostructure has several benefits: it keeps the smart * I dust from agglomerating, protects the SERS-active nanostructure * * * from contact with what is being probed, and allows the ‘smart dust’ * * to conform to different contours of samples. Given the availability of * II portable Raman spectrometers, our very simple method can be * widely applied to probe surface composition, adsorption and pro- * * cesses of diverse objects and morphologies, from single-crystal sur- III Yeast cell faces to cell walls, from semiconductors to fruits. The concept of shell-isolated-nanoparticle enhancement may also be applicable to IV SiO2 more general spectroscopy such as infrared spectroscopy, sum fre- 1,445 1,600 1,648 quency generation and fluorescence. We anticipate that SHINERS V Au will be developed into a simple, fast, non-destructive, flexible and portable characterization tool widely used in basic and applied 1,200 1,400 1,600 Raman shift (cm–1) research in the natural sciences, industry and even daily life. Figure 3 | In situ probing of biological structures by SHINERS. a, Curves I, METHODS SUMMARY II and III, SHINERS spectra obtained from different spots at the wall of an We first chemically synthesized Au nanoparticles with diameters of about 55 nm yeast cell incubated with Au/SiO2 nanoparticles. Curve IV, the spectrum as cores using a standard sodium citrate reduction method20, onto which ultrathin recorded from a substrate coated with Au/SiO2 but without a yeast cell; curve 21 silica shells were coated to form Au core/silica shell (Au/SiO2) nanoparticles .To V, a normal Raman spectrum for yeast cells. The peaks marked with red accelerate the synthesis procedure and make the uniformly ultrathin silica shell, b asterisks are closely related to mannoprotein. , Schematic of a SHINERS we elevated the reaction temperature from room temperature to 90 uC (see experiment on living yeast cells. Laser power on the sample was 4 mW. The Supplementary Methods for detailed information) The Au/Al2O3 core–shell collected times were 60 s for curves I, II, III and IV and 600 s for curve V. nanoparticles were prepared mainly using the atomic layer deposition technique5. the probed surface and the measurement taken in situ. This approach The structure and surface morphology of the core–shell nanoparticles and the shell thicknesses were measured by high-resolution transmission electron micro- could be used for inspecting pesticide residues on food and fruit. scopy (HRTEM) (JEM 4000EX and Tecnai F30). Raman spectra were recorded Figure 4 shows that normal Raman spectra recorded on fresh orange on a LabRam I confocal microprobe Raman system (Jobin-Yvon). The excitation with clean pericarps (curve I) or contaminated by parathion (curve II) line was 632.8 nm from a He-Ne laser. show only two bands at about 1,155 cm21 and 1,525 cm21, attributed to carotenoid molecules contained in citrus fruits29. By spreading Received 13 November 2009; accepted 8 February 2010. shell-isolated nanoparticles on the same surface, we can clearly 1. Nie, S. M. & Emory, S. R. Probing single molecules and single nanoparticles by detect two bands at 1,108 cm21 and 1,341 cm21 (curve III) that are surface enhanced Raman scattering. Science 275, 1102–1106 (1997). characteristic bands of parathion residues30. The same result can 2. Kneipp, K. et al. Single molecule detection using surface-enhanced Raman scattering (SERS). Phys. Rev. Lett. 78, 1667–1670 (1997). also be obtained by using a portable Raman spectrometer (Sup- 3. Moskovits, M. Surface-enhanced spectroscopy. Rev. Mod. Phys. 57, 783–826 plementary Fig. 14). This demonstrates that SHINERS could have (1985). tremendous scope as a simple-to-use, field-portable and cost- 4. Kneipp, K., Moskovits, M. & Kneipp, H. (eds) Surface-enhanced Raman effective analyser. Scattering–Physics and Applications (Springer, 2006). 5. Camden, J. P., Dieringer, J. A. & Van Duyne, R. P. Controlled plasmonic By changing the working mode from direct contact (SERS) or non- nanostructures for surface-enhanced spectroscopy and sensing. Acc. Chem. Res. contact (TERS) to the shell-isolated mode, SHINERS avoids the 41, 1653–1661 (2008). 6. Baumberg, J. J. et al. Angle-resolved surface-enhanced Raman scattering on abS metallic nanostructured plasmonic crystals. Nano Lett. 5, 2262–2267 (2005). CH O 3 OP 7. Cao, Y. W. C., Jin, R. C. & Mirkin, C. A. Nanoparticles with Raman spectroscopic NO2 CH3O fingerprints for DNA and RNA detection. Science 279, 1536–1540 (2002). 8. Chen, Z. et al. Protein microarrays with carbon nanotubes as multicolor Raman 1,346 labels. Nature Biotechnol. 26, 1285–1292 (2008). 1,109 Solid 9. Jain, P. K., Huang, X., El-Sayed, I. H. & El-Sayed, M. A. Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, IV sensing, biology, and medicine. Acc. Chem. Res. 41, 1578–1586 (2008). 10. Jackson, J. B. & Halas, N. J. Surface-enhanced Raman scattering on tunable 1,341 Au/SiO2 NP plasmonic nanoparticle substrates. Proc. Natl Acad. Sci. USA 101, 17930–17935 1,108 (2004). III 11. Graham, D., Thompson, D. G., Smith, W. E. & Faulds, K. Control of enhanced Raman scattering using a DNA-based assembly process of dye-coded nanoparticles. Nature Nanotechnol. 3, 548–551 (2008). 12. Qian, X. et al. In vivo tumor targeting and spectroscopic detection with surface- Methyl parathion enhanced Raman nanoparticle tags. Nature Nanotechnol. 26, 83–90 (2008). 1,525 II 13. Anker, J. N. et al. Biosensing with plasmonic nanosensors. Nature Mater. 7, 1,155 442–453 (2008). 14. Tian, Z. Q., Ren, B., Li, J. F. & Yang, Z. L. Expanding generality of surface-enhanced Raman spectroscopy with borrowing SERS activity strategy. Chem. Commun. 34, I Orange 3514–3534 (2007). 15. Nie, S. M. & Zare, R. N. Optical detection of single molecules. Annu. Rev. Biophys. 1,200 1,400 1,600 Biomed. 26, 567–596 (1997). –1 Raman shift (cm ) 16. Park, S., Yang, P., Corredor, P. & Weaver, M. J. Transition metal-coated Figure 4 | In situ inspection of pesticide residues on food/fruit. a, Normal nanoparticle films: vibrational characterization with surface-enhanced Raman scattering. J. Am. Chem. Soc. 124, 2428–2429 (2002). Raman spectra on fresh citrus fruits. Curve I, with clean pericarps; curve II, 17. Tian, Z. Q. & Ren, B. Adsorption and reaction at electrochemical interfaces as contaminated by parathion. Curve III, SHINERS spectrum of contaminated probed by surface-enhanced Raman spectroscopy. Annu. Rev. Phys. Chem. 55, orange modified by Au/SiO2 nanoparticles. Curve IV, Raman spectrum of 197–229 (2004). solid methyl parathion. Laser power on the sample was 0.5 mW, and the 18. Sto¨ckle, R. M., Suh, Y. D., Deckert, V. & Zenobi, R. Nanoscale chemical analysis by collected times were 30 s. b, Schematic of the SHINERS experiment. tip-enhanced Raman spectroscopy. Chem. Phys. Lett. 318, 131–136 (2000). 394 ©2010 Macmillan Publishers Limited. 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19. Pettinger, B., Ren, B., Picardi, G., Schuster, R. & Ertl, G. Nanoscale probing of 29. Schulte, F., Mader, J. & Kroh, L. W. Panne, U. & Kneipp, J. Characterization of adsorbed species by tip-enhanced Raman spectroscopy. Phys. Rev. Lett. 92, pollen carotenoids with in situ and high-performance thin-layer chromatography 096101–096104 (2004). supported resonant Raman spectroscopy. Anal. Chem. 81, 8426–8433 (2009). 20. Wu, D. Y., Li, J. F., Ren, B. & Tian, Z. Q. Electrochemical surface-enhanced Raman 30. Lee, D. et al. Quantitative analysis of methyl parathion pesticides in a spectroscopy of nanostructures. Chem. Soc. Rev. 37, 1025–1041 (2008). polydimethylsiloxane microfluidic channel using confocal surface-enhanced 21. Frens, G. Controlled nucleation for regulation of particle-size in monodisperse Raman spectroscopy. Appl. Spectrosc. 60, 373–377 (2006). gold suspension. Nature 241, 20–22 (1973). Supplementary Information is linked to the online version of the paper at 22. Liz-Marza´n, L. M., Michael, G. & Mulvaney, P. Synthesis of nanosized gold-silica www.nature.com/nature. core-shell particles. Langmuir 12, 4329–4335 (1996).

23. Lu, Y., Yin, Y., Li, Z. Y. & Xia, Y. Synthesis and self-assembly of Au@SiO2 core-shell Acknowledgements We thank P. Bartlett for suggestions and editing of the English colloids, Nano Lett. 2, 785–788 (2002). while writing the paper. We thank R. Zare, N. Zheng, B. Mao, U. K. Sur and L. Yang 24. Mulvaney, S. P., Musick, M. D., Keating, C. D. & Natan, M. J. Glass-coated, for discussions. We also thank Y. Yu, Y. Wu, M. Zhuang, X. Wang and A. Wang for analyte-tagged nanoparticles: a new tagging system based on detection with assistance in experiments. This work was supported by MOST, China surface-enhanced Raman scattering. Langmuir 19, 4784–4790 (2003). (2009CB930703, 2007DFC40440 and 2007CB935603), the NSF of China 25. Smith, W. E. Practical understanding and use of surface enhanced Raman (20620130427 and 20533040), the BES DOE (DE-FG02-07ER46394) and the US scattering/surface enhanced resonance Raman scattering in chemical and NSF (DMS 0706436, CMMI 0403671). biological analysis. Chem. Soc. Rev. 37, 955–964 (2008). Author Contributions Z.Q.T., Z.L.W., J.F.L. and B.R. conceived and designed the 26. Sherry, L. J. et al. Localized surface plasmon resonance spectroscopy of single experiments, analysed the results and participated in writing the manuscript. J.F.L., silver nanocubes. Nano Lett. 5, 2034–2038 (2005). Y.F.H., Y.D., S.B.L., X.S.Z., F.R.F., W.Z. and Z.Y.Z. performed the experiments and 27. Ren, B. et al. In situ monitoring of Raman scattering and photoluminescence analysed the results. Z.L.Y. and D.Y.W contributed to theoretical calculations. from silicon surfaces in HF aqueous solutions. Appl. Phys. Lett. 72, 933–935 (1998). Author Information Reprints and permissions information is available at 28. Sujith, A. et al. Surface enhanced Raman scattering analyses of individual silver www.nature.com/reprints. The authors declare no competing financial interests. nanoaggregates on living single yeast cell wall. Appl. Phys. Lett. 92, 103901 Correspondence and requests for materials should be addressed to Z.Q.T. (2008). ([email protected]) and Z.L.W. ([email protected]).

Isotope fractionation in silicate melts by thermal diffusion

F. Huang1{*, P. Chakraborty1*, C. C. Lundstrom1, C. Holmden2, J. J. G. Glessner1, S. W. Kieffer1 & C. E. Lesher3

The phenomenon of thermal diffusion (mass diffusion driven by a to thermal diffusion. In the steady state, these two fluxes balance each temperature gradient, known as the Ludwig–Soret effect1,2) has other, thereby leading to9,15 been investigated for over 150 years, but an understanding of its dC=C~{S dT ð1Þ underlying physical basis remains elusive. A significant hurdle in T studying thermal diffusion has been the difficulty of characterizing where ST, the Soret coefficient, equals DT/D and is the single parameter it. Extensive experiments over the past century have established that determines the magnitude of the resultant concentration gradient that the Soret coefficient, ST (a single parameter that describes in the steady state. Extensive experimental data over the last century the steady-state result of thermal diffusion), is highly sensitive to show that ST is markedly sensitive to conditions. For instance, the 3–9 many factors . This sensitivity makes it very difficult to obtain a magnitude and sign of ST can change drastically as a function of a robust characterization of thermal diffusion, even for a single variety of parameters4,6,8–10 including, but not limited to, the chemical material. Here we show that for thermal diffusion experiments that composition of the system, the size and charge of the particle, and the span a wide range in composition and temperature, the difference temperature. In many cases, the functional form of ST is unknown. in ST between isotopes of diffusing elements that are network The sensitive and unknown functional dependence of ST poses a sig- modifiers (iron, calcium and magnesium) is independent of the nificant practical problem: similar systems may be characterized by composition and temperature. On the basis of this finding, we different values of ST, requiring that the value of ST be individually propose an additive decomposition for the functional form of determined for each system. ST and argue that a theoretical approach based on local thermo- Our focus is on isotope fractionation through thermal diffusion in dynamic equilibrium3,5,10 holds promise for describing thermal silicate melts. We limit our attention to the steady state of thermal diffusion in silicate melts and other complex solutions. Our results diffusion and, furthermore, to the isotopic fractionation of iron, lead to a simple and robust framework for characterizing isotope calcium and magnesium (which break up the polymerization of fractionation by thermal diffusion in natural and synthetic the silicate melt and are thus termed network modifiers), and do systems. not consider network formers (for example silicon and oxygen, In 1856, Carl Ludwig discovered an intriguing phenomenon1 (that which form tetrahedron networks in silicate melts). Although multi- was tested in 1879 by Charles Soret2): when a homogeneous salt component silicate melts are different from the simple binary solution in a column is subjected to a temperature gradient, the salt mixtures discussed above, they are analysed as a pseudo-binary preferentially diffuses against the temperature gradient, from the hot system6,16–18 in the framework of equation (1). In this system, the end to the cold end. This phenomenon—mass diffusion driven by a silicate melt is the host liquid, albeit with a complex structure, and temperature gradient—is called thermal diffusion or the Ludwig– the isotopes are the guest particles in the melt. Similar to previous Soret effect2. Thermal diffusion has been suggested to have a role studies of thermal diffusion in other systems3,4,8,9,19,20, thermal in a number of phenomena, including the origin of life, via migration diffusion experiments on silicate melts show the marked sensitivity 11 and concentration of DNA along temperature gradients ; the sea- of ST to the details of the system. For instance, in the case of thermal sonal temperature-driven fractionation of nitrogen and argon iso- diffusion of potassium, changing the bulk composition of the melt 12,13 23 topes in the air trapped in polar ice ; and the differentiation of from basalt to rhyolite changes the value of ST from 25.5 3 10 to igneous rocks14. Furthermore, thermal diffusion shows great poten- 0.89 3 1023 uC21 (refs 6, 16). Furthermore, there exists no theo- 5,8,11 tial in emerging technological applications from nanotechnology retical framework for obtaining a functional form of ST for thermal to biotechnology. Despite the long history and wide importance of diffusion in silicate melts17,18,21–23. Instead, such systems are charac- this phenomenon, the physics of thermal diffusion remains poorly terized using empirical formulae; see, for example, refs 6, 16–18. Here understood3,7–10. we seek to use the properties of isotopes, namely their almost ident- Consider a dilute, homogeneous concentration of guest particles ical chemical properties but distinctly different masses, to bypass the suspended in a host liquid (a simple binary mixture), for example salt complexities of chemical interactions and characterize isotopic frac- in water. When a temperature gradient, =T, is applied across the tionation through thermal diffusion in a simple framework. mixture, thermal diffusion causes the particles to migrate (usually Ultimately, our objective is to propose a robust method for char- from the hot to the cold end) with a mass flux 2DTC=T, where DT acterizing isotopic thermal diffusion in silicate melts and gain insight is the thermal diffusion coefficient and C is the concentration. The into the functional dependence of ST. migration of the particles sets up a concentration gradient, which in We measured iron, magnesium and calcium isotope ratios of micro- turn causes chemical diffusion of particles with a mass flux D=C, drilled samples from different positions (and hence at different tem- where D is the chemical diffusion coefficient, in the opposite direction peratures) in elongate capsules of quenched silicate melt produced in

1Department of Geology, University of Illinois, Urbana, Illinois 61801, USA. 2Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada. 3Department of Geology, University of California, Davis, California 95616, USA. {Present address: Institute of Geochemistry and Petrology, ETH Zurich, CH-8092 Zurich, Switzerland. *These authors contributed equally to this work. 396 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

Table 1 | Iron, calcium and magnesium isotope data from thermal diffusion samples 56 57 25 26 44 d FeIRMM-014 2s d FeIRMM-014 2s N d MgDSM-3 2s d MgDSM-3 2s N d CaSW 2s NT (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (uC) ZM70, 46 h, ZM70A 22.45 0.22 23.70 0.77 2 23.40 0.02 26.59 0.13 3 ———1,518 Mt Hood andesite, ZM70B 20.18 0.04 20.24 0.15 2 21.39 0.04 22.70 0.03 3 ———1,485 DTA–C 5 164 uC, T0 5 1,430 uC, ZM70C 1.65 0.07 2.45 0.30 2 1.76 0.06 3.46 0.13 3 ———1,354 21 21 VFe 5 0.0125% uC AMU , Starting 0.06 0.05 0.09 0.05 4 20.20 0.05 20.40 0.09 4 ———— 21 21 VMg 5 0.0307% uC AMU material ZM71, 168 h, ZM71A 23.00 0.09 24.38 0.15 5 25.15 0.07 29.98 0.08 4 ———1,550 Mt Hood andesite, ZM71B 21.52 0.05 22.22 0.14 5 22.01 0.15 23.90 0.09 4 ———1,503 DTA–D 5 270 uC, T0 5 1,445 uC, ZM71C 1.07 0.06 1.60 0.12 4 0.96 0.07 1.82 0.08 5 ———1,402 21 21 VFe 5 0.0113% uC AMU , ZM71D 3.10 0.06 4.64 0.15 4 3.42 0.17 6.74 0.32 4 ———1,280 21 21 VMg 5 0.0309% uC AMU ZM98, 264 h, ZM98A 22.84 0.18 24.21 0.29 3 25.46 0.04 210.70 0.04 4 28.26 0.06 1 1,648 mid-ocean-ridge basalt, ZM98B 20.38 0.10 20.60 0.21 3 21.15 0.08 22.28 0.10 4 21.73 0.04 1 1,560 DTA–C 5 274 uC, T0 5 1,531 uC, ZM98C 4.91 0.05 7.29 0.14 3 5.49 0.04 10.74 0.08 4 8.38 0.03 1 1,374 21 21 VFe 5 0.0142% uC AMU , Starting 0.24 0.05 0.36 0.13 4 20.15 0.09 20.33 0.07 4 20.95 0.03 1 — 21 21 VMg 5 0.0387% uC AMU , material 21 21 VCa 5 0.0152% uC AMU ZM99, 207 h, ZM99A 21.82 0.05 22.70 0.09 3 ————————1,645 0.46:0.8:0.46 fayalite:leucite:quartz, ZM99B 0.86 0.07 1.27 0.09 3 ————————1,537 DTA–C 5 247 uC, T0 5 1,553 uC, ZM99C 3.30 0.06 4.91 0.08 3 ————————1,398 21 21 VFe 5 0.0104% uC AMU Starting 0.33 0.06 0.48 0.10 4 ————————— material ZM100, 166 h, ZM100A 22.25 0.28 23.48 0.46 2 ————————1,645 pantellerite, ZM100B 21.67 0.04 22.51 0.18 2 ————————1,566 DTA–C 5 194 uC, T0 5 1,536 uC, ZM100C 3.24 0.21 4.82 0.48 4 ————————1,451 21 21 VFe 5 0.0141% uC AMU Starting 0.25 0.13 0.40 0.13 4 ————————— material The d values reported here are computed relative to a standard: IRMM-014 for iron, DSM-3 for magnesium and natural sea water (SW) for calcium. We note that d relative to starting material equals d relative to standard minus d of starting material (relative to standard). Temperature is estimated assuming a parabolic temperature profile. T0 is the temperature of the position in the thermal diffusion experiment where the isotope composition is identical to that of the starting material. To compute T0 for any sample, we calculate the temperature for which the best-fit straight line in the plot of d (relative to starting material) versus temperature difference (relative to the hot end) of each isotope pair passes through d 5 0 and average this temperature over the different isotope pairs in the sample. The respective DT values indicate the temperature differences between the micro-drilled positions. N, number of repeat measurements.

ð previously performed and characterized thermal diffusion experi- X T 6,16 ( M)T ments (Methods). These experiments spanned a wide range of ~ exp { (S )X dT ð3Þ (X M) T M chemical compositions of starting materials and temperatures T0 T0 (Table 1). where ST is an unknown function of X, the melt composition, the To assess the timescale on which the steady-state distribution of 6,8–11,24 temperatures T0 and T, and other parameters .IfST were a isotopes is reached, we compared the results of two experiments with constant, the concentration of XM would be exponentially distributed identical andesitic starting composition but different run durations: 46 h (ZM70) and 168 h (ZM71). Although the shorter run was only 60% evolved to the steady state in terms of its major-element com- 6,16 position , the two experiments did not differ significantly in terms 0.04 100 of thermal-diffusion isotopic sensitivity, V (the magnitude of iso- Ω Fe Ω Mg topic fractionation per temperature offset, expressed as parts per Concentration steady state Concentration steady state (%) 12,18 thousand per degree Celsius per atomic mass unit ; Fig. 1). 0.03

Thus, the system reached the isotopic steady state within 46 h, mean- ) –1 ing that isotope fractionation induced by thermal diffusion in these 80 AMU experiments is not a transient feature. –1

Typically, the data from thermal diffusion experiments are pre- 0.02

sented in plots of concentration versus temperature. We, however, (‰ ºC start discussing our data in a context more typical of isotope frac- Ω tionation experiments—plots of isotope ratio versus isotope ratio 0.01 ZM71 60 (Fig. 2). In this convention, the data are reported in d notation: "# ZM70 (X M=Y M) dX M~ sample {1 |1,000% ð2Þ 0.00 (X M=Y M) 0 50 100 150 200 starting material Duration (h) X Y X Y Here M/ M is the ratio of isotopes M and M (by convention, Figure 1 | Thermal-diffusion isotopic sensitivity and concentration steady X . Y). This approach leads to a striking observation: despite signifi- state of time-series experiments (ZM70 and ZM71)6. The error bars cant differences in the compositions of the starting materials (from (,10% relative error, 1s) were calculated on the basis of errors in iron and 45 to 70 wt% SiO2), a wide span of average experimental tempera- magnesium isotope analyses and the temperature offset. The data labelled tures and even the presence of coexisting mineral phases, the data, to ‘concentration steady state’ corresponds to the fraction of the major- first order, are linearly correlated (Fig. 2). This observation suggests element concentration relative to its steady-state value6. We note that the that isotopic fractionation through thermal diffusion in silicate melts approach to a steady-state isotopic distribution occurs faster than the can be treated in a robust framework. compositional rearrangement of major elements in the silicate melt. This observation might be expected because isotopic diffusion is known to be To proceed, we cast our experimental observations in a theoretical faster than chemical diffusion and homogenization of isotope ratios occurs framework. First, we integrate equation (1) to determine the concen- roughly according to the self-diffusivity of the element26–29. Assuming X 211 2 21 tration distribution of an isotope M from temperature T0 (at the magnesium self-diffusivities of 7 3 10 m s in basaltic melt25, the reference location) to temperature T (at the location of interest), diffusion distance in 46 h is ,3.4 mm, which is similar to the length of the obtaining sample. 397 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a 12 We now consider the concentration distribution of the ratio of two X Y ZM70 isotopes, M and M. From equations (2) and (3), and using the fact ZM71 that dXM = 1, we obtain 8 ZM98 SRT4 (X M=Y M) TM dX M< ln T 4 (X M=Y M) T0 ð ð4Þ 0 (X ) (Y ) T ~ M T { M T ~{ Mg (‰) ln ln DS dT δ26Mg = 1.9δ57Fe X Y T 26 ( M) ( M)

δ T0 T0 T0 –4 R2 = 0.93 ~ { where DST (ST)X M (ST)Y M (Supplementary Information). –8 Notably, we find that in contrast to individual isotope concentrations (Fig. 3a–c), to first order each isotope ratio has a single exponential distribution as a function of DT for all experiments (or, equivalently, –12 –3 0 3 6 in d notation the data for each isotope is, to first order, linearly dis- δ57Fe (‰) tributed as a function of DT; Fig. 3d–f). This finding indicates that b DST is independent of the melt composition and temperature. Thus, DST is a constant for pairs of isotopes of the same element. With a 8 X constant DST, equation (4) implies that d M is linearly proportional to DT, which in turn manifests as the linear correlation in Fig. 2 4 (Supplementary Information). This constancy of DST for isotope ratios bears some similarity with its behaviour under thermal diffusion in simple binary liquid mixtures of benzene and cyclohexane: the differ- 0

Ca (‰) δ44Ca = 1.5δ57Fe ence in ST of such mixtures with and without isotopic substitution is 44 19,20 δ R2 = 0.97 observed to be independent of concentration and temperature . –4 Given the composition and temperature independence of DST,we now turn to the problem of the functional form of ST. We postulate –8 the following additive decomposition of ST:

ST(m, composition, T, ...)~ –3 0 3 6 ð5Þ δ57Fe (‰) fm(m, ...)zfc(composition, T, ...) c Here m and T are atomic mass and temperature, respectively. The 8 function fm depends on m and other parameters (except melt composition and T), and the function fc, which encapsulates the chemical effects, depends on melt composition, T and other parameters 4 (except m). We now consider the significance of this equation for isotope fractionation through thermal diffusion. Because isotopes 0 have nearly identical chemical properties and differ only in atomic Ca (‰) mass (X and Y), two isotopes of an element have different values of f 44 m δ δ44Ca = 0.79δ26Mg but the same value of fc. Thus DST 5 fm(X,…)2 fm(Y, …), wherein –4 2 R = 0.99 the chemical effects (fc terms) have cancelled out. The functional form of equation (5) provides a strong constraint for a general theory –8 of thermal diffusion in silicate melts—any theoretical prediction of the dependence of ST on mass, composition and temperature can be –8 –4 0 4 8 decomposed according to equation (5). We note that the functional δ26Mg (‰) 3,4,8,24 form of ST for thermal diffusion in other systems follows an Figure 2 | Cross-correlations of magnesium, iron and calcium isotope additive decomposition similar to equation (5). Furthermore, the ratios for both the thermal diffusion experiments of refs 6, 16 and previous decomposition suggested in equation (5) provides guidance on seek- work. The data form linear relationships for experiments spanning a wide ing empirical formulae for ST. For example, assuming that fm range of melt compositions and temperatures, even in the presence of depends on m and Z2/a (the field strength, which correlates well with coexisting minerals. a, d26Mg versus d57Fe for ZM70, ZM71 and ZM98. the self-diffusivity25,26; Z is the ionic charge and a is the ionic radius), Slopes defined by the previous studies (refs 6 and 17, 18 (thermal diffusion our experimental data are best described by f / m21/4(Z2/a)9/20 30 m experiment, SRT4) and (thermal migration experiment, TM)) are the (Supplementary Information). same. b, c, d44Ca versus d57Fe (b) and d44Ca versus d26Mg (c) for ZM98 and SRT4. Symbols as in a. Most error bars (2s; Table 1) are smaller than the Finally, we outline an approach for further theoretical work. Our symbols. R2, coefficient of determination. examination of isotope ratios during thermal diffusion in silicate melts shows that they follow an exponential distribution that is characterized by a constant DST. Considerable theoretical progress has been made in as a function of the temperature difference, DT 5 T 2T0 (the plot of understanding the thermal diffusion of particles in aqueous solu- the concentration of XM versus DT on a log–linear scale would be tions3,5,10, in which the concentration follows an equilibrium-like expo- linear). We, however, find that the concentrations of the isotopes nential distribution. This non-equilibrium process can be described in (even for the same melt composition) are not distributed in this the framework of local thermodynamic equilibrium—a theoretical way (Fig. 3a–c). Instead, on the log–linear plot of the concentration expression for ST is obtained by relating the exponential distribution of XM versus DT, the data from the different experiments are widely of concentration to the local Boltzmann law3,5,10.Wesubmitthata scattered (with the concentration in one sample changing by an order similar approach, in which the variation of isotope ratio and the cor- of magnitude) in the form of nonlinear, intersecting lines. Thus, the responding DST are analogous to the concentration of particles and the concentration distribution of isotopes in silicate melts cannot be char- corresponding ST, respectively, may yield further insights into the acterized by a constant ST. physics of thermal diffusion in silicate melts. 398 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

a d 6 TM 4 SRT4 0

1 T T

Fe) Fe) 2 54 54 Fe/ Fe/

57 57 0 ( ( Fe (wt%) 57 0.1 –2 ZM70 ZM71 1,000In 57 –5 ZM98 –4 δ Fe = –3.6 × 10 ΔT ZM99 R2 = 0.93 ZM100 b e 0.2 0

T T 5 Ca) Ca) 40 40 ZM70

Ca/ Ca/ 0 44 44 ( 0.1 ( Ca (wt%) ZM71 44 –5 1,000In δ44Ca = –6.1 × 10–5ΔT R2 = 0.99 c 1 f 10 SRT8 0 T T 5 Mg) Mg) 24 24 0 Mg/ Mg/ Mg (wt%) 26 26 ( ( 26 –5

0.1 1,000In δ26Mg = –6.8 × 10–5ΔT –10 R2 = 0.94

–200 –100 0 100 200 –200 –100 0 100 200 ΔT (ºC) ΔT (ºC) Figure 3 | Distribution of isotope concentrations and isotope ratios against (57Fe/54Fe, 44Ca/40Ca and 26Mg/24Mg) or, equivalently, approximate isotope DT for thermal diffusion experiments with a wide range of melt ratios in delta notation (1,000d57Fe, 1,000d44Ca and 1,000d26Mg) versus DT. 25 21 compositions and bulk temperatures. We choose T0 to be the temperature From the slopes of the best-fit lines, we find that DST 5 (3.6 3 10 ) uC of the location where the isotope ratio of the sample equals that of the for 57Fe/54Fe, (6.1 3 1025) uC21 for 44Ca/40Ca and (6.8 3 1025) uC21 for starting material. a–c, Isotope concentrations 57Fe, 44Ca and 26Mg 26Mg/24Mg. The systematic steepening of data points close to the hot end (logarithmic scale) versus DT. The solid lines depict the concentration may be due to higher-order effects of temperature on DST. SRT4 and SRT8 distribution as measured using an electron microprobe6,16, and the symbols are from refs 17, 18. Most error bars (2s; Table 1) are smaller than the depict the locations where we have micro-drilled the sample to measure the symbols. Symbols throughout are as defined in a, d and f. isotope ratios shown in panels d–f. d–f, Logarithm of isotope ratios

The discovery that isotopic fractionation of iron, calcium and (Bio-Rad AG50-X12). Magnesium and iron procedural blanks were negligible. magnesium is independent of melt composition and temperature Yields and matrix in purified solution were checked before isotope measurement. considerably simplifies the practical application of these isotope sys- Iron isotopes were measured using high resolution multi-collector inductively- tems as tools for elucidating temperature-gradient processes in the coupled-plasma mass spectroscopy (Nu Plasma HR, Nu Instruments) in static mode using a sample-standard bracketing method with resolutions .8,000 to evolution of the Earth. In fact, workers concerned with the origin of resolve isobaric inferences. IRMM-014 solutions with an iron concentration equi- igneous rocks in the early 1900s seized on the newly discovered ther- valent to that of the samples (1.5 or 0.4 p.p.m.) were used as the bracketing mal diffusion phenomenon driven by the Earth’s ever-present geo- standard. Magnesium isotopes were measured at a resolution of ,400 using a thermal gradient as a possible mechanism for producing the sample-standard bracketing method relative to an in-house magnesium standard 26 25 compositional diversity of the continental crust. Although the idea with d MgDSM-3 53.314 6 0.234% and d MgDSM-3 5 1.700 6 0.137% (over has since lost favour to modern concepts of fractional crystallization five months; n 5 28). Samples were diluted to approximately 0.15 p.p.m. and and partial melting, there may yet be a role for thermal diffusion in introduced using a DSN-100 Desolvation Nebuliser System (Nu Instruments), 24 the origin of some granites and the continental crust14. Unlike 150 producing a Mg signal of ,16 V. Calcium isotopes were measured on a thermal years ago, however, new investigations can be guided, and the role of ionization mass spectrometer (Thermo Finnigan TRITON) in the Saskatchewan Isotope Laboratory, University of Saskatchewan, using a 43Ca–42Ca double spike thermal diffusion possibly identified, by this definitive isotopic sig- to correct for instrumental mass fractionation. nature of thermal diffusion, which has thus far been documented only in the laboratory. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. METHODS SUMMARY Received 15 October 2009; accepted 13 January 2010. We micro-drilled samples from silicate glass in thermal diffusion experi- ments6,16, using tungsten carbide drill bits. The width of the drilling bands was 1. Ludwig, C.Diffusion zwischenungleich erwa¨rmtenOrten gleich zusammengesetzter ,0.4–0.5 mm. Temperature offsets between the drilling areas were estimated Lo¨sungen. Sitz. Math. Naturwiss. Classe Kaiserlichen Akad. Wiss. 20, 539 (1856). assuming first a linear and then a parabolic temperature profile across the ther- 2. Soret, C. Sur l’e´tat d’e´quilibre que prend au point de vue de sa concentration une dissolution saline primitivement homohe´ne dont deux parties sont porte´es a` des mal diffusion experiment. The difference in temperature estimation from the tempe´ratures diffe´rentes. Arch. Sci. Phys. Nat. 2, 48–61 (1879). two methods is ,5%. 3. Astumian, R. D. Coupled transport at the nanoscale: the unreasonable Samples were digested in a 3:1 mixture of concentrated hydrofluoric acid and effectiveness of equilibrium theory. Proc. Natl Acad. Sci. USA 104, 3–4 (2007). HNO3. After iron was separated by anion resin (Bio-Rad AG1-X8), magnesium 4. Debuschewitz, C. & Ko¨hler, W. Molecular origin of thermal diffusion in benzene 1 and calcium were separated from most matrix interferences using cation resin cyclohexane mixtures. Phys. Rev. Lett. 87, 055901 (2001). 399 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

5. Duhr, S. & Braun, D. Thermophoretic depletion follows Boltzmann distribution. 24. Reith, D. & Muller-Plathe, F. On the nature of thermal diffusion in binary Lennard- Phys. Rev. Lett. 96, 168301 (2006). Jones liquids. J. Chem. Phys. 112, 2436–2453 (2000). 6. Lesher, C. E. & Walker, D. Solution properties of silicate liquids from thermal 25. LaTourrette, T., Wasserburg, G. J. & Fahey, A. J. Self diffusion of Mg, Ca, Ba, Nd, diffusion experiments. Geochim. Cosmochim. Acta 50, 1397–1411 (1986). Yb, Ti, Zr, and U in haplobasaltic melt. Geochim. Cosmochim. Acta 60, 1329–1340 7. Platten, J. K. The Soret effect: a review of recent experimental results. J. Appl. (1996). Mech. 73, 5–15 (2006). 26. Lesher, C. E. Kinetics of Sr and Nd exchange in silicate liquids theory, experiments, 8. Putnam, S. A., Cahill, D. G. & Wong, G. C. L. Temperature dependence of and applications to uphill diffusion, isotopic equilibration, and irreversible mixing thermodiffusion in aqueous suspensions of charged nanoparticles. Langmuir 23, of magmas. J. Geophys. Res. 99, 9585–9604 (1994). 9221–9228 (2007). 27. van der Laan, S., Zhang, Y., Kennedy, A. K. & Wyllie, P. J. Comparison of element 9. Tyrrell, H. J. V. Diffusion and Heat Flow in Liquids (Butterworth, 1961). and isotope diffusion of K and Ca in multicomponent silicate melts. Earth Planet. 10. Duhr, S. & Braun, D. Why molecules move along a temperature gradient. Proc. Natl Sci. Lett. 123, 155–166 (1994). Acad. Sci. USA 103, 19678–19682 (2006). 28. Lesher, C. E. Decoupling of chemical and isotopic exchange during magma mixing. 11. Braun, D. & Libchaber, A. Thermal force approach to molecular evolution. Phys. Nature 344, 235–237 (1990). Biol. 1, 1–8 (2004). 29. Zhang, Y. A modified effective binary diffusion model. J. Geophys. Res. 98, 12. Severinghaus, J. P., Grachev, A. & Battle, M. Thermal fractionation of air in polar 11901–11920 (1993). firn by seasonal temperature gradients. Geochem. Geophys. Geosyst. 2, 1048 30. Huang, F. et al. Chemical and isotopic fractionation of wet andesite in a (2001). temperature gradient: experiments and models suggesting a new mechanism of 13. Severinghaus, J. P. et al. Timing of abrupt climate change at the end of the Younger magma differentiation. Geochim. Cosmochim. Acta 73, 729–749 (2009). Dryas interval from thermally fractionated gases in polar ice. Nature 391, 141–146 (1999). Supplementary Information is linked to the online version of the paper at 14. Lundstrom, C. C. Hypothesis for the origin of convergent margin granitoids and www.nature.com/nature. Earth’s continental crust by thermal migration zone refining. Geochim. Cosmochim. Acta 73, 5709–5729 (2009). Acknowledgements This work is supported by US National Science Foundation 15. Groot, S. R. D. Thermodynamics of Irreversible Processes 111–118 (North-Holland, grants NSF EAR 0609726 and NSF EAR 0944169 (C.C.L.), and NSF EAR 0943991 1959). (C.E.L.). The multi-collector inductively-coupled-plasma mass spectrometry 16. Lesher, C. E. & Walker, D. in Diffusion, Atomic Ordering, and Mass Transport (ed. laboratory at the University of Illinois at Urbana-Champaign is supported by NSF Ganguly, J.) 396–451 (Adv. Phys. Geochem. 8, Springer, 1991). EAR 0732481. P.C. acknowledges support from a Roscoe G. Jackson II Research 17. Richter, F. M. et al. Isotopic fractionation of the major elements of molten basalt by Fellowship. P.C. and S.W.K. acknowledge support from S.W.K.’s Walgreen Chair chemical and thermal diffusion. Geochim. Cosmochim. Acta 73, 4250–4263 (2009). funds. We thank B. Fouke for use of his micro-drilling system, Z. Zhang and X. Li for 18. Richter, F. M. et al. Magnesium isotope fractionation in silicate melts by chemical analytical assistance, V. Kariwala for help with references and Y. Zhang for a review and thermal diffusion. Geochim. Cosmochim. Acta 72, 206–220 (2008). of this work. 19. Wittko, G. & Kohler, W. Universal isotope effect in thermal diffusion of mixtures Author Contributions F.H. led the analytical effort and P.C. led the theoretical containing cyclohexane and cyclohexane-d12. J. Chem. Phys. 123, 014506 (2005). treatment of the results. C.E.L. performed the laboratory thermal diffusion 20. Wittko, G. & Kohler, W. On the temperature dependence of thermal diffusion of experiments. J.J.G.G. assisted with iron and magnesium isotope analyses and C.H. liquid mixtures. Europhys. Lett. 78, 46007 (2007). measured calcium isotopes. P.C. and F.H. wrote the manuscript and 21. Furry, W. H., Jones, R. C. & Onsager, L. On the theory of isotope separation by Supplementary Information with contributions from C.C.L., C.H., S.W.K., J.J.G.G. thermal diffusion. Phys. Rev. 55, 1083–1095 (1939). and C.E.L. 22. Kyser, T. 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METHODS as the bracketing standard. Samples were diluted to 1.5 p.p.m. iron (ZM71, Thermal diffusion experiments. Silicate melts of variable bulk composition ZM98 and ZM99) or to 0.4 p.p.m. iron (ZM70 and ZM100). Iron solutions were introduced into the mass spectrometer with a desolvating nebulizer (DSN-100, were placed at above liquidus temperatures in the temperature gradient of a 56 Nu Instruments) in 2% HNO3, producing a Fe signal sensitivity of ,10 V per piston cylinder apparatus for the times given in Table 1. The chemical composi- 1 1 tions of the starting materials ranged from mid-ocean-ridge basalt (ZM98) and p.p.m. Interferences of ArN and ArO were adequately resolved with resolu- 57 56 54 andesite (ZM70 and ZM71) to rhyolitic pantellerite (ZM100) and synthetic melt tions greater than 8,000. Isotope beams of Fe, Fe and Fe were measured in Faraday cups with 1011-V resistor channels. The measured 56Fe/54Fe and Fe2SiO4–KAlSiO4–SiO2 (ZM99) (Table 1). We also included data from a recent 57 54 54 53 thermal migration study on andesite, which involved coexisting silicate melt and Fe/ Fe ratios were corrected for the isobaric interference of Cr using Cr minerals in a temperature gradient30, and other thermal diffusion experiments simultaneously measured on detector L5. The long-term (six-month) average 17,18 56 using basalt as starting material . In addition to the wide range in composi- d FeIRMM-014 values from 28 analyses of ETH hematite and 55 analyses of in- tion, the data also span a wide range in temperature: the temperature at the hot house standard (UIFe) are 0.585 6 0.049% and 0.708 6 0.093% (2s), respect- 56 end ranged from 950 to 1,650 uC and that at the cold end ranged from 350 to ively. The error in the d FeIRMM-014 values of the samples is up to 0.28% (for the 1,415 uC. Generally, in thermal diffusion experiments iron, calcium and mag- solutions with 0.4 p.p.m. iron), which is insignificant relative to the isotopic nesium migrate to, and become enriched at, the cold end of the experiments, variation (.5%) of the thermal experiments. whereas silicon behaves oppositely6,16. All experiments except ZM70 were of long Magnesium isotopes were measured using the Nu Plasma HR mass spectro- enough duration to evolve to within 95% of the steady-state condition in terms meter at resolution of about 400, using a sample-standard bracketing method 26 25 of concentration changes of major elements. relative to an in-house magnesium standard. The d MgDSM-3 and d MgDSM-3 Iron, magnesium and calcium isotope analyses. Samples were micro-drilled values of the in-house bracketing standard over five months are 3.314 6 0.235% from silicate glass in thermal diffusion experiments6,16. To avoid contamination and 1.700 6 0.137% (2s, n 5 28), respectively. Samples were diluted to approxi- of iron by the micro-drilling process, we used tungsten carbide drill bits to drill mately 0.15 p.p.m. and introduced using the DSN-100, producing a 24Mg signal 0.4–0.5-mm-wide bands across the silicate glass in the polished surface mount of ,16 V. Isotope beams of 26Mg, 25Mg and 24Mg were measured simulta- section of the experiments reported in refs 6, 16. These widths translate into a neously. Matrix and yields were checked before each run, with 23Na/Mg and temperature range of ,10 uC. Temperature differences between the drilling areas 27Al/Mg generally less than 5%. Details of the magnesium analytical procedure were estimated assuming both a linear and a parabolic temperature profile across are available in ref. 31. the thermal diffusion experiment. The difference in temperature estimated by Calcium isotopes were measured on a thermal ionization mass spectrometer the two methods is negligible (the values are within 5% of each other). (Thermo-Finnigan TRITON) in the Saskatchewan Isotope Laboratory using a After micro-drilling, the silicate glass powder was digested in 7-ml Teflon 43Ca–42Ca double spike to correct for instrumental mass fractionation (see beakers using a 2-ml 3:1 mixture of concentrated hydrofluoric acid and ref. 32). Before mass spectrometric analysis, calcium was purified from matrix HNO3. Iron and magnesium were separated by anion resin (Bio-Rad AG1-X8) elements, including potentially interfering 40K, using conventional cation and cation resin (Bio-Rad AG50-X12), respectively. The iron and magnesium exchange chromatography. The data are reported in the conventional delta blanks for the chemical separation process had masses of ,10 ng and 3 ng, 44 44 40 44 40 notation as d CaSW 5 ( Ca/ Casample)/( Ca/ CaSW) 2 1. The standard is respectively. These are negligible in comparison with the amounts of iron and natural sea water. The external precision of d44Ca values is 60.07% (2s)on magnesium in the samples (.2 mg). The iron and magnesium blanks for the the basis of repeated analyses of sea water and an internal laboratory CaF2 micro-drilling process had masses of ,7 ng and 1 ng, respectively. Yields and standard. The average d44Ca value for NIST SRM 915a measured in the matrix were checked before isotopic measurements to ensure excellent recovery Saskatchewan Isotope Laboratory is 21.86%. and purification. Iron isotopes were measured by high-resolution multi-collector inductively- 31. Huang, F. et al. Magnesium isotopic composition of igneous rock standards coupled-plasma mass spectroscopy (Nu Plasma HR, Nu Instruments) in the measured by MC-ICP-MS. Chem. Geol. 268, 15–23 (2009). Department of Geology at the University of Illinois at Urbana-Champaign, in 32. Holmden, C. & Be´langer, N. Ca isotope cycling in a forested ecosystem. Geochim. static mode using a sample-standard bracketing method relative to IRMM-014 Cosmochim. Acta 74, 995–1015 (2010).

Post-copulatory sexual selection and sexual conflict in the evolution of male pregnancy

Kimberly A. Paczolt1 & Adam G. Jones1

Male pregnancy in seahorses, pipefishes and sea dragons (family documented opportunity for sexual selection in females of any taxon23. Syngnathidae) represents a striking reproductive adaptation that In nature, males normally mate with a single female per pregnancy23,24, has shaped the evolution of behaviour and morphology in this group whereas the most successful females can mate with several males, of fishes1–4. In many syngnathid species, males brood their offspring resulting in a limited supply of receptive males. Previous studies of in a specialized pouch, which presumably evolved to facilitate male post-copulatory processes in a related pipefish (Syngnathus typhle) parental care5,6. However, an unexplored possibility is that brood show that larger eggs experience higher survivorship, but fail to identify pouch evolution was partly shaped by parent–offspring or sexual male-mediated effects or address between-pregnancy trade-offs25,26. conflict, processes that would result in trade-offs between current Gulf pipefishes provide an excellent system in which to investigate such and future pregnancies. Here we report a controlled breeding experi- trade-offs while eliminating potential complications from multiple ment using the sexually dimorphic Gulf pipefish, Syngnathus mating within a pregnancy. scovelli, to test for post-copulatory sexual selection within broods Our experimental design involved mating each focal male with a and for trade-offs between successive male pregnancies as functions single female, allowing the male to carry his first brood to term, mating of female attractiveness. Offspring survivorship within a pregnancy the male with a second female (Fig. 1) and then monitoring embryo was affected by the size of a male’s mate, the number of eggs trans- mortality in the second brood. We photographed each brood imme- ferred and the male’s sexual responsiveness. Significantly, we also diately after egg transfer, near the pregnancy midpoint and just before found that embryo survivorship in a current pregnancy was nega- parturition (Fig. 1a, b). Embryos failing to develop can be distin- tively related to survivorship in the prior pregnancy, clearly dem- guished from viable embryos by their diminished size and colour at onstrating fitness trade-offs between broods. Overall, our data day seven of the pregnancy (which typically lasts 12–14 days), so we indicate that post-copulatory sexual selection and sexual conflict measured brood reduction by dividing the number of inviable occur in Gulf pipefishes. The conflict seems to be mediated by a embryos by the number of eggs initially received. Offspring survivor- strategy of cryptic choice in which males increase rates of offspring ship is defined as one minus brood reduction. We controlled the size abortion in pregnancies from unattractive mothers to retain of the females with which males mated to ensure that males sometimes resources for future reproductive opportunities. Hence, the male mated with females that differed from one another in size and some- brood pouch of syngnathid fishes, which nurtures offspring7–9,also times mated with females similar to one another in size. Most of the seems to have an important role as an arbiter of conflict between the analyses presented here are based on the second brood for each male sexes. Male pregnancy, a phenomenon unique to seahorses and their a Prior brood Current brood relatives, is facilitated by an elaborate brood pouch into which a Mating Pregnancy Birth Mating Pregnancy Birth female deposits eggs during mating10,11. Far from being a passive bag for offspring (Fig. 1), the male’s pouch provides aeration, protection, osmoregulation and nutrition to the developing offspring during a pregnancy that can last several weeks7–9,12–14. The brood pouch is usually viewed as a structure whose main role is to nurture Brood offspring. Recent work has established that nutrients move both from pouch father to offspring7–9 and from offspring to father15 during the pregnancy, raising the hitherto unexplored possibility that the brood b c OF d C pouch may have a role in modulating post-copulatory sexual selec- E tion and sexual conflict16–18. Such conflict could occur as a consequence of either males withholding (or taking) resources from Y some broods to save resources for future pregnancies19,20 or females 1 mm transferring substances during mating that stimulate males to invest 0.2 mm more resources in the current pregnancy21,22. Whether sexual conflict Figure 1 | Experimental design and brood pouch morphology. a, Each focal is driven by male- or female-mediated effects, the demonstration of a male mated with a single female for each of two successive pregnancies. b,We role for the brood pouch in such processes would precipitate a major documented the development of offspring during each pregnancy by c shift in thought regarding adaptive mechanisms responsible for the photographing the brood through the transparent pouch. , Scanning electron micrograph illustrating the close connection between father and evolution of male pregnancy and parent–offspring interactions in offspring; OF, outer pouch flap; C, chorion; E, embryonic tissue; Y, yolk. d,A this group of fishes. honeycomb of paternal tissue is present in the brood pouch when the We focus here on post-copulatory processes in the sexually embryos are removed at about the half-way point of a pregnancy. (Images, dimorphic, sex-role-reversed Gulf pipefish, which has the highest N. Ratterman and C. Partridge.)

(N 5 22), which we refer to as the ‘current brood’ (Fig. 1). The To ascertain the extent to which interbrood trade-offs are involved purpose of the first brood (which we call the ‘prior brood’) was to in Gulf pipefish reproduction, we examined brood reduction in light establish a known mating history for each male. First, we addressed of a male’s mating history. If sexual conflict occurs, we expect to see whether or not males practice pre-copulatory mate choice; second, we trade-offs in subsequent broods. We addressed this hypothesis by quantified post-copulatory sexual selection by examining relation- using a stepwise regression and path analysis to study the effects of ships between offspring survivorship and female size; third, we current and prior brood characteristics on offspring survivorship in examined factors affecting offspring survivorship within broods, with the current brood (see Methods for statistical details). Our main result a particular emphasis on trade-offs between broods; and, finally, we with respect to post-copulatory sexual selection and sexual conflict is used an individual-based model to investigate the circumstances that current offspring survivorship is dependent on characteristics of under which a brood reduction strategy would be adaptive in nature. the prior brood (Fig. 3). In particular, current offspring survivorship is With respect to pre-copulatory choice, our results indicate that negatively correlated with prior female size and prior brood size, males prefer to mate with larger females. Each mating trial was a no- suggesting that valuable (and energetically expensive) previous broods choice preference experiment, and we found that males’ reluctance to hinder a male’s ability to invest in current broods (Fig. 3). Similarly, mate was significantly shorter for larger females (Fig. 2a), a result that current offspring survivorship is negatively correlated with prior off- was even more pronounced when we considered female size relative spring survivorship (Fig. 3), suggesting that males are capable of redu- to male size (Fig. 2b). Thus, pre-copulatory mate choice favours cing investment in some broods to save resources for future larger females, and the pattern is very strong, as might be expected reproduction and that investment in a valuable brood decreases sur- for a highly sexually dimorphic species such as Gulf pipefishes23. vivorship in subsequent broods. Our second goal was to investigate the nature of post-copulatory In terms of the post-copulatory process, the simplest explanation for sexual selection in Gulf pipefishes. Our results indicated that pregnancies our results is that broods with larger mothers are energetically more showed substantial variation with respect both to the number of eggs costly for males. These costs could be manifested in two ways: either transferred per copulation (range, 4–42; mean, 22.5; variance, 89.8) and males are selected to invest more resources in embryos from large, to survivorship of eggs in the brood pouch (range, 0–1; mean, 0.71; attractive mothers as an adaptive strategy of cryptic male choice, or larger variance, 0.12), setting the stage for post-copulatory sexual selection. If females (or their offspring) have evolved a mechanism that induces post-copulatory sexual selection occurs in pipefishes, then one very greater reproductive investment by males (see Supplementary Infor- important question is whether it reinforces or opposes pre-copulatory mation for additional data bearing on the second possibility). Overall, sexual selection27,28. Other studies of post-copulatory sexual selection in the hypothesis of cryptic male choice is most consistent with our data, a wide range of taxa have provided mixed results on this issue27.Our because all of our observations can be explained by discrimination results show that males prefer to mate with larger females (who also tend against smaller females at every phase of pre- and post-copulatory to have more pronounced secondary sexual characters) and that post- sexual selection. Moreover, the mechanism would be relatively simple, copulatory sexual selection acts in the same direction. We observed a as males could reduce the rate at which resources are transferred strong positive correlation between the number of eggs transferred and from them to the broods from less attractive females, increasing com- female size (Fig. 2c), and saw the same pattern for offspring survivorship petition among siblings and reducing offspring survivorship. (Fig. 2d). In short, larger females transferred more eggs per mating and the resulting embryos experienced a greater probability of surviving to parturition. Thus, post-copulatory sexual selection reinforces pre- Prior female copulatory sexual selection in Gulf pipefishes. total length

5 a 50 c Prior b′ = –0.260 4 40 Prior brood P = 0.007 size reluctance 3 30 b′ = –0.283 to mate 2 20 P = 0.004

1 10 b′ = –0.245

Number of eggs transferred P = 0.012 0 0 90 100 110 120 90 100 110 120 b′ = –0.359 Female length (mm) Female length (mm) Current brood offspring P < 0.001 Prior brood offspring survivorship survivorship 5 b 1 d 4 0.8

3 0.6 b′ = 0.293 P = 0.003 b′ = 0.311 2 0.4 b′ = 0.588 P = 0.001 Natural logarithm of time elapsed before mating (d) 1 0.2 P < 0.001 b′ = –0.406 0 0 P = 0.032 Current –10 0102030 90 100 110 120 Current male brood Current female Female length – male length (mm) Number of viable offspring per egg Female length (mm) total length size total length Figure 2 | Evidence for pre-copulatory mate choice and post-copulatory sexual selection by male Gulf pipefishes. a, We observed a strong negative Figure 3 | Results of a path analysis showing the effects of variables from correlation between a female’s total length and the time that elapsed before the prior and current broods on current offspring survivorship. Solid mating took place for male–female pairs (linear regression: N 5 22, arrows represent significant partial regressions; dashed arrows show r2 5 0.23, P 5 0.025). b, The pattern was more pronounced when we took relationships that were included in the analysis but were not statistically into account the female’s length relative to the male’s length (N 5 22, significant. For each significant relationship, we label the corresponding r2 5 0.53, P , 0.001). c, We observed a significantly positive relationship solid arrow with the partial regression coefficient, b9, and the P value. The between the female’s total length and the number of eggs transferred to the key result illustrated in this figure is that the survival of embryos in the male’s pouch (N 5 22, r2 5 0.20, P 5 0.038). d, Males also experienced current brood depends on features of the previous brood; in particular, higher levels of offspring survivorship when they mated with larger as current offspring survivorship was negatively correlated with prior offspring opposed to smaller females (N 5 22, r2 5 0.56, P 5 0.007). survivorship and prior female length. 402 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

This conclusion is further bolstered by recent observations in a (length, 93–106 mm) female. We inspected male brood pouches daily. The brood related pipefish, S. typhle, which show that amino acids originating in pouches of pregnant males were photographed under a dissecting microscope on the eggs pass from the embryos through the male’s brood pouch to be days one, seven and 11 after mating. On these days, embryos were counted and incorporated into his liver and muscle tissue15. Thus, male pipefishes undeveloped eggs were noted. We calculated brood reduction by dividing the possess a mechanism to take resources from their broods, and the number of undeveloped eggs on day seven by the total number of eggs transferred by the female. Offspring survivorship was calculated as one minus brood male-mediated strategy of brood reduction that our data illustrate is reduction. Males were paired with their second mate within 24 hours after par- an example of sexual conflict, analogous to infanticide or filial can- turition of the first brood, and this brood also was photographed and scored on 18 nibalism . Males are partly, or in some cases completely, aborting the days one, seven and 11 after mating. At the end of each trial, we measured male offspring from smaller females, at the expense of the reproductive and female body size (total length, snout–vent length, depth and weight). Our interests (and Darwinian fitness) of those females. This strategy statistical analyses focused on the second brood for each male. We examined how appears to be beneficial to the males, as their future broods, which both prior and current brood (and mate) characteristics affected mating latency, should have larger mothers on average, have enhanced offspring brood size and offspring survivorship. survivorship. This clash of reproductive interests of the sexes is the Full Methods and any associated references are available in the online version of 18 crux of sexual conflict , so our results raise the hypothesis that the paper at www.nature.com/nature. females should be selected to somehow resist this male strategy. If males are capable of manipulating female broods, as our data Received 13 October 2009; accepted 22 January 2010. suggest, then a key question is whether or not such a strategy is likely 1. Berglund, A., Rosenqvist, G. & Svensson, I. Mate choice, fecundity and sexual to be adaptive in natural populations. Intuitively, the best strategy for dimorphism in two pipefish species (Syngnathidae). Behav. Ecol. Sociobiol. 19, males would be to mate with large, attractive females every pregnancy. 301–307 (1986). However, several lines of evidence suggest that this strategy may be 2. Vincent, A., Ahnesjo¨, I., Berglund, A. & Rosenqvist, G. Pipefishes and seahorses–are they all sex-role reversed? Trends Ecol. Evol. 7, 237–241 (1992). impossible. For example, the average potential reproductive rates of 3. Jones, A. G., Rosenqvist, G., Berglund, A., Arnold, S. J. & Avise, J. C. The Bateman 29 females are only about twice those of males and pipefish sex ratios gradient and the cause of sexual selection in a sex-role-reversed pipefish. Proc. R. fluctuate dramatically in nature, often showing a large excess of males30. Soc. Lond. B 267, 677–680 (2000). Thus, attractive females prepared to mate often will be in short supply. 4. Wilson, A. B., Ahnesjo¨, I., Vincent, A. C. J. & Meyer, A. The dynamics of male brooding, mating patterns, and sex roles in pipefishes and seahorses (family Moreover, results from our individual-based model show that under a Syngnathidae). Evolution 57, 1374–1386 (2003). wide range of circumstances, males are expected to benefit from the 5. Harlin-Cognato, A., Hoffman, E. A. & Jones, A. G. Gene co-option without cryptic choice strategy observed in our experiments, especially when duplication during the evolution of a male pregnancy gene in pipefish. Proc. Natl large females prepared to mate are in short supply (Supplementary Acad. Sci. USA 103, 19407–19412 (2006). Information). The fitness of the cryptic choice strategy increases when 6. Sto¨lting, K. N. & Wilson, A. B. Male pregnancy in seahorses and pipefish: beyond the mammalian model. Bioessays 29, 884–896 (2007). the preference threshold is high, as might be expected for a strongly 7. Berglund, A., Rosenqvist, G. & Svensson, I. Reversed sex roles and parental energy sexually selected species such as the Gulf pipefish, and when the sex ratio investment in zygotes of two pipefish (Syngnathidae) species. Mar. Ecol. Prog. Ser. is biased towards an excess of males, which is a common occurrence in 29, 209–215 (1986). nature30. In addition, the model predicts that males are reluctant to mate 8. Ripley, J. L. & Foran, C. M. Differential parental nutrient allocation in two with small females, as we observed. Males that are too eager to mate with congeneric pipefish species (Syngnathidae: Syngnathus spp.). J. Exp. Biol. 209, 1112–1121 (2006). small females suffer fitness costs by missing opportunities to mate with 9. Ripley, J. L. & Foran, C. M. Direct evidence for embryonic uptake of paternally- females above the preference threshold. Overall, the results of the model derived nutrients in two pipefishes (Syngnathidae: Syngnathus spp.). J. Comp. indicate that the cryptic choice strategy implied by our empirical results Physiol. B 179, 325–333 (2009). should be adaptive in natural populations of pipefishes. 10. Dawson, C. E. Indo-Pacific Pipefishes (Gulf Coast Research Lab, 1985). 11. Herald, E. S. From pipefish to seahorse–a study of phylogenetic relationships. Proc. The results of our study are significant at two levels. First, post- Calif. Acad. Sci. 29, 465–473 (1959). copulatory sexual selection has been virtually unexamined in sex- 12. Partridge, C., Shardo, J. & Boettcher, A. Osmoregulatory role of the brood pouch in role-reversed taxa. If cryptic choice is a general mechanism of sexual the euryhaline Gulf pipefish, Syngnathus scovelli. Comp. Biochem. Physiol. A 147, selection, then we should expect it to evolve in sex-role-reversed species 556–561 (2007). as well as in the more commonly studied species with conventional sex 13. Ripley, J. L. Osmoregulatory role of the paternal brood pouch for two Syngnathus species. Comp. Biochem. Physiol. A 154, 98–104 (2009). roles. Our results show that such a mechanism has evolved in Gulf 14. Berglund, A. & Rosenqvist, G. Sex role reversal in pipefish. Adv. Stud. Behav. 32, pipefishes, despite the fact that this species has effective mate choice 131–167 (2003). before mating, implying that cryptic choice is indeed a process of 15. Sagebakken, G., Ahnesjo¨, I., Mobley, K. B., Braga Gonc¸alves,I. & Kvarnemo, C. fundamental significance. Second, our results bear on the adaptive Brooding fathers, not siblings, take up nutrients from embryos. Proc. R. Soc. Lond. B. significance of the brood pouch. On casual inspection, the pouch seems doi:10.1098/rspb.2009.1767. 16. Trivers, R. L. in Sexual Selection and the Descent of Man (ed. Campbell, B.) 136–179 to be a structure that evolved to nurtureand providefor offspring,and it (Aldine, 1972). does serve this function. However, it also may grant the male better 17. Parker, G. A., Royle, N. J. & Hartley, I. R. Intrafamilial conflict and parental control over reproduction. Males seem to be able to adaptively affect investment: a synthesis. Phil. Trans. R. Soc. Lond. B 357, 295–307 (2002). their investment in broods as a function of the value of the pregnancy. 18. Arnqvist, G. & Rowe, L. Sexual Conflict (Princeton Univ. Press, 2005). Perhaps they simply invest fewer resources in broods originating from 19. Burley, N. The differential allocation hypothesis–an experimental test. Am. Nat. 132, 611–628 (1988). smaller females (resulting in competition among siblings within the 20. Sheldon, B. C. Differential allocation: tests, mechanisms and implications. Trends pouch for resources), but males also have the ability to exploit the Ecol. Evol. 15, 397–402 (2000). reproductive contributions of some females by actively absorbing 21. Wolfner, M. F. The gifts that keep on giving: physiological functions and evolutionary embryos from less valuable broods15. Thus, the brood pouch serves a dynamics of male seminal proteins in Drosophila. Heredity 88, 85–93 (2002). 22. Zeh, D. W. & Zeh, J. A. Reproductive mode and speciation: the viviparity-driven more complicated purpose in pipefish reproduction than previously conflict hypothesis. Bioessays 22, 938–946 (2000). believed, certainly providing parental care but also participating in a 23. Jones, A. G., Walker, D. & Avise, J. C. Genetic evidence for extreme polyandry and conflict between the sexes. extraordinary sex-role reversal in a pipefish. Proc. R. Soc. Lond. B 268, 2531–2535 (2001). METHODS SUMMARY 24. Jones, A. G. & Avise, J. C. Microsatellite analysis of maternity and the mating system in the Gulf pipefish Syngnathus scovelli, a species with male pregnancy and We collected Gulf pipefishes from seagrass meadows near Port Aransas, Texas, sex-role reversal. Mol. Ecol. 6, 203–213 (1997). maintained them in 9.5-l salt-water tanks and fed them Artemia nauplii twice 25. Ahnesjo¨, I. Apparent resource competition among embryos in the brood pouch of daily. Each experimental male mated twice in the lab, producing two successive a male pipefish. Behav. Ecol. Sociobiol. 38, 167–172 (1996). pregnancies with one mate per pregnancy. For each mating event, the male was 26. Partridge,C.etal.Theeffectofperceivedfemaleparasiteloadonpost-copulatorymale randomly assigned to mate with either a large (length, 108–122 mm) or small choice in a sex-role-reversed pipefish. Behav. Ecol. Sociobiol. 63, 345–354 (2009). 403 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

27. Andersson, M. & Simmons, L. W. Sexual selection and mate choice. Trends Ecol. Acknowledgements This work was supported by grant IOS-0455927 from the US Evol. 21, 296–302 (2006). National Science Foundation. We thank S. Scobell and R. Carter for help with 28. Hunt, J., Breuker, C. J., Sadowski, J. A. & Moore, A. J. Male-male competition, animal husbandry. We are also grateful to S. Arnold, A. Berglund, M. Giresi, female mate choice and their interaction: determining total sexual selection. N. Ratterman, E. Rose, G. Rosenthal, C. Small and D. Zeh for comments on the J. Evol. Biol. 22, 13–26 (2009). manuscript. 29. Scobell, S. K., Fudickar, A. M. & Knapp, R. Potential reproductive rate of a sex-role Author Contributions K.A.P. performed the experiments. A.G.J. and K.A.P. reversed pipefish over several bouts of mating. Anim. Behav. 78, 747–753 (2009). designed the experiments, analysed the data and wrote the manuscript. 30. Brown, J. D. A Comparative Life History Study of Four Species of Pipefishes (Family Syngnathidae) in Florida. PhD thesis, Univ. Florida (1972). Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Supplementary Information is linked to the online version of the paper at Correspondence and requests for materials should be addressed to K.A.P. www.nature.com/nature. ([email protected]).

METHODS length, snout–vent length, weight and standard depth at this time. At the end of the experiment, male total length ranged from 87 to 105 mm. Male and female total Animal collection and maintenance. We collected Gulf pipefishes near Port 2 Aransas, Texas, on three occasions between June and August 2007, transported lengths were not correlated in this experiment (N 5 22, r 5 0.13, P 5 0.107). them to the Texas A&M University campus in College Station and housed them Brood monitoring. On days one, seven and 11 after mating, we counted in a flow-through system until they were used in the experiment. Collection date embryos and assessed the progress of development. The male was transferred had no significant effect on brood reduction, brood size or time spent in the lab to a small amount of water and examined under a dissecting microscope. (Tukey–Kramer HSD test; a # 0.05). Most males were pregnant at the time of Embryos are visible through the brood pouch epithelium and can be counted collection, so we housed them in groups until they gave birth. Males usually were by eye. All broods were counted at least twice to verify accuracy. Unusual-looking added into the mating experiment less than 24 hours after releasing their field- eggs were noted, and eggs that showed no obvious development (in comparison conceived brood. Females were housed in female-specific tanks until they were with other embryos in the brood) counted towards brood reduction. used in the mating experiment. Undeveloped eggs often shrank and became more transparent during the brood- Mating experiment. We housed males in 9.5-l aquaria equipped with sponge ing period. We produced a time series of brood development by photographing filters and artificial seagrass, and fed them Artemia nauplii twice daily. Each male the broods on these days using a digital camera (Zeiss AxioCam MRc 5) attached was maintained in the same tank for the duration of the trial, and tanks were to a stereo microscope (Zeiss Stemi 2000C). thoroughly cleaned between trials. Males were randomly assigned to mate with Analysis. We calculated brood reduction by dividing the number of either a large (length, 108–122 mm) or small (length, 93–106 mm) female. The undeveloped eggs on day seven by the total number of eggs transferred by the reproductive status of females was assessed by the presence of the female sec- female. Offspring survivorship was calculated as one minus brood reduction. ondary sexual ornament, which appears as silvery-blue lateral bars on the trunk. The photographic time series of the brood was consulted if a question arose as to We presented each male with a randomly assigned female of the appropriate size the fate of a particular egg. class immediately upon addition to the experiment. Male brood pouches were Because of the large number of variables measured in this study, we used visually inspected daily. Of the 48 males that started the experiment, 34 accepted stepwise regression as an exploratory tool to evaluate which variables should eggs from their first mate. The remaining 14 males were excluded on the basis of the be included in a path analysis. The response variable was current brood reduc- following criteria: the death of either individual in the pair, mating outside the tion and all included variables had individual P values of less than 0.05 in the intended pair or a failure to mate before the experiment end date (4 November stepwise regression analysis. On the basis of these results, we kept variables 2007). Two more males were excluded because they died before the brood emerged. describing adult total length, reluctance to mate, brood size and prior brood Broods were monitored during development (see below). We maintained males reduction. All variables were transformed to fit a normal distribution before and females together until the male was moved into a brooding chamber 11 days statistical analysis. Univariate and stepwise regressions were performed in JMP 7. after mating. On the day the fry emerged, the female was killed by administration of In our path analysis, all variables retained from the stepwise regression analysis an overdose of tricaine methanesulphonate (MS222), photographed and preserved were connected to our focal variable, namely current offspring survivorship. in formalin solution. We made measurements of female total length, snout–vent Other connections were included to represent all likely biological interactions 29 length, weight, standard depth , maximum depth and dorsal fin area. Up to five between the measured variables. We estimated partial regression coefficients, eggs were removed from each female, photographed and measured for diameter. standardized to 1 s.d., by using the maximum-likelihood procedure implemented The 30 males still in the experiment after the first pregnancy were each presented in the computer program AMOS 5.0.1. The path analysis model resulted in a x2 with a second mate within 48 hours of giving birth. Males were again randomly value of 13.05, leading to a P value of 0.522, which implies that the hypothesis of a assigned to either a large or a small female, and the pairs were monitored as perfect fit of the model cannot be rejected. This model explained 84% of the previously described.Nofemales were re-used during the experiment, sothe second variance in current offspring survivorship. The model fit had a root mean squared mates were independent of the first mates. Twenty-three of the 30 males accepted error of approximation of ,0.001, which is less than the value, 0.05, expected for a eggs from the second mate and 22 males survived to maturity of the brood. The model with excellent fit31. Overall, the measures of fit for the path analysis suggest remaining seven males, which did not mate, were excluded from the experiment. that the estimated model is an accurate representation of our data. On the day of parturition of the second brood, both themale and female in each pair were killed using MS222, photographed and preserved in formalin. Females were 31. Steiger, J. H. & Lind, J. C. Statistically based tests for the number of common measured as previously described. We also made measurements of male total factors. Proc. Ann. Spring Meeting Psychometric Soc. (1980).

ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C

Jacques Fellay1*, Alexander J. Thompson2*, Dongliang Ge1*, Curtis E. Gumbs1, Thomas J. Urban1, Kevin V. Shianna1, Latasha D. Little1, Ping Qiu3, Arthur H. Bertelsen3, Mark Watson3, Amelia Warner3, Andrew J. Muir2, Clifford Brass3, Janice Albrecht3, Mark Sulkowski4, John G. McHutchison2 & David B. Goldstein1

Chronic infection with the hepatitis C virus (HCV) affects The SNPs showing a genome-wide significant association with 170 million people worldwide and is an important cause of liver- quantitative week-4 Hb reduction were spread over a 250 kilobase related morbidity and mortality1. The standard of care therapy (kb) region that contains five different protein-coding genes (Fig. 1). combines pegylated interferon (pegIFN) alpha and ribavirin We tested the independence of the top association signals in the (RBV), and is associated with a range of treatment-limiting adverse European-American population using nested linear regression mod- effects2. One of the most important of these is RBV-induced els, in which individual SNPs were added after inclusion of haemolytic anaemia, which affects most patients and is severe rs6051702, and found evidence for multiple independent signals of enough to require dose modification in up to 15% of patients. association: the most strongly associated SNPs after accounting for Here we show that genetic variants leading to inosine triphospha- the contribution of rs6051702 have P values of 1.4 3 1029 tase deficiency, a condition not thought to be clinically important, (rs2295547) and 2.3 3 1024 (rs6051855). The persistence of strongly protect against haemolytic anaemia in hepatitis-C-infected patients significant association in the region after accounting for the discovery receiving RBV. variant suggests the possibility of multiple causal variants and/or Using DNA from consenting participants of the IDEAL study2,we rarer causal variants9. performed a genome-wide association study (GWAS) of determi- To identify candidate causal sites we searched the region for known nants of treatment-related anaemia in individuals with chronic geno- functional variants. We focused first on the inosine triphosphatase type 1 hepatitis C. A total of 1,602 DNA samples were genotyped in the context of a previously reported study of anti-HCV treatment Table 1 | Clinical characteristics of the study population 3 response . Following quality control steps (Supplementary Informa- Population tion I) 1,286 individuals, who were classified into three ethnic groups European- African- Hispanics (988 European-Americans, 198 African-Americans, 100 Hispanics), Americans Americans were available for analyses (Table 1). Our primary analysis focused on n 988 198 100 the quantitative change in haemoglobin (Hb) levels from baseline to Sex (F/M) 378/610 78/120 36/64 the fourth week of treatment—historically the point at which many Age (yrs) 47.3 (7.4) 49.7 (6.6) 44.8 (9.3) 2 patients begin erythropoietin treatment to stimulate red blood cell BMI (kg m2 ) 27.9 (4.5) 29.7 (5.0) 29.3 (5.4) Baseline weight (kg) 83.3 (16.1) 88.7 (14.3) 83.0 (16.7) production. We tested each of 565,759 single nucleotide polymor- Baseline liver fibrosis stage* (n,%) phisms (SNPs) passing quality control measures in a linear regression Minimal (F0–2) 876 (88.7%) 182 (91.9%) 86 (86.0%) model incorporating significant clinical covariates including baseline Advanced (F3–4) 112 (11.3%) 16 (8.1%) 14 (14.0%) Hb levels. Initial daily RBV dose{ (n,%) 800 mg 88 (8.9%) 4 (2.0%) 6 (6.0%) Several SNPs on chromosome 20 (20p13 region) were found to be 1,000 mg 377 (38.2%) 63 (31.8%) 41 (41.0%) strongly associated with treatment-induced reduction in Hb at week 1,200 mg 460 (46.6%) 117 (59.1%) 45 (45.0%) 4 (Fig. 1), with the European-American sample showing overwhelm- 1,400 mg 63 (6.4%) 14 (7.1%) 8 (8.0%) ing genome-wide significance (P 5 1.1 3 10245) for an association Peg-interferon treatment{ PegIFNa-2a 330 (33.4%) 66 (33.3%) 31 (31.0%) between quantitative Hb reduction and the SNP rs6051702. Asso- PegIFNa-2b 1.0 333 (33.7%) 69 (34.9%) 32 (32.0%) ciations with smaller effect sizes but in the same direction were PegIFNa-2b 1.5 325 (32.9%) 63 (31.8%) 37 (37.0%) observed in both the African-American and Hispanic samples Baseline Hb value (g dl21) 15.1 (1.2) 14.6 (1.2) 15.2 (1.3) Hb reduction at week 4, mean (g dl21) 2.9 (1.5) 2.4 (1.3) 3.2 (1.5) (Table 2). 1 Severe anaemia, Hb ,10 gdl2 at 89 (9.0%) 18 (9.1%) 11 (11.0%) Further association signals were detected in the hexokinase 1 gene week 4 (n,%) 5 3 27 (HK1) on chromosome 10 (P 5.3 10 for the intronic SNP Data shown are mean with s.d. in parentheses unless otherwise indicated. BMI, body mass rs10159477 in European-Americans; Supplementary Information index. II). This result is not genome-wide significant, but supported by * Liver biopsies were evaluated for METAVIR fibrosis staging. The METAVIR scoring system classifies fibrosis on a 5-point scale: F0, no fibrosis; F1, portal fibrosis without septa; F2, few other lines of evidence: rare HK1 mutations cause severe haemolytic septa; F3, numerous bridging septa without cirrhosis; F4, cirrhosis. anaemia in both humans4–6 and mice7; in a recent GWAS, HK1 SNPs { Initial daily RBV dose was weight-based on a sliding scale in subjects’ baseline weight, from 40–125 kg. associated with differences in Hb concentration and haematocrit in { PegIFNa-2b 1.0 and PegIFNa-2b 1.5 refer to 1.0 mgkg21 week21 and 1.5 mgkg21 week21 Europeans8. interferon, respectively.

1Institute for Genome Sciences & Policy, Center for Human Genome Variation, Duke University, Durham, North Carolina 27708, USA. 2Duke Clinical Research Institute and Division of Gastroenterology, School of Medicine, Division of Gastroenterology, Duke University, Durham, North Carolina 27705, USA. 3Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. 4Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. *These authors contributed equally to this work. 405 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

2 4 6 8 10 12 14 16 18 20 22 Y 13579111315171921XM 100% 50 90% ( P )

10 25 80% Wild type both variants

–log 0 70% Heterozygous rs7270101 60% Chromosome 20 ideogram Heterozygous rs1127354 50% Combined heterozygous 0 Mb 10 Mb 20 Mb 30 Mb 40 Mb 50 Mb 60 Mb Homozygous rs7270101 40% Homozygous rs1127354 3,090 kb 3,133 kb 3,176 kb 3,219 kb 3,262 kb 3,305 kb 30% 50 20% ( P )

10 25 10% 0% –log 0 CC AC AA AL121891.1 RP5 DDRGK1ITPA SLC4A11 C20orf194 rs6051702 genotype UBOX5 Figure 2 | Two ITPA polymorphisms known to be responsible for inosine FASTKD5 triphosphatase deficiency co-segregate with the rs6051702 C allele that strongly associates with protection against Hb reduction in European- Americans. In the European-American population included in the study, the low-activity ITPA variants rs1127354 (missense P32T) and rs7270101 (intronic, splicing-altering) are found almost exclusively on chromosomes that also carry the rs6051702 minor allele C (Supplementary Table 4). The graph shows, for each rs6051702 genotype, the percentages of individuals r2 that are either heterozygous or homozygous for at least one ITPA variant. 0.0 2 0.5 region, the highest r is 0.65, which is what is observed for our discovery variant (Supplementary Table 2). 1.0 This observation suggested the possibility that these two known functional ITPA variants are responsible for part or all of the observed association and confer protection against anaemia. To test this possibility, we first sequenced the entire coding region of the gene Figure 1 | Genomic overview of the 20q13 region including the genome- in a subset of 168 samples, and found no other obvious reduced wide significant associated variants and the ITPA gene. Indicated are the function mutations (Supplementary Table 3). We then genotyped P values [2log10(P)] of all genotyped SNPs in the region and the structures the known functional SNPs rs1127354 and rs7270101 in our entire of the surrounding genes. The SNPs that show genome-wide significant cohort. We observed that in European-Americans, the two func- association with quantitative reduction in haemoglobin levels are marked in tional ITPA variants were found almost exclusively on chromosomes 27 red. The results were annotated using the WGAViewer software . that also carry the rs6051702 minor allele C (Fig. 2 and Supplemen- tary Table 4). Moreover, when these two variants were incorporated (ITPA) gene, which encodes a protein that hydrolyses inosine tripho- into a regression model, they entirely explained the association signal sphate (ITP). Several gene mutations have been described that lead to initially identified (Supplementary Table 5). Finally, in European- ITPA deficiency, a benign red cell enzymopathy characterized by the Americans, each functional variant was strongly independently asso- accumulation of ITP in erythrocytes and increased toxicity of purine ciated with protection (Table 3). A similar picture was observed in analogue drugs10,11. In particular, reduced ITPA activity has been African-Americans and in Hispanics: the SNP that has the strongest documented for a missense variant in exon 2 (rs1127354, resulting association with treatment-induced Hb reduction in the region was in a proline-to-threonine substitution denoted P32T) and a splicing- different between populations (Table 2), but the signal is explained altering SNP located in the second intron (rs7270101)12–15. Several in each case by the associations between the most significant SNP studies have documented and replicated the functional effects of and the ITPA low-activity variants. When the evidence for each these polymorphisms, showing that both minor alleles independently functional SNP is combined across the populations, we find that reduce ITPA activity and that homozygosity for the P32T mutation the P values strengthen considerably to 1.7 3 10258 for rs1127354 results in non-detectable ITPA activity and strong accumulation of and 5.9 3 10226 for rs7270101 (Table 3). Therefore, several lines of ITP in red blood cells (Supplementary Table 1)12,16–18. Both SNPs can evidence confirm that the two known functional variants confer- therefore be considered validated functional variants. ring reduced ITPA activity are responsible for the protection against Using HapMap data from CEU parents (Utah residents with anaemia identified in the original GWAS. ancestry from northern and western Europe)19 we found both of To assess the affect of both functional alleles on anaemia, we con- these polymorphisms to be associated with rs6051702, with the sidered the combined low-activity allele described earlier in an addit- low-activity variants preferentially associating with the protective ive regression model. We found that the combined allele has an rs6501702 C allele. We can directly test whether the association signal association of P 5 2.2 3 10291 and that the resulting difference in reflects the functional alleles by considering them equivalent and ITPA function explains 28.7% of the variability in quantitative Hb defining a new allele as a ‘low-activity allele’ made up of either func- reduction in the European-American sample, 19.1% in African- tional variant (Supplementary Information III). We tested this allele Americans and 23.2% in Hispanics. for association with surrounding polymorphisms and found that of We made a direct assessment of the clinical relevance of these all the HapMap SNPs located in the surrounding 1 megabase (Mb) variants by considering the proportion of patients suffering clinically

Table 2 | GWAS of quantitative Hb reduction at week 4 SNP Sample in which the SNP shows the P value in European-Americans P value in African-American P value in Hispanics strongest association (n 5 988) (n 5 198) (n 5 100) rs6051702 European-Americans 1.1 3 10245 1.9 3 1021 9.5 3 1023 rs3810560 African-Americans 2.6 3 1022 1.2 3 1024 3.0 3 1021 rs11697114 Hispanics 2.0 3 1026 2.8 3 1022 2.1 3 1024 The 20p13 region SNPs showing the strongest association in each of the three study populations are reported, together with their P values in the different ethnic groups. 406 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

Table 3 | ITPA variants protect against Hb reduction ITP competes with RBV-TP in whatever cellular processes are affected ITPA variant Population MAF (%) P value Independent by RBV-TP, and thereby protects cells from the lytic effects of RBV- P value TP. An alternative explanation is that ITPA activity and/or ITP levels rs1127354 European-Americans 7.64.6 3 10252 2.3 3 10268 directly or indirectly influence RBV pharmacokinetics. We did not, African-Americans 4.62.7 3 1027 5.1 3 1027 however, observe any significant association between the ITPA- 23 25 Hispanics 4.01.2 3 10 5.6 3 10 deficiency variants and early or late anti-HCV treatment outcomes All (combined) 6.91.7 3 10258 5.9 3 10226 rs7270101 European-Americans 12.36.8 3 10222 3.6 3 10238 (Supplementary Information IV), which suggests that RBV pharma- African-Americans 7.93.0 3 1025 6.6 3 1025 cokinetics is not significantly altered in hepatic cells. We also evaluated Hispanics 8.03.8 3 1024 1.9 3 1025 global differences in the frequencies of the ITPA variants. We found 276 243 All (combined) 11.28.5 3 10 2.6 3 10 that although each of the two functional variants showed strong geo- The two ITPA functional SNPs rs1127354 and rs7270101 show strong independent association graphic differences in frequency (Supplementary Table 6), these are with protection against treatment-induced Hb reduction. Independent P values were calculated in models in which the other functional variant was already included. Combined P values for not correlated, and the predicted overall ITPA activity levels do not all three populations were obtained using the weighted Z-method26. MAF, minor allele seem to show important variation. This suggests that these variants do frequency. not create strong population-level differences in susceptibility to anaemia, as was observed for the effect of IL28B variation on treatment significant anaemia, which we defined as either a decline in Hb of response3. .3gdl21 or Hb levels ,10 g dl21, which is the threshold at which Two related features of these observations are worth emphasizing. RBV dose reduction is recommended. As depicted in Fig. 3, we First, the ITPA variants constitute a clear example of a synthetic asso- modelled this using both the individual genotypes and the degree ciation in which the effects of rarer functional variants are observed as of ITPA deficiency, predicted from the residual enzymatic activity an association for a more common variant present on a whole- measured in the presence of the two functional variants12,16–18.We genome genotyping chip: indeed, the minor-allele frequency is higher found that of 184 patients predicted to have deficient ITPA function for the top-associated SNP rs6051702 (19.4%) than for the causal corresponding to less than one-third of the normal enzymatic activity, variants rs1127354 (7.6%) and rs7270101 (12.3%) in European- none was observed to have Hb levels ,10 g dl21, and only seven Americans. Second, ITPA deficiency seems to behave as a classical (3.8%) had a decline in Hb of .3gdl21 at week 4. On the other hand, pharmacogenetic trait as first described by Motulsky22, in which of the 863 patients with predicted normal ITPA function, 11.7% had genetic variation that is otherwise innocuous confers a strong drug Hb levels ,10 g dl21 and 55.9% had a decline in Hb of .3gdl21. response phenotype. It is interesting to note that, in contrast with The mechanism of RBV-induced haemolytic anaemia remains many common diseases23, relatively common genetic variants have poorly understood20, but clearly involves an accumulation of active been identified by GWAS that explain very significant proportions forms of RBV in red blood cells, including the triphosphate form of the population variation in drug responses, including the ITPA (RBV-TP). Similarly, it has been well-documented that inosine tri- variants described here and the IL28B variants reported recently3.It phosphatase deficiency leads to an accumulation of ITP in red blood is possible that this difference stems in part from the fact that drug cells16,21. A simple model to explain these observations would be that responses represent new environmental challenges, and that variants with a strong effect would not necessarily be purged by selection, as Hb decline >3 g dl–1 Hb level <10 g dl–1 would be the case for variants affecting relatively early-onset diseases. 60 60 We note, however, that like many common diseases, drug responses 50 47.6 50 48.4 are also likely to be heavily influenced by variants that are too rare for effective representation in GWAS23. 40 40 30 30 Finally, because ITPA deficiency seems to be a benign condition, it 18.7 20 20 may be possible to protect against RBV-induced anaemia by phar- Percentage 9.3* Percentage 10* macological intervention against ITPA. The identification of inosine 10 4.5 10 0 0 0.8 0 0 triphosphatase deficiency as a major projective factor against RBV- AA CA CC CC AC AA induced haemolytic anaemia therefore not only provides a valuable n = 10 n = 156 n = 1,114 n = 18 n = 252 n = 1,010 pharmacogenetic diagnostic, but also a window into red blood cell rs1127354 rs7270101 biology and the processes governing lysis. 60 55.9 METHODS SUMMARY 50 All participants were included in the IDEAL study2, which compared the effec- 40 tiveness of three anti-HCV treatment regimens including RBV. The genotype 30 data used was described previously (Supplementary Information I)3. 20.4 20 Haemoglobin values were measured at baseline, at weeks 2, 4, 8 and 12, and Percentage 11.7* then every 6 weeks up to treatment completion (48 weeks). Follow-up measure- 10 4.6 0 0.9 ments were obtained 4, 12 and 24 weeks after treatment. We considered Hb 0 +++ ++ + None decline at week 4 to be a clinically important time point at which significant n = 31 n = 153 n = 230 n = 863 anaemia had occurred but no growth factor therapy had been instituted. We excluded patients who were ,80% adherent to pegIFN or RBV up to week 4 Predicted ITPA deficiency (n 5 95), and patients with missing week-4 Hb data (n 5 21). Three phenotypes Figure 3 | ITPA deficiency protects against clinically significant decline in were considered: (1) absolute reduction in Hb, (2) a reduction in Hb of Hb concentration induced by HCV anti-viral treatment. Percentages of .3gdl21, and (3) a reduction in Hb levels to ,10 g dl21. The threshold of Hb treated subjects with Hb decline of .3gdl21 (blue) or Hb concentrations reduction of .3gdl21 was chosen as a clinically significant Hb decline; the ,10 g dl21 (red) at week 4 are shown for the two ITPA low function variants threshold of Hb ,10 g dl21 was chosen because it is the level at which RBV dose rs1127354 and rs7270101 (top), and for different degrees of predicted ITPA reduction is recommended according to package insert. deficiency (bottom). Severity of ITPA deficiency was estimated from refs 12, Our association tests involved single-marker genotype trend tests of asso- 16–18: compared to wild type, ITPA activity decreased to 60% with ciation between each SNP and the phenotypes, using linear and logistic regres- rs7270101 heterozygosity (1); to 30% with rs1127354 heterozygosity or sion models implemented in PLINK24. Covariates included age, gender, weight, rs7270101 homozygosity (11); and to very low residual activity with fibrosis severity on pretreatment liver biopsy, baseline Hb level, RBV dose, and combined heterozygosity or rs1127354 homozygosity (111). Asterisk type/dose of pegIFN. To control for the possibility of spurious associations denotes that six subjects had Hb concentrations ,10 g dl21 at week 4 but Hb resulting from population stratification, we used a modified EIGENSTRAT decline of ,3gdl21. method25 (Supplementary Information I): we defined three ethnic groups, in 407 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

which separate analyses were run, and corrected for population ancestry within 17. Atanasova, S. et al. Analysis of ITPA phenotype-genotype correlation in the each group. Combined P values for all three populations were obtained using the Bulgarian population revealed a novel gene variant in exon 6. Ther. Drug Monit. 29, Stouffer’s weight Z-method17. We assessed significance with Bonferroni correc- 6–10 (2007). tion using the total number of tests (565,759) as the denominator for the cal- 18. Maeda, T. et al. Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency in the Japanese population. Mol. Genet. Metab. 85, 271–279 (2005). culation (P cutoff 5 8.8 3 1028). 19. The International HapMap Consortium. A haplotype map of the human genome. Received 13 November 2009; accepted 11 January 2010. Nature 437, 1299–1320 (2005). Published online 21 February 2010. 20. Russmann, S., Grattagliano, I., Portincasa, P., Palmieri, V. O. & Palasciano, G. Ribavirin-induced anemia: mechanisms, risk factors and related targets for future 1. Lavanchy, D. The global burden of hepatitis. Liver Int. 29, 74–81 (2009). research. Curr. Med. Chem. 13, 3351–3357 (2006). 2. McHutchison, J. G. et al. Peginterferon a-2b or a-2a with ribavirin for treatment of 21. Fraser, J. H., Meyers, H., Henderson, J. F., Brox, L. W. & McCoy, E. E. Individual hepatitis C infection. N. Engl. J. Med. 361, 580–593 (2009). variation in inosine triphosphate accumulation in human erythrocytes. Clin. 3. Ge, D. et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral Biochem. 8, 353–364 (1975). clearance. Nature 461, 399–401 (2009). 22. Motulsky, A. G. Drug reactions enzymes, and biochemical genetics. J. Am. Med. 4. van Wijk, R. et al. HK Utrecht: missense mutation in the active site of human Assoc. 165, 835–837 (1957). hexokinase associated with hexokinase deficiency and severe nonspherocytic 23. Goldstein, D. B. Common genetic variation and human traits. N. Engl. J. Med. 360, hemolytic anemia. Blood 101, 345–347 (2003). 1696–1698 (2009). 5. de Vooght, K. M. K., van Solinge, W. W., van Wesel, A. C., Kersting, S. & van Wijk, 24. Purcell, S. et al. PLINK: a tool set for whole-genome association and population- R. First mutation in the red blood cell-specific promoter of hexokinase combined based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007). with a novel missense mutation causes hexokinase deficiency and mild chronic 25. Price, A. L. et al. Principal components analysis corrects for stratification in hemolysis. Haematologica 94, 1203–1210 (2009). genome-wide association studies. Nature Genet. 38, 904–909 (2006). 6. Bonnefond, A. et al. A genetic variant in HK1 is associated with pro-anemic state 26. Whitlock, M. C. Combining probability from independent tests: the weighted and HbA1c but not other glycemic control related traits. Diabetes 58, 2687–2697 Z-method is superior to Fisher’s approach. J. Evol. Biol. 18, 1368–1373 (2005). (2009). 27. Ge, D. et al. WGAViewer: software for genomic annotation of whole genome 7. Peters, L. L. et al. Downeast anemia (dea), a new mouse model of severe association studies. Genome Res. 18, 640–643 (2008). nonspherocytic hemolytic anemia caused by hexokinase (HKI) deficiency. Blood Cells Mol. Dis. 27, 850–860 (2001). Supplementary Information is linked to the online version of the paper at 8. Ganesh, S. K. et al. Multiple loci influence erythrocyte phenotypes in the CHARGE www.nature.com/nature. Consortium. Nature Genet. 41, 1191–1198 (2009). 9. Dickson, S. P., Wang, K., Kranz, I., Hakonarson, H. & Goldstein, D. B. Rare variants Acknowledgements We are indebted to the IDEAL principal investigators, the create synthetic genome-wide associations. PLoS Biol. 8, e1000294 (2010). study coordinators, nurses and patients involved in the study. We also thank 10. Bierau, J., Lindhout, M. & Bakker, J. A. Pharmacogenetic significance of inosine E. Gustafson, P. Savino, D. Devlin, S. Noviello, M. Geffner, E. L. Heinzen, A. C. Need triphosphatase. Pharmacogenomics 8, 1221–1228 (2007). and E. T. Cirulli for their contributions to the study. This study was funded by the 11. Stocco, G. et al. Genetic polymorphism of inosine triphosphate pyrophosphatase Schering-Plough Research Institute, Kenilworth, New Jersey. A.J.T. receives is a determinant of mercaptopurine metabolism and toxicity during treatment for funding support from the National Health and Medical Research Council of acute lymphoblastic leukemia. Clin. Pharmacol. Ther. 85, 164–172 (2009). Australia and the Gastroenterological Society of Australia. 12. Sumi, S. et al. Genetic basis of inosine triphosphate pyrophosphohydrolase Author Contributions J.F., A.J.T. and D.G. contributed equally. D.B.G., J.G.M., J.A., deficiency. Hum. Genet. 111, 360–367 (2002). A.J.T. and J.F. defined the clinical phenotypes. J.F., A.J.T., D.G. and T.J.U. performed 13. Cao, H. & Hegele, R. A. DNA polymorphisms in ITPA including basis of inosine the statistical and bioinformatical analyses. K.V.S. performed the genotyping. triphosphatase deficiency. J. Hum. Genet. 47, 620–622 (2002). C.E.G. and L.D.L. performed the sequencing experiments. P.Q., A.H.B., M.W., A.W., 14. Arenas, M., Duley, J., Sumi, S., Sanderson, J. & Marinaki, A. The ITPA c.94C.A A.J.M., M.S., C.B. and J.A. collected and analysed the clinical data. A.J.M., M.S., and g.IVS2121A.C sequence variants contribute to missplicing of the ITPA gene. J.G.M. and D.B.G designed the study. J.F., A.J.T. and D.B.G. wrote the manuscript Biochim. Biophys. Acta 1772, 96–102 (2007). with critical input from all authors. 15. Stepchenkova, E. I. et al. Functional study of the P32T ITPA variant associated with drug sensitivity in humans. J. Mol. Biol. 392, 602–613 (2009). Author Information Reprints and permissions information is available at 16. Shipkova, M., Lorenz, K., Oellerich, M., Wieland, E. & von Ahsen, N. Measurement www.nature.com/reprints. The authors declare competing financial interests: of erythrocyte inosine triphosphate pyrophosphohydrolase (ITPA) activity by details accompany the full-text HTML version of the paper at www.nature.com/ HPLC and correlation of ITPA genotype-phenotype in a Caucasian population. nature. Correspondence and requests for materials should be addressed to D.B.G. Clin. Chem. 52, 240–247 (2006). ([email protected]) or J.G.M. ([email protected]).

Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice

Axel Visel1,2, Yiwen Zhu1, Dalit May1, Veena Afzal1, Elaine Gong1, Catia Attanasio1, Matthew J. Blow1,2, Jonathan C. Cohen3, Edward M. Rubin1,2 & Len A. Pennacchio1,2

Sequence polymorphisms in a 58-kilobase (kb) interval on chro- Despite compelling genetic evidence for association, the mechanism mosome 9p21 confer a markedly increased risk of coronary artery by which 9p21 sequence polymorphisms confer an increased CAD risk disease (CAD), the leading cause of death worldwide1,2. The variants is unknown, preventing the development of pharmacological or beha- have a substantial effect on the epidemiology of CAD and other life- vioural intervention strategies. The variants are not associated with threatening vascular conditions because nearly one-quarter of established CAD risk factors such as plasma lipoprotein levels, hyper- Caucasians are homozygous for risk alleles. However, the risk tension or diabetes, suggesting that they influence CAD pathogenesis interval is devoid of protein-coding genes and the mechanism link- through a previously unappreciated pathway1,2. The CAD-associated ing the region to CAD risk has remained enigmatic. Here we show single nucleotide polymorphisms (SNPs) are located within a 58-kb that deletion of the orthologous 70-kb non-coding interval on linkage disequilibrium block on chromosome 9p21.3 that does not mouse chromosome 4 affects cardiac expression of neighbouring contain any known protein-coding genes. Several expressed sequence genes, as well as proliferation properties of vascular cells. tags of apparently non-coding transcripts, including a proposed long Chr4D70kb/D70kb mice are viable, but show increased mortality both non-coding RNA, have been mapped to the risk interval, but their during development and as adults. Cardiac expression of two genes functional relevance remains elusive11,12. Gene expression studies in near the non-coding interval, Cdkn2a and Cdkn2b,isseverely human peripheral blood cells and in vitro reporter assays have provided reduced in chr4D70kb/D70kb mice, indicating that distant-acting gene support for the notion that gene regulatory elements might be located regulatory functions are located in the non-coding CAD risk inter- within the risk interval, but there is conflicting evidence as to whether val. Allele-specific expression of Cdkn2b transcripts in heterozygous the CAD risk variants are associated with increased13 or decreased14 mice showed that the deletion affects expression through a cis- regulatory activity. More importantly, it is unclear whether altered acting mechanism. Primary cultures of chr4D70kb/D70kb aortic gene regulation would result in cellular or physiological phenotypes smooth muscle cells exhibited excessive proliferation and dimi- that are relevant to CAD pathogenesis, highlighting the need to study nished senescence, a cellular phenotype consistent with accelerated the function of this non-coding interval in a suitable in vivo system. CAD pathogenesis. Taken together, our results provide direct To create a mouse model for investigating the function of the evidence that the CAD risk interval has a pivotal role in regula- human 58-kb non-coding CAD risk interval, we sought to generate tion of cardiac Cdkn2a/b expression, and suggest that this region a severe (null) allele for this locus by its targeted removal from the affects CAD progression by altering the dynamics of vascular cell mouse genome. Human–mouse orthology could be unambiguously proliferation. established as 50% of the base pairs in the human region are alignable Each day, cardiovascular disease causes 2,400 deaths in the United to mouse15 and synteny with flanking genes is preserved (Fig. 1a–c). States alone, more than cancer, accidents and diabetes combined3. The The mouse interval is 70 kb in size and thus 20% longer than the largest proportion of this mortality is due to CAD, which causes human orthologous region, partially due to increased repetitive approximately 1 in every 5 deaths in the United States. CAD has a sequence content. Owing to the large size of the interval of interest, complex aetiology and there is strong evidence that both environ- its targeted deletion was accomplished through a sequential double- mental and genetic factors are major determinants of disease risk3. targeting strategy followed by Cre-mediated recombination (Fig. 1d However, identifying the genomic loci associated with increased CAD and Supplementary Figs 1–3). Chr4D70 kb/D70 kb mice are viable and susceptibility has been a challenge, and most of the known risk loci fertile. Although most of the live-born homozygous animals survive explain only small proportions of CAD cases (for example, ref. 4). to weaning and beyond without obvious morphological or beha- Genome-wide association studies have recently identified common vioural phenotypes, we did observe reduced embryonic, postnatal sequence variants on human chromosome 9p21 that confer an and adult survival due to the deletion (for details see Supplemen- increased risk for CAD and myocardial infarction1,2. These associa- tary Material and Supplementary Fig. 4). Clinical analysis of adult tions have been confirmed in several other cohorts5–9 and were animals at 7 months of age showed no significant general aberrations extended to other severe arterial diseases10. Even in homozygous indi- in urine and blood chemistry markers, differential blood cell counts, viduals, the variants increase the relative risk of CAD only moderately or histopathological appearance of internal organs including heart, by a factor of 1.3 to 2. However, because the risk alleles are very liver, lung, kidney, spleen and gastrointestinal tract. However, in a common, they contribute substantially to the epidemiology of CAD. larger cohort of chr4D70kb/D70kb mice on which gross necropsy was Between 20% and 25% of Caucasians are homozygous for risk alleles, performed between 7 and 14 months of age (or at time of premature resulting in estimates of 10–31% population attributable risk, depend- death), 9 out of 20 (45%) animals were found to have internal neo- ing on cohort and cases considered1,2. plasms or tumours of various types (see Supplementary Material),

1Genomics Division, MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 2US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA. 3Department of Molecular Genetics, Department of Internal Medicine, and Center for Human Nutrition, UT Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA. 409 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 a is required for appropriate expression of Cdkn2a and Cdkn2b in the 5 kb heart. Owing to the known roles of these genes in several disease- SNPs rs12555547 rs10757274 rs2383206 rs10965244 related pathways16–19, these results support the possibility of a D70kb Non-coding CAD risk interval (58 kb) regulation-mediated mechanism by which the chr4 deletion b CDKN2B Human MTAP CDKN2A DMRTA1 might affect cellular, physiological and pathological processes. (chr 9p21) CDKN2BAS To test whether the observed regulatory effect on gene expression c occurs through a cis- or trans-acting mechanism, we performed Risk interval orthologue allele-specific expression analysis. We used for this purpose crosses Mouse wild type (70 kb) (chr 4qC4/5) AK148321 of mice with the deletion linked to the Cdkn2b allele of the 129Sv Mtap Cdkn2a Cdkn2b Dmrta1 strain (in which the deletion was originally created) and wild-type d Chr4Δ70kb C57BL/6 strain mice. Strains C57BL/6 and 129Sv are distinguished by Mouse chr4Δ70kb 50 kb several transcribed SNPs in the Cdkn2b gene that can reveal quanti- Figure 1 | Deletion of the non-coding region orthologous to the 58-kb CAD tative differences in expression from the two alleles. Direct sequen- risk interval on human chromosome 9p21. a, Non-coding CAD risk interval cing of the PCR product from tail genomic DNA confirmed that the with SNPs found to be most significantly associated with CAD in genome- mice were heterozygous for the expected SNPs and the two alleles wide association studies1,2 and SNPs defining the boundaries of the linkage disequilibrium block. b, Overview of the human locus including were detected at the expected 1:1 ratio (Fig. 3a, b). As a control, in neighbouring genes (blue; intron/exon structure not shown). A non-coding complementary DNA derived from tissues of C57BL/6 (wild RNA of unknown function transcribed from this locus12 is shown in green type) 3 129Sv (wild type), the two alleles were also expressed at (CDKN2BAS, also known as ANRIL; GenBank accession NR_003529.3). indistinguishable levels, confirming the absence of general strain- c, Orthologous region on mouse chromosome 4. The exon structure of a specific differences in Cdkn2b expression levels (Fig. 3b). In contrast, non-coding transcript of unknown function, AK148321, is shown in green. d, Chr4D70kb after successful targeting and deletion of the 70-kb CAD risk interval. a rs4224578rs4224579 Cdkn2b C57BL/6 (wild type) compared to none in a cohort of age-matched wild-type controls CC 70 kb non-coding (P 5 0.0012, Fisher’s exact test). In addition to this increased tumour D70kb/D70kb Cdkn2b incidence, both male and female chr4 mice that were fed Δ TT 129Sv (chr4 70kb) standard mouse chow ad libitum gained weight significantly faster than wild-type controls, resulting in a 17% increased body mass by Heterozygous Δ 30 weeks of age (Supplementary Fig. 5). (C57BL/6 × 129Sv 70kb) To investigate the effects of the deletion in more detail, we examined Tail (genomic DNA) the possibility that the CAD risk interval is required for distant-acting regulation of gene expression. To compare messenger RNA expression Heart (cDNA) levels of surrounding genes between wild-type and chr4D70kb/D70kb mice, we isolated mRNA from hearts and other adult mouse tissues Liver (cDNA) and performed reverse transcription followed by quantitative PCR D70kb/D70kb (qRT–PCR). In heart tissue, chr4 mice had substantially Aorta (cDNA) depressed expression levels of the neighbouring Cdkn2a and Cdkn2b genes, but no significant alteration in expression levels of two other b neighbouring genes, Mtap and Dmrta1 (Fig. 2a, b). Cardiac expression C57BL/6 allele 129Sv allele of Cdkn2a and Cdkn2b was more than tenfold decreased compared C57BL/6 (WT) Genomic × NS Expression to wild-type controls. These results indicate that the CAD risk interval 129Sv (WT) (all tissues) P = 6 × 10–52 C57BL/6 (WT) Genomic a × P = 3 × 10–6 Δ Expression 129Sv (chr4 70kb) Wild-type littermates (n = 6) (all tissues) 8 Δ70kb/Δ70kb 0 255075100 7 Chr4 (n = 6) c Cdkn2b allele ratio (%) 6 C57BL/6 allele 129Sv allele 5 Aorta 4 Heart 3 NS C57BL/6 (WT) × Leukocyte Δ 2 NS 129Sv (chr4 70kb) Liver 1 ** Adult heart RNA expression 0 Spleen Mtap Cdkn2a Cdkn2b Dmrta1 0 255075100 Expressed Cdkn2b allele ratio by tissue (%) b 50 kb Figure 3 | Deletion of the CAD risk interval affects gene expression through a cis-regulatory mechanism. a, Two of seven transcribed SNPs that were used to distinguish expression of the C57BL/6 and 129Sv strain alleles Mtap Cdkn2a Cdkn2b CAD risk interval Dmrta1 of the Cdkn2b gene. The locus is not shown to scale. Representative (deleted in chr4Δ70kb) electropherograms from direct Sanger sequencing of PCR product are Figure 2 | Deletion of the CAD risk interval affects expression of shown. b, No differences between alleles are observed in genomic DNA or neighbouring genes Cdkn2a and Cdkn2b.a, Expression levels of Cdkn2a tissue-derived cDNA in chr41(C57BL/6)/1(129Sv) mice or in genomic DNA of (INK splice variant) and Cdkn2b are significantly reduced in heart tissue of chr41(C57BL/6)/D70kb(129Sv) mice. In contrast, in tissues of chr4D70kb/D70kb mice. In contrast, expression of the neighbouring genes Mtap chr41(C57BL/6)/D70kb(129Sv) mice, the C57BL/6 allele is expressed fourfold and Dmrta1 is not significantly altered. Expression levels were normalized to higher than the 129Sv allele (all tissues combined). WT, wild type. c, Allelic the b-actin gene. NS, not significant. b, Overview of locus with locations of expression differences shown by individual tissues. Error bars denote s.d. genes. Error bars are s.e.m.; *P # 0.005 (t-test, one-tailed). (b) and s.e.m. (c). P values were determined by t-test, two-tailed. 410 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS direct sequencing of the RT–PCR product derived from RNA isolated The risk interval affects human CAD through a mechanism that from chr41(C57BL/6)/D70kb(129Sv) hearts and other tissues showed that seems to be independent of plasma lipid levels and other known risk Cdkn2b was predominantly expressed from the wild-type C57BL/6 factors1,2. To study possible in vivo effects of the chr4D70kb allele on allele and expression from the chr4D70kb 129Sv allele was strongly plasma lipids and the early stages of atherogenesis, we placed 40 diminished (Fig. 3b). Among five organs and cell types examined, chr4D70kb/D70kb mice and 40 wild-type controls in an isogenic 129Sv the most severe allele-specific downregulation was observed for the background on a high-fat, high-cholesterol (‘western’) diet for heart and aorta (Fig. 3c). These results support the idea that the CAD 20 weeks21. As expected, this diet caused substantial alterations in risk interval controls gene expression in cardiac and other tissues plasma lipid levels; however, no significant differences in this physio- D D through a distant-acting cis-regulatory mechanism. logical response were observed between wild-type and chr4 70kb/ 70kb The proteins encoded by Cdkn2a, Cdkn2b and other cyclin- mice (Supplementary Fig. 6; see Supplementary Material for details). dependent kinase inhibitor genes have been implicated in cellular Furthermore, we did not observe significant differences in fatty lesion phenotypes including regulation of proliferation and cellular formation (Supplementary Fig. 7). Nevertheless, the high-fat, high- senescence16,18,19. Given the severe effect of the chr4D70kb deletion cholesterol nutrition caused substantially increased mortality among D70kb/D70kb on cardiac expression of Cdkn2a and Cdkn2b, we tested whether cell chr4 mice compared to isogenic wild-type controls, proliferation and senescence are affected in chr4D70kb/D70kb mice. To indicating an overall increased susceptibility to detrimental effects test for an effect on cell proliferation, we measured the proliferation of this noxious diet (Supplementary Fig. 8). Studies in complementary background strains and further genetic manipulations of the rates of primary cultures of aortic smooth muscle cells (aSMCs) and D70kb/D70kb mouse embryonic fibroblasts (MEFs) during early passages. In both lipid metabolism in chr4 mice may be required to mimic 21–23 cell types, cells derived from chr4D70kb/D70kb mice proliferated the full course of human atherosclerosis . Such data could also excessively compared to wild-type controls, with daily proliferation help to distinguish whether diet-induced increase in mortality is rates nearly twofold higher in aSMCs and nearly threefold higher in due to cardiovascular phenotypes other than the aortic fatty lesions examined here, or to extra-cardiovascular phenotypes that may be MEFs (Fig. 4a, b). During later passages of these primary cultures, D70kb/D70kb wild-type aSMCs and MEFs became senescent, whereas present in chr4 mice. Irrespective of the underlying aeti- chr4D70kb/D70kb-derived cells that had been isolated and cultured ology, these observations indicate that the CAD risk interval is not under identical conditions continued to proliferate and did not show required for maintenance of normal plasma lipid levels in mice, con- signs of senescence (Fig. 4c, d). These cellular phenotypes are consistent with the observation that variation in the human interval influ- sistent with known and proposed functions of Cdkn2a, Cdkn2b and ences CAD risk independent of altered lipid levels. other cyclin-dependent kinase inhibitors16,18–20. The chromosome 9p21 common haplotype linked to CAD represents an important but particularly puzzling risk interval, and the present study provides new insights about the in vivo a b function of this non-coding region. We have demonstrated that aSMC ** 80 MEF proliferation proliferation ** the precise orthologous mouse interval, despite its large distance 30 from any protein-coding genes, is critically required for normal 60 * cardiac expression of two cell cycle inhibitor genes, Cdkn2a and Cdkn2b. These observations raise the question as to what type of 20 40 underlying molecular mechanism mediates these regulatory effects. Subregions of the 58-kb non-coding risk interval increase transcriptional activity in cell-based in vitro transfection assays13, but the 10 20 location and function of distinct small enhancer sequences with relevant in vivo activities remains to be established (see Supplementary Increase in cell count per day (%) Increase in cell count per day (%) Baseline Baseline Material). Alternatively, our results are also consistent with models Δ70kb/Δ70kb Wild type Chr4 Wild type 0kb 7 Δ70kb Δ in which the non-coding interval shields the Cdkn2a/b genes from +/ Δ70kb/ the influence of very distal negative regulatory elements, either by Chr4 Chr4 spacing effects or owing to presence of insulator elements. In con- cdMEF, P8 trast, any mechanism mediated by freely diffusible RNA molecules is aSMC ** not expected to result in the allele-selective regulation observed in senescence chr4D70kb/D70kb mice (Fig. 3). Support for an RNA-mediated mech- anism11,12,24 is therefore restricted to models in which the transcriptional activity itself affects local chromatin state or in which RNA 20,000 Baseline cell number 200 μm at 0 days Wild type X-Gal molecules remain tethered to the chromosome from which they are transcribed. We have also shown that the aberrations of in vivo MEF, P8 expression of Cdkn2a and Cdkn2b coincide with abnormal regula-

Viable cells at 4 days tion of vascular cell proliferation and senescence. These phenotypes are reminiscent of mouse models in which the Cdkn2a/b genes 0 Wild type Chr4Δ70kb/Δ70kb themselves have been deleted. Specifically, primary cultures of 200 μm Cdkn2a-orCdkn2b-deficient fibroblasts16,19 and Cdkn2a-deficient Δ70kb/Δ70kb Chr4 X-Gal aSMCs25 show increased proliferation rates. Hence, a parsimonious Figure 4 | Deletion of the CAD risk interval disrupts normal dynamics of explanation for the cellular phenotypes observed in chr4D70kb/D70kb cellular proliferation and senescence. a, Increased proliferation of primary mice is that the non-coding CAD risk interval affects vascular cell aSMC cultures. b, Increased proliferation of primary MEF cultures. c, Failure proliferation and senescence by modulating the expression levels of of normal cellular senescence of primary aSMC cultures in late passages. D70kb/D70kb Cdkn2a and Cdkn2b. Altered proliferation rates of vascular cells Cells derived from wild-type and chr4 mice were grown to resulting from genetic manipulation of other cyclin-dependent senescence under identical conditions, seeded at equal densities and cell d D70kb/D70kb kinase inhibitors have been closely linked to the dynamics of CAD counts were determined after 4 days. , Chr4 -derived MEFs fail to 26 enter cellular senescence, as evident from absence of senescence staining by pathogenesis , and Cdkn2a deficiency causes altered vascular injury 25 X-Gal in comparison to wild-type MEFs30. P, passage. Mean daily responses in a mouse model of CAD . Moreover, sequence poly- proliferation rates are shown in a and b, viable cell count at day 4 is shown in morphisms in the promoters of at least two other human cyclin- c. Error bars indicate s.e.m. *P , 0.05, **P , 0.01; t-test. dependent kinase inhibitor genes have been implicated in increased 411 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 cardiovascular disease risk27,28. Thus, variation in distant-acting 11. Guttman, M. et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223–227 (2009). regulatory sequences required for cardiovascular expression of 12. Pasmant, E. et al. Characterization of a germ-line deletion, including the entire CDKN2A and CDKN2B provides a plausible mechanistic model for INK4/ARF locus, in a melanoma-neural system tumor family: identification of the increased CAD risk associated with the 9p21 region indepen- ANRIL, an antisense non-coding RNA whose expression coclusters with ARF. dently of lipid levels and other known risk factors. Cancer Res. 67, 3963–3969 (2007). 13. Jarinova, O. et al. Functional analysis of the chromosome 9p21.3 coronary artery METHODS SUMMARY disease risk locus. Arterioscler. Thromb. Vasc. Biol. 29, 1671–1677 (2009). 14. Liu, Y. et al. INK4/ARF transcript expression is associated with chromosome 9p21 Targeted deletion of the 70-kb non-coding interval was performed by two variants linked to atherosclerosis. PLoS One 4, e5027 (2009). sequential targeting steps, followed by Cre-mediated recombination 15. Karolchik, D. et al. The UCSC Genome Browser Database: 2008 update. Nucleic (Supplementary Figs 1–3). See Supplementary Table 1 for primer sequences used Acids Res. 36, D773–D779 (2008). for vector construction and genotyping. For quantitative real-time RT–PCR, 16. Serrano, M. et al. Role of the INK4a locus in tumor suppression and cell mortality. total RNA was extracted from wild-type and knockout mouse tissues using Cell 85, 27–37 (1996). Trizol Reagent (Invitrogen) following the manufacturer’s instruction. Total 17. Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997). RNA was treated with RNase-free DNase and first-strand cDNA was synthesized 18. Sharpless, N. E., Ramsey, M. R., Balasubramanian, P., Castrillon, D. H. & DePinho, by standard methods. RT–PCR was performed by standard methods; primer R. A. The differential impact of p16INK4a or p19ARF deficiency on cell growth and sequences are provided in Supplementary Table 2. For allele-specific expression tumorigenesis. Oncogene 23, 379–385 (2004). profiling, RNA extraction from tissues, DNase treatment and first-strand cDNA 19. Latres, E. et al. Limited overlapping roles of P15INK4b and P18INK4c cell cycle synthesis were done as described earlier. Genomic DNA was extracted from the inhibitors in proliferation and tumorigenesis. EMBO J. 19, 3496–3506 (2000). tails of the same mice used for RNA extraction by standard methods. PCR 20. Besson, A., Assoian, R. K. & Roberts, J. M. Regulation of the cytoskeleton: an products were generated using gene-specific primers containing M13 primer oncogenic function for CDK inhibitors? Nature Rev. Cancer 4, 948–955 (2004). tags (M13 240 forward or M13 220 reverse) and Platinum Taq DNA 21. Paigen, B., Morrow, A., Brandon, C., Mitchell, D. & Holmes, P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 57, Polymerase (Invitrogen). Primer sequences are provided in Supplementary 65–73 (1985). Table 2. PCR products were gel purified using QiaGen MinElute Gel 22. Plump, A. S. et al. Severe hypercholesterolemia and atherosclerosis in Extraction Kit (Qiagen) and sequenced using M13 primers. For proliferation apolipoprotein E-deficient mice created by homologous recombination in ES cells. and senescence assays, primary MEFs were isolated from embryonic day (E) Cell 71, 343–353 (1992). 12.5–14.5 chr4D70kb/D70kb, wild-type and heterozygous littermates. Each embryo 23. Zhang, S. H., Reddick, R. L., Piedrahita, J. A. & Maeda, N. Spontaneous was disaggregated in 0.25% trypsin, and fragments were cultured in DMEM with hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 10% FBS. Aortic smooth muscle cells were isolated from thoracic aorta of 258, 468–471 (1992). 4-week-old chr4D70kb/D70kb mice and wild-type littermates as previously 24. Broadbent, H. M. et al. Susceptibility to coronary artery disease and diabetes is described29. Cells were counted at each passage and re-cultured in constant encoded by distinct, tightly linked SNPs in the ANRIL locus on chromosome 9p. Hum. Mol. Genet. 17, 806–814 (2008). concentrations (2 3 104 per well). For senescence assays, cells were grown to 4 25. Gizard, F. et al. PPARa inhibits vascular smooth muscle cell proliferation senescence (passage 12), trypsinized and plated at 2 3 10 per well on day 0. underlying intimal hyperplasia by inducing the tumor suppressor p16INK4a. J. Clin. Cell counts were determined after 4 days. Senescence staining by X-Gal was done Invest. 115, 3228–3238 (2005). as previously described30. 26. Die´z-Juan, A. & Andres, V. The growth suppressor p27Kip1 protects against diet- induced atherosclerosis. FASEB J. 15, 1989–1995 (2001). Full Methods and any associated references are available in the online version of 27. Gonza´lez, P. et al. A single-nucleotide polymorphism in the human p27kip1 gene the paper at www.nature.com/nature. (-838C.A) affects basal promoter activity and the risk of myocardial infarction. BMC Biol. 2, 5 (2004). Received 29 August 2009; accepted 1 January 2010. 28. Rodrı´guez, I. et al. Role of the CDKN1A/p21, CDKN1C/p57, and CDKN2A/p16 Published online 21 February 2010. genes in the risk of atherosclerosis and myocardial infarction. Cell Cycle 6, 620–625 (2007). 1. Helgadottir, A. et al. A common variant on chromosome 9p21 affects the risk of 29. Yoshida, T., Kaestner, K. H. & Owens, G. K. Conditional deletion of Kruppel-like myocardial infarction. Science 316, 1491–1493 (2007). factor 4 delays downregulation of smooth muscle cell differentiation markers but 2. McPherson, R. et al. A common allele on chromosome 9 associated with coronary accelerates neointimal formation following vascular injury. Circ. Res. 102, heart disease. Science 316, 1488–1491 (2007). 1548–1557 (2008). 3. Lloyd-Jones, D. et al. Heart disease and stroke statistics—2009 update. A report 30. Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and from the American Heart Association Statistics Committee and Stroke Statistics in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). Subcommittee. Circulation 119, e21–e181 (2009). 4. Topol, E. J., Smith, J., Plow, E. F. & Wang, Q. K. Genetic susceptibility to myocardial Supplementary Information is linked to the online version of the paper at infarction and coronary artery disease. Hum. Mol. Genet. 15, R117–R123 (2006). www.nature.com/nature. 5. Schunkert, H. et al. Repeated replication and a prospective meta-analysis of the association between chromosome 9p21.3 and coronary artery disease. Circulation Acknowledgements We thank G. Owens for critical suggestions and discussion, 117, 1675–1684 (2008). T. Ley for providing vector pTURBO-Cre, and D. Lee for help with plasma lipid 6. Hinohara, K. et al. Replication of the association between a chromosome 9p21 analysis. L.A.P., E.M.R. and J.C.C. were supported by the National Heart, Lung, and polymorphism and coronary artery disease in Japanese and Korean populations. J. Blood Institute, D.M. and C.A. by EMBO long-term fellowships and L.A.P. by the Hum. Genet. 53, 357–359 (2008). National Human Genome Research Institute. Research was conducted at the E.O. 7. Lemmens, R. et al. Variant on 9p21 strongly associates with coronary heart Lawrence Berkeley National Laboratory and performed under Department of disease, but lacks association with common stroke. Eur. J Hum. Genet. 17, Energy Contract DE-AC02-05CH11231, University of California. Plasma lipid 1287–1293 (2009). analysis at the University of Cincinnati Mouse Metabolic Phenotyping Center was 8. Shen, G. Q. et al. Association between four SNPs on chromosome 9p21 and supported by MMPC DK59630. All animal work was reviewed and approved by myocardial infarction is replicated in an Italian population. J. Hum. Genet. 53, the LBNL Animal Welfare and Research Committee. 144–150 (2008). Author Contributions A.V. and L.A.P. wrote the manuscript. All authors contributed 9. Zhou, L. et al. Associations between single nucleotide polymorphisms on to data collection and analysis and provided comments on the manuscript. chromosome 9p21 and risk of coronary heart disease in Chinese Han population. Arterioscler. Thromb. Vasc. Biol. 28, 2085–2089 (2008). Author Information Reprints and permissions information is available at 10. Helgadottir, A. et al. The same sequence variant on 9p21 associates with www.nature.com/reprints. The authors declare no competing financial interests. myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm. Correspondence and requests for materials should be addressed to L.A.P. Nature Genet. 40, 217–224 (2008). ([email protected]).

D70kb/D70kb METHODS mortality among chr4 animals on high-fat diet compared to wild-type Targeted deletion of 70-kb non-coding interval. Two targeting vectors were controls was observed (P 5 0.006, Kaplan–Meier log-ranked survival test). generated for the deletion. See Supplementary Table 1 for primer sequences used High-fat diet, plasma lipid and aortic fatty lesion analysis. Deletion and wild- 21 for vector construction and genotyping. One targeting vector, containing a type mice at 6 weeks of age were fed with high-fat diet containing about 15.8% homologous region at the 59 end of the region of interest (proximal to fat, 1.25% cholesterol and 0.5% sodium cholate (Harlan, TD.88051). Mice were Cdkn2a/b), was in the ploxPneoTK-2 vector. The second targeting vector, con- under the diet for 18–22 weeks. The control group was fed with chow diet containing a homologous region at the 39 end of the region (distal from Cdkn2a/b), taining about 6.5% fat (Labdiet, Formulab Diet). For plasma lipid analysis, mice was in a ploxPhygTK vector backbone. Both ploxPneoTK-2 and ploxPhygTK were fasted overnight (approximately 15–17 h). Whole blood was collected into were generated in this laboratory. an EDTA Capillary Blood Collection tube (Fisher) by tail bleeding. Blood cells Homologous arms were generated by PCR from W4/129S6 ES cell genomic were removed by centrifugation at 4 uC. Clear plasma was transferred to a new DNA. The PCR product of the 59 homologous arm (with BamHI and EcoRI tags tube and frozen at 280 uC until analysis. Plasma lipids were measured by standard on primers) was cloned into ploxPneoTK-2 BamHI/EcoRI sites next to a loxP assays at the Cincinnati Mouse Metabolic Phenotyping Center. Aortic fatty lesion site, generating vector pCHD59neo for 59 targeting. The PCR product of the analysis was done as previously described31. In brief, mouse hearts were excised, 39-homologous arm (with NotI and BglII tags on primers) was cloned into and the upper third, including the proximal aorta, was embedded in optimal ploxPhygTK NotI/BamHI sites at the 39 side of the PGK terminator of the cutting temperature (OCT) compound. Serial 10-mm thick cryosections were PGKtk cassette, generating targeting vector pCHD39hyg for 39 targeting. cut in the region extending from the appearance to the disappearance of the aortic Targeting vector pCHD59neo was electroporated into W4/129S6 ES cells valves. Sections were mounted on Superfrost (VWR) slides, fixed in 10% neutral (Taconic Farms). Neomycin-resistant clones were picked, screened by PCR buffered formalin vapour, and stained with Oil-Red O in PEG and Gill’s III and confirmed by Southern hybridization (Supplementary Fig. 1). Successfully haematoxylin. Lesion areas were determined using a calibrated eyepiece at 59-targeted clones were pooled and electroporated with the 39 targeting vector 3200 magnification. pCHD39hyg. Hygromycin-resistant clones were picked, screened by PCR and Real-time RT–PCR. Total RNA was extracted from wild-type and knockout confirmed by Southern hybridization (Supplementary Fig. 2). mouse tissues using Trizol Reagent (Invitrogen) following the manufacturer’s Double-targeted clones (Hygr/Neor (hygromycin- and neomycin-resistant); instruction. Total RNA was treated with Promega RQ1 RNase-Free DNase. First- note that the two loxP sites could be in cis or in trans) were pooled together, strand cDNA was synthesized using SuperScript First-Strand Synthesis System expanded and electroporated with about 20 mg of Cre-recombinase-expressing for RT–PCR (Invitrogen). Real-time RT–PCR was performed using the Applied plasmid pTURBO-Cre. When loxP sites were present in cis, the loxP bracketed Biosystems SYBR Green PCR Master Mix and run on a 7500 Fast Real-Time PCR sequence included: (1) the region chr4: 89,054,800–89,126,878 (mm9) to be System (Applied Biosystems). Primer Sequences are provided in Supplementary deleted, and (2) the PGKhyg, PGKneo and HSV-tk cassettes, which can be Table 2. deleted by Cre-recombinase-mediated loxP recombination (Supplementary Allele-specific expression profiling. RNA extraction from tissues, DNase treat- Fig. 3). When loxP sites were present in trans, a translocation could be generated ment and first-strand cDNA synthesis were done as described earlier. Genomic that resulted in one deleted and one duplicated allele of the region of interest. DNA was extracted from the tails of the same mice used for RNA extraction. Embryonic stem (ES) cells that underwent cis-recombination and deletion were Tails were digested overnight at 50 uC in a buffer containing 1% SDS and identified by selecting for neomycin or hygromycin sensitivity and negative 100–200 mgml21 Proteinase K. The lysate was heated at 95–100 uC for 5 min, selection of HSV-tk for 1-(2-deoxy-2-fluoro-b-D-arabinofuransyl)-5-iodouracil and diluted for PCR. PCR products were generated using gene-specific primers (FIAU) resistance. Cells surviving this selection were screened by PCR for the containing M13 primer tags (M13 240 forward or M13 220 reverse) and predicted deletion using a primer outside the deleted region and T7 primer Platinum Taq DNA Polymerase (Invitrogen). Primer sequences are provided within the vector backbone left on the chromosome after deletion. The predicted in Supplementary Table 2. PCR products were gel purified using QiaGen deletion was further confirmed by PCR using primers outside the deleted region, MinElute Gel Extraction Kit (Qiagen) and sequenced using M13 primers. negative PCR of neo or hyg primers, and by Southern blot analysis using a probe Histopathology, blood and urine analysis. Complete clinical blood chemistry outside the deletion. profiles, haematological analysis, urine analysis, general histopathology, and Assessment of survival. To determine embryonic survival, embryos from timed histopathological analysis of neoplasms and tumours were performed at pregnancies were dissected between E9.5 and E15.5. Embryos whose size and Charles River Research Animal Diagnostic Services. appearance was normal for the respective stage were considered as surviving and Proliferation and senescence assays. Primary MEFs were isolated from genotyped by PCR. E12.5–14.5 chr4D70kb/D70kb, wild-type and heterozygous littermates. Each embryo To determine the survival from birth to weaning, live-born pups resulting was disaggregated in 0.25% trypsin, and fragments were cultured in DMEM with from wild type 3 wild type, chr41/D70kb 3 chr41/D70kb and chr4D70kb/D70kb 3 D70kb/D70kb 10% FBS. Aortic smooth muscle cells were isolated from thoracic aorta of 4-week- chr4 crosses were counted at postnatal day (P) 0 or 1 and the same old chr4D70kb/D70kb mice and wild-type littermates as previously described29. Cell litters were counted again at weaning at day P20 or P21. Any dead or missing counts were determined at each passage using a haemocytometer and re-cultured pups were considered as having died between birth and weaning. Pups from in constant concentrations (2 3 104 per well). Figure 4a shows mean daily pro- heterozygous crosses were counted regardless of their genotype because missing liferation rates over seven early passages in aSMC cultures derived from three pups could not generally be recovered for genotyping. animals per genotype. Figure 4b shows mean daily proliferation rates over four For adult survival analysis on standard chow and on high-fat diet, animals that early passages of MEFs derived from four (heterozygous) to six (wild-type and were found dead, met euthanasia criteria, or had an expected remaining survival homozygous) animals per genotype. For senescence assays, cells were grown to time of less than 7 days (based on progression of pre-mortal symptoms in ani- senescence, trypsinized and plated at 2 3 104 per well on day 0. Figure 4c shows mals that had previously died, which included severe weight loss and general senescence data from primary aSMC cultures in late passages (passage 12), inactivity) when euthanized for histological analysis were all considered as being derived from five animals per genotype. Cells were grown to senescence under in the ‘animals that died’ category. Tick marks in survival plots indicate ‘cen- identical conditions, seeded at equal densities and cell counts were determined sored’ animals, which includes animals that were healthy when removed from after 4 days. Senescence staining by X-Gal in MEFs (Fig. 4d) was done as previ- the study for histological analysis. To exclude the possibility of bias in assessing ously described30 after eight passages. euthanasia criteria, we also performed survival analysis for animals on the high- fat diet up to 120 days. All animals that died during this initial study phase were 31. Ueda, Y. et al. Relationship between expression levels and atherogenesis in found dead and no animals were euthanized or removed for histological analysis scavenger receptor class B, type I transgenics. J. Biol. Chem. 275, 20368–20373 before day 120. Consistent with the full study duration, a significantly increased (2000).

An intrinsic vasopressin system in the olfactory bulb is involved in social recognition

Vicky A. Tobin1*, Hirofumi Hashimoto1*, Douglas W. Wacker1, Yuki Takayanagi2, Kristina Langnaese3, Celine Caquineau1, Julia Noack3,4, Rainer Landgraf5, Tatsushi Onaka2, Gareth Leng1, Simone L. Meddle1,6, Mario Engelmann3 & Mike Ludwig1

Many peptides, when released as chemical messengers within the the accessory bulbs contained ,1,000 neurons. These large ovoid brain, have powerful influences on complex behaviours. Most neurons (,15 mm in diameter) are mostly located in the external plexi- strikingly, vasopressin and oxytocin, once thought of as circulat- form layer close to the glomeruli (the structures in the bulb that directly ing hormones whose actions were confined to peripheral organs, receive inputs from olfactory receptor cells). Each has several large are now known to be released in the brain, where they have fun- dendrites, one of which penetrates a single glomerulus, where it gives damentally important roles in social behaviours1. In humans, rise to many small branches. This suggests that the neurons receive disruptions of these peptide systems have been linked to several direct inputs from olfactory nerve afferents. Other dendrites travel neurobehavioural disorders, including Prader–Willi syndrome, laterally to the external zones around neighbouring glomeruli affective disorders and obsessive–compulsive disorder, and poly- (Fig. 1a, b). Using immunocytochemistry, we showed that these cells morphisms of V1a vasopressin receptor have been linked to indeed synthesize vasopressin (Fig. 1c, d), and we confirmed their autism2,3. Here we report that the rat olfactory bulb contains a presence in wild-type rats (Fig. 1e). We alsoconfirmed that they express large population of interneurons which express vasopressin, that vasopressin messenger RNA, using in situ hybridization (Fig. 1f), and blocking the actions of vasopressin in the olfactory bulb impairs that vasopressin is released from olfactory bulb explants in vitro in the social recognition abilities of rats and that vasopressin agonists response to depolarization using K1 in high concentration (release and antagonists can modulate the processing of information by increased from 0.65 6 0.19 to 4.88 6 1.88 pg per sample; P , 0.01, olfactory bulb neurons. The findings indicate that social informa- n 5 9). The total bulb vasopressin content was 42.9 6 2.6 pg mg21 tion is processed in part by a vasopressin system intrinsic to the wet weight (n 5 12; mixed sex). olfactory system. Unlike periglomerular cells and short axon cells (two other cell Complex social behaviour often depends on individual recog- populations in the same region), the vasopressin cells are immunone- nition, and most mammals distinguish individuals by their olfactory gative for GABA (c-aminobutyric acid), calretinin and calbindin-D28k signatures. Some individuals are accorded a particular status, such as (Fig. 1g–i), but like external tufted cells9,10, they are immunoreactive for when a bond is formed between a mother and offspring, or between glutamate (Fig. 1j–l). No cells were immunoreactive for oxytocin. sexual partners in monogamous species. In these cases, an olfactory Whole-cell patch-clamp recordings from olfactory bulb slices showed memory is forged in the olfactory bulb, partly as a result of the actions that the vasopressin cells have electrophysiological characteristics like of peptides4. For example, oxytocin released in the mother’s brain those of external tufted cells10. They show spontaneous bursts of during parturition helps to establish the olfactory signatures of the action potentials (1.5 bursts per second) arising at the start of a slow offspring as memorable5. depolarizing potential envelope (6.5 6 0.5 mV; n 5 5) that grows The converse of social attachment is rejection of, or aggression from a resting membrane potential of 255 6 2 mV, and have an input towards, individuals who are recognized as intruders or competitors6. resistance of 189 6 38 MV.Thisburstingisvoltagedependent(Fig.1s), For this, vasopressin, a peptide closely related to oxytocin, is important and injection of depolarizing current converts bursts of action potentials through its actions at V1 receptors, and mice without functional to an irregular firing pattern. accessory olfactory systems show many of the same behavioural deficits Unlike most external tufted cells, most vasopressin cells do not as mice that lack V1 receptors. This suggests that vasopressin is project outside the olfactory bulb. Microinjections of the retrograde involved in the processing and/or integration of olfactory stimuli, tracer Fluorogold into the cortical amygdala, the piriform cortex and that it couples socially relevant olfactory cues to an appropriate or the olfactory tubercle (major projection sites of olfactory bulb behavioural response7. efferents11) resulted in labelling of most mitral cells (the main output We have identified a hitherto unreported population of vasopressin neurons) but no vasopressin cells (Fig. 1m–o), whereas injections neurons in the olfactory bulb (Fig. 1). We first saw these cells in a into the anterior olfactory nucleus produced a very small number transgenic rat line in which enhanced green fluorescent protein of labelled cells (data not shown). If the vasopressin cells do not (eGFP) was targeted to the vasopressin secretory pathway, resulting project outside the olfactory bulb, any effects of the vasopressin that in its co-packaging with vasopressin in secretory vesicles8.Themain they release on olfactory information flow must be reflected by olfactory bulb contains similar numbers of eGFP-expressing cells in changes in the activity of other output cells. Vasopressin may be males and females (99 6 14 and 103 6 10 cells per section, respectively; released from their axon terminals, but the dendrites may be a more n 5 16 in each case), giving an estimated 5,000–7,000 neurons per bulb; important source as they are densely filled with vasopressin, and in

1Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK. 2Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan. 3Institute of Biochemistry and Cell Biology, 4Centre for Cellular Imaging and Innovative Disease Models, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany. 5Max Planck Institute of Psychiatry, 80804 Munich, Germany. 6The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, UK. *These authors contributed equally to this work. 413 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

acGCL MCL EPL bdCalbindin/GFP GFP Vasopressin s

–45 mV +90 pA

e f

–58 mV GL 0 pA GL GL

GL 20 mV 500 ms

g Calbindin/GFP hiCalretinin/GFP GABA/GFP mnFluorogold GFP o Fluorogold/GFP

GL MCL

jklGlutamate GFP Glutamate/GFP pqrV1a/GFP V1a/GFP V1b/GFP

GL MCL 20 µm

Figure 1 | Vasopressin neurons in the olfactory bulb. a, Most vasopressin glutamate (j–l). m–o, Fluorogold-labelling after injection into the anterior cells in eGFP transgenic rats (3,39-diaminobenzidine staining) are in the olfactory nucleus in mitral and periglomerular cells, but not vasopressin periglomerular region throughout the main olfactory bulb. b, An apical cells. p–r, V1a receptors are expressed on mitral cells and many dendrite ramifies into a glomerulus (blue staining, periglumerular cell- periglomerular neurons (p), but not on vasopressin cells (q), whereas some marker calbindin-D28k). c–e, Confirmation using antibodies against vasopressin cells express V1b receptors (r). s, Patch-clamp recordings GFP (c) and vasopressin (d) in transgenic rats and vasopressin in wild-type indicate firing patterns (spontaneous and depolarized) like those of external rats (e). f, In situ hybridization for vasopressin mRNA. g–l, Vasopressin cells tufted cells10. GCL, granule cell layer; MCL, mitral cell layer; EPL, external do not co-express calbindin, calretinin or GABA (red; g–i), but do contain plexiform layer; GL, glomerular layer. Scale bars, 20 mm. the hypothalamus vasopressin is released from dendrites in both an juvenile was retained (analysis of variance: F(3, 39) 5 3.34, P 5 0.026; activity-dependent manner and in an activity-independent way by Fig. 2a, b). agents that mobilize intracellular calcium12. To test involvement of the V1a receptor subtype specifically, we Vasopressin receptors are widespread in the main and accessory used infusions of a small interference RNA (siRNA) targeted against olfactory bulbs13. We found no immunoreactivity for V1a receptors V1a receptor mRNA (siRNA has previously been used to silence gene on vasopressin cells, but some for V1b receptors (Fig. 1p–r), so vaso- expression successfully, including silencing the V2 receptor in mouse pressin may act as an autocrine regulator through this receptor kidney18); these infusions produced transfection in the olfactory bulb subtype14. Many other cells in the periglomerular region were but not in the septum (Supplementary Fig. 1). The effects of siRNA immunoreactive for both subtypes, as were mitral cells in the main treatment were similar to those obtained with antagonist (treatment: and accessory bulbs. F(1, 16) 5 17.86, P , 0.01; factor interaction: F(3, 48) 5 4.37, We tested the hypothesis that olfactory bulb vasopressin is P , 0.01; Fig. 2c). Control rats (but not siRNA-treated rats) could involved in social recognition. It has already been shown that infu- recognize juveniles by their complex individual olfactory fingerprint sion of vasopressin into the bulb can enhance social recognition in even in the presence of distracting monomolecular odours (Sup- rats. In those experiments, a peptide V1 receptor antagonist had no plementary Fig. 2a). Treatment with siRNA impaired habituation/ significant effect15. Here we used a non-peptide V1 receptor antagonist dishabituation to juvenile cues (control: F(4, 32) 5 3.42, P , 0.02; (OPC-21268) that diffuses more readily, and which is effective in siRNA: F(4, 32) 5 0.47, P 5 0.76), but not to volatile odours (con- antagonizing the actions of dendritically released vasopressin in the trol: F(4, 32) 5 5.672, P , 0.01; siRNA: F(4, 32) 5 4.09, P , 0.01) or hypothalamus16. We injected the antagonist bilaterally into the olfac- object recognition (Fig. 3a–c), and did not affect open-field beha- tory bulb and tested social discrimination17. In this test, a juvenile rat is viour (Supplementary Fig. 2b, c). placed in the home cage of an adult male and the time that the adult We also used a transgenic rat line in which the human diphtheria spends investigating it is measured. Later, the same juvenile and an toxin receptor is inserted into the vasopressin promoter region. In unfamiliar juvenile are introduced into the cage. Normally the adult these rats, infusion of diphtheria toxin19,20 results in a local, selective investigates the familiar juvenile only briefly, and pays most attention destruction of vasopressin cells. Transgenic rats pretreated with toxin to the unfamiliar juvenile; this memory is short lasting (,40 min) and infusions into the olfactory bulb showed a similar impairment of is based on olfactory characteristics. In these experiments, we gave the juvenile recognition (treatment: F(1, 29) 5 14.95, P , 0.01; factor adults a microinjection of the antagonist just before the juvenile was interaction: F(1, 29) 5 4.99, P 5 0.033; Fig. 2d), again with no first presented. When retested, the adults did not discriminate between impairment of object recognition (Fig. 3c), locomotor activity or the familiar and unfamiliar juveniles, indicating that no memory of the anxiety-related behaviours (Supplementary Fig. 2d–i). 414 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

a a Olfactory habitation/dishabituation J J 20 Volatile scent d5 J A A Control vector Sampling Choice 15 siRNA b 80 Untreated Treated Untreated, extinction (180 min) 10 60 * * 5 *

40 Investigation duration (s) *

0 20 b 16 16 16 16 25 Juvenile d7 0 Vehicle V1 receptor antagonist

c d0 d4 d8 d16 20 80 * 15 60 *

** 10 40 Investigation duration (s)

Preference index Preference 20 5 1234Novel Trial juvenile 9 9 9 9 9 999 0 Control vector siRNA c Object recognition d4 d22 80 d 80 d0 d11

60 60 ** 40 40

Preference index Preference 20 17 14 17 14 0 Wild type Transgenic 919152 0 Figure 2 | Effects of V1a receptor blockade and vasopressin cell Control vector Wild type destruction on social recognition. a, A juvenile (J) is presented to an adult siRNA Transgenic male (A) for 4 min. This juvenile is removed and after 30 or 180 min is re- presented together with a non-familiar juvenile, and the preference index Figure 3 | Specificity of effects on social recognition. a, Control rats and (Methods Summary) is calculated. b, Administration of V1 receptor siRNA-treated rats show similar habituation and dishabituation to volatile antagonist results in performance similar to that after extinction of short- scents. b, Control rats also show habituation and dishabituation to juveniles, term discrimination (after 180 min). c, V1a receptor siRNA similarly impairs whereas siRNA-treated rats show neither. c, Neither siRNA nor diphtheria discrimination after 4, 8 and 16 d of treatment. d, Selective destruction of toxin injection affects object recognition. Data shown, mean 1 s.e.m.; vasopressin cells by means of diphtheria toxin injection results in a similar *P , 0.05 versus trial 4 and same treatment; n shown for each group in c. impairment of discrimination in transgenic rats, but not in wild-type rats. mean modal interspike interval, 3.2 6 0.4 ms) and at ,50 Hz (mean Data shown, mean 1 s.e.m; *P , 0.05 and **P , 0.001 versus control; for 6 each group, n is shown at the bottom of the corresponding column. modal interspike interval, 18 1 ms). The doublets are noteworthy as it is believed that only high-frequency firing episodes are back- Finally, we investigated vasopressin-dependent changes in olfac- propagated into the distal dendrites22. Topical administration of tory bulb output. Current theories of glomerular function propose vasopressin or the V1 receptor antagonist onto the exposed bulb that olfactory nerve afferents activate external tufted cells, which dorsal to the recording site modified the electrical activity of mitral activate short axon cells and periglomerular interneurons of the same cells. Vasopressin reduced the proportion of time they were active, glomerulus. This amplifies the olfactory nerve input and imposes on and particularly reduced doublet firing, whereas the antagonist had it a bursting pattern10, and this signal is transmitted to mitral cells. the opposite effect (P , 0.05, paired t-tests; Fig. 4c–e). We recorded signals from mitral cells antidromically identified by In 16 experiments, each involving a long recording from an iden- electrical stimulation of the lateral olfactory tract in freely breathing, tified mitral cell, we identified an odour to which that cell was par- anaesthetized rats (Fig. 4a, b and Supplementary Fig. 3). ticularly responsive, established the repeatability of that response in Under urethane anaesthesia, most mitral cells displayed patterned basal conditions and then retested the response to stimulation after discharge comprising prolonged intermittent bursts of action poten- topical application of vasopressin. In every case, the response to the tials21 (burst duration, 159 6 10 s; interburst time, 97 6 13 s; intra- odour was suppressed after vasopressin, whereas topical application burst firing rate, 6.4 6 0.5 Hz; n 5 94; Supplementary Fig. 3). Within of artificial cerebrospinal fluid had no effect on the responses of seven these bursts, the firing activity is modulated by the respiratory cells tested (Fig. 4f–j). rhythm. In addition, many mitral cells (57 of 94) displayed a bimodal These findings suggest that vasopressin is a retrograde signal that interspike interval distribution, reflecting the frequent occurrence of filters activation of the mitral cells. Its effects may involve presynaptic spike doublets within bursts. Thus, within bursts, these cells fired at modulation of noradrenaline or acetylcholine release, both of which two distinct instantaneous frequencies: at 100–250 Hz (doublets; are increased by retrodialysis of vasopressin in the olfactory bulbs of 415 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a f g Vasopressin

0.5 mV 25 15 5 ms 150 10 5 Spikes

Firing rate (Hz) 0 0 0 25 150 150 100 50 Spikes Spikes per 0.1 s (4 tests) 0 0 5 10 15 20 25 30 0 0 b –5 –3 –1 1 3 5 300 V1 receptor antagonist Time (s) 200 h 100 20 ACSF Vasopressin Instantaneous frequency (Hz) Instantaneous frequency 0 0 5 10 15 20 10 Time (min)

c d Firing rate (Hz) 0 1.0 * 0.08 1,000 1,500 2,000 2,500 3,000 )

* * –1 i 100 100 ACSF 0.5 50 50 Hazard (ms Hazard Activity quotient 0 0 13 6 7 11 7 77 0204060 0 0 700 750 800 e j Interval (ms) 1,400 1,450 Vasopressin Recovery 800 * 120 Before 100 100 ** ACSF 80 Vasopressin (4 ng) 50 50 400 Vasopressin (40 ng) V1 receptor

* 40 (Hz) Instantaneous frequency antagonist 0 * Recovery 0

00Spike change (%) 1,850 1,900 3,000 3,050 3,100 Number of doublets 13 6 77 7 71616 10 Time (s) Figure 4 | Vasopressin effects on mitral cells. a, b, Effects of showing activity modulated by respiratory rhythm 5 s before and 5 s after vasopressin (a) and V1 receptor antagonist (b) on firing rate (a) and odour. Middle and bottom: cumulative spikes from four tests during bursts instantaneous frequency in a single, representative mitral cell (a, b). The before (middle) and after (bottom) vasopressin. g, Number of spikes 5 s inset in a shows raw waveform traces of spike activity. c, The activity before (white) and 5 s after (black) odour for the tests in f. h, Responses of quotient decreases in six cells treated with 4 ng vasopressin and seven cells another cell to odour (arrows) before, during and after artificial treated with 40 ng vasopressin, and increases in seven cells treated with the cerebrospinal fluid (ACSF) and vasopressin. i, Magnified instantaneous vasopressin antagonist. d, The hazard function overlay (light line, before frequency records of the cell shown in h. j, Mean odour response in 16 cells vasopressin; heavy line, after vasopressin) shows the reduction in doublet tested with vasopressin, seven of which were also tested with ACSF, and in firing in a typical cell. e, Change in the number of doublets (intervals ten of which recordings were maintained long enough to observe recovery. ,10 ms), quantified for all doublet cells. f, Top: spike activity in a mitral cell, Data shown, mean 1 s.e.m.; *P , 0.05, **P , 0.01. ewes23. Because this filtering is important for social recognition, it To test effects on social discrimination, we bilaterally infused the V1 receptor seems that the vasopressin release must depend on previous olfactory antagonist or vehicle into the olfactory bulbs of adult rats15. A juvenile was experience. In the hypothalamus, activity-dependent dendritic vaso- introduced into the adult’s cage for 4 min and the duration of investigation by pressin release can be conditionally regulated (‘primed’) by recent the adult was recorded; either 30 or 180 min later, the juvenile was reintroduced experience12,24; such a mechanism in the olfactory bulb may therefore with another unfamiliar juvenile and the preference index ((time investigating unfamiliar juvenile)/(time investigating familiar juvenile 1 time investigating mediate conditional changes in olfactory recognition. unfamiliar juvenile) 3 100) was measured17. Olfactory habituation and dishabi- Thus, the olfactory bulb contains vasopressin cells that process tuation29 was tested by exposing rats to four 1-min trials separated by 10 min. olfactory signals relevant to social discrimination, and dendritic During a fifth dishabituation trial, the rats were exposed to a novel stimulus. In vasopressin release may be involved in filtering out familiar signals. rats injected with siRNA directed against V1a receptors (or control vectors), Genetic variations in brain vasopressin signalling are associated with behaviours were tested 4, 8 and 16 d after injection. differences in social behaviours in humans25,26 as well as in animal For conditional ablation of vasopressin neurons, we used transgenic rats with models. We are not suggesting that social recognition in humans a mutated human heparin-binding epidermal growth factor-like growth factor30 depends on olfactory signals; vasopressin affects social behaviour at gene (HBEGF) under the control of vasopressin promoter. Diphtheria toxin was many other sites as well as at the olfactory bulb27,28, and in humans microinjected into the olfactory bulb in rats anaesthetized with tribromoethanol. olfactory recognition probably has only a small role. However, these In urethane-anaesthetized rats, electrical activity of mitral cells was recorded studies suggest a mechanism by which experience-dependent vaso- before and after administration of vasopressin or V1 receptor antagonist onto a small exposure of the bulb. Cells were tested with odours applied for 2 s in an air pressin release can facilitate social recognition, and this mechanism stream directed at the nose. For in vitro electrophysiology, whole-cell current- may be common to several sites of action. clamp recordings were made from GFP-expressing cells in 300-mm horizontal olfactory bulb slices. METHODS SUMMARY Animal experimental procedures were conducted with regulatory approval and Full Methods and any associated references are available in the online version of ethics committee approvals in the UK, Germany and Japan. the paper at www.nature.com/nature. We processed brains immunocytochemically to detect cells expressing vaso- Received 9 April 2009; accepted 12 January 2010. pressin, GFP, calbindin-D28k, calretinin, GABA, glutamate and V1a and V1b Published online 24 February 2010. receptors, and by in situ hybridization to detect vasopressin mRNA and eGFP mRNA. Eleven eGFP rats were stereotaxically microinjected with the retrograde 1. Donaldson, Z. R. & Young, L. J. Oxytocin, vasopressin, and the neurogenetics of tracer Fluorogold at various sites to detect cells projecting from the olfactory sociality. Science 322, 900–904 (2008). bulb. Vasopressin content and potassium-stimulated release from olfactory bulb 2. Hammock, E. A. D. & Young, L. J. Oxytocin, vasopressin and pair bonding: explants was measured by radioimmune assay. implications for autism. Phil. Trans. R. Soc. B 361, 2187–2198 (2006). 416 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

3. Frank, E. & Landgraf, R. The vasopressin system–from antidiuresis to changes with maternal experience and effects on acetylcholine, gamma- psychopathology. Eur. J. Pharmacol. 583, 226–242 (2008). aminobutyric acid, glutamate and noradrenaline release. Brain Res. 669, 197–206 4. Ferguson, J. N. et al. Social amnesia in mice lacking the oxytocin gene. Nature (1995). Genet. 25, 284–288 (2000). 24. Ludwig, M. et al. Intracellular calcium stores regulate activity-dependent 5. Brennan, P. A. & Kendrick, K. M. Mammalian social odours: attraction and neuropeptide release from dendrites. Nature 418, 85–89 (2002). individual recognition. Phil. Trans. R. Soc. B 361, 2061–2078 (2006). 25. Meyer-Lindenberg, A. et al. Genetic variants in AVPR1A linked to autism predict 6. Winslow, J. T., Hastings, N., Carter, C. S., Harbaugh, C. R. & Insel, T. R. A role for amygdala activation and personality traits in healthy humans. Mol. Psychiatry 14, central vasopressin in pair bonding in monogamous prairie voles. Nature 365, 968–975 (2000). 545–548 (1993). 26. Walum, H. et al. Genetic variation in the vasopressin receptor 1a gene (AVPR1A) 7. DeVito, L. M. et al. Vasopressin 1b receptor knock-out impairs memory for associates with pair-bonding behavior in humans. Proc. Natl Acad. Sci. USA 105, temporal order. J. Neurosci. 29, 2676–2683 (2009). 14153–14156 (2008). 8. Ueta, Y. et al. Transgenic expression of enhanced green fluorescent protein 27. Ferguson, J. N., Young, L. J. & Insel, T. R. The neuroendocrine basis of social enables direct visualization for physiological studies of vasopressin neurons and recognition. Front. Neuroendocrinol. 23, 200–224 (2002). isolated nerve terminals of the rat. Endocrinology 146, 406–413 (2005). 28. Caldwell, H. K., Lee, H. J., Macbeth, A. H. & Young, W. S. III. Vasopressin: 9. Kosaka, K. & Kosaka, T. Synaptic organization of the glomerulus in the main behavioral roles of an ‘‘original’’ neuropeptide. Prog. Neurobiol. 84, 1–24 (2008). olfactory bulb: compartments of the glomerulus and heterogeneity of the 29. Bielsky, I. F., Hu, S. B., Ren, X., Terwilliger, E. F. & Young, L. J. The V1a vasopressin periglomerular cells. Anat. Sci. Int. 80, 80–90 (2005). receptor is necessary and sufficient for normal social recognition: a gene 10. Hayar, A., Karnup, S., Ennis, M. & Shipley, M. T. External tufted cells: a major replacement study. Neuron 47, 503–513 (2005). excitatory element that coordinates glomerular activity. J. Neurosci. 24, 30. Furukawa, N., Saito, M., Hakoshima, T. & Kohno, K. A diphtheria toxin receptor 6676–6685 (2004). deficient in epidermal growth factor-like biological activity. J. Biochem. 140, 11. Brennan, P. A. & Zufall, F. Pheromonal communication in vertebrates. Nature 444, 831–841 (2006). 308–315 (2006). 12. Ludwig, M. & Leng, G. Dendritic peptide release and peptide-dependent Supplementary Information is linked to the online version of the paper at behaviours. Nature Rev. Neurosci. 7, 126–136 (2006). www.nature.com/nature. 13. Ostrowski, N. L., Lolait, S. J. & Young, W. S. III. Cellular localization of vasopressin V1a receptor messenger ribonucleic acid in adult male rat brain, pineal, and brain Acknowledgements We thank V. Bishop, T. Gillespie, K. Richter and R. Murau for vasculature. Endocrinology 135, 1511–1528 (1994). technical support; E. Mohr for vectors containing the total vasopressin sequence 14. Gouze`nes, L., Desarme´nien, M. G., Hussy, N., Richard, P. & Moos, F. C. and a partial sequence; H. Gainer for vasopressin and oxytocin antibodies; Y. Ueta Vasopressin regularizes the phasic firing pattern of rat hypothalamic for the vasopressin–eGFP transgenic rats; K. Kohno and E. Kobayashi for the magnocellular neurons. J. Neurosci. 18, 1879–1885 (1998). complementary DNA for the diphtheria toxin receptor and support for the 15. Dluzen, D. E., Muraoka, S., Engelmann, M. & Landgraf, R. The effects of infusion of production of transgenic rats, respectively; and D. Murphy for the DNA for the arginine vasopressin, oxytocin, or their antagonists into the olfactory bulb upon vasopressin promoter. This work was supported by grants from the Biotechnology social recognition responses in male rats. Peptides 19, 999–1005 (1998). and Biological Sciences Research Council (M.L., S.L.M.), the Japan Ministry of 16. Ludwig, M. & Leng, G. Autoinhibition of supraoptic nucleus vasopressin neurons Education, Culture, Sports, Science and Technology (T.O., Y.T.), the Deutsche in vivo - a combined retrodialysis/electrophysiological study in rats. Eur. J. Forschungsgemeinschaft (M.E., J.N.) and the German Academic Exchange Neurosci. 9, 2532–2540 (1997). Service/Academic Research Collaboration (M.L., M.E.), and by a fellowship from 17. Engelmann, M., Wotjak, C. T. & Landgraf, R. Social discrimination procedure: an the Japan Society for the Promotion of Science awarded to H.H. alternative method to investigate juvenile recognition abilities in rats. Physiol. Author Contributions M.L., G.L., S.L.M. and M.E. designed the experiments. V.A.T. Behav. 58, 315–321 (1995). performed the immunohistochemistry and in vitro electrophysiology (Fig. 1). 18. Hassan, A. et al. Small interfering RNA-mediated functional silencing of D.W.W., S.L.M. and C.C. performed tracer injections (Fig. 1m–o). M.E. and J.N. vasopressin V2 receptors in the mouse kidney. Physiol. Genomics 21, 382–388 performed the receptor antagonist and siRNA study with behavioural analysis (2005). (Figs 2b, c and 3 and Supplementary Figs 1 and 2a, b). Y.T. and T.O. produced 19. Saito, M. et al. Diphtheria toxin receptor-mediated conditional and targeted cell diphtheria toxin receptor transgenic rats and performed behaviour experiments ablation in transgenic mice. Nature Biotechnol. 19, 746–750 (2001). (Figs 2d and 3c and Supplementary Fig. 2d–i). K.L. performed in situ hybridization 20. Chen, H., Kohno, K. & Gong, Q. Conditional ablation of mature olfactory sensory and R.L. performed the vasopressin radioimmune assay. H.H. performed in vivo neurons mediated by diphtheria toxin receptor. J. Neurocytol. 34, 37–47 (2005). electrophysiology (Fig. 4 and Supplementary Fig. 3). M.L., M.E., T.O., G.L. and H.H. 21. Jiang, M., Griff, E. R., Ennis, M., Zimmer, L. A. & Shipley, M. T. Activation of locus performed general data processing and statistical analyses. M.L. and G.L. wrote the coeruleus enhances the responses of olfactory bulb mitral cells to weak olfactory paper. nerve input. J. Neurosci. 16, 6319–6329 (1996). 22. Debarbieux, F., Audinat, E. & Charpak, S. Action potential propagation in dendrites Author Information Reprints and permissions information is available at of rat mitral cells in vivo. J. Neurosci. 23, 5553–5560 (2003). www.nature.com/reprints. The authors declare no competing financial interests. 23. Le´vy, F., Kendrick, K. M., Goode, J. A., Guevara-Guzman, R. & Keverne, E. B. Correspondence and requests for materials should be addressed to M.L. Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: ([email protected]).

METHODS with diphtheria toxin in the elevated plus maze. The apparatus had two open arms (50 cm 3 10 cm) and two enclosed arms of the same size with 40-cm-high Immunohistochemistry. Brains of eGFP and wild-type Sprague Dawley rats transparent walls (O’Hara & Co.). The arms and central square were constructed were perfusion fixed with 4% paraformaldehyde, post-fixed and cryoprotected31. of black plastic and elevated to 65 cm. The rat was placed on the central platform Free-floating frozen bulb sections (52 mm) were immunostained with primary with its head facing a closed arm. The number of entries into open arms, the time antibodies (Supplementary Table 1) for 24 to 48 h at 4 uC. Immunoreactivity spent in open arms and locomotor activity were measured using the public- was visualized using either biotinylated secondary antibodies and Alexa- domain US National Institutes of Health IMAGE IMAGEJ (version 1.36b; fluorophore-conjugated streptavidin or Alexa-fluorophore-conjugated secondary http://rsb.info.nih.gov/ij/) programs (modified by O’Hara & Co)35. antibodies, at 1:500 for 2 h at room temperature (20 uC). Sections were imaged as siRNA treatment. We used purified SMARTvector siRNA lentiviral particles previously described31. targeting the rat vasopressin V1a receptor (NM053019, SH-092092-02-10) and In situ hybridization. Six adult Wistar rats were decapitated under deep iso- SMARTvector empty vector control particles (S-004000-01), from Thermo flurane anaesthesia. Brains were frozen at 240 uC in methylbutane and stored at 8 Fisher Scientific. The titre of all solutions was .10 transforming units per 280 C. Cryosections (16 mm) were thaw-mounted on RNA-free slides and u millilitre. Lentiviral solution (2 ml) was infused through a fused silica capillary stored at 280 C. In situ hybridization was performed on sagittal sections using u into each olfactory bulb. Behaviour was tested 4, 8 and 16 d later. [35S]-UTP-labelled ribonucleotide probes32. The pcDNA3 plasmid, containing To confirm transfection by the SMARTvector lentiviral particles, the vasopressin precursor cDNA (595-bp insert), was linearized with XbaI; the TurboGFP reporter protein (Thermo Fisher Scientific) was visualized immuno- pBluescript KS plasmid, containing the vasopressin specific 39 end (230-bp histochemically 20 d after injection. Rats were anaesthetized using a mixture of insert) was linearized with HindIII. Both were in vitro transcribed with T7 0.1 ml Ketavet (Pharmacia) and 0.06 ml Dormitor (Bayer Vital), and transcar- RNA polymerase to yield an antisense complementary RNA probe. To generate dially perfused with physiological saline for 2 min and then 4% formaldehyde an eGFP-specific riboprobe, the cDNA encoding GFP was subcloned into diluted in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed for 20 h in 4% pBluescript SK. The eGFP fragment of peGFP-N3 (Clontech) was isolated using formaldehyde diluted in phosphate buffer and transferred to 0.5 M sucrose in the BamHI and NotI sites and subcloned via the same sites into pBluescript SK. phosphate buffer for 6 h and to 1 M sucrose in phosphate buffer for ,40 h. The construct was verified by sequencing. For the eGFP riboprobe, this plasmid Brains were shock-frozen in isopentanol at 250 uC and stored at 280 uC until was linearized with BamHI and in vitro transcribed with T3 polymerase. The cryosectioning. Floating frontal sections (25 mm) were incubated with anti- , 8 m 21 specific activity of the probes was 10 cpm g . The radioactivity was adjusted TurboGFP antibody (AB511, Evrogen; dilution, 1:10,000). Immunoreactive cells 3 6 m to 3.3 10 cpm per 100 l hybridization buffer. The slides were dipped in were visualized using the ABC-method using AxioVision software (version 4.7.1; Kodak NTB emulsion (Eastman Kodak) diluted 1:1 with 0.5% glycerol and 32 Zeiss) and counterstained with Nissl staining. Immunostaining was observed in exposed for 6 d at 4 uC, developed and counterstained . cells that surrounded the track of the injection cannula (data not shown) and in Vasopressin release. Sprague Dawley rats (100–200 g) were decapitated and one cells and fibres in the glomerular, external plexiform and mitral cell layers of the half of an olfactory bulb was incubated in oxygenated ACSF. We applied 50 mM 1 bulb. K for 5 min and measured vasopressin in 5-min samples. The other half was Conditional ablation of vasopressin neurons. We used transgenic Lewis rats sonicated (5 3 4 s) and centrifuged (10,000g, 5 min), and vasopressin content with a mutated HBEGF gene under the control of vasopressin promoter36. The was determined in the supernatant by radioimmune assay (sensitivity, 0.1 pg; HBEGF mutant has a high affinity to diphtheria toxin but no EGF-like activity30. cross-reactivity to related peptides including oxytocin, ,0.7%; intra- and inter- Diphtheria toxin binds to the extracellular domain of HBEGF, is incorporated assay variability, 6–9%). into cells by receptor-mediated endocytosis, inactivates eukaryotic elongation Antagonist, siRNA and diphtheria toxin injection and behaviour studies. The factor 2 through ADP-ribosylation and thereby inhibits protein synthesis, lead- 33 V1 receptor antagonist (60 ng in 3 ml ACSF, OPC-21268; Otsuka America ing to cell death19. Diphtheria toxin (D0564, Sigma-Aldrich; 300 pg per 3 ml) Pharmaceutical) or ACSF was infused under brief isoflurane anaesthesia over was microinjected bilaterally into the bulb of transgenic or wild-type rats 2 min through bilateral guide cannulae (7 mm anterior to bregma and 1.3 mm anaesthetized with tribromoethanol (200 mg kg21 intraperitoneal). Transgenic 15 lateral to midline, 3 mm deep, implanted 5 d earlier) as previously . Rats rats injected intracerebroventricularly with the toxin showed polyuria consistent received both vehicle and antagonist in a balanced crossover design. For with diabetes insipidus, but no polyuria was seen in rats after intrabulbar injec- 34 object recognition tests , rats were exposed to a laboratory glass bottle (Schott tions, indicating no significant loss of hypothalamic vasopressin cells (data not or Boro), a polyethylene terephthalate bottle (Nalgene) or a tube rack shown). (W. Krannich) for 4 min. This was removed and 30 min later was reintroduced Retrograde tracing. We stereotaxically microinjected eGFP rats under for 4 min with the other object (the preference index was calculated as in Hypnorm–Hypnovel anaesthesia (Janssen Pharmaceutica) with Fluorogold Methods Summary). For volatile scent habituation, a stainless-steel tea egg (Fluorochrome; 1 ml, 1%) into the cortical amygdala (22.1 mm posterior to (50 mm 3 35 mm) hanging from the top of the cage and containing filter paper bregma and 4 mm lateral to midline, 9.2 mm deep; n 5 3)37, anterior olfactory soaked with 5 ml carvone ((R)-(2)-carvone diluted 1:100 in diethylphtalat, nucleus (3.2 mm anterior to bregma and 1.5 mm lateral to midline, 8 mm deep; Merck) was presented in trials 1–4. For dishabituation, the filter paper was n 5 2), piriform cortex (0.7 mm anterior to bregma and 4 mm lateral to midline, soaked with 5 ml carvone mixed with isoamyl acetate. For juvenile habituation, 8 mm deep; n 5 4) or olfactory tubercle (2.7 mm anterior to bregma and 2 mm the same juvenile was presented during trials 1–4 and a new juvenile was used lateral to midline, 8 mm deep; n 5 2) over 20 min. One week later, rats were during the dishabituation test. All behaviours were measured by experimenters perfused transcardially with 4% paraformaldehyde and brains were processed for blind to identity of treatment. immunohistochemistry. Social discrimination with an additional scent was tested by scenting the Electrophysiology. For in vitro electrophysiology, glass microelectrodes juvenile with carvone (5 ml; (R)-(2)-carvone diluted 1:100 in diethylphtalat, (8–12 MV) were filled with 134 mM K-gluconate, 4 mM MgCl2,10mM Merck) during the first session and with isoamyl acetate (5 ml, Sigma-Aldrich; HEPES, 3 mM Mg2ATP, 0.3 mM Tris-GTP, 4 mM NaCl and 0.2 mM EGTA diluted 1:100 in diethylphtalat) in the second session; the new juvenile was scented (pH 7.3 adjusted with KOH, 270 mosM). In urethane-anaesthetized Sprague with carvone. Open-field behaviour was tested in two different laboratories using Dawley rats (250–300 g), extracellular recordings were made using glass micro- different equipment. In both cases, the rats were naive to the apparatus and the electrodes filled with 0.9% NaCl (tip diameter, ,1 mm). Recordings were made exposure duration was 10 min; exposure started with the rat placed in one corner from single mitral cells in the main olfactory bulb (,7 mm anterior to bregma of the apparatus. Behaviour was recorded using a video camera mounted over the and 1.3 mm lateral to midline) identified antidromically (collision test) by open field and behaviour was scored. Rats (Sprague Dawley) that received the electrical stimulation of the ipsilateral lateral olfactory tract21 (1.4 mm posterior siRNA were placed in an open-field apparatus (100 cm 3 100 cm 3 50 cm) 19 d to bregma and 3.2 mm lateral to midline, 9.5 mm deep, latencies in the range after treatment. Locomotor activity was scored by counting the number of crossed 1.0–5.8 ms)37. Activity quotients (ratio of time active and total time) were cal- lines on a grid (10 cm 3 10 cm) on the floor of the open field. Anxiety-related culated, and the hazard function was plotted38 (5-ms bins; hazard in bin behaviour was monitored by measuring how long the rat spent within the centre [t, t 1 5 ms] equals number of intervals in bin [t, t 1 5 ms] divided by the num- area (80 cm 3 80 cm), expressed as a percentage of the total duration of the open- ber of intervals of length .t). Odours were applied through a polythene cannula field testing. (0.1-mm diameter) placed 3 mm in front of the nose. Cells were tested with a Transgenic rats (Lewis rats with a mutated HBEGF gene under the control of range of odours including heptanal, hexanal, lemon and peppermint, using a vasopressin promoter; see below) were tested 15–18 d after treatment with diph- 1-ml syringe filled with odour-saturated air: 0.1 ml air was ejected over 2 s for theria toxin in an open-field apparatus measuring 60 cm 3 60 cm 3 40 cm. The each test; clean air had no effect. total distance travelled and the time spent in the centre area (40 cm 3 40 cm) Statistics. Data were analysed by analysis of variance followed by Fisher’s least- were recorded. The time spent in the centre area was expressed as a percentage of significant-difference test for planned comparisons, the Mann–Whitney U-test the total duration of testing. Transgenic rats were tested 16–19 d after treatment or unpaired Student’s t-tests. P , 0.05 was considered significant.

31. Tobin, V. A. et al. The effects of apelin on the electrical activity of hypothalamic 35. Ikeda, K. et al. Degeneration of the amygdala/piriform cortex and enhanced fear/ magnocellular vasopressin and oxytocin neurons and somatodendritic peptide anxiety behaviors in sodium pump alpha2 subunit (Atp1a2)-deficient mice. J. release. Endocrinology 149, 6136–6145 (2008). Neurosci. 23, 4667–4676 (2003). 32. Orlando, G. F., Langnaese, K., Schulz, C., Wolf, G. & Engelmann, M. Neuronal nitric 36. Murphy, D. & Wells, S. In vivo gene transfer studies on the regulation and oxide synthase gene inactivation reduces the expression of vasopressin in the function of the vasopressin and oxytocin genes. J. Neuroendocrinol. 15, 109–125 hypothalamic paraventricular nucleus and of catecholamine biosynthetic (2003). enzymes in the adrenal gland of the mouse. Stress 11, 42–51 (2008). 37. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates 3rd edn 33. Yamamura, Y. et al. OPC-21268, an orally effective, nonpeptide vasopressin V1 (Academic, 1986). receptor antagonist. Science 252, 572–574 (1991). 38. Sabatier, N., Brown, C. H., Ludwig, M. & Leng, G. Phasic spike patterning in 34. Ennaceur, A. & Delacour, J. A new one-trial test for neurobiological studies of rat supraoptic neurones in vivo and in vitro. J. Physiol. (Lond.) 558, 161–180 memory in rats. 1: Behavioral data. Behav. Brain Res. 31, 47–59 (1988). (2004).

Differential innate immune signalling via Ca21 sensor protein kinases

Marie Boudsocq1{, Matthew R. Willmann1{, Matthew McCormack1, Horim Lee1, Libo Shan1{, Ping He1{, Jenifer Bush1, Shu-Hua Cheng1{ & Jen Sheen1

Innate immunity represents the first line of inducible defence in plant innate immune responses, we developed an in-gel kinase assay against microbial infection in plants and animals1–3. In both king- to detect flg22-induced endogenous Ca21-dependent protein kinase doms, recognition of pathogen- or microbe-associated molecular activities using histone as a general substrate12,15. We observed tran- patterns (PAMPs or MAMPs, respectively), such as flagellin, initi- sient activation of several putative CDPKs between 5 and 30 min after ates convergent signalling pathways involving mitogen-activated flg22 elicitation (Fig. 1a and data not shown). The putative 60-kDa protein kinase (MAPK) cascades and global transcriptional CDPKs are distinct, in molecular mass and kinetics, from the long- changes to boost immunity1–4. Although Ca21 has long been recog- lasting (10–180 min) CDPK induced by Avr9–Cf9 interaction in cell nized as an essential and conserved primary mediator in plant death control in tobacco16,17, but similar in size to the potato (Solanum defence responses, how Ca21 signals are sensed and relayed into tuberosum) StCDPK4 and StCDPK5 that were recently shown to early MAMP signalling is unknown5,6. Using a functional genomic mediate oxidative burst by directly phosphorylating NADPH oxidase screen and genome-wide gene expression profiling, here we show RBOHB (respiratory burst oxidase homologue)18. Notably, flg22 acti- 21 that four calcium-dependent protein kinases (CDPKs) are Ca - vation of CDPKs was abolished in the fls2 mutant and by Ca21 blockers sensor protein kinases critical for transcriptional reprogramming (La31 and BAPTA) (Supplementary Fig. 2). To test whether transient in plant innate immune signalling. Unexpectedly, CDPKs and H O flg22 MAPK cascades act differentially in four MAMP-mediated regu- a flg22 b 2 latory programs to control early genes involved in the synthesis of +– Min 0 30 60 120 30 60 120 kDa NHL10 defence peptides and metabolites, cell wall modifications and 72 + Histone UBQ5 60 redox signalling. Transcriptome profile comparison suggests that cd NHL10-LUC NHL10-LUC CDPKs are the convergence point of signalling triggered by most 12 72 18 – Histone MAMPs. Double, triple and quadruple cpk mutant plants display 60 progressively diminished oxidative burst and gene activation 8 12 72 induced by the 22-amino-acid peptide flg22, as well as compro- + EGTA 4 6 60 Promoter activity Promoter mised pathogen defence. In contrast to negative roles of calmodulin 0 activity Promoter 0 7,8 flg22 – ––+++ flg22 –––+–+++ and a calmodulin-activated transcription factor in plant defence , Coomassie 21 WT ﬂs2 ﬂs2 Ctrl La Gd K the present study reveals Ca signalling complexity and demon- staining FLS2 –– + strates key positive roles of specific CDPKs in initial MAMP sig- Anti-HA e nalling. NHL10-LUC Plants and animals sense invasion of potential microbial patho- 20 gens using pattern recognition receptors (PRRs) for diverse MAMPs, 15 and launch cascades of innate immune responses that are critical for 10 1–3 5 fitness and survival . Several MAMPs seem to trigger similar early activity Promoter 0 21 responses by different PRRs, including Ca influxes, MAPK cascade CPKac –12345678910111213152122232627282930313233 kDa 64 activation, oxidative burst and transcriptional reprogramming in Anti-Flag various plants1,3–6. The bacterial flagellin epitope flg22 and other 32 21 MAMPs can induce potent Ca signatures in both the cytoplasm Figure 1 | Functional genomic screen for CDPKs in early flg22 signalling. and nucleus within minutes9,10. It has been shown that lipopolysac- a, Flg22 stimulates endogenous CDPK activities. Calcium-dependent charide (LPS)-mediated NO production is dependent on Arabidopsis histone phosphorylation, abolished by EGTA, is stimulated by flg22 at calmodulin (CAM)-like protein CML24 (ref. 11). However, no Ca21 15 min. b, NHL10 is an early flg22-responsive gene. c, Flg22 activation of sensors have been identified in flg22 or other MAMP signalling, and NHL10-LUC is dependent on the FLS2 receptor. The fls2 mutant is their immediate downstream responses remain elusive. complemented by FLS2–HA. Error bars, s.d. (n 5 8). WT, wild type. d, Flg22 signalling requires calcium and protein kinase activity. Inhibitors: 1 mM There are three major types of known Ca21 sensors in plants: LaCl3 (La), 0.5 mM GdCl3 (Gd), 2 mM K252a (K). Ctrl, control. Error bars, CAMs and CAM-like proteins, calcineurin B-like proteins, and s.d. (n 5 6). e, Specific CPKac can activate NHL10-LUC. Error bars, s.d. 11–14 CDPKs . CDPKs represent a plant innovation encoded by a large (n 5 6). Mesophyll protoplasts (a–d) were treated with 100 nM flg22. The gene family of 34 members in Arabidopsis12,15 (Supplementary Fig. 1). expression level of FLS2–HA and CPKac–Flag was monitored by To investigate the potential link between Ca21 signatures and CDPKs immunoblot.

1Department of Genetics, Harvard Medical School and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Massachusetts 02114, USA. {Present addresses: Institut des Sciences du Ve´ge´tal, UPR2355 CNRS, 1 Avenue de la Terrasse, 91198 Gif s/ Yvette Cedex, France (M.B.); Department of Biology, University of Pennsylvania, 433 South University Avenue, Philadelphia, Pennsylvania 19104, USA (M.R.W.); Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, Texas 77843, USA (L.S., P.H.). {Deceased. 418 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

Ca21 signatures9 and CDPK activation could be correlated with tran- multiple algorithms, stringent filtering, and marker gene validation by scriptional activation of early flg22-responsive genes, we first selected a qRT–PCR (Figs 2, 3, 4a, Supplementary Fig. 6, Supplementary Table 2 marker gene, NHL10 (NDR1/HIN1-LIKE 10 also known as YLS9)19, and Supplementary Methods). Notably, most (70%, 171 out of 244) of that is highly induced by flg22 within 30 min (Fig. 1b). To simplify the the CPK5ac and CPK11ac early target genes were also regulated by NHL10 transcription activation assay, the promoter of NHL10 was flg22 within 30 to 60 min in mesophyll protoplasts, seedlings and fused to the firefly luciferase reporter gene (LUC) to generate a reporter leaves19 (Fig. 2a and Supplementary Tables 2–6). Many (81 out of for leaf protoplast transient assays—a well-established cell system for 171) of these flg22–CDPK early target genes are shared by CPK5ac MAMP signalling studies4,20. Importantly, flg22 activation of NHL and CPK11ac, suggesting functional redundancy. However, unique 10-LUC is absolutely dependent on the FLS2 receptor kinase (Fig. 1c), target genes for CPK5ac (59 out of 171) or CPK11ac (31 out of 171) and is abolished by the general protein kinase inhibitor K252a that were also observed. CPK5ac-specific target genes (72) showed even could potentially inhibit many protein kinases in flg22 signalling higher positive correlation (82%, 59 of the 72) with early flg22- 21 31 31 (Fig. 1d). The Ca channel blockers (La ,Gd ) that prevent influx responsive genes. CPK11ac but not CPK5ac seemed to repress genes of external calcium effectively diminished NHL10-LUC activation by in early flg22 signalling (Fig. 2a and Supplementary Tables 4 and 5). 21 flg22 (Fig. 1d), despite a possible Ca release from internal stores. Notably, many flg22–CDPK target genes encode peptides, receptor Thus, these data suggest that putative CDPKs could be activated by kinases and enzymes that modulate defence-related metabolites (cyto- flg22 and involved in early transcriptional control downstream of the chrome P450, methyltransferase, tryptophan and phenylpropanoid FLS2 receptor. pathway), cell wall (glycoside hydrolase, glucosyltransferase and pec- We designed a functional genomic screen to identify CDPK can- tinesterase) and redox signalling (peroxidase, oxidoreductase and didates and determine their biological functions in innate immune glutathione transferase) (Supplementary Tables 3–5). For instance, signalling. In Arabidopsis, the 34 CDPK members can be classified recent biochemical and genetic evidence supports the involvement into four subgroups (I–IV) on the basis of sequence similarity12,15 (Supplementary Fig. 1). We first determined the expression patterns ab c of Arabidopsis CPK genes using quantitative real-time reverse tran- CPK5 and CPK11 CPK5 and CPK11 CPK5 and CPK11 flg22 diverse MAMPs microbial infection scriptase PCR (qRT–PCR) (Supplementary Fig. 3 and Supplemen- tary Table 1) and public microarray data21. The 25 members

expressed in leaves were tested in a functional screen for their ability CPK5 CPK11 P30 P60 L60 sd30 sd60 CPK5 CPK11 elf26 chitin harpin NPP1 LPS PGN ABA CPK5 CPK11 Psp Pi Bc to activate the Ca21-dependent flg22 reporter NHL10-LUC in mesophyll protoplasts. A constitutively active form of each CDPK (CPKac) was generated by deleting the carboxy-terminal Ca21 regulatory and auto-inhibitory domains22,23, while retaining the amino- terminal variable sequences potentially important for subcellular localization12,15. When each CPKac was co-expressed with NHL10- LUC in a protoplast transient expression assay, we discovered that only specific CPKac could induce NHL10-LUC more than fivefold, mimicking flg22 (Fig. 1e). Remarkably, five of them (CPKac4, 5, 6, 11 and 26) belong to a closely related clade in subgroup I (Supplemen- tary Fig. 1), suggesting potential redundancy along with the functional specificity. This finding was unexpected because the best studied CDPK known to have a critical role in plant defence is the tobacco NtCDPK2 involved in gene-for-gene (Avr9–Cf9) fungal resi- stance17, but its Arabidopsis orthologues, CPK1ac and CPK2ac, did not significantly induce NHL10-LUC expression (Fig. 1e) despite their relatively high kinase activity (Supplementary Fig. 4). Thus, different CDPKs even within the same subgroup may have distinct roles in plant defence signalling. NHL10-LUC induction by CPK3ac, which belongs to subgroup II (Supplementary Fig. 1), might be 121 genes of the associated with its higher protein expression and kinase activity 171 (71%) (Fig. 1e and Supplementary Fig. 4). The five CDPK candidates in subgroup I showed predicted molecular masses matching the putat- 142 genes of the 171 (83%) ive 60-kDa CDPKs activated by flg22 in leaf cells15 (Fig. 1a), but CPK26 showed relatively low endogenous expression in leaves, compared to the others (Supplementary Fig. 3 and Supplementary Table –2 0 +2 1). Thus, we focused our studies on CPK4, 5, 6 and 11 and demon- log ratio strated that their kinase activity was required to induce NHL10-LUC 171 genes 2 expression (Supplementary Fig. 5a). Their full-length forms that Figure 2 | Transcriptome profiling of CPK5ac and CPK11ac target genes in 21 absolutely required Ca for activation could not activate NHL10- flg22, multiple MAMP and microbial signalling. a, Hierarchical clustering 21 LUC in the absence of flg22 unless Ca was artificially introduced analysis of CPK5ac and CPK11ac target genes and early flg22-responsive into the cells using the Ca21 ionophore A23187 to partially mimic genes. The genes were identified from protoplasts transiently expressing flg22 signalling22 (Supplementary Fig. 5b–d). constitutively active CPK5 or CPK11, protoplasts (P30 and P60), leaves To determine the function of CPK4, CPK5, CPK6 and CPK11 in (L60) and seedlings (sd30 and sd60) treated with flg22 for 30 and 60 min. flg22 signalling further, we used ATH1 whole-genome GeneChips to b, Diverse MAMPs activate CPK5ac and CPK11ac target genes. ABA c identify potential CDPK early target genes24. CPK5 and CPK11 were treatment was used as a control. , Different microbes activate CPK5ac and CPK11ac target genes. Bc, Botrytis cinerea; Pi, Phytophthora infestans; Psp, selected as representative of the closely related gene pairs CPK5– Pseudomonas syringae pv. phaseolicola. Genes are clustered into three CPK6 and CPK4–CPK11 (Supplementary Fig. 1), and constitutively groups: shared by CPK5 and CPK11 (top panel), CPK5-specific (middle active forms were transiently expressed in protoplasts to carry out panel) and CPK11-specific (bottom panel). The number of genes co- RNA expression profiling. Potential target genes of CPK5ac and regulated by CPK5ac and/or CPK11ac is indicated at the bottom of each CPK11ac were identified by extensive microarray data analyses using panel. 419 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a 15 FRK1 24 PHI-1 NHL10 PER62 PER4 CYP82C2 CYP81F2 WAK2 FOX 24 80 45 120 18 10 18 60 30 90 12 12 5 15 30 6 6 20 Fold induction 0 0 0 00 0 CPKac – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – – 4 5 6 11 – MKK4a – – – – – + – – – – – + – – – – – + – – – – – + – – – – – + – – – – – + – – – – – + – – – – – + – – – – – +

MAPK- CDPK- CDPK–MAPK specific specific CDPK–MAPK synergistic effect MAPK-dominant effect b 160 9 FRK1 PHI-1 NHL10 PER62 PER4 CYP82C2 CYP81F2 WAK2 FOX 15 90 300 120 6 10 60 200 80 3 5 30 100 40 Fold induction 0 00 00 CPK5ac – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – CPK5acKM – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + MKK4a – – – + + + – – – + + + – – – + + + – – – + + + – – – + + + – – – + + + – – – + + + – – – + + + – – – + + + Figure 3 | Interaction between CDPK and MAPK cascades in flg22 co-expressing CPKac and MKKa (b). CPKac and MKK4a are constitutively signalling. a, b, CDPKs and MAPKs differentially regulate four flg22- active kinases, CPK5acKM is an inactive kinase mutant. Error bars, s.d. responsive gene programs. The induction of endogenous genes was (n 5 6). Marker gene details are shown in the Supplementary Information. monitored by qRT–PCR in protoplasts expressing individual kinases (a)or of CYP81F2 (cytochrome P450 monooxygenase), PEN2 (myrosinase) and no significant marker genes for abscisic acid (ABA), methyljas- and the transcription factor MYB51 in glucosinolate and callose meta- monate, ethylene or salicylate could be identified28 (Supplementary bolism in innate immune responses25,26. Three genes (PROPEP1, Tables 3–5 and data not shown). CDPKs may have a crucial involve- PROPEP2 and PROPEP3) encoding endogenous defence peptide sig- ment in rapid transcriptional reprogramming by directly regulating nals were also activated early by flg22, CPK5ac and CPK11ac27 the activity of transcription factors rather than their expression29.Iden- (Supplementary Tables 3–5). Notably, only a few transcription factors tification of early flg22-responsive genes as CPK5ac and CPK11ac

a FRK1 PHI-1 NHL10 PER62 PER4 CYP82C2 CYP81F2 WAK2 FOX 45 45 45 150 30 45 450

20 30 30 300 30 30 100

10 15 15 150 15 15 50 Fold induction 0 0 0 0 0 0 0 flg22 –+–+–+ –+ –+–+–+ –+ –+–+ –+–+–+–+ –+–+ –+–+ –+–+ –+–+ –+–+ –+–+ –+–+–+–+ –+–+ –+–+ –+–+ WT WT WT WT WT WT WT WT WT VIGS VIGS VIGS VIGS VIGS VIGS VIGS VIGS VIGS cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4 cpk5 cpk6 cpk11 cpk4

bcdMAMPs/flg22 WT Ca2+ channel H2O2 cpk5 cpk6 PRRs/FLS2 BAK1 cpk5 cpk6 cpk11 7.0 cpk5 cpk6 cpk11 cpk4VIGS * bak1 * ﬂs2 6.5 * * NADPH Ca2+ oxidase 24 MAPKKKs ? 6.0 * MKK4/5 CPK4/11 RLU) 3

(c.f.u. per seedling) CPK5/6 Bacterial number 16 10 5.5 MPK3/6 log

5.0 8 flg22 –+++++ TF TF TF TF TF WT ﬂs2 ROS production (10 ROS production bak1 0 FRK1 CYP81F2 NHL10 PHI-1 0102030 Time (min) cpk5 cpk6 MAPK- MAPK- Synergistic CDPK- specific dominant effect specific cpk5 cpk6 cpk11 Figure 4 | CDPKs are critical positive regulators in flg22 signalling. a, Flg22 Pst DC3000. Seedlings pre-treated with flg22 were inoculated with Pst induction of gene expression is diminished in cpk mutants. Gene expression DC3000 3 days before bacterial count. c.f.u., colony-forming units. Error was analysed by qRT–PCR in protoplasts treated with 2 nM flg22 for 30 min. bars, s.d. (n 5 6). *P , 0.001, significant difference from wild type plus flg22. Error bars, s.d. (n 5 8). b, The cpk mutants exhibit impaired flg22-induced d, Model for convergent activation of specific CDPKs and MAPK cascades in oxidative burst. RLU, relative light units. Error bars, s.d. (n 5 8). c, The cpk initial MAMP signalling. TF, transcription factors. mutants are impaired in flg22-induced resistance to the bacterial pathogen 420 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS target genes further supports potential roles of specific CDPKs in and data not shown). We obtained the cpk5 cpk6 double and primary flg22 signalling. cpk5 cpk6 cpk11 triple mutants by genetic crosses and generated the Because Ca21 influxes are common early events downstream of cpk4 cpk5 cpk6 cpk11 quadruple mutant by virus-induced gene silen- multiple MAMP perception9,10, we compared the flg22–CDPK early cing (VIGS)24 (Supplementary Fig. 9). Single and higher order cpk target genes (Fig. 2a and Supplementary Tables 3–5) to published mutants did not exhibit apparent phenotypes under normal growth MAMP microarray data sets generated with ATH1 GeneChips conditions (data not shown). Consistent with the functional redund- (Fig. 2b, Supplementary Table 7 and Supplementary Methods). In ancy of subgroup I CDPKs (Figs 1e, 2 and 3a), we did not observe these experiments, Arabidopsis seedlings or leaves were treated with altered flg22 responses or pathogen susceptibility in single cpk mutants diverse MAMPs from bacteria (elf26—a 26-amino-acid peptide of (Supplementary Fig. 10 and data not shown). Further mutant char- elongation factor EF-Tu, harpin, LPS and peptidoglycan (PGN)), fungi acterization was performed with double, triple and quadruple cpk (chitin) and oomycetes (NPP1, necrosis-inducing Phytophthora mutants (Fig. 4a–c). Although cpk5 cpk6 and cpk5 cpk6 cpk11 lost most protein)10,20. The results clearly showed that elf26, harpin, chitin and flg22 activation of 60-kDa CDPKs (Supplementary Fig. 11), MAPK NPP1 activated similar early genes as flg22, CPK5ac and CPK11ac activation by flg22 was not affected in cpk5 cpk6 and cpk5 cpk6 cpk11 (Fig. 2b). Thus, in addition to the MAMP-activated convergent mutants (Supplementary Fig. 12), confirming the gain-of-function MAPK cascades4,20, specific CDPKs also have a crucial role in early results (Supplementary Fig. 8). Thus, CDPKs and MAPK cascades 3,9,10 MAMP signalling downstream of diverse PRRs . Intriguingly, LPS are probably activated independently downstream of the FLS2 recep- activated very few flg22–CDPK target genes (19%), and PGN stimu- tor (Fig. 4d). 21 lated only 79 (46%), presumably owing to eliciting distinct Ca sig- Using qRT–PCR, we confirmed that flg22 induction of the MAPK- 10,11 natures . As a control, we showed that ABA activated hardly any specific target gene FRK1 was not altered, whereas all eight predicted flg22–CDPK target genes despite its role in defence gene regulation28 21 flg22–CDPK target genes showed differentially impaired flg22 induc- (Fig. 2b). Thus, Ca influx induced by elicitors, MAMPs, Avr effec- tion in double, triple and quadruple cpk mutants (Fig. 4a). CPK4, tors, and hormones may activate similar as well as different down- CPK5, CPK6 and CPK11 were all important for regulation of PHI-1, stream defence responses. Future investigations will reveal more 21 NHL10, PER62 and PER4 as their flg22 induction decreased progres- complex and diverse Ca signalling mechanisms in plants. sively in the double, triple and quadruple cpk mutants (Fig. 4a). For To gain new insight into potential CDPK activation during plant– CYP81F2, WAK2 and FOX activation by flg22, CPK5/6 and the microbe interactions, we compared the flg22–CDPK target genes MAPK cascades had equal but independent roles (Figs 3 and 4a). (Fig. 2a and Supplementary Tables 3–5) with published microarray Notably, CPK5 and CPK6 were essential for flg22 activation of data sets of genes activated during the infection processes by bacteria CYP82C2, which uniquely required the synergistic CDPK and (Pseudomonas syringae pv. phaseolicola), fungi (Botrytis cinerea)and MAPK functions (Figs 3b and 4a). The oxidative burst induced by oomycetes (Phytophthora infestans) (Fig. 2c, Supplementary Table 8 flg22 was gradually reduced in cpk5 cpk6, cpk5 cpk6 cpk11 and and Supplementary Methods) (http://arabidopsis.org/info/expression/ VIGS cpk5 cpk6 cpk11 cpk4 mutants to a similar level as in the flg22 ATGenExpress.jsp). Identification of co-regulated genes indicated that signalling mutant bak1 (Fig. 4b), suggesting a role for these CDPKs CDPK activation of early gene expression is probably a conserved in regulating reactive oxygen species (ROS) production, potentially natural response when plants encounter various microbes and launch by directly phosphorylating the NADPH oxidase RBOHB18.We MAMP-mediated innate immune signalling. evaluated the role of CDPKs in Arabidopsis plant susceptibility to a To uncover possible connections between CDPK signalling and bacterial pathogen, and showed increased growth of Pseudomonas MAPK cascades3,4,10,30, we analysed by qRT–PCR the expression of syringae pv. tomato (Pst) DC3000 in cpk5 cpk6 and cpk5 cpk6 cpk11 selected flg22-inducible genes in protoplasts expressing constitu- mutants (Supplementary Fig. 13), probably due to impairment in tively active CPKac or MKK4a. Unexpectedly, our analysis showed several MAMP signalling pathways20 (Fig. 2a, b). Moreover, flg22- at least four regulatory programs for early flg22-responsive genes (Fig. 3a). Genes such as FRK1 (FLG22-INDUCED RECEPTOR induced immunity against Pst DC3000 (refs 19, 20) was also compro- KINASE1) were MAPK-specific4, whereas PHI-1 (PHOSPHATE- mised in a seedling pathogen assay in cpk5 cpk6 and cpk5 cpk6 cpk11 INDUCED 1) seemed to be CDPK-specific. Other flg22 early genes mutants compared to those observed in bak1 and fls2 mutants (Fig. 4c). Interestingly, all four CPK–GFP (green fluorescent protein) were either activated equally by both CPKac and MKK4a, such as 29 NHL10, PER62 (PEROXIDASE62, also known as At5g39580) and fusions were localized in both the cytoplasm and nucleus (Sup- PER4 (At1g14540), or were induced to much higher levels by MKK4a, plementary Fig. 14), supporting their potentially versatile functions for example, CYP82C2, CYP81F2, WAK2 (WALL-ASSOCIATED in response to flg22, including the activation of NADPH oxidase and KINASE 2) and FOX (FAD-LINKED OXIDOREDUCTASE, At1g26380). the modulation of gene expression (Fig. 4d). When CPK5ac and MKK4a were co-expressed in protoplasts, CDPK- By integrating a functional genomic screen, transcriptome profiling and MAPK-specific genes were partially antagonized by the introduc- and reverse genetics, we have presented compelling evidence for the tion of the other pathway (Fig. 3b). In contrast, for genes co-regulated identification of a specific subgroup of CDPKs that regulate MAMP- by CDPK and MAPK signalling, the two pathways could act either triggered immunity in Arabidopsis. Further characterization of the synergistically or independently. Unlike in other defence signalling functions of CDPK and MAPK target genes, such as peptides, receptor pathways5,6,Ca21 blockers reduced but did not eliminate MAPK or kinases and enzymes involved in antimicrobial chemical production, NHL10-LUC activation by flg22 (Supplementary Fig. 7 and Fig. 1d). cell wall modifications and redox signalling, will help elucidate the Consistently, CPKac did not activate MPK3 or MPK6 when co- diverse primary responses in innate immune signalling. Because expressed in protoplasts (Supplementary Fig. 8). These data un- CDPKs are evolutionarily conserved, these findings might offer new ravelled previously unrecognized complexity and dynamics in early tools to enhance disease resistance in crop plants. The apparent func- transcriptional reprogramming mediated by differential activities of tional redundancy of closely related CDPKs and their complex inter- CDPKs and MAPK cascades in plant innate immunity. actions with MAPK cascades illustrate the challenges in dissecting the 1 In parallel to the comprehensive gain-of-function approach, we MAMP signalling network. The different Ca2 signatures associated identified loss-of-function cpk single mutants from the T-DNA inser- with diverse MAMPs9,10 may potentially be decoded by distinct tion collections (Supplementary Fig. 9a), and generated higher order CDPKs and other Ca21 sensors, such as CML24 (ref. 11), in various cpk mutants to conduct molecular and physiological analyses of flg22 subcellular locales and partially account for differential MAMP res- signalling. Although T-DNA insertion into CPK5, CPK6 and CPK11 ponses. We provide genomic evidence that CDPK activities are the created null mutants, all the putative cpk4 mutant lines we examined convergence point of signalling triggered by several but not all showed only partially reduced transcript levels (Supplementary Fig. 9b MAMPs. 421 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

METHODS SUMMARY 15. Cheng, S. H., Willmann, M. R., Chen, H. C. & Sheen, J. Calcium signaling through DNA constructs. The full-length and truncated Arabidopsis CPK constructs protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol. 129, 469–485 (2002). were fused to the Flag tag or GFP at the C terminus in a plant expression 4,20,22 16. Romeis, T., Piedras, P. & Jones, J. D. G. Resistance gene-dependent activation of a vector (Supplementary Tables 9–11). A 1,632-base-pair (bp) promoter calcium-dependent protein kinase in the plant defense response. Plant Cell 12, region of NHL10 was fused to the LUC reporter gene to generate NHL10- 803–815 (2000). LUC. The effector constructs FLS2-HA (haemagglutinin), MKK4a-MYC, 17. Romeis, T., Ludwig, A. A., Martin, R. & Jones, J. D. G. Calcium-dependent protein MPK3-HA and MPK6-HA have been described4,20. kinases play an essential role in a plant defence response. EMBO J. 20, 5556–5567 Mesophyll protoplast transient expression assay. Protoplast isolation and tran- (2001). sient expression assays were carried out as described4,20. For promoter activities, 18. Kobayashi, M. et al. Calcium-dependent protein kinases regulate the production protoplasts were co-transfected with UBQ10-GUS (b-glucuronidase) as an of reactive oxygen species by potato NADPH oxidase. Plant Cell 19, 1065–1080 internal control. Protein expression was monitored by immunoblot using com- (2007). mercially available monoclonal antibodies raised against haemagglutinin 19. Zipfel, C. et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764–767 (2004). (Roche), MYC (Roche) and Flag (Sigma) epitope tags. In vitro and in-gel kinase 20. Shan, L. et al. Bacterial effectors target the common signaling partner BAK1 to 4 assays were carried out as described , using MBP and histone as substrates for disrupt multiple MAMP receptor-signaling complexes and impede plant MAPK and CDPK activities, respectively. immunity. Cell Host Microbe 4, 17–27 (2008). Gene expression analysis. Real-timeand semi-quantitative RT–PCR were carried 21. Zimmermann, P., Hennig, L. & Gruissem, W. Gene-expression analysis and out using the primers listed in Supplementary Tables 12 and 13. UBQ5 network discovery using Genevestigator. Trends Plant Sci. 10, 407–409 (2005). (At3g62250), TUB4 (At5g44340) and EIF4a (At3g13920) were used as control 22. Sheen, J. Ca21-dependent protein kinases and stress signal transduction in plants. genes. Global gene expression analyses were performed with Arabidopsis ATH1 Science 274, 1900–1902 (1996). GeneChip arrays (Affymetrix) and were compared to publicly available micro- 23. Uno, Y., Rodriguez Milla, M. A., Maher, E. & Cushman, J. C. Identification of array data. For details on data processing and analyses, see Supplementary proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis. Mol. Genet. Genomics 281, 375–390 (2009). Information. 24. Baena-Gonza´lez, E., Rolland, F., Thevelein, J. M. & Sheen, J. A central integrator of Analysis of cpk mutants. The loss-of-function cpk mutants were isolated from transcription networks in plant stress and energy signalling. Nature 448, 938–942 Arabidopsis Biological Resource Center (ABRC) resources (Supplementary (2007). Table 14). Higher order cpk mutants were generated either by crossing or by 25. Bednarek, P. et al. A glucosinolate metabolism pathway in living plant cells VIGS. Bacterial growth assays were performed in leaves and seedlings using Pst mediates broad-spectrum antifungal defense. Science 323, 101–106 (2009). DC3000. The oxidative burst was measured using a luminol-based assay3. 26. Clay, N. K., Adio, A. M., Denoux, C., Jander, G. & Ausubel, F. M. Glucosinolate metabolites required for an Arabidopsis innate immune response. Science 323, Full Methods and any associated references are available in the online version of 95–101 (2009). the paper at www.nature.com/nature. 27. Huffaker, A. & Ryan, C. A. Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response. Proc. Natl Acad. Received 24 August 2009; accepted 5 January 2010. Sci. USA 104, 10732–10736 (2007). Published online 17 February 2010. 28. Nemhauser, J. L., Hong, F. & Chory, J. Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses. Cell 126, 1. Nu¨rnberger, T., Brunner, F., Kemmerling, B. & Piater, L. Innate immunity in plants 467–475 (2006). and animals: striking similarities and obvious differences. Immunol. Rev. 198, 29. Zhu, S. Y. et al. Two calcium-dependent protein kinases, CPK4 and CPK11, 249–266 (2004). regulate abscisic acid signal transduction in Arabidopsis. Plant Cell 19, 3019–3036 2. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. (2007). Cell 124, 783–801 (2006). 30. Sheen, J. et al. in Biology of Plant Microbe Interactions Vol. 6, Ch. 86 (IS-MPMI 3. Boller, T. & Felix, G. A renaissance of elicitors: perception of microbe-associated Symposium Proceedings, 2008). molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379–406 (2009). Supplementary Information is linked to the online version of the paper at 4. Asai, T. et al. MAP kinase signalling cascade in Arabidopsis innate immunity. www.nature.com/nature. Nature 415, 977–983 (2002). 5. Lecourieux, D., Ranjeva, R. & Pugin, A. Calcium in plant defence-signalling Acknowledgements We thank K. H. Liu and E. Baena-Gonza´lez for contributing to pathways. New Phytol. 171, 249–269 (2006). the CPK clone collection, O.R.Patharkarforinitialmicroarraydata analysis, B.Mueller 6. Ma, W. & Berkowitz, G. A. The grateful dead: calcium and cell death in plant innate and G. Tena for offering qPCR primers, M. N. Soler and S. Bolte for assistance on immunity. Cell. Microbiol. 9, 2571–2585 (2007). confocal microscopy, C. Laurie`re for discussions, S. P. Dinesh-Kumar for VIGS 7. Kim, M. C. et al. Calmodulin interacts with MLO protein to regulate defence vectors, K. Schreiber and D. Desveaux for the seedling pathogen assay, and A. Gust against mildew in barley. Nature 416, 447–451 (2002). and T. Nu¨rnberger for the original PGN microarray data. We thank the Salk Institute, 8. Du, L. et al. Ca21/calmodulin regulates salicylic-acid-mediated plant immunity. Syngenta Biotechnology and A. Sessions for sharing the Arabidopsis T-DNA Nature 457, 1154–1158 (2009). collections, and the Arabidopsis Biological Resource Center for providing Arabidopsis 9. Lecourieux, D. et al. Proteinaceous and oligosaccharidic elicitors induce different mutant seeds. This research has been supported by a Marie Curie International calcium signatures in the nucleus of tobacco cells. Cell Calcium 38, 527–538 (2005). fellowship within the 6th European Community Framework Program to M.B., an NSF 10. Gust, A. A. et al. Bacteria-derived peptidoglycans constitute pathogen-associated predoctoral fellowship to M.R.W., and grants from the National Science Foundation molecular patterns triggering innate immunity in Arabidopsis. J. Biol. Chem. 282, and the National Institute of Health, and the MGH CCIB fund to J.S. 32338–32348 (2007). Author Contributions J.S. and M.R.W. initiated the project; M.B., M.R.W. and J.S. 11. Ma, W., Smigel, A., Tsai, Y. C., Braam, J. & Berkowitz, G. A. Innate immunity 21 designed the experiments; M.R.W., M.B. and S.-H.C. built the CPK clone collection; signaling: cytosolic Ca elevation is linked to downstream nitric oxide generation M.B., M.R.W., H.L., L.S., P.H. and J.S. conducted the experiments and analysed the through the action of calmodulin or a calmodulin-like protein. Plant Physiol. 148, data; M.M., J.S. and M.B. analysed microarray data; J.B. managed plants; M.B., J.S. 818–828 (2008). and M.M. prepared the manuscript with inputs from all co-authors. 12. Harper, J. F. & Harmon, A. Plants, symbiosis and parasites: a calcium signalling connection. Nature Rev. Mol. Cell Biol. 6, 555–566 (2005). Author Information All microarray data are available at the Gene Expression 13. Boudsocq, M. & Sheen, J. in Abiotic Stress Adaptation in Plants: Physiological, Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession number Molecular and Genomic Foundation (eds Pareek, A., Sopory, S. K., Bohnert, H. J. & GSE16557. Reprints and permissions information is available at www.nature.com/ Govindjee) Ch. 4 (Springer, 2009). reprints. The authors declare no competing financial interests. Correspondence 14. Luan, S. The CBL-CIPK network in plant calcium signaling. Trends Plant Sci. 14, and requests for materials should be addressed to M.B. 37–42 (2009). ([email protected]) or M.R.W. ([email protected]).

METHODS (Promega). RT–PCR was run for 30 cycles and UBQ5 (At3g62250) was used Plant material and growth conditions. We isolated homozygous T-DNA inser- as a control gene. qRT–PCR analysis was carried out with an iCycler iQ real-time tion mutants31, cpk5 (sail_657C06 from Syngenta), cpk6 (salk_025460) and cpk11 PCR system using iQ SYBR Green supermix (Bio-Rad), with TUB4 (At5g44340) (salk_054495) using seeds obtained from the ABRC. The T-DNA insertions were and EIF4a (At3g13920) as control genes. confirmed by sequencing (Supplementary Fig. 9a). Although T-DNA insertions Microarray analysis. Protoplasts transfected with control DNA, CPK5ac or abolished gene expression of CPK5, CPK6 and CPK11, VIGS of CPK4 strongly CPK11ac were collected after 6 h expression. For flg22 treatment, untransfected reduced gene expression of CPK4 (Supplementary Fig. 9b). Wild-type Col-0, fls2 protoplasts were incubated for 4 h before induction with 100 nM flg22 at 28 uC (salk_093905), bak1 (salk_116202) and cpk mutants were grown on soil in a for 30 and 60 min, and 8-day-old seedlings grown in liquid 0.53 MS were growth chamber at 23 uC with a 13 h photoperiod for 4 weeks before bacterial induced with 2 nM flg22 for 30 and 60 min. Total RNA was extracted using inoculation or protoplast isolation. The cpk5 cpk6 double and cpk5 cpk6 cpk11 RNeasy Plant Mini kit (Qiagen) according to the manufacturer’s protocol. triple mutants were generated by genetic crosses. For in-gel kinase assay with Eight micrograms of total RNA was amplified, biotinylated and fragmented seedlings, wild-type and cpk mutants were grown in liquid medium 0.53 MS, using the BioArray HighYield RNA transcript labelling kit (Enzo) according to 0.5% sucrose, for 10 days at 23 uC with a 13 h photoperiod. the manufacturer’s instructions. Hybridization to Arabidopsis ATH1 GeneChip Bacterial growth assays. The bacterial growth assays were performed as previ- arrays (Affymetrix) and scanning were conducted by the microarray facility of ously described32,33. In brief, Arabidopsis leaves of 4-week-old plants were infil- Harvard-Partners Center for Genetics and Genomics. Data were pre-processed trated with Pst DC3000 at 5 3 104 c.f.u. ml21 and bacterial growth was in GCOS 1.2 (Affymetrix GeneChip Operating Software) as well as being monitored 4 days after inoculation using serial dilution plating of ground leaf imported into FlexArray and processed with MAS5.0 (Affymetrix), dChip disks. For seedling assay, 5-day-old seedlings grown in liquid 0.53 MS were (http://www.dchip.org), and RMA (Bioconductor-R, http://www.bioconductor. treated with 100 nM flg22 for 1 day before co-cultivation with Pst DC3000 at org/) algorithms. Gene-level variability across duplicate and triplicate arrays was 1 3 107 c.f.u. ml21 for 3 days. Surface sterilized seedlings were ground for bac- then determined separately with Cyber-T, as well as SAM (significance analysis terial counting as above. The experiments were repeated three times with similar of microarrays) and t-test. Results of the different methods were compared on results. the basis of the validated marker genes (Figs 3 and 4a). Details for microarray Effector and reporter constructs. All primers used for cloning and mutagenesis are data set sources, data analyses and identification of CDPK target genes listed in Supplementary Tables 9–11 and PCR products were checked by sequencing. (Supplementary Tables 2–5) are described in Supplementary Methods. The ori- For CDPK effector constructs, the coding region was amplified from Arabidopsis ginal GeneChip files will be available at Gene Expression Omnibus. 24,36 Col-0 complementary DNA, fused to a Flag epitope tag or GFP at the C terminus, Virus-induced gene silencing. VIGS was performed as described using and cloned between a 35S-derived promoter and a NOS terminator4,20,22,32,34. For the Arabidopsis wild-type, cpk5 cpk6 and cpk5 cpk6 cpk11 mutants. The control 24 NHL10-LUC reporter construct, the 1,632-bp sequence immediately upstream of VIGS construct using a GFP fragment has already been reported . The CPK4 the translation start codon of NHL10 was amplified by PCR from Arabidopsis (Col- VIGS construct was generated using a gene-specific 500-bp fragment. The 0) genomic DNA and fused to the luciferase coding region. mutant plants were identified by RT–PCR (Supplementary Table 13) and the Mesophyll protoplast transient expression assay. The detailed protocol was VIGS experiments were repeated four times with similar results. described previously34. Typically, 0.1 ml protoplasts at a density of 2 3 105 ml21 Oxidative burst measurement. The production of ROS was measured with a were transfected with 20 mg total DNA including different effector and reporter luminol-based assay37,38. Leaf disks from 4-week-old plants were floated on water constructs. The ratio of effector and reporter DNA was 1:1. For promoter activities, overnight and transferred to assay tubes with 100 ml reaction buffer (20 mM UBQ10-GUS was co-transfected as an internal control. The results were presented luminol, 1 mg horseradish peroxidase, 100 nM flg22). Luminescence was mea- as the LUC/GUS ratio and normalized to the values obtained without the treatment sured every 3 min for 30 min using an LB 9507 luminometer (Berthold tech- or effector expression. To induce flg22 responses, transfected protoplasts were nologies). The experiments were repeated four times with similar results. incubated for 30 min before elicitation with 100 nM flg22 for various time periods 31. Alonso, J. M. & Stepanova, A. N. T-DNA mutagenesis in Arabidopsis. Methods Mol. as indicated. To examine the effect of various effectors (for example, CDPKs) on Biol. 236, 177–188 (2003). reporter expression, transfected protoplasts were incubated for 6 h. For experi- 21 32. He, P. et al. Specific bacterial suppressors of MAMP signaling upstream of ments with full-length CPK5 and Ca /A23187, protoplasts were incubated for MAPKKK in Arabidopsis innate immunity. Cell 125, 563–575 (2006). 4 h to allow CDPK protein accumulation, and then cultured with the ionophore for 33. Schreiber, K., Ckurshumova, W., Peek, J. & Desveaux, D. A high-throughput 3 h. For protein expression and kinase assays, 0.2 ml protoplasts were transfected chemical screen for resistance to Pseudomonas syringae in Arabidopsis. Plant J. 54, with 40 mg total DNA and collected 6 h later. All experiments showed similar results 522–531 (2008). when repeated 3–5 times with duplicate or triplicate samples. 34. Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell Subcellular localization. Protoplasts from cell suspension were isolated and systemfor transientgene expression analysis. Nature Protocols 2, 1565–1572 (2007). transfected as reported35. GFP fluorescence was observed by confocal micro- 35. Boudsocq, M., Barbier-Brygoo, H. & Laurie`re, C. Identification of nine sucrose scopy (LEICA SP2 inverted confocal microscope). nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. J. Biol. Chem. 41758 Protein kinase assays. Kinase assays were carried out as previously described4,32. 279, –41766 (2004). 36. Burch-Smith, T. M., Schiff, M., Liu, Y. & Dinesh-Kumar, S. P. Efficient virus-induced The in-gel CDPK assay was modified from the MAPK assay using histone as the gene silencing in Arabidopsis. Plant Physiol. 142, 21–27 (2006). substrate without cold ATP and EDTA. 37. Felix, G., Duran, J. D., Volko, S. & Boller, T. Plants have a sensitive perception RT–PCR analysis. Primers used for qRT–PCR and semi-quantitative RT–PCR system for the most conserved domain of bacterial flagellin. Plant J. 18, 265–276 are listed in Supplementary Tables 12 and 13. Total RNA was isolated from (1999). protoplasts using RNeasy Plant Mini kit (Qiagen). The cDNA was synthesized 38. Chinchilla, D. et al. A flagellin-induced complex of the receptor FLS2 and BAK1 from 1 mg total RNA using oligo(dT) primer and reverse transcriptase initiates plant defence. Nature 448, 497–500 (2007).

Control of Arabidopsis apical–basal embryo polarity by antagonistic transcription factors

Zachery R. Smith1,2 & Jeff A. Long1

Plants, similarly to animals, form polarized axes during embryo- PHABULOSA (PHB) in this mutant, which we designate phb-14d. genesis on which cell differentiation and organ patterning pro- This mutation resides within a known microRNA (MIR)165/166 grams are orchestrated. During Arabidopsis embryogenesis, family binding site and is predicted to result in a loss of MIR165/ establishment of the shoot and root stem cell populations occurs 166-mediated regulation9,10. Notably, the observed increase in PHB at opposite ends of an apical–basal axis. Recent work has identified transcript abundance is less severe than previously described alleles the PLETHORA (PLT) genes as master regulators of basal/root (Supplementary Fig. 2). fate1–3, whereas the master regulators of apical/shoot fate have All five HD-ZIP III genes (PHB, PHAVOLUTA (PHV), REVOLUTA remained elusive. Here we show that the PLT1 and PLT2 genes (REV), INCURVATA4 (ICU4,alsoknownasCORONA and CNA), are direct targets of the transcriptional co-repressor TOPLESS and ARABIDOPSIS THALIANA HOMEOBOX-8 (ATHB-8)) are pre- (TPL) and that PLT1/2 are necessary for the homeotic conversion dicted to be regulated by MIR165/166 (refs 9–12). Semi-dominant of shoots to roots in tpl-1 mutants. Using tpl-1 as a genetic tool, we gain-of-function (GOF) mutations in the MIR binding site of PHB, identify the CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD- PHV, REV and ICU4 have been characterized previously for their role ZIP III) transcription factors as master regulators of embryonic in specifying adaxial/dorsal fate in lateral organs and vasculature11,13–16. apical fate, and show they are sufficient to drive the conversion of the embryonic root pole into a second shoot pole. Furthermore, abc genetic and misexpression studies show an antagonistic relationship between the PLT and HD-ZIP III genes in specifying the root and shoot poles. During early embryogenesis, the phytohormone auxin has a critical role in apical–basal axis formation4. Auxin-induced gene expression involves degradation of AUX/IAA transcriptional repressors5, and IAA12 (also known as BODENLOS) was shown to require the transcriptional co-repressor TPL for its function during embryonic root development6. In the dominant negative tpl-1 allele the embryonic d e f g shoot pole is transformed into a second root pole (Fig. 1a, b), indicating that root-specifying genes must be actively repressed in the apical half of the embryo for normal apical/basal patterning7,8. tpl-1 is temperature-sensitive and shows a high frequency of shoot to root transformation when embryos develop at 29 uC8. In wild-type plants, PLT1 and PLT2 are expressed in the root meristem throughout embryo development1 (Fig. 1d, f). In tpl-1 grown at 29 uC, both PLT1 and PLT2 are misexpressed in the apical domain, beginning at hi the heart stage (Fig. 1e, g). It has been shown that the PLT genes are 40 PLT1 promoter 6 PLT2 promoter sufficient to initiate ectopic roots when driven from an embryonic 35 5 1 30 promoter , indicating that the misexpression seen in tpl-1 is causative 25 4 of the double-root phenotype. In agreement with this, double-root 20 3 formation was never observed in tpl-1 plt1-5 plt2-1 embryos grown at 15 2 10

29 C(n . 1,000) (Fig. 1c). Thus, the PLT genes are necessary for Fold enrichment 1 u 5 Fold enrichment apical root formation in tpl-1. To assess whether the PLT genes are 0 0 direct targets of TPL repression, we performed chromatin immuno- –7000 –6000–5000 –4000 –3000 –2000 –1000 –3000 –2500 –2000 –1500 –1000 –500 0 Position from start codon Position from start codon precipitation (ChIP) on TPLp::TPL-HA dissected ovules containing globular to heart stage embryos. We observed enrichment of regions Figure 1 | Misregulation of PLT genes is necessary for tpl-1 apical to basal in both the PLT1 and PLT2 promoters in the TPL ChIP samples transformation. a–c, Seedlings from embryos grown at 29 uC. a, Wild-type (Fig. 1h, i), indicating that TPL acts in the apical region of the embryo seedling. b, tpl-1 double root. c, tpl-1 plt1-5 plt2-1 monocot. d–g, In situ by repressing PLT expression directly. hybridization with PLT1 and PLT2 antisense probe, embryos grown at 29 uC. d, PLT1 expression in wild type. e, PLT1 expression in tpl-1. f, PLT2 A second-site modifier screen on tpl-1 uncovered a semi-dominant expression in wild type. g, PLT2 expression in tpl-1. h, Graph of fold mutant that completely suppressed the formation of double-root enrichment at the PLT1 locus from ChIP of TPLp::TPL-HA. i, Graph of fold seedlings (Supplementary Fig. 2). Map-based cloning identified a enrichment at the PLT2 locus from ChIP of TPLp::TPL-HA. Scale bars, single missense mutation within the HD-ZIP III transcription factor 1mm(a–c) and 50 mm(d–g).

1Plant Biology Laboratory, The Salk Institute for Biological Studies, 2Department of Biology, University of California San Diego, La Jolla, California 92037, USA. 423 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

Although neither rev-10d nor icu4-1d display obvious embryonic pattern in tpl-1 phb-14d (Fig. 2i, l and Supplementary Fig. 3). We then patterning defects (Supplementary Fig. 2), they also completely asked whether the loss of HD-ZIP III apical expression in tpl-1 was suppress the shoot to root transformation seen in tpl-1 when grown due to expansion of MIR165/166 activity. Green fluorescent protein at 29 uC(n . 1,000) (Supplementary Fig. 2). These results suggest that (GFP)-based sensors for MIR165/166 activity, in which the sensor the HD-ZIP III genes have an important role in promoting apical fate is inactivated in all cells where MIR165/166 are active, accumulate in early embryogenesis. in a pattern similar to that of the HD-ZIP III messenger RNA and Consistent with the observation that the GOF mutations in HD- protein accumulation in the embryo (Fig. 2b, m, o and Supplementary ZIP III genes restore apical fate to tpl-1 embryos, PHB, PHV, REV and Figs 3 and 5). Notably, the sensor is cleared from the root meristem ICU4 are all expressed in an apical/central domain of the globular organizing centre from the globular stage onwards (Fig. 2m, o and embryo17 (Fig. 2a and Supplementary Fig. 3). By the heart stage, the Supplementary Fig. 5). If MIR165/166 were misexpressed in tpl-1,we expression of all four genes expands to the adaxial domain of the would expect to observe clearance of the sensor similar to that of the cotyledons and throughout the provascular tissue17 (Fig. 2b and Sup- mRNA of HD-ZIP III genes. However, the sensor continues to accu- plementary Fig. 3). In tpl-1 embryos grown at 29 uC, PHB, PHV, REV mulate in the apical domain of tpl-1 embryos grown at 29 uC (Fig. 2n, and ICU4 expression is identical to wild type at the globular stage, but p and Supplementary Fig. 5). Therefore, the loss of apical HD-ZIP III is absent from the apical domain by the heart stage (Fig. 2c and expression in tpl-1 is due to a mechanism independent of MIR165/166 Supplementary Fig. 3). Persistent PHB expression in the apical action and is probably at the level of transcriptional control. This domain in tpl-1 phb-14d embryos grown at 29 uC (Fig. 2d) indicates represents a novel aspect of the control of HD-ZIP III gene expression that increasing HD-ZIP III transcript abundance is sufficient to and indicates that HD-ZIP III genes are excluded from the root by restore apical fate in tpl-1. both transcriptional and post-transcriptional mechanisms. We then examined the radial organization of the apical domain of MIR165/166-independent loss of HD-ZIP III expression in tpl-1 these embryos at 29 uC by determining the expression patterns of the may be caused by PLT1/PLT2 misexpression in apical tissues. This FILAMENTOUS FLOWER (FIL) and WUSCHEL (WUS) genes. FIL idea is supported by the finding that PHB and REV expression is is normally expressed peripherally to the meristem in globular stage maintained in the apical domain of tpl-1 plt1-5 plt2-1 triple mutants wild-type embryos (Supplementary Fig. 3) and becomes restricted to (Fig. 2q, r), indicating that PLT1/PLT2 act as negative regulators of the abaxial/ventral side of the cotyledons in heart and later stages HD-ZIP III expression during embryogenesis. This is also consistent (Fig. 2g and Supplementary Fig. 3). In tpl-1, FIL expression expands with what is observed in mutants where PHB mRNA is thought to be throughout the apical domain from late globular to heart stage and is completely uncoupled from MIR165/166 regulation, such as phb-1d subsequently lost (Fig. 2h and Supplementary Fig. 3). WUS has a and serrate (se). PHB mRNA accumulates throughout a wide pattern critical role in SAM initiation and maintenance, and serves as a in phb-1d and se-5 embryos, and can lead to root defects and embry- central-apical marker throughout embryogenesis18.Intpl-1, WUS onic lethality. However, it is still restricted from the lenticular cell in is expressed correctly through the globular stage but is subsequently these mutant backgrounds15,19 (Fig. 2f), an area of high PLT1/PLT2 lost7 (Fig. 2j, k). These patterns of FIL and WUS misexpression in tpl-1 expression (Fig. 1d, f). are identical to what is seen in the triple loss-of-function mutant rev-9 Double-root formation in tpl-1 requires PLT1/PLT2 misexpres- phb-6 phv-5, and show that the apical portion of tpl-1 embryos lose sion and is suppressed by GOF HD-ZIP III mutations. Therefore, adaxial identity during the shoot to root transformation (Supplemen- we examined PLT1/PLT2 gene expression in tpl-1 phb-14d and tary Fig. 4). FIL and WUS expression are restored to the wild-type tpl-1 rev-10d double mutants grown at 29 uC. PLT1 and PLT2 are still

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m n o p q r

Figure 2 | Molecular characterization of tpl-1, phb-14d and tpl-1 phb-14d in (l). m–p, MIR165/166 sensor. GFP fluorescence in wild type (m) and tpl-1 embryos grown at 29 6C. a–f, PHB in situ hybridizations. Wild-type (n). GFP in situ hybridizations in wild type (o) and tpl-1 (p). q, PHB in situ globular (a) and early heart (b) stage, tpl-1 transition stage (c), tpl-1 phb-14d hybridizations in tpl-1 plt1-5 plt2-1. r, REV in situ hybridization in tpl-1 transition stage (d), phb-14d heart stage (e), and phb-1d heart stage plt1-5 plt2-1. Arrowheads indicate the root meristem organizing centre. (f). g–i, FIL in situ hybridizations. Wild type (g), tpl-1 (h) and tpl-1 phb-14d Scale bars, 50 mm(a–r). (i). j–l, WUS in situ hybridizations. Wild type (j), tpl-1 (k) and tpl-1 phb-14d. 424 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS misexpressed in the vascular tissue and abaxial regions of developing tissues (Fig. 3j and Supplementary Table 2). In addition, rev-10d plt1-5 cotyledons (Fig. 3a–d and Supplementary Fig. 4). However, we never plt2-1 show expansion of REV transcript into the root meristem observed PLT gene misexpression in the cells that would give rise to region (Fig. 3k), further indicating that the PLT genes have an active the shoot meristem. This indicates that the ability of PLT genes to role in repression of the HD-ZIP III genes, in addition to negative promote root meristem formation in tpl-1 is dependent on misregulation by MIR165/166. expression in the meristem, and the GOF HD-ZIP III alleles are able To investigate whether the HD-ZIP III genes could impart apical to repress the PLT pathway in these cells. polarity to basal tissue if misexpressed in the basal pole of the early Our genetic studies with the GOF HD-ZIP III alleles and tpl-1 embryo, we expressed MIR-resistant cDNAs of the HD-ZIP III implicate the HD-ZIP III genes in promotion of apical fate and anta- genes fused to the glucocorticoid receptor (GR) domain under the gonism of basal fate. phb-6 phv-5 rev-9 triple loss-of-function control of the PLT2 promoter. When induced with dexamethasone mutants produce pin-shaped seedlings11 similar to what is observed during early embryogenesis, plants harbouring PLT2p::REVDMIR- at low frequency in tpl-1 (ref. 8) (Supplementary Fig. 4). tpl-1 seed- GR, PLT2p::PHBDMIR-GR or PLT2p::ICU4DMIR-GR transgenes lings rarely form double roots at 24 uC (2%, n 5 682), whereas tpl-1 produce seedlings that show a complete transformation of the root rev-9 significantly enhances the double root phenotype (48%, pole into a second shoot pole (Fig. 4a–c, Supplementary Table 2 and n 5 355). Additionally, loss of PHB and PHV in the tpl-1 rev-9 back- Supplementary Fig. 6). ground further increases the frequency of the double-root phenotype To characterize these homeotic transformations further we examined (Supplementary Table 1). At 24 uC PLT1 and PLT2 are not broadly the expression of genes specifically associated with the apical and basal misexpressed in the apical domain of tpl-1 embryos (Fig. 3e, f). poles. PINFORMED4 (PIN4) is initially expressed in the embryo However, in tpl-1 rev-9 embryos grown at 24 uC, PLT1 and PLT2 proper, as well as the upper cells of the suspensor (Fig. 4d). It is then are misexpressed similarly to tpl-1 at 29 uC (Fig. 3g, h). These results restricted from the suspensor and expressed in the developing root show that at lower temperatures, the HD-ZIP III genes act to prevent meristem and provascular cells, and this expression has been shown the misexpression of PLT1/PLT2 in a tpl-1 background. to be dependent on the activity of PLT1 and PLT2 (ref. 20; Fig. 4e, f). In Given that GOF HD-ZIP III alleles suppress PLT1/PLT2-dependent globular through heart stage PLT2p::REVDMIR-GR embryos, PIN4 apical root formation in tpl-1, we investigated the genetic interactions mRNA is only detectable in aberrantly dividing suspensor cells of GOF HD-ZIP III mutants with PLT loss-of-function mutants. (Fig. 4g, h). The loss of PLT1/PLT2-dependent PIN4 expression in cells phb-14d and rev-10d have no discernible embryonic root defects ectopically expressing REV again illustrates the antagonistic action of (Supplementary Fig. 2) and plt1 plt2 have only a minor defect, result- these two classes of genes. ing in a properly organized seedling1. However, phb-14d plt1-5 plt2-1 In induced globular stage PLT2p::REVDMIR-GR embryos, WUS is triple mutant seedlings completely lacked a root and displayed only a misexpressed in the basal region coincident with the presumptive rudimentary hypocotyl structure (Fig. 3i and Supplementary Table 2), second shoot position, indicating that an additional shoot organizing similar to plt1 plt2 plt3 plt4/bbm segregants3. rev-10d plt1-5 plt2-1 centre has formed (Fig. 4i, j). Likewise, AINTEGUMENTA (ANT) seedlings showed an even more severe loss of both root and hypocotyl expression, which marks the cotyledon primordia in the wild type21 (Fig. 4k), is misexpressed in the basal region of transition stage embryos a bbcccdd (Fig. 4l). In older embryos, multiple basal foci of ANT misexpression

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Figure 4 | HD-ZIP III gene misexpression can initiate apical fate and acts antagonisticallyto PLT gene function. a, SEM image of PLT2p:REVDMIR-GR seedling. b, PLT2p:PHBDMIR-GR seedling. c, PLT2p::ICU4DMIR-GR Figure 3 | HD-ZIP III genes antagonize PLT function. a–c, In situ seedling. d–h, In situ hybridizations with anti-sense PIN4 probe. d–f,Wild- hybridizations with PLT1/ PLT2 in 29 uC-grown embryos. a, PLT1 in tpl-1 type 16-cell (d), globular (e)andheart(f)stage.g–h, PLT2p:REVDMIR-GR phb-14d. b, PLT1 in tpl-1 rev-10d. c, PLT2 in tpl-1 phb-14d. d, PLT2 in tpl-1 globular stage (g) and late heart stage (h) embryos after dexamethasone rev-10d. e–h, PLT1/PLT2 in situ hybridizations in 24 uC grown embryos. induction. i–j, In situ hybridizations with anti-sense WUS probe in e, PLT1 in tpl-1. f, PLT2 in tpl-1. g, PLT1 in tpl-1 rev-9. h, PLT2 in tpl-1 rev-9. PLT2p:REVDMIR-GR globular (i)andheart(j)stageembryosafterinduction. i, phb-14d plt1-5 plt2-1 seedling. j, Scanning electron micrograph (SEM) of k–m, In situ hybridizations with anti-sense ANT probe in wild-type transition rev-10d plt1-5 plt2-1 seedling. k, REV in situ hybridization in rev-10d plt1-2 stage (k)andPLT2p:REVDMIR-GR transition stage (l)andtorpedostage plt2-1. Scale bars, 50 mm(a–h, k) and 1 mm (i, j). (m) embryos after induction. Scale bars, 1 mm (a–c)and50mm(d–m). 425 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 accumulate (Fig. 4m). These results show that the alteration in embryo 8. Long, J. A., Woody, S., Poethig, S., Meyerowitz, E. M. & Barton, M. K. Transformation of shoots into roots in Arabidopsis embryos mutant at the polarity begins during the early globular stage of induced embryos. TOPLESS locus. Development 129, 2797–2806 (2002). Furthermore, these results indicate that establishment of apical fate 9. Tang, G., Reinhart, B. J., Bartel, D. P. & Zamore, P. D. A biochemical framework for by the HD-ZIP III genes precedes WUS and ANT expression (and RNA silencing in plants. Genes Dev. 17, 49–63 (2003). therefore meristem and cotyledon formation). 10. Mallory, A. C. et al. MicroRNA control of PHABULOSA in leaf development: Our results show the HD-ZIP III genes are master regulators of importance of pairing to the microRNA 59 region. EMBO J. 23, 3356–3364 (2004). apical fate in early embryogenesis. In addition, there is a clear anta- 11. Emery, J. F. et al. Radial patterning of Arabidopsis shoots by class III HD-ZIP and gonism between the HD-ZIP III and PLT gene families, both of which KANADI genes. Curr. Biol. 13, 1768–1774 (2003). are under multiple modes of regulation that ensures proper spatial 12. Floyd, S. K. & Bowman, J. L. Gene regulation: ancient microRNA target sequences distribution and apical–basal patterning (Supplementary Fig. 1). in plants. Nature 428, 485–486 (2004). 13. Ochando, I. et al. Mutations in the microRNA complementarity site of the Whether this antagonism is direct or is a more downstream con- INCURVATA4 gene perturb meristem function and adaxialize lateral organs in sequence of fate change will require further investigation. Arabidopsis. Plant Physiol. 141, 607–619 (2006). 14. Zhong, R., Taylor, J. J. & Ye, Z. H. Transformation of the collateral vascular bundles METHODS SUMMARY into amphivasal vascular bundles in an Arabidopsis mutant. Plant Physiol. 120, 53–64 (1999). Plants were grown on either soil or Petri dishes containing Linsmaier and Skoog 15. McConnell, J. R. et al. Role of PHABULOSA and PHAVOLUTA in determining radial salts medium. Percival growth chambers were used for controlled temperature patterning in shoots. Nature 411, 709–713 (2001). experiments. All other plants were grown under greenhouse conditions on a 16 h 16. McConnell, J. R. & Barton, M. K. Leaf polarity and meristem formation in light/8 h dark cycle. In situ hybridizations were detected with digoxigenin- Arabidopsis. Development 125, 2935–2942 (1998). labelled riboprobes using the method found at http://www.its.caltech.edu/ 17. Prigge, M. J. et al. Class III homeodomain-leucine zipper gene family members ,plantlab/protocols/insitu.htm. ChIP was performed as described22 with minor have overlapping, antagonistic, and distinct roles in Arabidopsis development. modifications. The Bio-Rad MyiQ, single colour, real-time PCR detection system Plant Cell 17, 61–76 (2005). was used with the MyiQ Optical System Software for analysis of SYBR Green I- 18. Mayer, K. F. et al. Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95, 805–815 (1998). stained amplification products. Embryos were cleared with Hoyer’s solution and 19. Grigg, S. P. et al. Repression of apical homeobox genes is required for embryonic differential interference contrast (DIC) images were collected using a Leica root development in Arabidopsis. Curr. Biol. 19, 1485–1490 (2009). DM5000B microscope. Confocal images were collected using a Leica DM IRE2 20. Friml, J. et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in laser scanning confocal microscope. Arabidopsis. Cell 108, 661–673 (2002). 21. Elliott, R. C. et al. AINTEGUMENTA,anAPETALA2-like gene of Arabidopsis with Full Methods and any associated references are available in the online version of pleiotropic roles in ovule development and floral organ growth. Plant Cell 8, the paper at www.nature.com/nature. 155–168 (1996). 22. Aoyama, T. & Chua, N. H. A glucocorticoid-mediated transcriptional induction Received 13 March 2009; accepted 8 January 2010. system in transgenic plants. Plant J. 11, 605–612 (1997). Published online 28 February 2010. Supplementary Information is linked to the online version of the paper at 1. Aida, M. et al. The PLETHORA genes mediate patterning of the Arabidopsis root www.nature.com/nature. stem cell niche. Cell 119, 109–120 (2004). 2. Blilou, I. et al. The PIN auxin efflux facilitator network controls growth and Acknowledgements We thank J. Sewell for technical assistance with plasmid patterning in Arabidopsis roots. Nature 433, 39–44 (2005). construction and microscopy; R. Biddick, B. Crawford, Y. Jaillais, N. Krogan, 3. Galinha, C. et al. PLETHORA proteins as dose-dependent master regulators of J. Posakony, B. VanSchooten and D. Kelley for discussions and reading of the Arabidopsis root development. Nature 449, 1053–1057 (2007). manuscript; E. York and SIO Unified Laboratory Facility for SEM analysis. This work 4. Friml, J. et al. Efflux-dependent auxin gradients establish the apical–basal axis of was supported by a Ray Thomas Edwards Foundation Career Development Award, Arabidopsis. Nature 426, 147–153 (2003). the National Institute of Health, NIGMS (J.A.L.), Z.R.S. is a USCD Plant Systems 5. Gray, W. M., Kepinski, S., Rouse, D., Leyser, O. & Estelle, M. Auxin regulates Biology NSF-IGERT Fellow. TIR1 SCF -dependent degradation of AUX/IAA proteins. Nature 414, 271–276 Author Contributions Z.R.S. collected the data. Z.R.S. and J.A.L. designed the study (2001). and wrote the manuscript. 6. Szemenyei, H., Hannon, M. & Long, J. TOPLESS mediates auxin dependent transcriptional repression during Arabidopsis embryogenesis. Science 319, Author Information Reprints and permissions information is available at 1384–1386 (2008). www.nature.com/reprints. The authors declare no competing financial interests. 7. Long, J. A., Ohno, C., Smith, Z. R. & Meyerowitz, E. M. TOPLESS regulates apical Correspondence and requests for materials should be addressed to J.A.L. embryonic fate in Arabidopsis. Science 312, 1520–1523 (2006). ([email protected]).

METHODS Plasmid construction. The MIR165/166 sensor was generated using comple- Plant growth and mutant alleles. Plants were grown on either soil or Petri dishes mentary 42-bp primers encompassing the MIR165/166 recognition sequence in containing Linsmaier and Skoog salts medium. Percival growth chambers were PHB and REV, which were annealed to generate double-stranded fragments with used for controlled temperature experiments. All other plants were grown under EcoRI compatible sites at each end. These fragments were then treated with T4 greenhouse conditions on a 16 h light/8 h dark cycle. All mutants, with the polynucleotide kinase in T4 DNA ligase buffer and cloned into a unique EcoRI exception of icu4-1d, are in the Landsberg erecta (Ler) ecotype. Germplasms site in the mERGFP5 sequence that lies between the endoplasmic reticulum (ER) used were as follows: plt1-5 and plt2-1 (ref. 1), rev-10d (ref. 11), phb-1d (ref. 16), localization signal and mGFP5. The modified mERGFP5 were then cloned as a BamHI fragments into a pBJ36 construct, 39 to a 925-bp promoter fragment phb-6 phv-5 rev-9 (ref. 11), icu4-1d (ref. 13). icu4-1d was isolated in the Enkheim-2 24 (En-2) and back crossed to tpl-1 four times. from the potato ubi3 gene . For the negative control, three silent mutations were In situ hybridization. In situ hybridizations were detected with digoxigenin- introduced within the 39 end of the MIR recognition sequence. Primers used are labelled riboprobes using the method found at http://www.its.caltech.edu/ listed in Supplementary Table 3. ,plantlab/protocols/insitu.htm. PHB, PHV, REV and FIL probes were made For construction of the PLT2p::HD-ZIPIII-GR constructs, a 4,380-bp genomic using 300–700-base-pair (bp) regions of coding sequence using the primers fragment 59 to the PLT2 start codon was cloned as a XhoI/SalI fragment into a SalI site of a pBJ36 vector containing the hormone-binding domain of the rat gluco- listed in Supplementary Table 3. PLT1, PLT2, ICU4, WUS, ANT, PIN4 and 22 GFP probes were generated using full-length cDNAs. corticoid receptor . HD-ZIP III MIR-resistant cDNAs were generated by indu- Chromatin immunoprecipitation. ChIP was performed as described23 with the cing three silent mutations within the 39 end of the MIR recognition sequence by following modifications. Ovules were dissected from siliques containing globu- site-directed mutagenesis using the primers listed in Supplementary Table 3. lar to heart stage embryos. Tissue was fixed in 2% formaldehyde/PBS under Microscopy. Excised ovules were mounted in Hoyer’s solution for analysis of vacuum for 2 h, replacing vacuum every 30 min. For each ChIP sample 500 mg embryonic morphology. Embryos were imaged using a Leica DM5000B micro- of starting material was used. The anti-HA monoclonal antibody HA.11 scope, seedlings using a Leica MZ FLIII microscope. For GFP analysis, ovules were (Covance) and M-280 sheep anti-mouse IgG Dynabeads (Invitrogen) were used dissected into 0.53 LS medium (Caisson Laboratories), vacuum infiltrated in 4% to immunoprecipitate TPL–HA fusion. Two negative controls were performed, paraformaldehyde, rinsed with water, vacuum infiltrated with 2% SCRI including a no antibody sample and a ChIP reaction performed on wild-type (no Renaissance 2200 (Renaissance Chemicals) and 4% DMSO, then washed twice transgene) tissue. and mounted in 20% glycerol. Embryos were imaged using a Leica DM IRE2 laser Real-time PCR. The Bio-Rad MyiQ, single colour, real-time PCR detection scanning confocal microscope. SR2200 was excited with the ultraviolet diode system was used with the MyiQ Optical System Software for analysis. SYBR 405 nm line, and emission was measured between 420 and 470 nm. GFP was Green I was used as an intercalating fluorescent dye. The standard curve method excited with a 488 nm argon laser line and emission was measured at 500–535 nm. was used to determine reaction efficiency for each primer pair and determine 23. Bowler, C. et al. Chromatin techniques for plant cells. Plant J. 39, 776–789 (2004). fold enrichment by comparing the Ct (threshold cycle) values of immunopreci- 24. Garbarino, J. E. & Belknap, W. R. Isolation of a ubiquitin-ribosomal protein gene pitated (IP) and negative control which were normalized normalize by calculat- (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol. ing input(IP)/input(control) when appropriate. Biol. 24, 119–127 (1994).

RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF

Poulikos I. Poulikakos1, Chao Zhang2, Gideon Bollag3, Kevan M. Shokat2 & Neal Rosen1

Tumours with mutant BRAF are dependent on the RAF–MEK– HER2 dependent9. The HER2 inhibitor lapatinib abolished basal and ERK signalling pathway for their growth1–3. We found that ATP- PLX4032-induced ERK signalling in these cells (Supplementary Fig. 5a). competitive RAF inhibitors inhibit ERK signalling in cells with In 293H cells, induction of MEK and ERK phosphorylation by either mutant BRAF, but unexpectedly enhance signalling in cells with PLX4032 or PLX4720 was barely detectable (referred to hereafter as wild-type BRAF. Here we demonstrate the mechanistic basis for these PLX4032/PLX4720 to indicate data obtained with both compounds). findings.Weusedchemicalgeneticmethodstoshowthatdrug- Haemagglutinin (HA)-tagged wild-type RAS overexpression resulted mediated transactivation of RAF dimers is responsible for para- in enhanced MEK/ERK activation by RAF inhibitor, which was more doxical activation of the enzyme by inhibitors. Induction of ERK pronounced when mutant RAS was overexpressed (Fig. 2a and Sup- signalling requires direct binding of the drug to the ATP-binding site plementary Fig. 5b). The results indicate that RAS activity is required for of one kinase of the dimer and is dependent on RAS activity. Drug MEK/ERK activation by RAF inhibitors. In contrast, in 293H cells binding to one member of RAF homodimers (CRAF–CRAF) or expressing Flag-tagged BRAF(V600E), ERK signalling was inhibited heterodimers (CRAF–BRAF) inhibits one protomer, but results in by PLX4032 (Supplementary Fig. 5c). These results indicate that RAF transactivation of the drug-free protomer. In BRAF(V600E) tumours, inhibitors will inhibit the growth of tumours with mutant BRAF, but not RAS is not activated, thus transactivation is minimal and ERK sig- those with wild-type BRAF, including those with RAS mutation. This is nalling is inhibited in cells exposed to RAF inhibitors. These results a indicate that RAF inhibitors will be effective in tumours in which pMEK pERK MEK μ BRAF is mutated. Furthermore, because RAF inhibitors do not inhi- 0 0.010.030.10.3 1 3 10 30 0 0.010.030.10.3 1 3 10 30 0 0.010.030.1 0.3 1 3 10 30 ( M) bit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than GW5074 mitogen-activated protein kinase (MEK) inhibitors, but could also L779450 cause toxicity due to MEK/ERK activation. These predictions have PLX4720 been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization Lysates Sorafenib by elevation of wild-type RAF expression or RAS activity could lead to ZM336372 drug resistance in mutant BRAF tumours. In agreement with this b c Time (min): 25100 PLX4720 BRAF(WT) pMEK prediction, RAF inhibitors do not inhibit ERK signalling in cells that KRAS(Q61K) BRAF(V600E) coexpress BRAF(V600E) and mutant RAS. (Calu-6) (Malme-3M) pERK

Six distinct ATP-competitive RAF inhibitors induced ERK activa- – + – + PLX4720 Lysates MEK pMEK tion in cells with wild-type BRAF but inhibited signalling in mutant d BRAF(V600E) cells (Fig. 1a, b, Supplementary Fig. 2a, b and data not pERK ++++ +– ––PLX4720 shown; structures of compounds are shown in Supplementary Fig. 3, p380-RSK –––– –+++Vehicle except that of PLX4032, which is unavailable). PLX4720 (ref. 6), and p573-RSK 0 60 70 90 120 0 60 70 90 120 Time (min) pMEK its analogue in clinical trial PLX4032, were studied in more detail. MEK PLX4032 inhibited ARAF, BRAF and CRAF immunoprecipitated ERK pERK Lysates from 293H cells (Supplementary Fig. 4) and purified catalytic domains ARAF Lysates MEK 112345 6789 0 of BRAF(V600E), wild-type BRAF and CRAF (half-maximum inhibi- BRAF Medium Medium tory concentration (IC50) values of 35, 110 and 48 nM, respectively) CRAF (Supplementary Table 1). PLX4032 was assayed against 62 additional change change kinases that span the kinome, and had IC50 values of 1–10 mM against Figure 1 | RAF inhibitors rapidly activate MEK/ERK in cells with wild-type eight of these and greater than 10 mM against the rest (G.B., un- BRAF. a, Calu-6 cells (wild-type BRAF (BRAF(WT))/KRAS(Q61K)) were published data). Induction of ERK signalling by PLX4720 was rapid treated with increasing doses of the indicated RAF inhibitors and the effects on (Fig. 1c), reversible (Fig. 1d) and associated with increased phosphor- ERK signalling were determined by immunoblotting for phosphorylated MEK ylation of the ERK substrate RSK (Fig. 1b). MEK and ERK phosphor- (pMEK) and phosphorylated ERK (pERK). b, Cells with wild-type BRAF (Calu-6) or mutant BRAF (Malme-3M) were treated with vehicle or PLX4720 ylation were induced at intermediate concentrations of RAF inhibitor, (1 mM for 1 h). Phosphorylation and expression of the indicated proteins were and inhibited at much higher doses (Fig. 1a). assayed by immunoblotting. c, Calu-6 cells treated with 1 mMPLX4720forthe Physiological induction of ERK signalling depends on upstream indicated time points. d, Calu-6 cells were treated with 1 mM PLX4720 for activation of RAS by receptor-induced signalling7,8. PLX4032 induced 60 min, then medium was replaced with medium containing 1 mMPLX4720 ERK signalling in SKBR3 breast cancer cells, in which RAS activation is (lanes 3–5) or vehicle (lanes 8–10) for the indicated time points.

1Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. 2Howard Hughes Medical Institute & Department of Cellular and Molecular Pharmacology, University of California San Francisco, California 94158, USA. 3Plexxikon Inc., 91 Bolivar Drive, Berkeley, California 94710, USA. 427 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a b c catC(T421M)

–/– –/– +/+ Braf Craf Sorafenib (μM) 0 0.06250.125 0.2500.5 1 Control RAS(G12V)catC catC(T421M) 0551 051 10 PLX4720 (μM) – +– +– +– + +– +– – +– PLX4720 pMEK pMEK pMEK

pERK pERK pERK HA BRAF

Lysates V5 Lysates Lysates V5 CRAF MEK MEK MEK

Figure 2 | MEK/ERK activation requires binding of drug to the catalytic knockout (Braf2/2) or CRAF knockout (Craf2/2) MEFs were treated with domain of RAF. a, 293H cells transfected with EGFP (control), HA-tagged the indicated concentrations of PLX4720 for 1 h. c, Sorafenib inhibits the RAS(G12V), the catalytic domain of CRAF (V5-tagged catC) and catC gatekeeper mutant catC(T421M) protein in vitro (Supplementary Fig. 8c) carrying a mutation at the gatekeeper residue (V5-tagged catC(T421M)), and activates MEK/ERK in cells expressing it. 293H cells overexpressing treated with vehicle or PLX4720 (1 mM for 1 h). Lysates were subjected to catC(T421M) were treated with the indicated concentrations of sorafenib for immunoblot analysis for pMEK and pERK. b, Wild-type (1/1), BRAF 1 h. Lysates were subjected to analysis for pMEK and pERK. indeed the case: MEK-dependent tumours with RAS mutation are un- (Fig. 3d). The data suggest that binding of PLX4032/PLX4720 to CRAF affected by PLX4032 (N.R., unpublished data). induces activation of the enzyme and, subsequently, ERK signalling. The BRAF and CRAF kinases form homo- and heterodimers on RAS result seems paradoxical: binding of ATP-competitive inhibitors to the activation10–12. PLX4032/PLX4720 induced pronounced phosphory- catalytic domain of CRAF activates its function. lation of MEK and ERK in wild-type mouse embryonic fibroblasts RAF isoforms form dimers in cells10–12,16. Because binding of both (MEFs) and Braf2/2 MEFs. The response was diminished markedly in the drug and ATP to the catalytic domain would be required for Craf2/2 (also called Raf1) MEFs (Fig. 2b and Supplementary Fig. 6a). activation and cannot occur simultaneously on the same molecule, Coexpression of CRAF and active RAS in Craf2/2 MEFs reconstituted we hypothesized that RAF inhibitors activate CRAF dimers in trans the wild-type phenotype (Supplementary Fig. 6b, c). We conclude that (Supplementary Fig. 1b). To test this model, we generated mutant BRAF is dispensable for MEK/ERK activation by PLX4032/PLX4720, catC(S428C) that binds to 6-acrylamido-4-anilinoquinazolines17, and that CRAF expression is required for significant induction. We whereas catC does not. Two inhibitors, JAB13 (ref. 17) and JAB34 therefore investigated the mechanism of CRAF-dependent induction (also called PD-168393)18, both inhibited catC(S428C), but up to of ERK signalling in response to the drug. 30 mM had no effect in vitro on catC (Supplementary Fig. 10a, b). Autoinhibition of RAF by its amino-terminal domain13 is relieved JAB13 and JAB34 selectively affected ERK signalling in cells expressing on binding to activated RAS7. We asked whether overexpression of an catC(S428C) and were inactive in those expressing catC (Supplemen- N-truncated form of CRAF would bypass the requirement for RAS tary Fig. 11). Like the other inhibitors (Fig. 1a), lower doses (40 nM to activity. In 293H cells expressing the catalytic domain of CRAF a c

(catC), PLX4032/PLX4720 caused marked induction of MEK and Pretreatment: 0 0.25 1 4 16 PLX4720 (μM) T421M WT D486N V5–CRAF ERK phosphorylation (Fig. 2a and Supplementary Fig. 7a). We focused – + – + – + PLX4720 mechanistic investigations on catC, in which PLX4032/PLX4720- pMEK p338-CRAF induced MEK/ERK activation is RAS independent. To test whether IP: catC MEK p621-CRAF direct binding of PLX4032/PLX4720 to CRAF is required for induc- V5 V5 Lysates

tion of signalling, we generated a catC carrying a mutation at the Kinase assays gatekeeper position (T421) in the kinase domain (mutations used MEK and their properties are in Supplementary Fig. 1a). Structural studies6 b d predict that the T421M mutation should prevent drug binding and p338-CRAF catC(T421M) was indeed resistant to inhibition by PLX4032/PLX4720 Pretreatment: – + PLX4720 μ in vitro (Supplementary Fig. 8a, b). ERK signalling was not induced by pMEK 0 0.010.030.1 0.3 1 3 10 30 ( M) PLX4032/PLX4720 in cells expressing catC(T421M) (Fig. 2a and IP: BRAF MEK Supplementary Fig. 7b). Thus, activation of MEK/ERK by PLX4032/ BRAF GW5074 PLX4720 depends on its direct binding to the RAF kinase active site. L779450 Sorafenib inhibited catC(T421M) in vitro (Supplementary Fig. 8c) and pMEK PLX4720 induced ERK signalling in cells expressing catC(T421M) (Fig. 2c), Kinase assays demonstrating that this mutant is capable of inhibitor-induced IP: CRAF MEK Lysates Sorafenib MEK/ERK activation. Thus, direct binding of an ATP-competitive CRAF ZM336372 inhibitor to CRAF is required for induction of ERK signalling. Figure 3 | RAF inhibitor induces the active, phosphorylated state of wild- Recent work shows that binding of ATP-competitive inhibitors to type and kinase-dead RAF. a, 293H cells overexpressing catC were treated AKT and protein kinase C inhibits their activity, but induces the active, with the indicated amounts of PLX4720 for 1 h. Cells were lysed, catC was 14,15 phosphorylated state of these kinases .WashedcatCimmuno- immunoprecipitated (IP), washed extensively and subjected to kinase assay. precipitated from PLX4032/PLX4720-treated cells was more active Kinase activity was determined by immunoblotting for pMEK. b, Calu-6 cells than that isolated from untreated cells (Fig. 3a and Supplementary were treated with PLX4720 (1 mM for 1 h). Endogenous BRAF and CRAF were Fig. 9a). The same was true for endogenous BRAF and CRAF immuno- immunoprecipitated, washed and assayed for kinase activity. c, Treatment precipitated from Calu-6 cells (Fig. 3b and Supplementary Fig. 9b). with RAF inhibitor results in elevated phosphorylation at activating Phosphorylation of CRAF at S338 and S621 has been correlated with phosphorylation sites on RAF. V5-tagged wild-type CRAF or kinase-dead CRAF(D486N) were overexpressed in 293H cells. After 24 h cells were treated its activation7. PLX4032/PLX4720 caused increased phosphorylation of with vehicle or PLX4720 (5 mM for 1 h) and lysates were immunoblotted for both sites on wild-type and kinase-dead CRAF in 293H cells. In contrast, p338-CRAF and p621-CRAF. The gatekeeper mutant CRAF(T421M) was it did not affect the phosphorylation of the PLX4032/PLX4720-resistant used as negative control. d, Samples as in Fig. 1a, immunoblotted for pS338- CRAF(T421M) mutant (Fig. 3c and Supplementary Fig. 9c). All RAF CRAF. Note that phosphorylation at S338 steadily increased, even when inhibitors tested induced phosphorylation at p338 of endogenous CRAF concentrations were reached that inhibited MEK/ERK. 428 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

1 mM) induced ERK signalling (Supplementary Fig. 11), whereas in severe loss of both expression and activity (data not shown). We higher doses (10 mM) caused inhibition (Fig. 4a). The specificity of therefore mutated R401 to alanine in V5–catC(S428C) and Flag– this system allows us to test the dimer transactivation model. catC. This mutation diminished dimerization (Supplementary Fig. 13) We coexpressed a V5-tagged, JAB-sensitive, kinase-dead catC, V5– but retained expression and activity. In cells coexpressing these mutants, catC(S428C/D486N), and Flag-tagged catC in 293H cells. V5– JAB34 failed to induce ERK signalling (Fig. 4b, lanes 7, 8). Thus, a catC(S428C/D486N) is deficient in catalytic activity; it can bind to mutation that affects dimerization prevents transactivation. the inhibitor (JAB34) but cannot phosphorylate MEK, whereas Flag– The transactivation model explains the observation that inhibitors catC is catalytically active, but cannot bind JAB34. Treatment of of RAF activate ERK signalling at low concentrations, but inhibit at cells expressing both constructs with a concentration of JAB34 that higher concentrations in wild-type BRAF cells. Binding of an ATP- inhibited ERK signalling in cells expressing catC(S428C) alone competitive inhibitor to one protomer within a RAF dimer results in (10 mM JAB34, Fig. 4a) resulted in marked induction of ERK signal- both abolition of the catalytic activity of the inhibitor-bound RAF ling (Fig. 4b, lanes 5, 6). Thus, binding of JAB34 to kinase-dead, and transactivation of the other. Transactivation of RAF homo- and V5–catC(S428C/D486N) transactivated the catalytically competent heterodimers is therefore responsible for induction of MEK/ERK Flag–catC. When the catalytically active drug-binding mutant V5– phosphorylation by RAF inhibitors in cells with wild-type BRAF. catC(S428C) is coexpressed with catalytically inactive catC (Flag– Our model explains the paradoxical phenomenon of ERK activation catC(D486N)), 10 mM JAB34 inhibited, rather than activated, ERK by RAF inhibitors, previously reported by others20–22. Other kinases signalling (Fig. 4b, lanes 9, 10). When both constructs were insensitive that exist in dimeric or multimeric complexes may behave in a similar to JAB, JAB34 had no effect on ERK signalling (Fig. 4b, lanes 1, 2). manner. Recently, another model to explain these phenomena has When both constructs were catalytically active, we observed moderate been proposed23. That study reports that only selective BRAF MEK/ERK activation, probably resulting from inhibition of V5– inhibitors activate CRAF and ERK signalling, whereas pan-RAF catC(S428C) and transactivation of Flag–catC (Fig. 4b, lanes 3, 4). inhibitors do not. Our data that all RAF inhibitors activate ERK Transactivation from CRAF to BRAF can occur as well. JAB34 signalling at low concentrations, that the phenomenon occurs in activated ERK signalling in cells coexpressing Flag–BRAF and V5– BRAF-null cells and that binding to CRAF activates CRAF- and catC(S428C/D486N) (Fig. 4c). Finally, JAB34 induced ERK activa- BRAF-dependent ERK signalling render that model unlikely. tion in cells coexpressing full-length V5–CRAF(S428C/D486N) and Nevertheless, the clinical utility of these inhibitors depends on their full-length Flag–CRAF, confirming that our model is valid in the inhibition of ERK signalling in tumour cells with BRAF(V600E). context of full-length CRAF (Supplementary Fig. 12). Because transactivation of wild-type RAF requires dimerization and Thus, activation of RAF by ATP-competitive inhibitors can be depends on RAS activity, we hypothesized that the levels of RAS explained by transactivation: binding of drug to one RAF in the dimer activity in BRAF(V600E) mutant tumours may not be sufficient to activates the other. This is consistent with the enhancement of induc- support transactivation. If so, activation of RAS in BRAF(V600E) cells tion by active RAS, which promotes homo- and heterodimerization of should prevent inhibition of ERK signalling by RAF inhibitors. In BRAF and CRAF10,12. Our model suggests that transactivation will be 293H cells overexpressing BRAF(V600E) and in HT29 tumour cells dependent on formation of RAF dimers. A side-to-side dimer of the with endogenous BRAF(V600E), ERK signalling was inhibited by kinase domain is observed in crystal structures of BRAF11, and the either PLX4032/PLX4720 or a MEK inhibitor. In contrast, when residues at the dimer interface are conserved in all RAF isoforms. On mutant RAS was coexpressed with BRAF(V600E) in either cell, ERK the basis of the BRAF crystal structures, we identified a conserved Arg signalling became resistant to PLX4032/PLX4720, but remained sensi- (R509) at the centre of the dimer interface. Structural analysis predicts tive to the MEK inhibitor (Fig. 4d and Supplementary Fig. 14a, b). that mutation of R509 will diminish contacts between the two interact- The data are consistent with the idea that RAF inhibitors suppress ing proteins and reduce dimer formation, as also recently reported19.In ERK signalling in BRAF(V600E) tumours because the level of RAS acti- that study, mutation of BRAF at R509 to histidine resulted in a marked vation in these cells is insufficient to support transactivation of wild-type loss of activity. The corresponding mutation in catC (R401H) results RAF and inhibition of BRAF(V600E) activity becomes the dominant ab cd

V5–catC WT S428C S428C/D486NS428C/R401AS428C WT WT WT R401A D486N Flag–catC V5–catC Control RAS(G12V) catC catC(S428C) – + – + – + – + – + JAB34 (10 μM) D486N S428C/D486N μ Control WT WT Flag–BRAF – + – + PLX4720 – + – + JAB34 (10 M) pMEK pMEK – + – + – + JAB34 (10 μM) pMEK pERK pMEK pERK pERK V5 pERK

Lysates pMEK long exp.

Lysates HA MEK FLAG

pERK long exp. Lysates Lysates V5 MEK V5 MEK MEK 165432

Flag 118765432 9 0 Figure 4 | MEK/ERK activation occurs via transactivation of RAF dimers. alone. c, Activation in the context of the heterodimer BRAF–CRAF occurs in a, Similarly to RAF inhibitors, JAB34 inhibits MEK/ERK at higher trans. 293H cells coexpressing Flag-tagged wild-type BRAF and V5-tagged concentrations. 293H cells expressing V5-tagged catC or catC(S428C) were kinase-dead catC (catC(D486N)) (lanes 3, 4) or JAB34-sensitive/kinase-dead treated with either vehicle or 10 mM JAB34 for 1 h. b, Coexpression of drug- catC (catC(S428C/D486N)) (lanes 5, 6) treated with vehicle or 10 mM JAB34 sensitive V5-tagged catC with drug-resistant Flag–catC reveals that for 1 h are shown. d, HT-29 cells (colorectal; BRAF(V600E)) were transfected activation in the homodimer occurs in trans. 293H cells expressing the with EGFP or HA-tagged NRAS(G12V) and treated with PLX4720 (1 mM for indicated mutants V5-tagged catC and Flag-tagged catC were treated with a 1 h). Lysates were blotted for pMEK and pERK. dose of JAB34 (10 mM for 1 h) that inhibits catC(S428C) when expressed 429 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 effect of the drug. The findings indicate that increases in RAS activation 2. McDermott, U. et al. Identification of genotype-correlated sensitivity to selective or RAF dimerization may be sufficient to cause drug resistance. kinase inhibitors by using high-throughput tumor cell line profiling. Proc. Natl Acad. Sci. USA 104, 19936–19941 (2007). The clinical implications of these findings are profound. 3. Wellbrock, C. et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, BRAF(V600E) tumours and some with RAS mutation are dependent 2338–2342 (2004). on ERK signalling. However, in clinic, a MEK inhibitor had only a 4. Chapman, P. et al. Early efficacy signal demonstrated in advanced melanoma in a 12% response rate in melanomas with BRAF mutation24. MEK inhi- phase I trial of the oncogenic BRAF-selective inhibitor PLX4032. Eur. J. Cancer 7 bitors block ERK signalling in all tumour and normal cells and the (suppl.), 5 (2009). 5. Flaherty, K. et al. Phase I study of PLX4032: Proof of concept for V600E BRAF dose of the drug that can be administered is limited by toxicity. RAF mutation as a therapeutic target in human cancer. J. Clin. Oncol. 27 (suppl.), abstr. inhibitors and MEK inhibitors might have been expected to have 9000 (2009). similar biological effects. Our findings show otherwise. RAF inhibi- 6. Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with tors will be useful for the treatment of tumours driven by potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008). BRAF(V600E), but could have deleterious effects in some contexts 7. Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nature Rev. Mol. Cell Biol. 5, 875–885 (2004). due to ERK activation. However, the absence of ERK inhibition in 8. Young, A. et al. Ras signaling and therapies. Adv. Cancer Res. 102, 1–17 (2009). normal cells may allow administration of high doses of RAF inhibi- 9. Konecny, G. E. et al. Activity of the dual kinase inhibitor lapatinib (GW572016) tors and thus more complete inhibition of ERK signalling in against HER-2-overexpressing and trastuzumab-treated breast cancer cells. BRAF(V600E) tumours than is possible with MEK inhibitors. Cancer Res. 66, 1630–1639 (2006). The recent phase I clinical trial of PLX4032 in metastatic melanoma 10. Weber, C. K., Slupsky, J. R., Kalmes, H. A. & Rapp, U. R. Active Ras induces 4,5 heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001). strikingly confirmed these predictions . High serum levels of drug 11. Wan, P. T. et al. Mechanism of activation of the RAF-ERK signaling pathway by were achieved with modest toxicity and resulted in profound inhibi- oncogenic mutations of B-RAF. Cell 116, 855–867 (2004). tion of ERK signalling in tumours. Tumour regression was observed in 12. Rushworth, L. K., Hindley, A. D., O’Neill, E. & Kolch, W. Regulation and role of Raf- more than 90% of patients with BRAF(V600E) mutation, with 64% 1/B-Raf heterodimerization. Mol. Cell. Biol. 26, 2262–2272 (2006). achieving a partial response by RECIST criteria. We believe that the 13. Cutler, R. E. Jr, Stephens, R. M., Saracino, M. R. & Morrison, D. K. Autoregulation of the Raf-1 serine/threonine kinase. Proc. Natl Acad. Sci. USA 95, 9214–9219 (1998). remarkable activity of this drug, compared to that of MEK inhibitors, 14. Okuzumi, T. et al. Inhibitor hijacking of Akt activation. Nature Chem. Biol. 5, is due to its ability to inhibit ERK signalling in tumours more com- 484–493 (2009). pletely because of the absence of ERK inhibition in normal tissue. 15. Cameron, A. J., Escribano, C., Saurin, A. T., Kostelecky, B. & Parker, P. J. PKC Resistance to PLX4032 does develop, with a median time to disease maturation is promoted by nucleotide pocket occupation independently of progression of 8–9 months5. Potential mechanisms include gatekeeper intrinsic kinase activity. Nature Struct. Mol. Biol. 16, 624–630 (2009). 16. Karreth, F. A., DeNicola, G. M., Winter, S. P. & Tuveson, D. A. C-Raf inhibits mutations in BRAF and activating mutations in parallel signalling path- MAPK activation and transformation by B-Raf(V600E). Mol. Cell 36, 477–486 ways. Our results indicate the possibility of novel mechanisms as well. (2009). Lesions that activate RAS or, as recently reported, overexpression of 17. Blair, J. A. et al. Structure-guided development of affinity probes for tyrosine wild-type RAF isoforms25 could result in inability of RAF inhibitors to kinases using chemical genetics. Nature Chem. Biol. 3, 229–238 (2007). suppress ERK signalling in the tumour and thus lead to resistance. 18. Sun, Y. et al. Growth inhibition of nasopharyngeal carcinoma cells by EGF receptor tyrosine kinase inhibitors. Anticancer Res. 19, 919–924 (1999). 19. Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F. & Therrien, M. A METHODS SUMMARY dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, Compounds and cell culture. PLX4032 and PLX4720 were obtained from 542–545 (2009). Plexxikon. PD0325901 was synthesized in the MSKCC Organic Synthesis Core 20. Hall-Jackson, C. A. et al. Paradoxical activation of Raf by a novel Raf inhibitor. Facility by O. Ouerfelli. Sorafenib was synthesized using published procedures26. Chem. Biol. 6, 559–568 (1999). JAB13 and JAB34 were synthesized as previously described17. All other drugs 21. King, A. J. et al. Demonstration of a genetic therapeutic index for tumors were obtained from Calbiochem. Drugs were dissolved in DMSO and stored at expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 66, 220 uC. Cells were maintained in either DMEM or RPMI, supplemented with 11100–11105 (2006). 2/2 22. Hoeflich, K. P. et al. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is 2 mM glutamine, antibiotics and 10% fetal bovine serum. Wild-type, Braf V600E and Craf2/2 MEFs were provided by M. Baccarini. 293H cells were from predicted by BRAF mutational status and sustained extracellular signal- Invitrogen. All other cell lines were from the American Type Culture Collection. regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer Res. 69, 3042–3051 (2009). Antibodies. Western blot analysis was performed as described1. The following 23. Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive antibodies were used: p217/p221-MEK (pMEK), p202/p204-ERK (pERK), p338- tumor progression through CRAF. Cell 140, 209–221 (2010). CRAF, p380-RSK, p573-RSK, MEK, ERK, Myc tag (Cell Signaling), p621-CRAF, V5 24. Dummer, R. et al. AZD6244 (ARRY-142886) vs temozolomide (TMZ) in patients tag (Invitrogen), ARAF, BRAF (Santa Cruz Biotechnology), Flag tag (Sigma), CRAF (pts) with advanced melanoma: An open-label, randomized, multicenter, phase II (BD Transduction Laboratories), HA tag (Covance). For immunoprecipitations of study. J. Clin. Oncol. 26 (suppl.), abstr. 9033 (2008). tagged proteins, the following reagents were used: anti-V5 agarose affinity gel, anti- 25. Montagut, C. et al. Elevated CRAF as a potential mechanism of acquired Flag M2 affinity gel, anti-c-Myc agarose affinity gel (all from Sigma). resistance to BRAF inhibition in melanoma. Cancer Res. 68, 4853–4861 (2008). Plasmids. Plasmids encoding HA-tagged wild-type and mutant NRAS were 26. Bankston, D. et al. A scaleable synthesis of BAY 43-9006: A potent Raf kinase obtained from Biomyx. Plasmids for wild-type BRAF and BRAF(V600E) were inhibitor for the treatment of cancer. Org. Process Res. Dev. 6, 777–781 (2002). provided by W. Kolch and were used as template to create Flag-tagged con- Supplementary Information is linked to the online version of the paper at structs. All other plasmids were created using standard cloning methods, with www.nature.com/nature. pcDNA3.1 (Invitrogen) as a vector. Mutations were introduced using the site- directed Mutagenesis Kit (Stratagene). The catalytic domain of CRAF (catC) was Acknowledgements We are grateful to W. Kolch for the BRAF plasmids and created by truncating the first 305 amino acids of CRAF. M. Baccarini for the RAF knockout MEFs. We thank J. Blair for synthesis of JAB compounds and A. Dar, S. Chandarlapaty and D. Solit for discussions. This work has Kinase assays. RAF kinase assays were conducted in the presence of 100 mM been funded by the Melanoma Research Alliance, the Starr Cancer Consortium, an ATP, at 30 uC for 20 min. Recombinant inactive K97R MEK (Millipore) was used NIH/NCI P01 grant (1P01CA129243-02) and by Joan’s Legacy: United Against Lung as a substrate and kinase activity was estimated by immunoblotting for pMEK. Cancer Foundation (P.I.P., N.R.). K.M.S. would like to thank NIH-2R01EB001987, The Transfections. Cells were seeded on 35 mm or 100 mm plates and transfected the Children’s Tumor Foundation and the Waxman Foundation for funding. following day using Lipofectamine 2000 (Invitrogen). Author Contributions P.I.P. and C.Z. designed research, performed experiments, Full Methods and any associated references are available in the online version of analysed data and co-wrote the paper. G.B. provided reagents, analysed data and the paper at www.nature.com/nature. co-wrote the paper. K.M.S. and N.R. designed research, analysed experiments and co-wrote the paper. Received 13 November 2009; accepted 16 February 2010. Published online 23 February 2010. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. 1. Solit, D. B. et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 439, Correspondence and requests for materials should be addressed to N.R. 358–362 (2006). ([email protected]).

METHODS Plasmids. Plasmids encoding HA-tagged wild-type and mutant NRAS were Compounds and cell culture. PLX4032 and PLX4720 were obtained from obtained from Biomyx. Plasmids expressing Myc-tagged wild-type BRAF and Plexxikon. PD0325901 was synthesized in the MSKCC Organic Synthesis Core BRAF(V600E) were provided by W. Kolch and were used as templates to create Facility by O. Ouerfelli. Sorafenib was synthesized using published procedures26. Flag-tagged constructs. All other plasmids were created using standard cloning JAB13 and JAB34 were synthesized as previously described17. All other methods, with pcDNA3.1 (Invitrogen) as a vector. Mutations were introduced drugs were obtained from Calbiochem. Drugs were dissolved in DMSO to yield using the site-directed Mutagenesis Kit (Stratagene). The catalytic domain of 10 mM stock and stored at 220 uC. Cells were maintained in DMEM (MEFs, CRAF (catC) was created by truncating the first 305 amino acids of CRAF. 293H, NIH3T3 and HeLa) or RPMI (all other cell lines), supplemented Immunoprecipitations and kinase assays. Cells were lysed in lysis buffer with 2 mM glutamine, antibiotics and 10% fetal bovine serum. Wild-type, (50 mM Tris, pH 7.5, 1% NP40, 150 mM NaCl, 10% glycerol, 1 mM EDTA) Braf2/2 and Craf2/2 MEFs were provided by M. Baccarini. 293H cells were supplemented with protease and phosphatase inhibitor cocktail tablets from Invitrogen. All other cell lines were from the American Type Culture (Roche). Immunoprecipitations were carried out at 4 uC for 4 h, followed by Collection. three washes with lysis buffer and, in cases of subsequent kinase assay, one extra 1 Antibodies. Western blot analysis was performed as described . The following wash with kinase buffer (25 mM Tris, pH 7.5, 10 mM MgCl2). RAF kinase assays antibodies were used: p217/p221-MEK (pMEK), p202/p204-ERK (pERK), were conducted in the presence of 100 mM ATP, at 30 uC for 20 min. p338-CRAF, p380-RSK, p573-RSK, MEK, ERK, Myc tag (Cell Signaling), Recombinant inactive K97R MEK (Millipore) was used as a substrate and the p621-CRAF, V5 tag (Invitrogen), ARAF, BRAF (Santa Cruz Biotechnology), reaction was terminated with the addition of sample buffer and boiling. Kinase Flag tag (Sigma), CRAF (BD Transduction Laboratories), HA tag (Covance). activity was estimated by immunoblotting for pMEK. For immunoprecipitations of tagged proteins, the following reagents were used: Transfections. Cells were seeded on 35 mm or 100 mm plates and transfected the anti-V5 agarose affinity gel, anti-Flag M2 affinity gel, anti-c-Myc agarose affinity following day using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, gel (all from Sigma). cells were collected for subsequent analysis.

RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

Georgia Hatzivassiliou1, Kyung Song1, Ivana Yen1, Barbara J. Brandhuber2, Daniel J. Anderson1, Ryan Alvarado1, Mary J. C. Ludlam1, David Stokoe1, Susan L. Gloor2, Guy Vigers2, Tony Morales2, Ignacio Aliagas1, Bonnie Liu1, Steve Sideris1, Klaus P. Hoeflich1, Bijay S. Jaiswal1, Somasekar Seshagiri1, Hartmut Koeppen1, Marcia Belvin1, Lori S. Friedman1 & Shiva Malek1

Activating mutations in KRAS and BRAF are found in more than evident in control mice (Supplementary Fig. 3). Interestingly, erythema 30% of all human tumours and 40% of melanoma, respectively, has been previously reported in mice treated with chemically unrelated thus targeting this pathway could have broad therapeutic effects1. RAF inhibitors6. Immunohistochemistry of serial skin sections showed Small molecule ATP-competitive RAF kinase inhibitors have increased staining for the proliferation marker Ki67 in the keratinocyte potent antitumour effects on mutant BRAF(V600E) tumours but, compartment of sections from inhibitor- but not vehicle-treated ani- in contrast to mitogen-activated protein kinase kinase (MEK) inhi- mals, co-localized with increased cytoplasmic phospho-ERK staining bitors, are not potent against RAS mutant tumour models, despite (Fig. 1c), which is intriguing in light of the recent clinical reports of RAF functioning as a key effector downstream of RAS and ab upstream of MEK2,3. Here we show that ATP-competitive RAF inhi- GDC-0879 PLX4720 20 * ***** * bitors have two opposing mechanisms of action depending on the MEXF514 (BRAF(V600E)) tumour xenograft

(µM) 15 400 Vehicle cellular context. In BRAF(V600E) tumours, RAF inhibitors effec- 50 100 mg kg–1 GDC-0879 10 tively block the mitogen-activated protein kinase (MAPK) signal- 300 ling pathway and decrease tumour growth. Notably, in KRAS 5

RAF inh. EC 200 mutant and RAS/RAF wild-type tumours, RAF inhibitors activate 0

624 P < 0.0001 HLE MT3 LoVo PL45 A375 100 H226 * KM12 SW48 MeWo H2122 the RAF–MEK–ERK pathway in a RAS-dependent manner, thus AsPC1 CaCo-2 HCT-15 HCT116 SKMEL30 enhancing tumour growth in some xenograft models. Inhibitor COLO201 COLO205 0 0 5 10 15 20 25 binding activates wild-type RAF isoforms by inducing dimeriza- MDA-MB-468 BRAF(V600E) RAF/RAS-WT RAS-MT LXFA983 (KRAS-MT) tumour xenograft tion, membrane localization and interaction with RAS–GTP. These 0.5 ) 3 1,400 (µM) 0.4 events occur independently of kinase inhibition and are, instead, 50 1,200 0.3 1,000 linked to direct conformational effects of inhibitors on the RAF 0.2 kinase domain. On the basis of these findings, we demonstrate that 0.1 800 600 ATP-competitive kinase inhibitors can have opposing functions as MEK inh. EC 0 400 624 HLE MT3 LoVo PL45 A375 H226 KM12 SW48 MeWo H2122 P = 0.042 inhibitors or activators of signalling pathways, depending on the AsPC1 200 CaCo-2 HCT-15

HCT116 * Mean tumour volume (mm SKMEL30 cellular context. Furthermore, this work provides new insights into COLO201 COLO205 0 048121620 MDA-MB-468 the therapeutic use of ATP-competitive RAF inhibitors. c –1 Vehicle 200 mg kg GDC-0879 LXFA1041 (KRAS-MT) tumour xenograft The RAS–RAF–MEK–ERK signalling pathway is an attractive target 100 µm 1,200 for therapeutic intervention in oncology, and several selective RAF 1,000 800

and MEK small molecule inhibitors are being tested at present in Ki67 phase I and phase II clinical trials. Although both RAF and MEK (also 600 400 known as MAP2K) inhibitors have excellent preclinical activity in * P = 0.009 tumour models with BRAF(V600E) mutations, their potencies in 200 0 0 4 8 12162024 BRAF wild-type (BRAF-WT) and KRAS mutant (KRAS-MT) tumour Days of treatment models unexpectedly diverge. The selective and chemically unrelated pERK RAF inhibitors GDC-0879 (refs 3, 4) and PLX4720 (ref. 5) both show specificity towards BRAF(V600E) tumour lines, unlike the MEK Figure 1 | RAF inhibitors are selective in inhibiting growth and proliferation inhibitor PD0325901 that inhibits proliferation of BRAF(V600E), of BRAF(V600E) lines. a, Half-maximum effective concentration (EC50) RAS/RAF-WT and KRAS-MT tumour lines (Fig. 1a). Unexpectedly, values (mM) in cellular proliferation assays of RAF inhibitors (inh.) (GDC- RAF inhibitors cause an increase in viable cell numbers (marked with 0879 and PLX4720) and MEK inhibitor (PD0325901) against a panel of an asterisk in Fig. 1a; see also Supplementary Fig. 2) in a subset of BRAF(V600E), RAS/RAF-WT and RAS-MT cell lines. Cell lines with BRAF-WT tumour cell lines (,50% of tested). Furthermore, although hyperproliferation are noted with asterisks. b, Tumour volume GDC-0879 is able to inhibit growth of BRAF(V600E) tumour xeno- measurements of MEXF514 (BRAF(V600E)), LXFA983 (KRAS-MT) and LXFA1041 (KRAS-MT) xenograft tumours treated with vehicle or grafts in vivo, it can increase the growth rate of KRAS-MT lung xeno- 100 mg kg21 GDC-0879 once a day. Error bars represent s.e.m. with n 5 10. grafts (Fig. 1b). In addition, histopathological examination of mice P values were obtained by Dunnett’s t-test. c, Ki67 (top) and phospho-ERK treated with GDC-0879 showed hyperkeratosis and acanthosis of (pERK, bottom) staining of skin sections from mice treated with vehicle or the epidermis, as well as inflammation in the dermis, which was not 200 mg kg21 GDC-0879 once per day for 21 days. All scale bars, 100 mm.

1Genentech, South San Francisco, California 94080, USA. 2Array BioPharma, Boulder, Colorado 80301, USA. 431 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 cutaneous squamous cell carcinoma and keratoacanthoma for two and low activity BRAF mutants can cause CRAF activation through selective RAF inhibitors7–9. heterodimerization11,14, we tested whether BRAF is required for CRAF RAF inhibitor treatment of tumour cell lines in vitro resulted in the activation and MEK phosphorylation. Treatment of isogenic induction of phospho-MEK and phospho-ERK levels in the RAS/RAF- BRAF1/1 and BRAF2/2 cell lines15,16 with RAF inhibitors resulted in WT (MeWo) and KRAS-MT (H2122) lines, versus their sustained a similar induction of CRAF specific activity as well as phospho-MEK inhibition in BRAF(V600E) (A375) cells (Fig. 2a). Knockdown of and phospho-ERK levels, demonstrating that BRAF is not essential CRAF (also known as RAF1), but not BRAF,inHCT116(KRAS- (Supplementary Figs 7 and 8). Notably, in addition to heterodimers, MT) cells10 was able to reverse the phospho-MEK induction observed RAF inhibitors induced CRAF homodimers (Supplementary Fig. 7c), after RAF inhibitor treatment, indicating that CRAF has the major providing a further mechanism for priming of CRAF activity17 when role in signalling to MEK (Fig. 2b). Notably, both BRAF and CRAF other isoforms are not expressed or heterodimer formation is kinase activities increased in a dose-dependent manner after GDC- impaired, as is the case for PLX4720 treatment. Kinase activation of 0879 treatment, selectively in non-BRAF(V600E) lines (Fig. 2c). The CRAF(T421N), a gatekeeper mutant that does not bind either GDC- PLX4720 RAF inhibitor showed more modest effects, inducing 0879 or PLX4720, was markedly impaired and phospho-MEK induc- moderate CRAF activity at a high concentration. Activation of ARAF tion after RAF inhibitor treatment in CRAF(T421N) transfectants was was also observed selectively after GDC-0879 treatment, and dual similar to untransfected controls, demonstrating that inhibitor bind- ARAF and CRAF knockdown was synergistic in decreasing inhibitor- ing to the CRAF nucleotide-binding pocket is required for effective induced phospho-MEK levels in HCT116 (KRAS-MT) cells (Sup- CRAF activation and downstream signalling (Fig. 2d, e and Sup- plementary Fig. 5). plementary Fig. 9b, c). RAF activation proceeds through the formation of homo- and To address the question of whether CRAF kinase activity was heterodimers11–13. The RAF inhibitor GDC-0879 induced B–C and required for phospho-MEK induction by RAF inhibitors, we charac- A–B heterodimers together with the induction of A-, B- and CRAF terized the chemically unrelated ATP-competitive RAF inhibitor AZ- kinase activities (Fig. 2c and Supplementary Figs 5 and 6). Because 628 (ref. 18), which binds to the inactive conformation of the RAF RAF heterodimers are expected to have higher specific kinase activity12 kinase active site motif Asp-Phe-Gly (DFG-out conformation), has

ab1 h 24 h Figure 2 | RAF inhibitors activate the MAPK GDC-0879 PLX4720 GDC-0879 PLX4720 –shBRAF +shBRAF –shBRAF +shBRAF pathway in non-BRAF(V600E) cells in a CRAF- GDC-0879 GDC-0879 PLX4720 PLX4720 dependent manner. a DMSO

DMSO , Immunoblot of lysates DMSO DMSO pMEK DMSO DMSO from A375, MeWo and H2122 cells treated with BRAF tMEK 0.1, 1 and 10 mM GDC-0879 or PLX4720. pMEK CRAF (and pERK) refer to phospho-MEK (and -ERK) A375 pERK levels, whereas tMEK (and tERK) refer to total

BRAF(V600E) pMEK tERK MEK (and ERK) levels. b, Immunoblot of lysates tMEK from HCT116 (KRAS-MT) cells carrying pMEK doxycycline (dox)-inducible short hairpin RNA tMEK –shCRAF +shCRAF –shCRAF +shCRAF (shRNA) directed against BRAF or CRAF. Cells GDC-0879 GDC-0879 PLX4720 PLX4720

MeWo pERK were treated with 0.1, 1 and 10 mM inhibitors for 2 RAF/RAS-WT tERK DMSO DMSO DMSO DMSO 1 h in the absence ( shRNA) or presence BRAF (1shRNA) of dox. c, CRAF and BRAF immunoprecipitation kinase assays from lysates pMEK CRAF of A375, MeWo and H2122 cells treated with tMEK pMEK inhibitor for 1 h. Error bars represent s.d. with pERK H2122 n 5 3; *P , 0.001 using a Student’s t-test.

KRAS-MT tMEK tERK d, e, HCT116 cells transiently transfected with control vector, Venus–CRAF-WT or c A375 MeWo H2122 Venus–CRAF(T421N) were treated with 1 and BRAF(V600E) RAF/RAS-WT KRAS-MT 10 mM GDC-0879 for 1 h. d, Venus-tagged CRAF 120 120 * * 120 GDC-0879 PLX4720 immunoprecipitation kinase assay (data 100 100 100 * * 80 80 80 normalized to DMSO control with error bars e 60 60 * 60 representing s.d. of n 5 3). , Immunoblot of

CRAF *

% pMEK 40 40 * 40 * lysates detecting endogenous CRAF, 20 20 20 Venus–CRAF and phospho-MEK. Data are 0 0 0 representative of at least two independent [Inhibitor] (µM): 0.1 1 10 0.1 1 10 0.1 1 10 experiments. 160 * 160 160 * 140 140 * 140 * 120 120 * 120 100 100 100 80 80 * 80 BRAF 60 60 60 % pMEK 40 40 40 20 20 20 0 0 0 [Inhibitor] (µM): 0.1 1 10 0.1 1 10 0.1 1 10

d Venus–CRAF e Venus–CRAF 12 Vector CRAF-WT CRAF(T421N) 10 GDC-0879 GDC-0879 GDC-0879 8 DMSO DMSO 6 DMSO activity 4 Venus–CRAF 2 CRAF Relative CRAF kinase 0 pMEK 110 110 110 tMEK

DMSO GDC-0879DMSO GDC-0879DMSO GDC-0879 Vector CRAF-WT CRAF(T421N)

432 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS high potency against CRAF and a very slow/irreversible off-rate membrane and cytosolic phospho-MEK levels is significantly impaired (Supplementary Figs 10 and 12). We proposed that, when primed (Supplementary Fig. 11b). Thus, as a consequence of inhibitor binding, CRAF is blocked through prolonged binding of a potent RAF inhibitor CRAF gets activated in a RAS–GTP-dependent manner, through to its active site, there should be no MEK phosphorylation if CRAF membrane translocation, Ser 338 phosphorylation and dimeriza- kinase activity is required. Indeed, CRAF complexes purified from tion20–22. The important role of RAS–GTP is further underscored by AZ-628-treated cells show no in vitro kinase activity, and AZ-628- the fact that transfection of BRAF(V600E) A375 cells with activated treated cells show no phospho-MEK induction or hyperproliferation mutant KRAS(G12D), but not wild-type KRAS, leads to CRAF–BRAF (Supplementary Figs 2 and 10d–f). In comparison, both GDC-0879 heterodimerization and CRAF kinase activation after inhibitor treat- and PLX4720 show very rapid off-rates and cannot persistently occupy ment (Supplementary Fig. 11d). the ATP-binding pocket to effectively block CRAF kinase activity and The conformation of the ATP-binding pocket is crucial for kinase phospho-MEK induction (Supplementary Fig. 10). This series of maturation and has an important role in the ability of small molecules experiments suggests that CRAF kinase activity is required for both to prime and induce the translocation of their target kinases to the phospho-MEK induction and cellular hyper-proliferation. membrane23–25. Membrane targeting of CRAF requires inhibitor bind- To gain a better understanding of the RAF activation sequence in ing to the ATP-binding pocket, because the gatekeeper threonine response to inhibitor priming, we characterized the topology of the mutant CRAF(T421N) (which does not bind to GDC-0879 and events and the role of RAS–GTP. Fractionation experiments demon- weakly to AZ-628; Supplementary Fig. 9a) remains in the cytoplasm strated that all RAF inhibitors tested, except PLX4720, induced a after inhibitor treatment (Fig. 3c). Furthermore, membrane targeting dose-dependent translocation to the membrane fraction of BRAF of CRAF does not seem to require kinase activity, as CRAF(D486A), a and CRAF, accompanied by increased CRAF(S338) phosphoryla- DFG-loop aspartate mutant devoid of kinase activity, is still able to tion19, selectively in non-BRAF(V600E) lines (Fig. 3a and Sup- translocate to the membrane (Fig. 3c) as long as it can bind the inhibi- plementary Fig. 11). Membrane targeting was RAS–GTP dependent, tor, as is the case for AZ-628 but not GDC-0879 (Supplementary because expression of dominant-negative KRAS(S17N) led to cyto- Fig. 12a). Thus, membrane targeting is not due to CRAF kinase inhibi- plasmic retention of CRAF after inhibitor treatment, versus the tion and does not proceed through an extrinsic mechanism, such as membrane localization observed with KRAS-WT (Fig. 3b). RAF inhi- feedback, but instead relies on inhibitor occupancy of the ATP-bind- bitors induce membrane and/or cytosolic phospho-MEK levels, ing pocket. together with their effects on CRAF localization, with the exception To test further whether priming was mediated by the inhibitors’ of AZ-628 as previously discussed (Fig. 3a and Supplementary Figs 10 conformational effects on the RAF kinase domain, we assayed BRAF– and 11). After KRAS(S17N) transfection, the induction of both CRAF heterodimerization biochemically. The CRAF kinase domain

a MeWo RAF/RAS-WT Membrane fraction Cytosolic fraction GDC-0879 PLX4720 AZ-628 GDC-0879 PLX4720 AZ-628 DMSO DMSO

BRAF

CRAF

pCRAF(S338)

pMEK1/2

tMEK1/2

b +KRAS-WT +KRAS(S17N) c DMSO GDC-0879 AZ-628 DMSO GDC-0879 AZ-628 DMSO GDC-0879 AZ-628 Venus–CRAF CFP–KRAS Overlay Overlay

20 µm CRAF(D486A)20 CRAF(T421N) µm CRAF-WT

Figure 3 | RAF inhibitors induce RAS-dependent translocation of wild-type treated with 10 mM GDC-0879 or AZ-628 for 4 h. Membrane translocation is RAF to the plasma membrane in non-BRAF(V600E) cells. a, Membrane blocked when the dominant-negative CFP-tagged KRAS(S17N) is (P100) and cytosolic (S100) fractionation of MeWo cells treated with transfected instead of KRAS-WT. c, Live cell imaging of Venus-tagged inhibitors at 0.1, 1 and 10 mM for 1 h and immunoblotted with the indicated CRAF-WT, CRAF(T421N) and CRAF(D486A) co-transfected with antibodies. pMEK1/2 refers to phospho- and tMEK1/2 refers to total MEK1/ CFP–KRAS in HEK293T cells. Cells were treated with 10 mM GDC-0879 or 2 levels, respectively. b, HEK293T cells were transiently transfected with AZ-628 for 4 h. Images are representative of three independent experiments Venus–CRAF (green), CFP–KRAS (red) and mCherry–H2B (blue). Venus- performed in triplicate. tagged CRAF co-localizes with CFP–KRAS on the plasma membrane in cells 433 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 forms a stable complex with the BRAF kinase domain in the absence GDC-0879 and AZ-628 when bound to BRAF, PLX4720 induces a of any inhibitor in an immunoprecipitation and western blot assay shift in the aC-helix in BRAF (cyan), bringing it closer to the dimer using purified recombinant proteins (Fig. 4a). Notably, both a interface, which may affect priming (Fig. 4c). non-hydrolysable analogue of ATP and PLX4720 destabilize, whereas Finally, to demonstrate formally that modulation of kinase AZ-628 and GDC-0879 significantly stabilize, the CRAF–BRAF domain dimerization by ATP-competitive RAF inhibitors underlies heterodimer kinase domain interaction (Fig. 4a). This result is con- their ability to activate CRAF and downstream signalling to MEK, we sistent with our findings from the cellular immunoprecipitation and generated CRAF and BRAF dimer interface mutants that either con- western blot experiments in non-BRAF(V600E) cell lines (Sup- stitutively heterodimerize (CRAF(E478K) and BRAF(E586K)) or are plementary Figs 6 and 10), and supports the model that dimerization defective in dimerization (CRAF(R401H) and BRAF(R509H))26. Co- is mediated directly through the inhibitors’ effects on the RAF kinase transfection of CRAF(E478K) and BRAF(E586K) in HCT116 KRAS- domain, rather than being a secondary effect. The inhibitors had no MT cells resulted in a basal increase of exogenous CRAF kinase activity effect on the basal interaction between the CRAF and BRAF(V600E) compared to wild-type controls (Fig. 4d). No further induction was kinase domains (Fig. 4a), in agreement with the cellular data demon- observed after RAF inhibitor treatment, confirming that dimerization strating lack of priming in BRAF(V600E) mutant lines (Fig. 2 and is the main driver for the inhibitors’ effects on CRAF kinase activity. Supplementary Fig. 6). In summary, our biochemical and cellular Constitutive heterodimerization by these mutants was confirmed data suggests that inhibitor occupancy of the ATP-binding pocket (Fig. 4d), suggesting that the interactions characterized by our assays can effect RAF dimerization and that the inhibitors’ effects are dif- were mediated at the level of kinase domain dimer interface. In con- ferent from ATP itself. trast, co-transfection of CRAF(R401H) and BRAF(R509H) lead to To probe further the structural basis of the inhibitors’ effects on decreased levels of basal CRAF activity and a significant impairment CRAF we solved the crystal structure of the CRAF kinase domain of CRAF activation by RAF inhibitors, consistent with the mutants’ complexed to a close analogue of GDC-0879 (ref. 4) (Supplemen- defective heterodimerization properties (Fig. 4d). Phospho-MEK tary Fig. 13 and Supplementary Table 1). The CRAF–inhibitor com- level induction by RAF inhibitors correlated with the efficiency of plex adopts a dimer conformation in the asymmetric unit, as previ- heterodimer formation and CRAF activation in all cases (Supplemen- ously described for BRAF26. Comparison of the CRAF and BRAF tary Fig. 15). homodimers shows an extremely well-conserved interface, the single In conclusion, we have discovered that ATP-competitive RAF inhi- difference among them residing in the most amino-terminal residue bitors have opposing roles as inhibitors and activators of the RAF– of CRAF resolved in the crystal structure, CRAF(Y340), within its MEK–ERK pathway, and that the outcome is dependent on cellular negative-charged regulatory region27 (Fig. 4b and Supplementary context and genotype. In BRAF(V600E) cells, the pathway is sensitized Table 2). Further amino acid residues that differ between BRAF and to ATP-competitive RAF inhibitors owing to the higher ATP apparent CRAF are highlighted in Fig. 4b. A heterodimer model of BRAF– Km (Km(app)) of the mutant BRAF(V600E) protein (Supplemen- CRAF illustrates that, in contrast to the conformation induced by tary Fig. 4) and lack of CRAF activation (Supplementary Fig. 1). In

a CRAF-KD–BRAF-KD heterodimer CRAF-KD–BRAF(V600E)-KD heterodimer Figure 4 | Modulation of kinase domain dimerization by ATP-competitive RAF inhibitors underlies their ability to activate CRAF. a, Biochemical dimerization assays DMSO DMSO AMP-PCPGDC-0879PLX4720AZ-628 DMSO DMSO AMP-PCPGDC-0879PLX4720AZ-628 (immunoprecipitation (IP) and western blot) CRAF + + + + + + CRAF + + + + + + BRAF – + + + + + BRAF – + + + + + using recombinant RAF proteins in the presence of DMSO, 10 mM inhibitors or 1 mM AMP-PCP. CRAF CRAF Bands were quantified and BRAF levels were normalized to captured GST–CRAF. KD, kinase BRAF BRAF domain. b, CRAF crystal structure with a GDC-

IP: GST–CRAF 1.0 0.2 2.7 0.2 2.0 IP: GST–CRAF 1.0 1.4 1.3 1.3 1.5 0879 analogue4 reveals a side-to-side dimer. Non- αC-helix conserved residues between BRAF and CRAF are b c shift coloured in violet. Conserved residues in the surface depiction are shown in green; residues in the dimer interface are shown in lime green. c, BRAF–CRAF heterodimer model derived from CRAF–GDC-0879 analogue (green) and BRAF–GDC-0879 analogue (yellow) or BRAF–PLX4720 (cyan). d, HCT116 cells were transfected with the indicated wild-type and dimerization mutant RAF constructs for 48 h and treated with 0.1, 1 and 10 mM GDC-0879 for 1 h. CRAF BRAF Immunoprecipitation and western blot (WB) for Venus–CRAF and associated BRAF (top) and d Venus–CRAF-WT Venus–CRAF(E478K) Venus–CRAF(R401H) wild-type or mutant Venus–CRAF Vector control mCherry–BRAF-WT mCherry–BRAF(E586K) mCherry–BRAF(R509H) immunoprecipitation kinase assay (bottom). GDC-0879 GDC-0879 GDC-0879 GDC-0879 Error bars represent s.d. of n 5 3. Data are representative of two independent experiments. WB DMSO DMSO DMSO DMSO Venus–CRAF – mCherry–BRAF –

IP: GFP BRAF –

(Venus–CRAF) 3

Relative 1

CRAF kinase activity 0

BRAF-WT cells, inhibitors trigger the CRAF activation sequence, 7. Flaherty, K. et al. Phase I study of PLX4032: proof of concept for V600E BRAF which can lead to MEK/ERK phosphorylation and in some cases to mutation as a therapeutic target in human cancer. J. Clin. Oncol. 27, abstract 9000 (2009). enhanced growth (Supplementary Fig. 1). This suggests that careful 8. Schwartz, G. K. et al. A phase I study of XL281, a selective oral RAF kinase inhibitor, assessment should be made of the functional consequences of other in patients (Pts) with advanced solid tumors. J. Clin. Oncol. 27, abstract 3513 ATP-competitive inhibitors with respect to priming, which may affect (2009). the inhibitors’ efficacy and safety profiles. On the basis of our findings, 9. Chapman, P. et al. Early efficacy signal demonstrated in advanced melanoma in a we propose that preventing activation of CRAF could potentially pre- phase I trial of the oncogenic BRAF-selective inhibitor PLX4032. Eur. J. Cancer, Suppl. 7, 5 (2009). vent undesirable effects of RAF inhibitors on normal tissue and non- 10. Jaiswal, B. S. et al. Combined targeting of BRAF and CRAF or BRAF and PI3K BRAF(V600E) tumours. This could be attained by an allosteric non- effector pathways is required for efficacy in NRAS mutant tumors. PLoS One 4, ATP competitive mechanism that would prevent RAF dimerization, e5717 (2009). or RAS–RAF RAS-binding domain (RBD) interaction. This data also 11. Garnett, M. J. et al. Wild-type and mutant B-RAF activate C-RAF through distinct highlights the need for BRAF(V600E) patient selection in V600E- mechanisms involving heterodimerization. Mol. Cell 20, 963–969 (2005). 12. Rushworth, L. K., Hindley, A. D., O’Neill, E. & Kolch, W. Regulation and role of Raf- selective RAF inhibitor clinical trials. 1/B-Raf heterodimerization. Mol. Cell. Biol. 26, 2262–2272 (2006). 13. Weber, C. K., Slupsky, J. R., Kalmes, H. A. & Rapp, U. R. Active Ras induces METHODS SUMMARY heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001). Chemicals and cell culture. PLX4720 and PD0325901 were purchased from 14. Wan, P. T. et al. Mechanism of activation of the RAF-ERK signaling pathway by Symansis, and EMD from CalBiochem. AZ-628 was synthesized according to oncogenic mutations of B-RAF. Cell 116, 855–867 (2004). the procedure described in patent number WO2006024834 (http://www.wipo. 15. Kim, J. S., Lee, C., Foxworth, A. & Waldman, T. B-Raf is dispensable for K-Ras- int/pctdb/en/wo.jsp?wo52006024834). mediated oncogenesis in human cancer cells. Cancer Res. 64, 1932–1937 (2004). Fluorescence microscopy. For CRAF sub-cellular localization experiments, 16. Yun, J. et al. Glucose deprivation contributes to the development of KRAS pathway HEK293T cells were transiently transfected with relevant cyan fluorescent mutations in tumor cells. Science 325, 1555–1559 (2009). 17. Zang, M. et al. Characterization of Ser338 phosphorylation for Raf-1 activation. J. protein (CFP)–KRAS and Venus–CRAF constructs and TagBFP or mCherry– Biol. Chem. 283, 31429–31437 (2008). H2B to identify nuclei. Compounds were added 20 h after transfection and live 18. Khazak, V., Astsaturov, I., Serebriiskii, I. G. & Golemis, E. A. Selective Raf inhibition cells were imaged 24 h after transfection. in cancer therapy. Expert Opin. Ther. Targets 11, 1587–1609 (2007). Tumour xenograft studies and immunohistochemistry. Tumour fragments 19. Goetz, C. A., O’Neil, J. J. & Farrar, M. A. Membrane localization, oligomerization, were obtained from MEXF514, LXFA983 and LXFA1041 xenografts. In vivo and phosphorylation are required for optimal raf activation. J. Biol. Chem. 278, studies were conducted at Oncotest. Immunohistochemistry were conducted 51184–51189 (2003). as described previously3. 20. Xiang, X. et al. Phosphorylation of 338SSYY341 regulates specific interaction In vitro kinase assays. Cell lysates were immunoprecipitated with anti-CRAF, between Raf-1 and MEK1. J. Biol. Chem. 277, 44996–45003 (2002). anti-BRAF or anti-GFP antibody (Millipore) and then incubated with 0.4 mg 21. Terai, K. & Matsuda, M. Ras binding opens c-Raf to expose the docking site for unactive MEK1 (Millipore) in 40 ml of kinase buffer (20 mM MOPS, pH 7.2, mitogen-activated protein kinase kinase. EMBO Rep. 6, 251–255 (2005). 25 mM b-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 22. Jelinek, T. et al. RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2. Mol. Cell. Biol. 14, 8212–8218 (1994). 1 mM dithiothreitol (DTT), 120 mM ATP, 18 mM MgCl ) for 30 min at 30 uC. 2 23. Cameron, A. J. et al. PKC maturation is promoted by nucleotide pocket occupation Samples were then loaded onto the Meso Scale Discovery Phospho (Ser 217/ independently of intrinsic kinase activity. Nature Struct. Mol. Biol. 16, 624–630 221)/Total MEK1/2 assay plates and quantified. (2009). Expression and crystallization of CRAF. The dual expression baculovirus con- 24. Okuzumi, T. et al. Inhibitor hijacking of Akt activation. Nature Chem. Biol. 5, struct pBac4x-1-HTH-CRAF(324-618):CDC37 was expressed in Hi5 cells. 484–493 (2009). CRAF was co-crystallized with inhibitor in hanging drops (12% PEG 8K, 25. Hindie, V. et al. Structure and allosteric effects of low-molecular-weight activators 100 mM Tris, pH 8.0, 10% tacsimate, grown at 12 uC). on the protein kinase PDK1. Nature Chem. Biol. 5, 758–764 (2009). RAF dimerization. His–BRAF-WT kinase domain or His–BRAF(V600E) kinase 26. Rajakulendran, T. et al. A dimerization-dependent mechanism drives RAF domain and glutathione S-transferase (GST)–CRAF kinase domain (Invitrogen) catalytic activation. Nature 461, 542–545 (2009). were incubated in the presence of a fixed concentration of compound or 27. Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. dimethylsulphoxide (DMSO) for 1 h and immunoprecipitated with a rabbit Nature Rev. Mol. Cell Biol. 5, 875–885 (2004). anti-GST antibody. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Acknowledgements We would like to thank the chemists at Genentech and Array BioPharma for providing GDC-0879. We thank Oncotest GmbH, L. Murray, Received 29 September 2009; accepted 18 January 2010. S. Gould, B. Alicke, W. Voegtli, K. Bock, H. Stern and the immunohistochemistry Published online 3 February 2010. core laboratory for technical support. We thank B. Vogelstein for HCT116 isogenic BRAF1/1 and BRAF2/2 cell lines, and T. Waldman for the HEC1A BRAF1/1 and 1. Schubbert, S., Shannon, K. & Bollag, G. Hyperactive Ras in developmental BRAF2/2 cell lines. Special thanks goes to A. Bruce for help with artwork. disorders and cancer. Nature Rev. Cancer 7, 295–308 (2007). 2. Yeh, J. J. et al. KRAS/BRAF mutation status and ERK1/2 activation as biomarkers Author Contributions G.H. and S.M. designed the studies, interpreted the data and for MEK1/2 inhibitor therapy in colorectal cancer. Mol. Cancer Ther. 8, 834–843 wrote the manuscript. K.S., I.Y., B.L., S.S. and D.S. conducted cellular experiments (2009). and dimerization assays. D.J.A., M.J.C.L. and R.A. conducted microscopy 3. Hoeflich, K. P. et al. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is experiments. B.J.B., G.V., T.M. and I.A. conducted crystallography and provided V600E predicted by BRAF mutational status and sustained extracellular signal- structural input. S.L.G. conducted enzymology. K.P.H. and H.K. conducted in vivo regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer experiments and immunohistochemistry. B.S.J. and S.S. generated inducible Res. 69, 3042–3051 (2009). shRNA cell lines. M.B. and L.S.F. interpreted the data and wrote the manuscript. 4. Hansen, J. D. et al. Potent and selective pyrazole-based inhibitors of B-Raf kinase. Bioorg. Med. Chem. Lett. 18, 4692–4695 (2008). Author Information Atomic and structural data are deposited in Protein Data Bank 5. Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with under accession number 3LB7. Reprints and permissions information is available at potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008). www.nature.com/reprints. The authors declare competing financial interests: 6. Malek, S. Selective inhibition of Raf results in downregulation of the RAS/RAF/ details accompany the full-text HTML version of the paper at www.nature.com/ MEK/ERK pathway and inhibition of tumor growth in vivo. Eur. J. Cancer, Suppl. 4, nature. Correspondence and requests for materials should be addressed to G.H. 184 (2006). ([email protected]) or S.M. ([email protected]).

METHODS In vitro kinase assays. Cells were plated and lysed as described in immunoblot- Chemicals and cell culture. PLX4720 and PD0325901 were purchased from ting method. Cell lysates were incubated with anti-CRAF, anti-BRAF, or anti- Symansis, and EMD from CalBiochem. AZ-628 was synthesized according to GFP antibody (Millipore) and 50 ml of Protein A agarose beads (Millipore) for the procedure described in patent number WO2006024834 (http://www.wipo. 2 h at 4 uC. After washing with lysis buffer plus protease and phosphatase inhibi- int/pctdb/en/wo.jsp?wo52006024834). All cells lines were obtained from the tor mixtures, protein A beads were incubated with 0.4 mg of unactive MEK1 American Type Culture Collection (ATCC) except for the isogenic pairs of cell (Millipore) in 40 ml of kinase buffer (20 mM MOPS, pH 7.2, 25 mM b-glycerol lines. Hec1a BRAF1/1 and Hec1a BRAF2/2 were obtained from T. Waldman’s phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 120 mM laboratory. HCT116 BRAF1/1 and HCT116 BRAF2/2 were obtained from ATP, 18 mM MgCl2) for 30 min at 30 uC. Samples were then loaded onto the Horizon Discovery. Meso Scale Discovery Phospho (Ser 217/221)/Total MEK1/2 assay plates and Enzymology assays. Full-length 63His-tagged human BRAF(V600E) co- quantified. expressed with human CDC37 (1–378), full-length 63His tagged human Expression and purification of CRAF for crystallization. The dual expression BRAF-WT, and full-length Flag-tagged human wild-type CRAF were expressed baculovirus construct pBac4x-1-HTH-CRAF(324-618):CDC37 was expressed in in baculovirus-infected insect cells and purified using standard affinity tag chro- Hi5 cells. Baculovirus pellets were homogenized in 25 mM Tris, pH 7.5, 0.5 M matographic methods. The enzymatic activity of was quantified by measuring NaCl, 10% glycerol, 10 mM imidazole, 10 mM b-mercaptoethanol, 0.035% Brij- the incorporation of radiolabel from [c-33P]ATP into full-length 63His-tagged 35 COMPLETE EDTA-free (Roche) and solubilized with a PANDA homogenizer. human MEK covalently modified with 59-p-fluorosulphonylbenzoyladenosine After centrifugation, the soluble fraction was mixed with Talon (Clontech) resin (FSBA). Standard enzyme reaction conditions have been described previously28. for 16 h at 4 uC. The protein was eluted with an imidazole-containing buffer and ATP Km(app) values were determined by monitoring the enzymatic activity over applied to a Q Sepharose column, followed by a Mono S column and then loaded 120 min at a constant amount of [c-33P]ATP (33 mCi ml21), while varying ATP. onto a Superdex 75 10/30 equilibrated with 25 mM HEPES, pH 7.0, 0.3 M NaCl, ATP Km(app) values were determined by fitting a hyperbolic model tothe enzymatic 15% glycerol, 0.25% CHAPS, 1 mM EDTA, 1 mM TCEP buffer. The monomeric 21 rate versus [ATP] data. Half-maximum inhibitory concentration (IC50)values CRAF pool was concentrated to 2.3 mg ml for crystallization trials. were calculated by fitting a standard four-parameter logistic model to the dose Crystallization and structure solution of CRAF. CRAF was co-crystallized with response curve plotted as the per cent of control versus concentration of com- Inhibitor 18 (ref. 4) in hanging drops (12% PEG 8K, 100 mM Tris, pH 8.0, 10% pound. The apparent inhibition constant (Ki(app)) and predicted IC50 values at tacsimate, grown at 12 uC). Crystals were cryoprotected in 10% PEG 8000, 10% 1 mM ATP were calculated using the Cheng–Prusoff relationship29 assuming com- tacsimate, 0.1 M Tris, pH 8.0, 25% glycerol and flash-frozen on an Oxford pounds are competitive with ATP. For off-rate experiments, BRAF(V600E) Cryosystems coldstage. The crystal was frozen at 100 K and data were collected protein was pre-incubated with 100-fold excess of enzyme and compound and in-house on an FR-E generator and Raxis-IV11 detector (Rigaku). The crystals the mixture was diluted 100-fold at the initiation of the kinase activity assay for diffracted to 4.0 A˚ . Diffraction data were processed with the CCP4 suite33. BRAF(V600E). The inhibitor off-rates were calculated from measuring the rate of Molecular replacement searches were carried out with Molrep, using a previous kinase activity rebound of the inhibitor-preincubated BRAF(V600E) compared to structure of BRAF co-crystallized with Inhibitor 18 (Protein Data Bank (PDB) non-preincubated control. accession code 3D4Q). Molrep successfully found solutions for two monomers, Fluorescent protein-fusion constructs. KRAS, BRAF, CRAF and H2B were in space group P41212. This gave a fractional occupancy 0.498 and no other amino-terminally tagged with fluorescent proteins using multisite Gateway monomers could be found. Because the crystal only diffracted to 4.0 A˚ , further cloning (Invitrogen). Relevant point mutations were introduced using site- precautions were taken during refinement in Refmac: non-crystallographic sym- directed mutagenesis. Each fusion was inserted into the CMV-driven pcDNA- metry (NCS) restraints were enforced on all residues of both chains and a low DEST vector, in which the carboxy-terminal GFP tag was not translated owing to X-ray weighting term of 0.0025 was used. Also, all atoms were given a fixed a stop codon following the inserted genes. Cells were transfected with isotropic B-factor or 68.0. No water atoms were added to the structure and, Lipofectamine2000 (Invitrogen), according to the manufacturer’s instructions. although there was weak density visible for the ligand, it was not modelled in. Microscopy. For CRAF sub-cellular localization experiments, HEK293T cells The final structure had reasonably good geometry (see Supplementary Table 1), were transiently transfected with relevant CFP-KRAS and Venus-CRAF con- and in the Ramachandran plot of the final structure only 8 out of 468 residues structs and TagBFP or mCherry-H2B (Clontech)30,31 to identify nuclei. (1.7%) are in disallowed regions. Compounds were added 20 h after transfection and live cells were imaged 24 h Immunoprecipitation and western blots for dimerization. In assay buffer after transfection using an SP5 point-scanning confocal microscope with a 340 (25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% (v/v) Triton X-100, 2 mM DTT, 1.25 numerical aperture (NA) HCX PL APO CS oil-immersion objective (Leica 0.25% DMSO), 500 nM of enzyme: Flag–CRAF full-length, His–BRAF-WT or Microsystems). B-RAF(V600E) kinase domain, or GST–CRAF kinase domain (Invitrogen) in the Tumour xenograft studies and immunohistochemistry. Tumour fragments presence of a fixed concentration of compound or vehicle was incubated for 1 h at obtained from MEXF514, LXFA983 and LXFA1041 xenografts (conducted at 3 room temperature. AMP-PCP was purchased from Sigma. Proteins were immu- Oncotest) were cut into 1–2-mm pieces and implanted subcutaneously into noprecipitated with rabbit anti-Flag (Sigma, F7425) or rabbit anti-GST (Cell NMRI nu/nu mice (Taconic). GDC-0879 compound was prepared fresh weekly Signaling Technology, 2622) antibodies bound to protein A agarose beads in 0.5% methylcellulose and 0.2% Tween-80 in water, stored at 4 uC, and adminis- (Millipore). Each sample was separated by SDS–PAGE and transferred to nitro- tered daily by oral gavage. Tumours were staged and measured as described previ- cellulose membranes. Membranes were incubated with anti-CRAF (BD 3 ously . Xenograft tissues were fixed for 24 h in 10% neutral buffered formalin and Biosciences, 610152) or anti-BRAF (Sigma, WH0000673M1) primary antibodies, were then processed and paraffin embedded. Immunohistochemistry was con- 3 and analysed by the addition of a secondary Alexa Fluor 488 goat anti-mouse IgG ducted as described previously . (H1L) antibody (Invitrogen Molecular Probes, A11029). Images were acquired Cell viability assays. Cell viability assays were carried out as described previ- 3 on the TyphoonTM Scanner (Amersham Bioscience), and quantified using ously . In brief, cells were seeded at 2,000 per well and treated with compound on ImageQuant TL v2005 Software. day 2. The relative numbers of viable cells were measured by luminescence using CellTiter-Glo (Promega). 28. Wallace, E. M. et al. Potent and selective mitogen-activated protein kinase kinase Membrane fractionation. Cells were lysed in buffer A (25 mM Tris, pH 7.5, 1 mM (MEK) 1,2 inhibitors. 1. 4-(4-bromo-2-fluorophenylamino)-1- methylpyridin- EDTA, 1 mM DTT, 10 mM NaCl, 25 mM NaF, protease inhibitors and phospha- 2(1H)-ones. J. Med. Chem. 49, 441–444 (2006). tase inhibitors) and mechanically disrupted using a Dounce homogenizer. 29. Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (KI) and Cytosolic (S100) and membrane (P100) fractions were separated as described the concentration of inhibitor which causes 50 per cent inhibition (I50)ofan previously32. enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973). Immunoblotting. Cells lysed in lysis buffer (0.5% NP40, 20 mM Tris, pH 7.5, 30. Lukyanov, K. A. et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 137 mM NaCl, 10% glycerol, 1 mM EDTA) plus protease inhibitor mixture- 25879–25882 (2000). complete mini (Roche Applied Science) and phosphatase inhibitor mix 31. Matz, M. V. et al. Fluorescent proteins from nonbioluminescent Anthozoa (Pierce). Ten-to-twenty micrograms of protein from each sample was separated species. Nature Biotechnol. 17, 969–973 (1999). by SDS–PAGE and transferred to nitrocellulose membranes. Membranes were 32. Stokoe, D. & McCormick, F. Activation of c-Raf-1 by Ras and Src through different incubated with the indicated primary antibodies and analysed either by the mechanisms: activation in vivo and in vitro. EMBO J. 16, 2384–2396 (1997). enhanced chemiluminescence (ECL) detection system (Amersham) or by the 33. Collaborative Computational Project, Number 4. The CCP4 suite: programs for addition of Alexa Fluor 647 goat anti-mouse (Molecular Probes Inc.). protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

Mad2-induced chromosome instability leads to lung tumour relapse after oncogene withdrawal

Rocio Sotillo1, Juan-Manuel Schvartzman1, Nicholas D. Socci2 & Robert Benezra1

Inhibition of an initiating oncogene often leads to extensive tumour under the cytomegalovirus (CMV) promoter induces lung tumours cell death, a phenomenon known as oncogene addiction1.Thishas in 35% of animals3, only 1 in 40 Mad2-overexpressing mice (TI-M) in led to the search for compounds that specifically target and inhibit a Kras wild-type background harboured a lung adenoma after 15 oncogenes as anticancer agents. However, there has been no sys- months on doxycycline (data not shown). This may be the result of tematic exploration of whether chromosomal instability generated Mad2 transgene expression being restricted to type II pneumocytes in as a result of deregulation of the mitotic checkpoint pathway2,3,a the CCSP–rtTA model compared with ubiquitous overexpression in frequent characteristic of solid tumours, has any effect on oncogene CMV–rtTA mice. Neither total nor exogenous levels of Mad2 in TI- addiction. Here we show that induction of chromosome instability KM animals were higher than in TI-M mice, making it likely that the by overexpression of the mitotic checkpoint gene Mad2 in mice increase in tumour burden is dependent on combined Kras and does not affect the regression of Kras-driven lung tumours when Mad2 overexpression (Supplementary Fig. 1a–c). Kras is inhibited. However, tumours that experience transient Mad2 overexpression and consequent chromosome instability a b c 0.8 TI-K 0.5 P = 0.006 ) recur at markedly elevated rates. The recurrent tumours are highly Mad2 P = 0.028 2 aneuploid and have varied activation of pro-proliferative pathways. –+Dox. 0.6 0.4 0.3 Thus, early chromosomal instability may be responsible for tumour Exo. 0.4 TI-KM relapse after seemingly effective anticancer treatments. Endo. 0.2 Weight (g) 0.2 Chromosomal instability (CIN) is commonly found in human solid 0.1 0 Tumour area (mm 0 tumours, and extensive studies with mouse models suggest that it is TI-K TI-KM TI-K TI-KM 4–6 sufficient to initiate tumorigenesis in a variety of cellular contexts . d TUNEL We have previously reported that ubiquitous overexpression of Mad2, H&E Ki67 which induces whole-chromosome instability and rearrangements3, is sufficient for tumour initiation in multiple tissues including lung3. TI-K To determine whether CIN imparted by Mad2 overexpression could accelerate tumour formation driven by a classical oncogene, we generated cohorts of mice carrying a type II alveolar epithelial cell- specific doxycycline-inducible (CCSP-rtTA) transactivator transgene7 in addition to rtTA-responsive Mad2 and/or murine oncogenic K-Ras4bG12D transgenes8. Resulting progeny were divided into groups TI-KM maintained on normal or doxycycline diets at weaning. We refer to these mice as TI-K, TI-M and TI-KM for ‘tetracycline inducible’ Kras, e g Mad2 and Kras 1 Mad2, respectively. Eight weeks after induction, 150 f 150 100 western blot analysis of lungs confirmed that only those mice exposed P = 0.189 P = 0.024 –2 80 to doxycycline expressed exogenous Mad2 protein, resulting in levels 100 100 60 that were double those in control animals (Fig. 1a). Endogenous levels P < 0.0001 40 of Mad2 were not upregulated on KrasG12D activation (Fig. 1a and 50 50 Survival (%)

Nodules mm 20 TI-KM Supplementary Fig. 1a). TI-K

0 Ki67-positive cells/area 0 0 Eight weeks after transgene induction, the lungs of TI-KM mice TI-K TI-KM TI-K TI-KM 0 200 400 600 were nearly twice as large as those of TI-K mice (Fig. 1b) and total Time (days) tumour area was significantly increased (Fig. 1c). However, tumour Figure 1 | Mad2 overexpression cooperates with KrasG12D in lung nodule number was not increased (Fig. 1e), indicating that tumour tumorigenesis. a, Mad2 western blot of TI-KM lungs maintained with (1) initiation is not affected by Mad2 overexpression. Adenocarcinomas or without (2) doxycycline. b, Left: lung weights from TI-K and TI-KM mice in TI-KM mice showed a more aggressive morphology, with invasion on doxycycline for eight weeks. Right: representative macroscopic pictures into the pleura and higher proliferation rates (Fig. 1d–f) but no showing lung tumour-size (dotted lines). c, Total tumour area in TI-K and significant differences in apoptosis (Fig. 1d). Moreover, a statistically TI-KM lungs. d, Haematoxylin/eosin staining of lung tissue from TI-K and TI-KM mice after eight weeks on doxycycline (left), Ki67 staining (middle) significant decrease in survival was seen in TI-KM versus TI-K mice. and TdT-mediated dUTP nick end labelling (TUNEL) (right). Scale bar, Whereas TI-K mice survived for 543 6 32 days (mean 6 s.e.m.), TI- 100 mm. e, Tumour nodules mm22 of lung tissue in TI-K and TI-KM KM mice survived for 258 6 11 days (mean 6 s.e.m.) (P , 0.0001) animals. f, Percentage of Ki67-positive cells. g, Kaplan–Meier curve of TI-K (Fig. 1g). All mice died of lung tumours, as determined by post- and TI-KM mice. Error bars represent means and s.e.m. from at least four mortem examination. Although overexpression of Mad2 by itself different mice. P values were determined with an unpaired t-test.

1Cancer Biology and Genetics Program, 2Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. 436 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

In both TI-K and TI-KM mice, lung adenocarcinomas were SP-C resonance imaging (MRI) were identified (Supplementary Table 1) positive, which is consistent with a type-II-like classification (Sup- and then switched to a regular diet. MRI performed two weeks after plementary Fig. 2a, b), and negative for Clara cell secretory protein de-induction revealed a striking decrease in tumour volume in both (CCSP)8 (Supplementary Fig. 2c, d), which was seen in normal cells TI-K and TI-KM mice (Fig. 2c and Supplementary Fig. 4). Exami- lining the airways (Supplementary Fig. 2e, f). Hence, combined lung- nation of lungs from all mice confirmed normal histology with areas specific Mad2 and Kras overexpression leads to the formation of lung of pleural and parenchymal fibrosis. Thus, CIN associated with Mad2 adenocarcinomas that are more aggressive than, although histologi- overexpression is not sufficient to prevent tumour regression after cally similar to, those seen with Kras alone. abrogation of Kras and Mad2 expression. It is likely that these We have previously reported that ubiquitous overexpression of tumours regress in response to mutant Kras withdrawal and not Mad2 leads to tumour initiation through the acquisition of a CIN Mad2, because, as stated above, de-induction of Mad2 in the phenotype3. To determine whether the CIN generated by high levels CMV-rtTA and tTA systems never leads to tumour regression by of Mad2 could be contributing to the more severe phenotype in TI- itself3. KM mice, fluorescence in situ hybridization (FISH) analysis was Next, we sought to determine whether CIN would affect the rate of performed on lung tissue, with chromosomes 12, 16 and 17 specific tumour recurrence. A total of 25 TI-K and 24 TI-KM mice main- probes. TI-K lung tumours showed higher levels of aneuploidy (13%) tained on doxycycline were monitored for tumour appearance by than normal lung (2%, not shown). However, TI-KM tumours MRI (Supplementary Table 2). When solid tumours were identified showed a threefold increase in aneuploid cells over TI-K (38% versus (between 12 and 28 weeks), doxycycline was removed from the diet 13%, P , 0.0001) (Fig. 2a, b). TI-M animals developed very low levels and the mice were periodically screened for tumour regrowth. of aneuploidy (5.7%), which is consistent with the observed lack of Whereas TI-K tumours never relapsed after de-induction of the tumour formation. Despite high rates of aneuploidy seen in TI-KM oncogene (even up to one year after withdrawal of doxycycline), primary tumours, array comparative genomic hybridization (CGH) tumours recurred in 11 out of 24 TI-KM mice killed 4–11 months analysis failed to detect any gross abnormalities (data not shown). after doxycycline withdrawal (Fig. 3a, b). These nodules were solid or This most probably reflects the absence of stable, clonally propagated well-defined proliferating papillary adenocarcinomas that were mor- events that contribute significantly to the CGH profile. phologically indistinguishable from the primary tumours (Fig. 3e). RNA microarray analysis confirmed the diversity of tumour sub- The presence of recurrences in TI-KM animals was not a result of the types in the Kras and Mad2 tumours. Whereas the majority of Kras larger primary tumour size, because even in TI-KM animals selected tumours clustered together, the TI-KM primary tumours were mark- for a small tumour volume a 50% recurrence was observed (Fig. 3c edly different from each other and from the cluster of mutant Kras and Supplementary Table 3). In fact, the average volume of primary overexpressors. These results suggest that Mad2 overexpression KM tumours that went on to recur in this selected population was induces a wide spectrum of chromosomal aberrations and expression significantly smaller than that of K and of KM primaries that never changes that are probably contributing to enhanced tumorigenesis. recurred (Fig. 3c). The overexpression of Mad2 alone led only to a The precise causative events have not been identified (Supplementary single tumour in 1 out of 40 animals after 15 months of induction, Fig. 3). making it likely that the KM relapses were dependent on having Continued expression of mutant Kras is required for tumour expressed both transgenes. maintenance8. In contrast to what is observed with classical oncogene It is, however, formally possible that these recurrences are de novo overexpression, continuous Mad2 overexpression is not required for tumours derived from cells that had expressed Kras and Mad2 but tumour maintenance, because primary tumours induced after Mad2 were not part of the initial tumour mass, a possibility that we consider overexpression in two different ubiquitous inducible model systems unlikely for the following reasons: first, no such events have been do not regress on transgene de-induction3. To determine whether the reported in other inducible mouse models of oncogene addiction9,10; CIN imparted by Mad2 overexpression affected Kras oncogene second, TI-KM animals maintained off doxycycline never develop addiction, TI-K or TI-KM mice with lesions evident by magnetic lung tumours in this model system; third, TI-KM mice that were on

a TI-M TI-K TI-KM

b c On doxycycline 18 weeks Off doxycycline 2 weeks P < 0.0001 50 P < 0.0001 T H H 40 TI-K T 30 P = 0.0013 20 T H 10 H T 0 TI-KM Frequency of aneuploidy (%) TI-M TI-K TI-KM normal tumour tumour Figure 2 | Mad2 overexpression increases aneuploidy but is not enough to aneuploidy in lung tissue from five individual mice in each group. c,MR overcome oncogene addiction. a, FISH images from TI-M normal lung and images and histological sections of lung tumours from TI-K and TI-KM mice from TI-K and TI-KM lung tumours showing aneuploid cells in yellow and after 18 weeks on doxycycline (left). After 2 weeks of doxycycline withdrawal, normal cells in white. blue, DNA; green, chromosome 12 probe; red, lung opacities disappeared from magnetic resonance images, and tumours chromosome 16 probe; orange, chromosome 17 probe. b, Frequency of regressed histologically. H, heart; T, tumour. Scale bar, 200 mm. 437 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a On 23 wk Off 6 wk Off 16 wk Off 22 wk acquired resistance to targeted therapies in other inducible mouse models12 and in human cancer13–15. In parallel to what was seen with the primary tumours, recurrent

TI-K tumours in TI-KM mice were highly aneuploid (39.4%). Lung tissue from TI-K animals after tumour regression showed very low levels of 3 0.047 cm aneuploidy (2.7%), similar to that in wild-type lung, suggesting that this baseline aneuploid level is insufficient to drive tumour recurrence. In contrast, elevated rates of aneuploidy were observed both in the TI- KM primary tumours (38%) and TI-KM lungs after regression (12.9%) before tumour relapse. This suggests that high levels of 3 3 0.0326 cm 0.0130 cm aneuploidy in the primary tumour and in cells remaining after de- On 28 wk Off 2 wk Off 14 wk Off 22 wk TI-KM induction of Kras and Mad2 enable survival after oncogene withdrawal, thereby facilitating relapse (Fig. 4a, b). KrasG12D lung tumours show positive staining for pErk, pStat3, 0.147 cm3 0.0121 cm3 pAkt and pS6, indicating that the mitogen-activated protein kinase b c e (MAPK), STAT3 and phosphoinositide 3-kinase pathways are all

) 16 100 3 0.5 TI-K SP-C stimulated by oncogenic Kras (Supplementary Fig. 7). Only five 80 0.4 TI-KM TI-KM R out of ten KM recurrences retained MAPK pathway activation, and 60 0.3 four out of ten showed low-level pStat3 activity (Fig. 4c and Sup- (%) 40 0.2 plementary Fig. 8). Ten out of ten recurrences scored positive for 20 TI-K 0.1 TI-KM AKT, though the different levels and patterns seen by immunohisto-

Tumour-free survival 0 0.0 Tumour volume (cm Ki67 0 102030405060 10 15 20 25 30 chemistry make this unlikely to be the sole determinant of recurrence. Time to recur (wk) Time on doxycycline (wk) For further characterization of the signalling pathways implicated in d On On/off tumour relapse, RNA microarray analysis was performed in six TI- KM recurrent tumours. Consistent (Supplementary Fig. 9) with the

TI-K TI-KM immunohistochemistry analysis, recurrent tumours were very hetero- Relapse 1 Relapse 2 Relapse 3 Normal 3 Relapse 4 Relapse 5 Relapse 6 Relapse 7 Relapse 8 Relapse 9 TI-K TI-KM HA geneous, different from each other and from the K or KM primary Mad2 tumours. Thus, recurrent tumours use alternative signalling pathways Kras to escape oncogene addiction, a result consistent with the notion that

Actin Mad2-mediated CIN in the primary tumour provides extensive diversity and therefore an evolutionary advantage to oncogene-driven Figure 3 | Chromosomal instability increases the likelihood of recurrence in solid tumours. Kras tumours. a, MRI from TI-K and TI-KM mice on doxycycline showing The role of aneuploidy in the initiation of human tumours has been lung tumours (yellow circles) (left panel), after 2–6 weeks of doxycycline modelled extensively in mice5,17,18. Overexpression of the mitotic withdrawal, showing complete regression (second panel) and after 14–16 checkpoint gene Mad2 is a very common occurrence in human weeks off doxycycline (third panel), showing recurrent tumours in TI-KM tumours, probably due to its direct activation by E2F when the retino- mice at the same anatomical site as the primary tumour. Right panel: 2 histological sections. b, Relapse-free survival curve of TI-K and TI-KM mice; blastoma tumour suppressor pathway is inhibited . Indeed, inducible censored events indicated by circles. c, Initial tumour volume of TI-K and overexpression of Mad2 in mice leads to modest levels of aneuploidy 3 TI-KM mice that never recurred and in TI-KM mice that underwent a and robust tumour induction in multiple tissue types . Extremely high relapse. d, RT–PCR from normal, TI-K and TI-KM lungs on doxycycline levels of aneuploidy are observed in cells with complete loss of mitotic showing the corresponding transgenes and from TI-K and TI-KM mice and checkpoint function19–21, but these cells are non-viable, a fact reflected relapses off doxycycline. e, SP-C, Ki67 and HA immunostaining of a relapse. in the observation that no mitotic checkpoint-null tumours have ever Black scale bars, 200 mm; blue scale bars, 100 mm. been reported, regardless of p53 status. Although loss of mitotic checkpoint function in some settings can suppress tumour formation doxycycline for 5 weeks, during which time no tumours developed, in vivo22–24, whether this is due to intolerable levels of aneuploidy and and switched to a regular diet for 35 additional weeks did not develop subsequent cell death or perhaps even non-cell autonomous effects on tumours (Supplementary Fig. 5); and fourth, in 3 out of 3 informa- the tumour microenvironment has not yet been examined. tive cases in which primary tumours were small enough to localize by The requirement for continued expression of an initiating onco- MRI, recurrences appeared at the same anatomical site as the primary genic stimulus for tumour cell survival (oncogene addiction1)isa tumour (Fig. 3a). puzzling phenomenon, because withdrawal of a deregulated activity Tumour relapse has been observed in several tetracycline-inducible might be predicted to lead to reversion of the cell to a normal state. mouse models of oncogene addiction11. Although many of these re- Nonetheless, its occurrence suggests that therapeutic targeting of curring tumours show doxycycline-independent re-expression of the oncogenes might lead to profound tumour regression. Indeed, transgene, additional mechanisms responsible for tumour recurrence inhibition of the deregulated kinase activity of bcr-abl with the drug have also been identified. We performed haemagglutinin (HA)- imatinib (Gleevec) in chronic myelogenous leukaemia is an extre- specific immunohistochemistry and/or RT–PCR analysis to detect mely effective targeted therapy25. Patients in the accelerated phase of the Mad2 and Kras transgenes in the primary tumours, recurrent the disease, in which multiple chromosomal anomalies are observed, lesions and tumour-free lung samples from TI-K and TI-KM mice still show a strong clinical response, although with significantly (Fig. 3d, e, and Supplementary Fig. 6b). None of the non-tumour- higher relapse rates26–28. Here we show that a similar phenomenon bearing lungs after doxycycline withdrawal re-expressed the inducible might take place in solid tumours. CIN induced by overexpression of transgenes. Only one of the recurrences re-expressed the Kras trans- the mitotic checkpoint gene Mad2 (observed in a wide variety of gene and 2 out of 11 re-expressed the Mad2 transgene (which by itself human tumours) does not alleviate addiction to Kras in a lung is insufficient to induce tumour formation), suggesting modes of tumour model, because profound tumour regression takes place escape that are independent of the initiating oncogene. RT–PCR and when Kras expression is inhibited whether or not CIN is present in sequencing of commonly mutated exons revealed that the recurrences the primary lesion. However, a marked increase in tumour recurrence were not a result of secondary point mutations in the endogenous occurs in the chromosomally unstable setting with emergent inde- Hras, Kras or Nras loci (data not shown), a common mechanism of pendence from the original oncogenic stimulus. Although resistance 438 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

a b TI-K (de-induced lung) TI-KM (de-induced lung) TI-KM (relapse) 50 P = 0.0045 40 30 P = 0.0053 20 Frequency of

aneuploidy (%) 10 0 TI-K TI-KM Relapse off off c Relapse 1 Relapse 3 Relapse 4 Relapse 7 Relapse 8 Relapse 9 pERK pStat3 pAKT pS6

Figure 4 | Recurrent tumours from TI-KM mice are heterogeneous and values were determined with an unpaired t-test. c, Immunohistochemistry highly aneuploid. a, FISH images from a TI-K and TI-KM regressed lung of pERK, pStat3, pAKT and pS6 (antibodies shown on the left) in tumour and a TI-KM tumour relapse, with aneuploid cells outlined in yellow and relapses, showing positive staining for all three pathways in relapses 1, 3 and normal cells in white. b, Frequency of aneuploidy in three independent TI-K 8, whereas relapses 4 and 7 are positive only for pAKT and pS6. Scale bar, and three TI-KM regressed lungs and three recurrent tumours of TI-KM 100 mm. mice, showing an increase in aneuploid cells in the recurrent tumours. P to targeted therapies is often initially due to point mutations or ampli- Full Methods and any associated references are available in the online version of fications of the intended target gene, CIN might provide an escape the paper at www.nature.com/nature. route even after sequential, successful inhibition of the altered target. Received 5 October 2009; accepted 7 January 2010. Moreover, CIN in the primary tumour may accelerate the disruption Published online 21 February 2010. of DNA repair pathways, thereby facilitating the generation of point 1. Weinstein, I. B. & Joe, A. K. Mechanisms of disease: oncogene addiction—a mutations. Thus, early CIN in tumours, which in some cases results rationale for molecular targeting in cancer therapy. Naure. Clin. Pract. Oncol. 3, directly from the loss of the retinoblastoma tumour suppressor path- 448–457 (2006). way and overactivation of the mitotic checkpoint, may provide the 2. Hernando, E. et al. Rb inactivation promotes genomic instability by uncoupling cell evolutionary fuel responsible for high relapse rates in human cancers cycle progression from mitotic control. Nature 430, 797–802 (2004). 3. Sotillo, R. et al. Mad2 overexpression promotes aneuploidy and tumorigenesis in that initially respond to targeted or perhaps even conventional che- mice. Cancer Cell 11, 9–23 (2007). motherapeutic regimens. Mouse models of cancer that often show 4. Schvartzman, J. M., Sotillo, R. & Benezra, R. Mitotic chromosomal instability and benign levels of CIN relative to human cancers may thereby over- cancer: mouse modelling of the human disease. Nature Rev. Cancer 10, 102–115 estimate the efficacy of clinical drug candidates. (2010). 5. Ricke, R. M., van Ree, J. H. & van Deursen, J. M. Whole chromosome instability and METHODS SUMMARY cancer: a complex relationship. Trends Genet. 24, 457–466 (2008). 6. Chang, S., Khoo, C. & DePinho, R. A. Modeling chromosomal instability and All mice (TI-K, TI-KM, TI-M, CCSP–rtTA) were of mixed 129/Bl6 background. epithelial carcinogenesis in the telomerase-deficient mouse. Semin. Cancer Biol. 11, Doxycycline (625 p.p.m.) was administered in impregnated food pellets (Harlan- 227–239 (2001). Teklad). For MRI analysis, mice were anaesthetized with isofluorane and images 7. Tichelaar, J. W., Lu, W. & Whitsett, J. A. Conditional expression of fibroblast were obtained by a low-resolution axial scout followed by a high-spatial resolution growth factor-7 in the developing and mature lung. J. Biol. Chem. 275, T2 weighted scan. FISH analysis was performed with three pericentromeric probes 11858–11864 (2000). from chromosomes 12, 16 and 17 amplified from corresponding bacterial artificial 8. Fisher, G. H. et al. Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor chromosome (BAC) clones and labelled by nick translation with SpectrumGreen, genes. Genes Dev. 15, 3249–3262 (2001). SpectrumRed and SpectrumOrange fluorophores. Quantification of aneuploidy 9. Chin, L. et al. Essential role for oncogenic Ras in tumour maintenance. Nature 400, was conducted in a minimum of 150 interphase nuclei counterstained with 4,6- 468–472 (1999). diamidino-2-phenylindole. Microarray expression analysis was performed on 10. Felsher, D. W. & Bishop, J. M. Reversible tumorigenesis by MYC in hematopoietic MOE430A 2.0 Affymetrix arrays with RNA extracted using the Qiagen RNeasy lineages. Mol. Cell 4, 199–207 (1999). kit. For CGH, DNA was extracted with a Qiagen DNeasy kit, whole-genome 11. Giuriato, S. & Felsher, D. W. How cancers escape their oncogene habit. Cell Cycle amplified and hybridized to normal genome controls on an Agilent mouse 244K 2, 329–332 (2003). array. RT–PCR was conducted with SuperScript III (Invitrogen) in accordance 12. Boxer, R. B., Jang, J. W., Sintasath, L. & Chodosh, L. A. Lack of sustained regression with the manufacturer’s instructions, and amplified with transgene-specific of c-MYC-induced mammary adenocarcinomas following brief or prolonged primers. Protein expression was assessed by western blot of 40 mg of tissue lysates MYC inactivation. Cancer Cell 6, 577–586 (2004). 13. Gorre, M. E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL incubated with antibodies against Mad2 (BD) and actin (Amersham). Histo- gene mutation or amplification. Science (New York, N. Y.) 293, 876–880 (2001). pathology and immunohistochemistry were performed on paraffin sections of 14. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to formalin-fixed tissue with Ki67 (Novocastra), anti-prosurfactant protein C gefitinib. N. Engl. J. Med. 352, 786–792 (2005). (Chemicon), anti-Clara cell protein (Chemicon), anti-phospho-AKT (Cell 15. Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib Signaling Technology), anti-phospho-Stat3 (Cell Signaling Technology), anti- is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 phospho-S6 (Cell Signaling Technology) and anti-HA (Roche). (2005). 439 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

16. Politi, K. et al. Lung adenocarcinomas induced in mice by mutant EGF receptors 27. Hochhaus, A. et al. Molecular and chromosomal mechanisms of resistance to found in human lung cancers respond to a tyrosine kinase inhibitor or to down- imatinib (STI571) therapy. Leukemia 16, 2190–2196 (2002). regulation of the receptors. Genes Dev. 20, 1496–1510 (2006). 28. O’Dwyer, M. E. et al. The impact of clonal evolution on response to imatinib 17. Baker, D. J., Chen, J. & van Deursen, J. M. The mitotic checkpoint in cancer and mesylate (STI571) in accelerated phase CML. Blood 100, 1628–1633 (2002). aging: what have mice taught us? Curr. Opin. Cell Biol. 17, 583–589 (2005). 18. Pe´rez de Castro, I., de Ca´rcer, G. & Malumbres, M. A census of mitotic cancer Supplementary Information is linked to the online version of the paper at genes: new insights into tumor cell biology and cancer therapy. Carcinogenesis 28, www.nature.com/nature. 899–912 (2007). Acknowledgements We thank H. Varmus and W. Pao for the CCSP–rtTA and 19. Dobles, M., Liberal, V., Scott, M. L., Benezra, R. & Sorger, P. K. Chromosome TetO–Kras mice; K. Politi, K. Podsypanina and M. Jechlinger for reagents, missegregation and apoptosis in mice lacking the mitotic checkpoint protein discussions and critical reading of the manuscript; C. Le and M. Lupu for MR Mad2. Cell 101, 635–645 (2000). imaging; M. Leversha, C. Kalyani and J. McGuire for FISH staining; K. M. D. La 20. Li, M., York, J. P. & Zhang, P. Loss of Cdc20 causes a securin-dependent metaphase Perle, M. Jiao and M. Squatrito for immunohistochemistry and pathological arrest in two-cell mouse embryos. Mol. Cell. Biol. 27, 3481–3488 (2007). analysis; A. Viale for CGH and Affymetrix; and Y. Chin, S. Curelariu and C. Coker 21. Kwon, M. et al. Mechanisms to suppress multipolar divisions in cancer cells with for excellent technical assistance. Support was provided to R.S. by the Charles extra centrosomes. Genes Dev. 22, 2189–2203 (2008). H. Revson Foundation, to J.M.S. by a Breast Cancer Research Program (BCRP) 22. Weaver, B. A., Silk, A. D., Montagna, C., Verdier-Pinard, P. & Cleveland, D. W. Predoctoral Traineeship Award from the US Department of Defense Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 11, (Congressionally Directed Medical Research Programs) and to R.B. by the 25–36 (2007). National Institutes of Health. 23. Rao, C. V. et al. Colonic tumorigenesis in BubR11/2ApcMin/1 compound mutant mice is linked to premature separation of sister chromatids and enhanced Author Contributions R.S., J.M.S. and R.B. designed the study. R.S. performed genomic instability. Proc. Natl Acad. Sci. USA 102, 4365–4370 (2005). experiments. R.S., J.M.S., N.S. and R.B. analysed the data. R.S., J.M.S. and R.B. wrote 24. Jeganathan, K., Malureanu, L., Baker, D. J., Abraham, S. C. & van Deursen, J. M. the paper. Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis. J. Cell Biol. 179, 255–267 (2007). Author Information The raw and processed microarray data are deposited at GEO 25. Druker, B. J. et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine under accession number GSE19753. Reprints and permissions information is kinase in chronic myeloid leukemia. N. Engl. J. Med. 344, 1031–1037 (2001). available at www.nature.com/reprints. The authors declare no competing financial 26. Cortes, J. & O’Dwyer, M. E. Clonal evolution in chronic myelogenous leukemia. interests. Correspondence and requests for materials should be addressed to R.B. Hematol. Oncol. Clin. North Am. 18, 671–684 (2004). ([email protected]).

METHODS differential expression, the LIMMA package (also from Bioconductor) was used Animal husbandry and genotyping. TetO–Mad2 transgenic mice and TetO– for a variant of the standard t-test, which adds a correction term to the group Kras and CCSP–rtTA mice were kept in pathogen-free housing under guidelines variance. The false discovery rate (FDR) method was used to correct for multiple approved by the MSKCC Institutional Animal Care and Use Committee and testing. Hierarchical clustering was performed with a distance function derived Research Animal Resource Center. Doxycycline was administered by feeding from the correlation between samples (distance 5 (1 2 r)/2, where r is the mice with food pellets impregnated with doxycycline (625 p.p.m.; Harlan- Pearson correlation coefficient). Teklad). Tail DNA was isolated with a Qiaprep Tail DNeasy isolation kit RNA and protein analysis. RNA was isolated with the RNeasy kit (Qiagen) and (Qiagen) in accordance with the manufacturer’s protocol. TetO–Mad2, TetO– treated with DNase I (Ambion) to eliminate any contaminating DNA. Reverse Kras and CCSP–rtTA transgenic mice were genotyped as described3,8. transcriptase reactions were performed with SuperScript III (Invitrogen) in Magnetic resonance imaging. Individual mice were subjected to MRI for detec- accordance with the manufacturer’s instructions, followed by PCR with primers tion of tumours. In brief, mice were anaesthetized with 2% isofluorane, and images specific for transgene amplification. Protein expression was assessed by immu- were obtained with a Bruker 4.7-T magnet (40 cm bore) with a commercial 7-cm noblotting with 40 mg of total tissue lysates. Blots were probed with antibodies inner diameter birdcage coil in the Animal Imaging MRCore Facility at Memorial directed against Mad2 (BD Transduction Laboratories). Horseradish peroxi- Sloan-Kettering Cancer Center. Low-resolution axial scout images were obtained dase-conjugated anti-mouse (Amersham) was used as a secondary antibody initially, followed by high-spatial-resolution T2-weighted axial images (repetition and proteins were revealed by enhanced chemiluminescence with the ECL detec- interval 3,800 ms, effective echo time 35 ms, eight echoes per phase encoding step, tion system (Amersham). For quantitative western blots, fluorochrome- spatial resolution 1.0 mm slice thickness 3 112 mm 3 112 mm in-plane resolution) conjugated secondary antibodies (LI-COR) were used in place of horseradish and four repetitions of data acquisition for 8–9 min of imaging time. peroxidase, and signals were detected with LI-COR Odyssey. Fluorescence in situ hybridization. Probes were synthesized from three peri- Histopathology. For immunohistochemistry analysis, representative sections were centromeric BAC clones of chromosome 12 (Chr 12-RP23-54G4, RP23-41E22 deparaffinized, rehydrated in graded alcohols and processed with the use of the and RP23 16809 5a), chromosome 16 (Chr 16-RP23-290E4, RP23-356A24 and avidin–biotin immunoperoxidase method. Sections were subjected to antigen RP24 258J4 4a) and chromosome 17 (Chr 17-RP23-354J18-6c, RP23-73N16 and retrieval by microwave oven treatment with standard procedures. Diamino- RP23-202G20) and labelled with SpectrumGreen-dUTP, SpectrumRed-dUTP benzidine was used as the chromogen, and haematoxylin to counterstain nuclei. and SpectrumOrange-dUTP (Vysis), respectively. The BAC DNAs were labelled The primary antibodies used in immunohistochemistry were Ki67 (NovoCastra), by nick translation in accordance with standard procedures. The number of anti-prosurfactant protein C (1:400 dilution, AB3786; Chemicon), anti-Clara cell hybridization signals for these probes was assessed in a minimum of 150 inter- protein (1:1,000, AB07623; Chemicon), anti-phospho-MAPK (1:100, catalogue no. phase nuclei with strong and well-delineated contours. 4376; Cell Signaling Technology), anti-phospho-Akt (1:100, catalogue no. 3787; RNA expression data. Raw data (CEL files) were normalized and quantified to Cell Signaling Technology), anti-phospho-Stat3 (1:100, catalogue no. 9145; Cell give log2 expression levels for each probe set, using the gcRMA method available Signaling Technology), anti-phospho-S6 (1:100, catalogue no. 2211; Cell Signaling in the Bioconductor library for the R statistical analysis system. To compute Technology) and anti-HA (1:100, clone 3F10, catalogue no. 11867423001; Roche).

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome

Heinz Neumann1*, Kaihang Wang1*, Lloyd Davis1, Maria Garcia-Alai1 & Jason W. Chin1

The in vivo, genetically programmed incorporation of designer would enhance misreading of the proteome. In contrast, orthogonal amino acids allows the properties of proteins to be tailored with ribosomes8, which are specifically addressed to the orthogonal message molecular precision1. The Methanococcus jannaschii tyrosyl- and are not responsible for synthesizing the proteome, may, in prin- 2,3 transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA) and ciple, be evolved to efficiently decode quadruplet codons on the ortho- the Methanosarcina barkeri pyrrolysyl-tRNA synthetase–tRNACUA gonal message (Supplementary Figs 1 and 2). To discover evolved 4–6 (MbPylRS–tRNACUA) orthogonal pairs have been evolved to orthogonal ribosomes that enhance quadruplet decoding we first incorporate a range of unnatural amino acids in response to the created 11 saturation mutagenesis libraries in the 16S ribosomal amber codon in Escherichia coli1,6,7. However, the potential of syn- RNA of ribo-X (an orthogonal ribosome previously evolved for effi- thetic genetic code expansion is generally limited to the low efficient amber codon decoding on an orthogonal message9); taken ciency incorporation of a single type of unnatural amino acid at a together these libraries cover 127 nucleotides that are within 12 A˚ of time, because every triplet codon in the universal genetic code is a tRNA bound in the decoding centre19 (Supplementary Fig. 3). We used in encoding the synthesis of the proteome. To encode effi- used ribo-X as a starting point for library generation because we hoped ciently many distinct unnatural amino acids into proteins we to discover evolved orthogonal ribosomes that gain the ability to effi- require blank codons and mutually orthogonal aminoacyl-tRNA ciently decode quadruplet codons while maintaining the ability to synthetase–tRNA pairs that recognize unnatural amino acids and efficiently decode amber codons on the orthogonal mRNA; thereby decode the new codons. Here we synthetically evolve an orthogonal maximizing the number of additional codons that can be decoded by ribosome8,9 (ribo-Q1) that efficiently decodes a series of quadruplet the orthogonal ribosome. codons and the amber codon, providing several blank codons on an To select orthogonal ribosomes that efficiently decode quadruplet orthogonal messenger RNA, which it specifically translates8.By codonsusing extended anticodon tRNAs we combined each orthogonal creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA (O)-ribosome library with a reporter construct (O-cat (AAGA 146)– Ser 2 pairs and combining them with ribo-Q1 we direct the incorpora- tRNAUCUU). The reporter contains a chloramphenicol acetyl trans- tion of distinct unnatural amino acids in response to two of the ferase gene that is specifically translated by O-ribosomes9, an in-frame Ser 2 new blank codons on the orthogonal mRNA. Using this code, we AAGA quadruplet codon and tRNAUCUU (a designed variant of genetically direct the formation of a specific, redox-insensitive, tRNASer 2 that is aminoacylated by E. coli seryl-tRNA synthetase and nanoscale protein cross-link by the bio-orthogonal cycloaddition decodes the AAGA codon9,20). The orthogonal cat gene is read in frame, 10 Ser 2 of encoded azide- and alkyne-containing amino acids . Because the and confers chloramphenicol resistance, only if tRNAUCUU efficiently synthetase–tRNA pairs used have been evolved to incorporate decodes the AAGA codon and restores the reading frame. Clones sur- numerous unnatural amino acids1,6,7, it will be possible to encode viving on chloramphenicol concentrations that kill cells containing more than 200 unnatural amino acid combinations using this ribo-X and the cat reporter have four distinct sequences. Clone ribo- approach. As ribo-Q1 independently decodes a series of quadruplet Q4 has double mutations: C1195A and A1196G, ribo-Q3 has the three codons, this work provides foundational technologies for the mutations: C1195T, A1196G and A1197G; ribo-Q2 and ribo-Q1 have encoded synthesis and synthetic evolution of unnatural polymers two mutations: A1196G and A1197G, ribo-Q2 also has eight other non- in cells. programmed mutations. Although the entire decoding centre was Because each of the 64 triplet codons are used to encode natural mutated, the selected mutations are spatially localized and might amino acids or polypeptide termination, new blank codons are required accommodate an extended anticodon–codon interaction in the decod- for cellular genetic code expansion. In principle, quadruplet codons ing centre (Fig. 1a). The chloramphenicol resistance of cells containing Ser 2 might provide 256 blank codons. Stoichiometrically aminoacylated tRNAUCCU and cat with two AGGA codons is greatly enhanced when extended anticodon tRNAs have been used to incorporate unnatural the cat gene is translated by ribo-Q ribosomes in place of unevolved amino acids in response to four-base codons with very low efficiency in ribosomes (Fig. 1b, c). Indeed the chloramphenicol resistance of cells in vitro systems11–13 and in limited in vivo systems, by the import of containing two AGGA codons read by the ribo-Q ribosomes previously aminoacylated tRNA14,15. In one case a four-base suppressor approaches that of a wild-type cat gene. This suggests that ribo-Q1 and amber codon have been used, in a non-generalizable approach, to may decode quadruplet codons with an efficiency approaching that encode two unremarkable amino acids with low efficiency16. Indeed, the for triplet decoding and with a much greater efficiency than the une- inefficiency with which natural ribosomes decode quadruplet codons volved ribosome. The enhancement in quadruplet decoding efficiency severely limits their utility for genetic code expansion. is maintained for a variety of quadruplet codon–anticodon interactions A ribosome must accommodate an extended anticodon tRNA in (Supplementary Fig. 4). its decoding centre to be able to decode it17,18. Natural ribosomes are Natural ribosomes decode triplet codons with high fidelity (error very inefficient at, and unevolvable for quadruplet decoding, which frequencies ranging from 1022 to 1024 errors per codon have been

1Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. *These authors contributed equally to this work. 441 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

ab Cm µg ml–1

tRNA O-cat 0

UCCU 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 O – WT WT – AGGA O – AGGA 16S rRNA libraries tRNA Rx – AGGA Q4 – AGGA Q3 – AGGA mRNA Q2 – AGGA Q1 – AGGA O + AGGA Rx + AGGA Q4 + AGGA Q3 + AGGA Q2 + AGGA A1197G Q1 A1196G + AGGA c O-ribosome – O Rx Q4 Q3 Q2 Q1 O Rx Q4 Q3 Q2 Q1 O Rx Q4 Q3 Q2 Q1 tRNAUCCU ––– –––– –– – –––++ ++++ 16S rRNA libraries O-catWT +++ ++++ –– – – – ––– ––– – O-catAGGA ––– –––– ++++++++ ++++

AcCm Cm

Ser 2 Figure 1 | Selection and characterization of orthogonal quadruplet The tRNAUCCU-dependent enhancement in decoding AGGA codons in the decoding ribosomes. a, Mutations in quadruplet decoding ribosomes form O-cat (AGGA 103, AGGA 146) gene was measured by survival on increasing a structural cluster close to the space potentially occupied by an extended concentrations of chloramphenicol (Cm). WT, wild type. c,Asinb, but anticodon tRNA. Selected nucleotides are shown in red. Image created using measuring CAT enzymatic activity directly by thin-layer chromatography. Pymol (http://www.pymol.org) and Protein Data Bank (PDB) accession AcCm, acetylated chloramphenicol; O, O-ribosome; Q1–Q4, ribo-Q1–Q4; 2J00. b, Ribo-Qs substantially enhance the decoding of quadruplet codons. Rx, ribo-X. reported21–23). To explicitly compare the fidelity of triplet decoding mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs. We 4,5 and quadruplet decoding for the evolved orthogonal ribosomes and demonstrated that the MbPylRS–tRNACUA pair and MjTyrRS– 2 the progenitor ribosome we used two independent methods: the tRNACUA pair , each of which have previously been evolved to incor- incorporation of 35S-cysteine into a protein, which contains no porate a range of unnatural amino acids1,6,7,25, are mutually orthogonal cysteine codons in its gene9, and variants of a dual luciferase system9,23 in their aminoacylation specificity (Supplementary Fig. 8). We created (Supplementary Fig. 5). We find that the triplet and quadruplet the AzPheRS*–tRNAUCCU pair, which is derived from the MjTyrRS– decoding translational fidelity is the same for the evolved ribosome tRNACUA pair, by a series of generally applicable directed evolution (ribo-Q1) and unevolved and wild-type ribosomes, and the fourth steps (Supplementary Figs 9–11). The MbPylRS–tRNACUA pair and base of the codon–anticodon interaction is discriminated equally AzPheRS*–tRNAUCCU pair are mutually orthogonal: they decode dis- well by all ribosomes (Supplementary Fig. 5). tinct codons, use distinct amino acids and are orthogonal in their To demonstrate that the enhanced amber-decoding properties of aminoacylation specificity (Supplementary Fig. 12). ribo-X are maintained in ribo-Q1 we compared the efficiency of To demonstrate the simultaneous incorporation of two useful incorporating p-benzoyl-L-phenylalanine (Bpa) into a recombinant unnatural amino acids into a single protein we combined the glutathione S-transferase–maltose binding protein (GST–MBP) fusion MbPylRS–MbtRNACUA pair, the AzPheRS* tRNAUCCU pair and in response to an amber codon on an orthogonal mRNA using orthogonal ribosomes and a previously evolved Bpa-tRNA synthetase– a 3 Ribosome WT Ribo-X Ribo-Q1 WT Ribo-X Ribo-Q1 tRNACUA pair (BpaRS–tRNACUA) (Fig. 2). Ribo-Q1 and ribo-X BpaRS–tRNA ++++++ ++++++ incorporate Bpa with a comparable and high efficiency in response CUA − + ++−− − + − ++− to the amber codons in the orthogonal mRNA (compare lanes 4 and Bpa gst(UAG)nmalE 11−−−− 22−−−− 6 and lanes 10 and 12 in Fig. 2a). Ribo-X and ribo-Q1 are substantially O-gst(UAG) malE −−11 11 −−22 22 more efficient than the wild-type ribosome at incorporating Bpa by n means of amber suppression (compare lanes 4 and 6 to lane 2, and lanes GST–MBP 10 and 12 to lane 8 in Fig. 2a). To demonstrate the utility of ribo-Q1 for incorporating unnatural 123456 7 89101112 amino acids in response to quadruplet codons we compared the b WT Ribo-Q1 efficiency of incorporating p-azido-L-phenylalanine (AzPhe) into a Ribosome AzPheRS–tRNAUCCU ++ +++++ + recombinant GST–MBP fusion in response to a quadruplet codon − ++− + − − + using ribo-Q1 or the wild-type ribosome. To direct the incorporation AzPhe gst(AGGA)nmalE 11 22−−−− of AzPhe we used the AzPheRS*–tRNAUCCU pair (a variant of the O-gst(AGGA) malE − − −−11 22 24 n pAzPheRS-7–tRNACUA pair derived from the MjTyrRS–tRNACUA pair for the incorporation of AzPhe as described later). We find that GST–MBP ribo-Q1 substantially increases the efficiency of incorporation of 12 3 4 5 67 8 AzPhe in response to a quadruplet codon, and even allows the incorporation of AzPhe in response to two quadruplet codons for Figure 2 | Enhanced incorporation of unnatural amino acids in response to the first time (compare lanes 2 and 6 and lanes 4 and 8, Fig. 2b). The amber and quadruplet codons with ribo-Q1. a, Ribo-Q1 incorporates Bpa as efficiently as ribo-X. The entire gel is shown in Supplementary Fig. 6. b, Ribo- site and fidelity of incorporation of AzPhe were further confirmed by Q1 enhances the efficiency of AzPhe incorporation in response to the AGGA analysis of tandem mass spectrometry (MS/MS) fragmentation series quadruplet codon using AzPheRS*–tRNAUCCU. The gel showing the ratio of of the relevant tryptic peptides (Supplementary Fig. 7). GST–MBP to GST as well as MS/MS spectra of the single and double AzPhe To take advantage of ribo-Q1 for the incorporation of multiple incorporations are shown in Supplementary Fig. 7. (UAG)n or (AGGA)n distinct unnatural amino acids in recombinant proteins, we required describes the number of amber or AGGA codons (n) between gst and malE. 442 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

ribo-Q1 in E. coli. We used these components to produce full-length a AzPhe – – ++ GST–calmodulin containing AzPhe and N6-[(2-propynyloxy)carbo- CAK – + +– nyl]-L-lysine (CAK; which we recently discovered is an efficient sub- 7 strate for MbPylRS ) (Fig. 3) in response to an AGGA and UAG GST–CaM–H6 codon in an orthogonal gene. Production of the full-length protein required the addition of both unnatural amino acids. We further confirmed the incorporation of AzPhe and CAK at the genetically b y y y y y y y y y y N programmed sites by MS/MS sequencing of a single tryptic fragment 100 9 8 7 6 5 4 3 2 1 6 3 y4 y5 y7 O O containing both unnatural amino acids (Fig. 3). D G Y* L Q I E K* G K Y*= NH H N 2 K*= To begin to demonstrate that emergent properties may be pro- b b b b b b b b b 1 2 3 4 5 6 7 8 9 O OH H N 50 2 grammed into proteins by combinations of unnatural amino acids a3 b y b y6+1 3 3 a4 6 y +1 7 b8 O OH y2 b4 b we genetically directed the formation of a triazole cross-link, by a b5 7 y8 b9 copper catalysed Huisgen [213] cycloaddition reaction (‘click reac- 0 10 Relative intensity (%) 200 400 600 800 1,000 1,200 tion’) . We first encoded AzPhe and CAK at positions 1 and 149 in m/z calmodulin (Fig. 4). After incubation of calmodulin incorporating the Figure 3 | Encoding an azide and an alkyne in a single protein by orthogonal azide (AzPhe) and alkyne (CAK) at these positions with Cu(I) for 5 min we observe a more rapidly migrating protein band in SDS– translation. a, Expression of GST–CaM–His6 (a GST–calmodulin–His6 fusion) containing two unnatural amino acids. An orthogonal gene polyacrylamide gel electrophoresis (SDS–PAGE). MS/MS sequencing producing a GST–CaM–His6 fusion that contains an AGGA codon at unambiguously confirms that the faster mobility band results from the position 1 and an amber codon at position 40 of calmodulin was translated product of a bio-orthogonal cycloaddition reaction between AzPhe by ribo-Q1 in the presence of AzPheRS*–tRNAUCCU and and CAK. Our results demonstrate the genetically programmed proxi- MbPylRS–tRNACUA. The entire gel is shown in Supplementary Fig. 13. mity acceleration of a new class of asymmetric, redox-insensitive b, LC–MS/MS analysis of the incorporation of two distinct unnatural amino cross-link that can be used to specifically constrain protein structure acids into the linker region of GST–MBP. Y*, AzPhe; K*, CAK.

O O a NH His6 His N N 6 GST N O O N NH O N O GST N O HN NH NH NH O

GST–CaM 1 Tyr 149 CAK ++ ––––––

GST–CaM 1 AzPhe 149 CAK – – –––+– + GST–CaM 1 AzPhe 149 BocK – – –+– –+– GST–CaM 1 AzPhe 40 CAK –– +––– +– Click reagents – + –+–+–+M

GST–CaM–H6 circ. GST-CaM–H6

c ×5 [M–128–18]2+ 1,153.8467 100 100 2+ GS A D K L T E 95 2 [M–128] + 1,208.8850 B –73 N N 90 2+ + 1,863.9752 NH [M–FK] y N 85 4 10 )

80 2 1,080.2944 80 + 75 + –3× H20 (M–) B

70 [M–y ] A G K -(CH 11 O + + 65 [A-E*-GGGK + H] y11 + y O 1,335.7220 [M–y ] 12 6060 517.3469 5 y11 55 + y NH + A y 11 50 y 10 + 4 [M–y+] y10 A 45 + 6 40 [M–y ] 40 + 10 [M–y+] 35 + y7 7 + y 0 [M–y ] 7 30 y ++ + 4 y7

3 y Relative intensity (%) y y 25 5 6 12 y6 405.3304 Q I A E F K 1,717.7955 y6 20 y 20 864.3882 1,588.8136 5 y 15 980.2118 y 5 718.4348 4 10 y 1,450.9149 4 5

0 400 800 1,200 1,600 2,000 m/z

Figure 4 | Genetically directed cyclization of calmodulin by a Cu(I)- lysine; circ., circularized protein. The first two lanes are from a separate gel. catalysed Huisgen’s [213]-cycloaddition. a, Structure of calmodulin M denotes molecular mass marker. c, LC–MS/MS confirms the triazole indicating the sites of incorporation of AzPhe and CAK and their triazole formation. The MS/MS spectra of a doubly charged peptide containing the product. Image created using Pymol (http://www.pymol.org) and Protein crosslink (m/z 5 1,226.6092, which is within 1.8 p.p.m. of the mass expected Data Bank (PDB) accession 4CLN. b, GST–CaM–His6 1 AzPhe 149 CAK for cross-linked peptide). e specifically cyclizes with Cu(I)-catalyst. BocK, N -tert-butyl-oxycarbonyl-L- 443 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 on the nanometre scale. Unlike existing protein cyclization methods for 10. Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise huisgen recombinant proteins26,27, these cross-links can be encoded at any spa- cycloaddition process: copper(I)-catalyzed regioselective ‘‘ligation’’ of azides and terminal alkynes. Angew. Chem. Int. Edn Engl. 41, 2596–2599 (2002). tially compatible sites in a protein, not just placed at the termini. In 11. Hohsaka, T. & Sisido, M. Incorporation of non-natural amino acids into proteins. contrast to the chemically diverse cyclization methods that can be Curr. Opin. Chem. Biol. 6, 809–815 (2002). accessed with peptides by solid-phase peptide synthesis28, these cross- 12. Ohtsuki, T., Manabe, T. & Sisido, M. Multiple incorporation of non-natural amino links can be encoded into proteins of essentially any size. Given the acids into a single protein using tRNAs with non-standard structures. FEBS Lett. importance of disulphide bonds in natural therapeutic proteins and 579, 6769–6774 (2005). 29 13. Murakami, H., Hohsaka, T., Ashizuka, Y. & Sisido, M. Site-directed incorporation hormones, the utility of peptide stapling strategies , the importance of of p-nitrophenylalanine into streptavidin and site-to-site photoinduced electron 30 peptide cyclization , and the improved stability of proteins cyclized by transfer from a pyrenyl group to a nitrophenyl group on the protein framework. native chemical ligation26, it will be interesting to investigate the J. Am. Chem. Soc. 120, 7520–7529 (1998). enhancement of protein function that may be accessed by combining 14. Rodriguez, E. A., Lester, H. A. & Dougherty, D. A. In vivo incorporation of multiple the encoding of these cross-links with directed evolution methods. By unnatural amino acids through nonsense and frameshift suppression. Proc. Natl Acad. Sci. USA 103, 8650–8655 (2006). combining the numerous variant MjTyrRS–tRNACUA and MbPylRS– 15. Monahan, S. L., Lester, H. A. & Dougherty, D. A. Site-specific incorporation of tRNACUA pairs reported for the incorporation of unnatural amino unnatural amino acids into receptors expressed in mammalian cells. Chem. Biol. acids1,6,7 (after appropriate anticodon conversion using the steps 10, 573–580 (2003). reported here) with ribo-Q1, it will be possible to encode more than 16. Anderson, J. C. et al. An expanded genetic code with a functional quadruplet 200 amino acid combinations in recombinant proteins. codon. Proc. Natl Acad. Sci. USA 101, 7566–7571 (2004). 17. Atkins, J. F. & Bjork, G. R. A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment. Microbiol. Mol. METHODS SUMMARY Biol. Rev. 73, 178–210 (2009). Methods for cloning, site-directed mutagenesis and library construction are 18. Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the described in the Supplementary Materials. Ribosome libraries were screened connection between translational accuracy and unconventional decoding. Trends for quadruplet suppressors using a modification of the strategy to discover Biochem. Sci. 27, 178–183 (2002). ribo-X9. 19. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. E. coli genehogs or DH10B were used in all protein expression experiments Science 313, 1935–1942 (2006). 20. Magliery, T. J., Anderson, J. C. & Schultz, P. G. Expanding the genetic code: using LB medium supplemented with appropriate antibiotics and unnatural selection of efficient suppressors of four-base codons and identification of amino acids. Proteins were purified by affinity chromatography using published ‘‘shifty’’ four-base codons with a library approach in Escherichia coli. J. Mol. Biol. standard protocols. 307, 755–769 (2001). Translational fidelity of evolved O-ribosomes was measured by mis- 21. Khazaie, K., Buchanan, J. H. & Rosenberger, R. F. The accuracy of Qb RNA 35 incorporation of S-labelled cysteine9. In brief, GST–MBP was produced by translation. 1. Errors during the synthesis of Qb proteins by intact Escherichia coli the O-ribosome in the presence of 35S-cysteine. The protein was purified, cleaved cells. Eur. J. Biochem. 144, 485–489 (1984). with thrombin, which cleaves the linker between GST and MBP, and analysed by 22. Laughrea, M., Latulippe, J., Filion, A. M. & Boulet, L. Mistranslation in twelve SDS–PAGE and phospho-imaging. A modified dual-luciferase assay was used to Escherichia coli ribosomal proteins. Cysteine misincorporation at neutral amino measure the fidelity of translation of O-ribosomes9. Luminescence from a luci- acid residues other than tryptophan. Eur. J. Biochem. 169, 59–64 (1987). ferase mutant containing an inactivating missense mutation in this assay is a 23. Kramer, E. B. & Farabaugh, P. J. The frequency of translational misreading errors in measure of translational inaccuracy of the ribosome. The dual luciferase reporter E. coli is largely determined by tRNA competition. RNA 13, 87–96 (2007). 24. Chin, J. W. et al. Addition of p-azido-L-phenylalanine to the genetic code of system was translated by the O-ribosome, extracted in the cold and luciferase Escherichia coli. J. Am. Chem. Soc. 124, 9026–9027 (2002). activity measured using the Dual-Luciferase Reporter Assay System (Promega). 25. Mukai, T. et al. Adding L-lysine derivatives to the genetic code of mammalian cells Liquid chromatography (LC)–MS/MS of proteins was performed by NextGen with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. Science. Proteins were excised from Coomassie-stained SDS–PAGE gels, 371, 818–822 (2008). digested with trypsin and analysed by LC–MS/MS. Total protein mass was 26. Camarero, J. A., Pavel, J. & Muir, T. W. Chemical synthesis of a circular protein obtained by electrospray ionization (ESI)-MS; purified protein was dialysed into domain: evidence for folding-assisted cyclization. Angew. Chem. Int. Ed. 37, 10 mM ammonium bicarbonate, pH 7.5, mixed 1:1 with 1% formic acid in 50% 347–349 (1998). methanol, and total mass was determined in positive ion mode. 27. Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production Cyclization reactions were performed for 5 min at room temperature on purified of cyclic peptides and proteins in vivo. Proc. Natl Acad. Sci. USA 96, 13638–13643 protein in 50 mM sodium phosphate, pH 8.3, in the presenceof 1 mM ascorbic acid, (1999). 1mMCuSO and 2 mM bathophenathroline.Details of all methodscan be found in 28. Li, P. & Roller, P. P. Cyclization strategies in peptide derived drug design. Curr. Top. 4 Med. Chem. 2, 325–341 (2002). the Supplementary Materials. 29. Walensky, L. D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 Received 26 June 2009; accepted 7 January 2010. helix. Science 305, 1466–1470 (2004). Published online 14 February 2010. 30. Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A. & Walsh, C. T. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 1. Xie, J. & Schultz, P. G. A chemical toolkit for proteins–an expanded genetic code. 407, 215–218 (2000). Nature Rev. Mol. Cell Biol. 7, 775–782 (2006). 2. Steer, B. A. & Schimmel, P. Major anticodon-binding region missing from an Supplementary Information is linked to the online version of the paper at archaebacterial tRNA synthetase. J. Biol. Chem. 274, 35601–35606 (1999). www.nature.com/nature. 3. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a Acknowledgements We are grateful to P. B. Kapadnis for synthesizing CAK and to photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl W. An for assistance with some experiments. J.W.C. is grateful to the ERC and the Acad. Sci. USA 99, 11020–11024 (2002). MRC for funding. K.W. is grateful to Trinity College, Cambridge for a fellowship. 4. Srinivasan, G., James, C. M. & Krzycki, J. A. Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002). Author Contributions K.W., H.N., L.D. and J.W.C. planned the experiments. K.W. 5. Polycarpo, C. et al. An aminoacyl-tRNA synthetase that specifically activates selected and characterized ribo-Q, with help from L.D. K.W. and L.D. characterized pyrrolysine. Proc. Natl Acad. Sci. USA 101, 12450–12454 (2004). amber and quadruplet incorporation by protein expression and mass e 6. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N -acetyllysine spectrometry. L.D. and M.G.-A. performed protein expression experiments. H.N. in recombinant proteins. Nature Chem. Biol. 4, 232–234 (2008). demonstrated the mutual orthogonality of synthetase tRNA pairs, evolved 7. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes synthetases, and characterized the double incorporation and protein cyclization, in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair and with help from M.G.-A. H.N., K.W., L.D. and J.W.C. analysed the data and wrote the click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009). paper. 8. Rackham, O. & Chin, J. W. A network of orthogonal ribosome?mRNA pairs. Nature Chem. Biol. 1, 159–166 (2005). Author Information Reprints and permissions information is available at 9. Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal www.nature.com/reprints. The authors declare no competing financial interests. ribosomes enhance the efficiency of synthetic genetic code expansion. Nature Correspondence and requests for materials should be addressed to J.W.C. Biotechnol. 25, 770–777 (2007). ([email protected]).

Structural basis for receptor recognition of vitamin-B12–intrinsic factor complexes Christian Brix Folsted Andersen1,2*, Mette Madsen1*, Tina Storm1, Søren K. Moestrup1 & Gregers R. Andersen2

Cobalamin (Cbl, vitamin B12) is a bacterial organic compound and observed in the loop regions interacting with CUB5–8. In parti- an essential coenzyme in mammals, which take it up from the diet. cular, the interaction of Cbl and IF is unchanged. The vitamin is not ˚ This occurs by the combined action of the gastric intrinsic factor (IF) directly recognized by CUB5–8 and does not approach closer than 13 A and the ileal endocytic cubam receptor formed by the 460-kilodalton to CUB5–8. Instead, because IF has a less compact structure than (kDa) protein cubilin and the 45-kDa transmembrane protein IF–Cbl8 and does not bind to cubilin1, the Cbl molecule seems to 1,2 amnionless . Loss of function of any of these proteins ultimately assemble these domains for interaction with the CUB6 and CUB8 leads to Cbl deficiency in man3,4. Here we present the crystal struc- domains. CUB domains 5–8 are structurally similar to other CUB ture of the complex between IF–Cbl and the cubilin IF–Cbl-binding- domains except that the loop 5 (L5) region of CUB6 lacks a disulphide 5 ˚ region (CUB5–8) determined at 3.3 A resolution. The structure pro- bridge. The CUB–CUB domain interfaces are dominated by van der vides insight into how several CUB (for ‘complement C1r/C1s, Uegf, Waals interactions and only a few hydrogen bonds are observed Bmp1’) domains collectively function as modular ligand-binding (Supplementary Fig. 6). regions, and how two distant CUB domains embrace the Cbl mole- ACa21 ion binds at the predicted site in all four CUB domains as cule by binding the two IF domains in a Ca21-dependent manner. verified by the anomalous signal in diffraction data with a maximum This dual-point model provides a probable explanation of how Cbl resolution of 4.2 A˚ (Supplementary Table 1) collected at a wavelength indirectly induces ligand–receptor coupling. Finally, the comparison of 1.8 A˚ (Fig. 1d). The calcium coordinating glutamate and the first of Ca21-binding CUB domains and the low-density lipoprotein aspartate residue are located in L5 of the CUB domain, whereas the (LDL) receptor-type A modules suggests that the electrostatic pairing second aspartate residue is located in loop 9 (L9). This triad of nega- of a basic ligand arginine/lysine residue with Ca21-coordinating tively charged residues is strictly conserved in all four CUB domains acidic aspartates/glutamates is a common theme of Ca21-dependent from six species (Supplementary Fig. 3). The Ca21 ion fixes the ligand–receptor interactions. conformation of surrounding loop regions, but in CUB6 and CUB8 Intestinal uptake of the ‘extrinsic factor’ Cbl is a highly specific the highly specific arrangement of the acidic residues associated with process dependent on IF, cubilin and amnionless. The uptake of the calcium ion serves a further purpose, as they interact by electro- IF–Cbl occurs by classical endocytosis, in which the cubam receptor static interactions with a conserved lysine/arginine finger from IF. constitutively recycles and the IF–Cbl sorts to lysosomes for degra- The CUB6-binding site on the a-domain of IF involves residues dation and release of Cbl for transport and use in the body. IF–Cbl from helices a7 and a9 and the loop region between a5 and a6 binds with high affinity to a region encompassed by four (CUB5–8)of (Fig. 2a, b and Supplementary Fig. 7). The CUB8-binding site on the 27 CUB domains in cubilin5 (Supplementary Fig. 1). The remain- the b-domain only involves residues from the loop between b1 and ing CUB domains are suggested to contain the binding sites for the b2 and between b5 and b6. In CUB6 and CUB8, only residues from L5 several cubilin ligands identified in kidney and other non-intestinal and L9 interact directly with IF. Lys 159 of IF extends from IF to the 6 tissues . calcium-binding site of CUB6, where it forms salt bridges with To uncover the basis for CUB domain recognition of IF–Cbl by Glu 1096 and Asp 1146. CUB6 Tyr 1102 engages in extensive hydro- cubilin, we determined the crystal structure of a complex containing phobic interactions with Pro 122, Pro 125 and Thr 160 from IF, ˚ CUB5–8 and IF–Cbl to 3.3 A resolution. The structure shows that and forms a hydrogen bond with Asn 164 (Fig. 2c and Sup- CUB5–8 forms an elongated structure interacting with both domains plementary Fig. 8). In addition, hydrogen bonds are formed between of IF (Fig. 1a and Supplementary Fig. 2). The CUB6 domain (Sup- the main-chain atoms of CUB6 Asn 1101 and IF Ser 123 and between plementary Fig. 3) directly contacts the a-domain of IF (residues the side chains of CUB6 Gln 1147 and IF Gln 201. The CUB8–IF 25–299, Supplementary Fig. 4), whereas the CUB8 domain interacts interface involves fewer residues than CUB6–IF (Fig. 2d, Supplemen- with the IF b-domain (residues 308–417). The CUB5 and CUB7 tary Fig. 8 and Supplementary Table 2). Like in CUB6, the positively domains do not interact with IF but seem to serve a structural role charged residue Arg 323 from IF forms electrostatic interactions with in the positioning of the two other CUB domains for interacting the calcium-coordinating residues Glu 1328 and Asp 1373 in CUB8 specifically with the closed Cbl-dependent conformation of IF. An and a hydrogen bond with Thr 1335. Otherwise, IF and CUB8 inter- N-glycan of IF partly fills the hole between CUB5–8 and IF and points act only through van der Waals interactions and hydrogen bonds towards a positively charged patch in the interface between CUB5 and from the main-chain nitrogen atoms of Val 325 and Gly 324 to the CUB7 (Fig. 1b, c). carbonyl of Ser 1334 and the carboxylate of Asp 1373. Except for the The structure of intact IF–Cbl (Supplementary Fig. 5a) bound to lysine/arginine residue interacting with the residues coordinating CUB5–8 is similar to the structure of unbound IF–Cbl (Supplementary the calcium ion, the interactions between the CUB domains and Fig. 5b)7 with the only difference that the latter is cleaved in the linker the individual domains of IF are quite different in terms of partici- region between the IF a- and b-domains. Only minor differences are pating structural elements (Fig. 2).

1Department of Medical Biochemistry, 2Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark. *These authors contributed equal to this work. 445 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010

a d Q1147 D1029 I1148 CUB8 S1028 IF β-domain D1146 D1027 L1030 CUB7 D1149

CUB5 CUB6 P1297 Cbl D988 E980 D1105 E1096

E1263 G1264 V1375 CUB6 G1374

D1262 D1373 Q1265 G1376

CUB IF α-domain 5 CUB7 CUB8 D1221 E1328 E1213 bc D1336 α-Man (2006) α-Man (2005) CUB7 β-Man (2004) CUB5 NAG CUB8 (2003) NAG (2002) CUB6 N413 N413

Figure 1 | Crystal structure of CUB5–8–IF–Cbl. a, Structure of the is present at Asn 413, it may interact with the positive patch in the interface CUB5–8–IF–Cbl complex (PDB accession 3KQ4). CUB5–8 is coloured in between CUB5 and CUB7 and thereby stabilize the complex further. blue, IF is in green. Calcium ions are shown as red spheres and glycosylations However, this glycan is not essential for the complex formation20. as grey sticks. Cbl and the cobalt atom are coloured pink and blue, d, Calcium-binding sites in CUB5–8. The three-fold averaged anomalous respectively. b, Electron density of the glycosylation linked to IF Asn 413 difference based electron density map is shown for the calcium ions. The generated from a 2Fo 2 Fc omit map contoured at 1s. Five glycosylation sites relative peak heights are 10.34s (CUB5), 22.91s (CUB6), 16.03s (CUB7) and 21 are predicted in IF, but significant density for the carbohydrates is only 17.72s (CUB8) and no other peaks can be interpreted as Ca ions. The observed at Asn 334 and Asn 413. The latter appears quite ordered and two calcium ions are coordinated by three negatively charged side chains (Glu, N-acetylglucosamines (NAG) and three mannoses (a-Man) have been fitted Asp and Asp) and a variable number of main-chain carbonyls, as previously into the density. c, Electrostatic potential mapped onto the surface of observed in the structures of C1s/C1r13, MASP-1/3 (ref. 14), MAp19 (ref. 21) 22 21 CUB5–8. Blue and red areas have positive and negative electrostatic potential, and neuropilin .ACa ion is expected to have six coordinating ligands respectively, whereas grey areas are neutral. Modelling suggests that if a arranged in a tetragonal bipyramid13. This includes waters that we do not complex-type N-glycan with terminal negatively charged sialic acid residues observe at the resolution of 3.3 A˚ .

a b c N164 Q201 CUB8 IF IF β-domain A163 322–326 P125 L5 1337 β 2 T160 1334–1335 K159 β1 P122 Q1147 S123 Y1102 D1146 1373–1376 382 β6 β5 R156 I1148 I1099 1328 N1101 CUB7 123–125 Ca2+ Cbl L9 163–164 CUB L5 6 E1096 L9 d 1101–1102 α 6 V325 L322 α 1099 5 IF α9 S1334 R323 G324 L5 156 1104 α 7 D1373 L9 CUB6 L9 T1335 V1375 1096 201 159–160 Ca2+ 194 L5 H382 CUB E1328 1146–1148 5 IF α-domain

CUB8

Figure 2 | Interactions between CUB5–8 and IF. a, Mapping of CUB6 and sticks and the cobalt atom as a blue sphere. c, d, Selected interactions ˚ CUB8 residues interacting with IF. Residues within 3.8 A distance from IF between CUB6 (c) or CUB8 (d) and IF. Residues from IF are shown in green are coloured blue. Calcium ions are shown as red spheres. b, IF residues and CUB5–8 residues in blue. Dashed lines represent electrostatic ˚ within 3.8 A distance from CUB6 and CUB8 are coloured green. IF in b is interactions or hydrogen bonds. related to CUB5–8 in a by a vertical rotation of 180u. Cbl is shown as purple 446 ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 | 18 March 2010 LETTERS

a In summary, negatively charged surface patches organized by cal- C1r CUB1 C1r CUB2 C1s CUB1 Cubilin CUB6 cium sites in two CUB domains lead to cooperative binding of IF–Cbl IF-K159 I1148 by two CUB domains. This is likely to cause a gain in functional H228 I1099 Q1147 Y56 D102 Y235 D273 E45 D98 D1146 affinity. Electrostatic interactions are the dominating binding force Y1102 E49 E1096 and binding is strongly pH-dependent. Native cubilin and CUB5–8 D226 show similar pH-dependence in IF–Cbl binding and do not bind to IF–Cbl below pH 5.3, as measured by surface plasmon analysis (data not shown). This indicates that the low endosomal pH exerts its MASP-1/3 CUB MASP-1/3 CUB MAp19 Cubilin CUB uncoupling effect on the CUB5–8 region alone. However, it is not 1 2 8 known whether pH mainly affects the electrostatic interactions at E106 S265 N264 IF-R323 21 S104 F103 Y225 E109 Y106 V1375 the Ca -binding sites or rather the conformation of CUB5–8 or E267 T1335 IF–Cbl. Y56 Y59 D105 D1373 D102 H218 E1328 A mutation in CUB8 changing Pro 1297 (Fig. 1a) to leucine E49 D60 9 10 decreases IF–Cbl affinity leading to inborn Cbl-deficiency disease . E80 E83 This proline is positioned in the L3 loop of CUB8 and is not directly involved in IF binding although located close to the calcium-binding site. However, together with the nearby Pro 1299, Pro 1297 stabilizes b IF K159 an unusual conformation of the L3 loop that keeps Tyr 1296 in a S109 conformation allowing it to interact with and stabilize the L9 loop I1148 D110 involved in IF binding. Q1147 The CUB5–8 region represents an essential but minor part of the RAP K270 entire cubilin structure, and how CUB5–8 relates to the remaining 23 E119 CUB plus 8 EGF domains and the amnionless protein unit anchored D1146 D1149 D108 in the membrane is a major future challenge to define. To further D112 complicate this, multicoil algorithms of a putative coiled–coil region in the amino-terminal part of cubilin and electron micrographs of F105 cubilin have suggested that the protein organizes into trimers11.By D118 D1105 E1096 gel filtration and crosslinking of cubilin mutant fragments encompassing the putative coiled–coil region, we have provided further c support of such a trimerization (Supplementary Fig. 9). The current D1373 CUB5–8–IF–Cbl crystal structure might therefore be present in up to E1328 R323 three copies in the ligand-saturated ,1.5 megadalton (MDa) cubilin–amnionless complex. D112 CUB K270 Our structure provides, to our knowledge, the first structural insight 8 LDLR 21 into how CUB domains function in Ca -dependent complexes with CUB7 RAP ligand proteins. Notably, both domains of IF specifically recognize D108 either CUB6 or CUB8 domains through a long and positively charged D151 side chain that forms electrostatic interactions with conserved gluta- K256 mate and aspartate residues in the Ca21-binding site of the CUB E1096 K159 domains. CUB domains are involved in a wide range of biological 21 functions12 and their Ca -dependent ligand binding has been D1146 D147 CUB observed in several proteins, suggesting a conserved recognition mech- 6 IF anism. Furthermore, sequence analysis suggests that about two-thirds of known CUB domains are expected to bind calcium13. The serine Figure 3 | Ligand-binding sites of CUB domains and LA modules. proteinases MASP-1/3, C1r and C1s—involved in activation of the a, Structures of C1r CUB1 and C1r CUB2 (modelled from MASP-1/3 using 3D Jigsaw23), C1s CUB (PDB accession 1NZI), cubilin CUB , MASP-1/3 complement systems—all contain the three-domain segment CUB1– 1 6 CUB1 (PDB accession 3DEM), MASP-1/3 CUB2 (PDB accession 3DEM), EGF–CUB2. In all these proteins, mutating residues either coordinat- 21 21 MAp19 CUB1 (PDB accession 1SZB) and cubilin CUB8. Mutations that ing Ca or located in the Ca -binding region interfered with ligand affect ligand binding are shown as orange sticks (spheres for homology binding (Fig. 3a). In the MASP-interaction partners (MBL, ficolin-A, 14,15,21 models) . Cubilin CUB6 and CUB8 side-chain residues interacting with H-ficolin and L-ficolin) a lysine has already been suggested to insert IF are shown as blue sticks. IF residues are shown as green sticks. 21 14 directly into the Ca -binding site , and very recently the C1s–C1r b, Comparison of calcium-binding sites in CUB6 (left) and LDLR (right, complex has been proposed to interact with lysine residues in the C1q PDB accession 2FCW). Residues from IF and RAP are shown as green sticks. 15 stem in a similar manner . These examples together with our structure c, Comparison of CUB5–8–IF–Cbl (left) and (LDLR LA3–4)–RAP (right) 21 of the CUB5–8–IF–Cbl complex strongly suggest that the Ca - interactions. Residues engaging in electrostatic interactions are shown as dependent interaction of CUB domains with other proteins involves sticks. a crucial interaction of a basic side chain from the binding partner with the acidic residues coordinating the Ca21 ion in the CUB domain. interact with various ligands in a similar manner. Interestingly, com- In the CUB5–8–IF–Cbl structure, further interactions are formed parison of the CUB5–8–IF and (LDLR LA3–4)–RAP complexes shows by residues from L5 and L9 (Fig. 2) but these residues are not con- marked similarities. In both cases, there is a two-site Ca21-dependent served within different CUB domains (Supplementary Fig. 10). attachment between the receptor and the ligand with relatively small Homologous modules/domains interacting in a Ca21-dependent intermolecular interfaces at each site, and in both cases a lysine or manner with ligands is a common theme of endocytic receptors such arginine side chain from the ligand is inserted directly into the neigh- as scavenger repeat receptors (for example, CD163)16, the LDL- bourhood of the receptor Ca21 sites (Fig. 3b, c and Supplementary receptor type-A (LA) module receptors such as the LDL receptor17 Fig. 11). Furthermore, by superimposing just four residues in the L9 and CD320 (ref. 18). The structure of a complex between two LA loop of a CUB domain (residues 1146–1149) with four residues in an LA 21 modules (LA3–4) of the LDL receptor and the third domain of the ligand module (residues 108–111), the Ca sites in the two otherwise very receptor associated protein (RAP)19 have suggested that LA modules different structural entities can be matched quite well (root mean 447 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 18 March 2010 square deviation 0.8 A˚ for the Ca atoms), and several essential atomic 7. Mathews, F. S. et al. Crystal structure of human intrinsic factor: cobalamin details of the Ca21 coordination are conserved (Fig. 3b and Supplemen- complex at 2.6-A resolution. Proc. Natl Acad. Sci. USA 104, 17311–17316 (2007). 8. Fedosov, S. N. et al. Assembly of the intrinsic factor domains and oligomerization tary Fig. 11a). Notably, the positive charge of the IF lysine/arginine of the protein in the presence of cobalamin. Biochemistry 43, 15095–15102 21 and the RAP lysines are located identically relative to the Ca ion, (2004). and both complexes use two (CUB) or three (LA) critical acidic side 9. Kristiansen, M. et al. Cubilin P1297L mutation associated with hereditary chains fixed in position by calcium coordination to form salt bridges at megaloblastic anemia 1 causes impaired recognition of intrinsic factor-vitamin B12 by cubilin. Blood 96, 405–409 (2000). each docking site between receptor and ligand. Another feature shared 10. Aminoff, M. et al. Mutations in CUBN, encoding the intrinsic factor-vitamin B12 by the CUB5–8–IF and (LDLR LA3–4)–RAP complexes, is that the cal- receptor, cubilin, cause hereditary megaloblastic anaemia 1. Nature Genet. 21, cium sites are well separated from the receptor ‘backbone’ where the 309–313 (1999). Ca21-binding modules/domains within the receptor are connected and 11. Lindblom, A. et al. The intrinsic factor-vitamin B12 receptor, cubilin, is assembled interact. CD320 was recently discovered as a receptor for Cbl in complex into trimers via a coiled-coil a-helix. J. Biol. Chem. 274, 6374–6380 (1999). 18 12. Blanc, G. et al. Insights into how CUB domains can exert specific functions while with transcobalamin, a plasma homologue of IF . Like the LDL recep- sharing a common fold: conserved and specific features of the CUB1 domain tor, CD320 contains two LA modules and possibly uses a similar mech- contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity. anism for ligand recognition. J. Biol. Chem. 282, 16924–16933 (2007). On the basis of the compatible information now available on 13. Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C. & Gaboriaud, 21 Ca21-dependent ligand binding by CUB domains and LA modules, C. X-ray structure of the Ca -binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement. J. Biol. Chem. 278, 32157–32164 we suggest that the insertion of ligand lysine/arginine side chains into (2003). 21 exposed Ca -binding sites on the receptor is a general principle that 14. Teillet, F. et al. Crystal structure of the CUB1-EGF-CUB2 domain of human MASP- may apply to other modular receptors interacting with their ligands 1/3 and identification of its interaction sites with mannan-binding lectin and in a Ca21-dependent manner. Furthermore, it might be a common ficolins. J. Biol. Chem. 283, 25715–25724 (2008). feature of modular proteins, as seen for cubilin and LDLR, that dual- 15. Bally, I. et al. Identification of the C1q binding sites of human C1r and C1s. A refined 21 three-dimensional model of the C1 complex of complement. J. Biol. Chem. 284, point interaction of two Ca -binding domains with the ligand pro- 19340–19348 (2009). vides a simple mechanism to increase functional ligand affinity. 16. Kristiansen, M. et al. Identification of the haemoglobin scavenger receptor. Nature 409, 198–201 (2001). METHODS SUMMARY 17. Blacklow, S. C. Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers. Curr. Opin. Struct. Biol. 17, 419–426 (2007). CUB was expressed in CHO cells and purified by IF–Cbl–ligand affinity 5–8 18. Quadros, E. V., Nakayama, Y. & Sequeira, J. M. The protein and the gene encoding chromatography. IF was expressed in Pichia pastoris and purified using phenyl- the receptor for the cellular uptake of transcobalamin-bound cobalamin. Blood sepharose followed by gel filtration. CUB5–8 and IF–Cbl were mixed in a 2:1 ratio 113, 186–192 (2009). and incubated at 4 uC overnight with Endo H. The assembled complex was 19. Fisher, C., Beglova, N. & Blacklow, S. C. Structure of an LDLR-RAP complex reveals purified by ion-exchange chromatography and crystallized using sitting-drop a general mode for ligand recognition by lipoprotein receptors. Mol. Cell 22, vapour diffusion against a reservoir containing 18% PEG MME 2k, 12% glycerol, 277–283 (2006). 40 mM MES, pH 6.2, and 160 mM KCl. Before freezing crystals were equilibrated 20. Gordon, M. M., Hu, C., Chokshi, H., Hewitt, J. E. & Alpers, D. H. Glycosylation is not against reservoir solution including 20% glycerol, and X-ray diffraction data was required for ligand or receptor binding by expressed rat intrinsic factor. Am. J. collected on the XO6SA beamline at the Swiss Light Source and the i911-3 Physiol. 260, G736–G742 (1991). beamline at MAX-lab. The structure was determined by molecular replacement 21. Gregory, L. A. et al. The X-ray structure of human mannan-binding lectin- associated protein 19 (MAp19) and its interaction site with mannan-binding lectin with human IF–Cbl (Protein Data Bank (PDB) accession 2PMV) and neuropi- and L-ficolin. J. Biol. Chem. 279, 29391–29397 (2004). lin-2 (PDB accession 2QQL) as search models. The oligomerization status of 22. Appleton, B. A. et al. Structural studies of neuropilin/antibody complexes provide affinity-purified full-length human cubilin and recombinantly expressed trun- insights into semaphorin and VEGF binding. EMBO J. 26, 4902–4912 (2007). cated rat cubilin fragments was analysed by gel filtration using Superose columns 23. Bates, P. A., Kelley, L. A., MacCallum, R. M. & Sternberg, M. J. Enhancement of equilibrated in 100 mM NaCl, 20 mM Tris, pH 8, including 2 or 6 M urea (for protein modeling by human intervention in applying the automatic programs 3D- full-length cubilin), or in PBS, pH 7.4, with/without 6 M urea (for the truncated JIGSAW and 3D-PSSM. Proteins 45, 39–46 (2001). fragments). Cross-linkage experiments were performed with the purified, trun- Supplementary Information is linked to the online version of the paper at cated rat cubilin fragments using glutaraldehyde. www.nature.com/nature. Full Methods and any associated references are available in the online version of Acknowledgements We are grateful to G. Biller, G. Ratz and G. Hartvigsen for the paper at www.nature.com/nature. technical assistance, the staff at MAX-lab and SLS beamlines for help with data collection, and S. Fedosov for advice on expression. The study was essentially Received 10 July 2009; accepted 2 February 2010. supported by a grant to S.K.M. from the Lundbeck foundation. G.R.A. was 1. Birn, H. et al. Characterization of an epithelial approximately 460-kDa protein that supported by the Danish Science Research Council and a Hallas-Møller stipend facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor- from the Novo-Nordisk foundation. M.M. was supported by the Novo Nordisk associated protein. J. Biol. Chem. 272, 26497–26504 (1997). Foundation. 2. Fyfe, J. C. et al. The functional cobalamin (vitamin B12)-intrinsic factor receptor is Author Contributions C.B.F.A.: cloning, expression, purification, crystallization, a novel complex of cubilin and amnionless. Blood 103, 1573–1579 (2004). data collection, structure determination and analysis, manuscript preparation; 3. Tanner, S. M. et al. Genetically heterogeneous selective intestinal malabsorption of M.M.: cloning, expression, purification, size-exclusion chromatographic analyses, vitamin B12: founder effects, consanguinity, and high clinical awareness explain manuscript preparation; T.S.: cloning, mutagenesis, expression, size-exclusion aggregations in Scandinavia and the Middle East. Hum. Mutat. 23, 327–333 (2004). chromatographic analyses, cross-linkage experiments; G.R.A. and S.K.M.: study 4. Tanner, S. M. et al. Hereditary juvenile cobalamin deficiency caused by mutations design and manuscript preparation. in the intrinsic factor gene. Proc. Natl Acad. Sci. USA 102, 4130–4133 (2005). 5. Kristiansen, M. et al. Molecular dissection of the intrinsic factor-vitamin B12 Author Information Atomic structure factors and coordinates are deposited at the receptor, cubilin, discloses regions important for membrane association and Protein Data Bank under accession 3KQ4. Reprints and permissions information is ligand binding. J. Biol. Chem. 274, 20540–20544 (1999). available at www.nature.com/reprints. The authors declare no competing financial 6. Christensen, E. I., Verroust, P. J. & Nielsen, R. Receptor-mediated endocytosis in interests. Correspondence and requests for materials should be addressed to renal proximal tubule. Pflugers Arch. 458, 1039–1048 (2009). S.K.M. ([email protected]).

33 34 METHODS prepared with PYMOL and ALINE . Five residues are in the disallowed region of the Ramachandran plot. Four of these are directly involved in Ca21 binding CUB5–8 and cubam protein expression and purification. A human cubilin cDNA fragment extended with enzyme restriction sites and encoding CUB and one, a cysteine residue, engages in disulphide bridge formation. The 21 ˚ 2 ˚ 2 domains 5–8 (base pairs (bp) 2808–4184) was amplified by PCR, purified by B-factors of the Ca ions of CUB6 (55 A ) and CUB8 (83 A ) are significantly 21 ˚ 2 ˚ 2 gel extraction, and subcloned into the pSecTag2B expression vector (Invitrogen) lower than the Ca ions of CUB5 (141 A ) and CUB7 (146 A ), probably owing using the BamHI and EcoRV sites9. Stable transfected CUB -expressing CHO to the direct interaction of calcium-coordinating side chains in CUB6 and CUB8 5–8 ˚ 2 ˚ 2 K-1 clones were established by limited dilution and selection with 500 mgml21 with IF. Likewise, the overall B-factors of CUB6 (95 A ) and CUB8 (112 A ) are ˚ 2 ˚ 2 Zeocin (Invitrogen). Clones were grown in serum-free medium for CHO cells also significantly lower than CUB5 (132 A ) and CUB7 (122 A ). 21 Size-exclusion chromatographic analyses of purified cubam. Samples of purified, with 300 mgml Zeocin. Human cubilin CUB5–8 protein was purified from conditioned medium of transfected cells by IF–Cbl–ligand affinity chromato- concentrated cubam complex were prepared for analytical size-exclusion chro- graphy, as previously described1. Purification of the cubam complex from post- matography. Using a 24-ml Superose 6 HR 10/30 column (GE Healthcare) con- ¨ mortem human kidney cortex membranes by IF–Cbl affinity chromatography nected to an Akta Explorer 10 (GE Healthcare) and equilibrated in running buffer was carried out as previously described2,24. (100 mM NaCl, 20 mM Tris, pH 8, and 2 or 6 M urea), each sample was subjected to size-exclusion. For each run, approximately 200–400 mg protein was applied. The Cubilin fragments I69–T449, R110–T449 and I69–T449(L90R/L97R). 21 The cubilin cDNA fragments I69–T449 (bp 265–1407, HindIII, EcoRI) and flow rate was 0.5 ml min , and fraction volumes of 0.5 ml were collected. Fractions R110–T449 (bp 388–1407, HindIII, EcoRI) extended with enzyme restriction were analysed by SDS–PAGE and silver staining. sites were amplified by PCR. PCR products were purified by gel extraction and Size-exclusion chromatographic analyses of recombinant proteins. Sets of two subcloned into pSecTag2B. I69–T449(L90R/L97R) (bp 265–1407, HindIII, homologous samples of I69–T449, R110–T449, and I69–T449(L90R/L97R) in EcoRI) was generated by site-directed mutagenesis using the I69–T449 construct concentrated conditioned medium or dialysed into PBS buffer, pH 7.4, includ- as a template. Stably expressing CHO K-1 clones were established and grown as ing 8 M urea, were prepared for analytical size-exclusion chromatography. Using ¨ described earlier. a 24-ml Superose 12 10/300 GL column (GE Healthcare) connected to an Akta Intrinsic factor. The gene encoding human IF (GIF) was cloned into the EcoRI Explorer 10 and equilibrated in running buffer (PBS, pH 7.4 with/without 6 M and XbaI sites of the pPICZaA vector and transformed into the P. pastoris X33 urea), each recombinant protein was subjected to size-exclusion in the absence and presence of urea. For each run, approximately 400 ml of 80–85-times con- cell line (Invitrogen). Colonies expressing recombinant IF were inoculated in 21 buffered glycerol-complex medium and incubated at 30 uC for 24 h. Cells were centrated, conditioned medium was applied. The flow rate was 0.5 ml min and transferred to buffered methanol-complex medium containing 0.5 mM vitamin fractions of 0.5 ml were collected. Fractions were analysed by SDS–PAGE and immunoblotting. Scanned blots were processed using Multi-Analyst, version B12 (Sigma) and grown for 72 h at 30 uC. Protein expression was induced by adding 1.5% methanol every 12 h. Secreted IF–Cbl was purified from the 1.0.2 (BioRad). medium by addition of solid ammonium sulphate to a final concentration of Cross-linkage analyses using glutaraldehyde. Peak fractions from size-exclusion 1.6 M, and the solution was then incubated overnight with phenylsepharose (GE chromatographic analyses were incubated for 15 min at room temperature with Healthcare) at 4 uC. The beads were washed with buffer containing 20 mM Tris- 0.125% of the crosslinker glutaraldehyde (Bie and Berntsen A/S). The cross-linking reaction was quenched by the addition of 1 M Tris-HCl, pH 7.5, for 15 min at room HCl, pH 7.6, and 2 mM CaCl2 to elute bound IF–Cbl, which was then further purified by Superdex 75 (GE Healthcare) gel-filtration chromatography using temperature. Samples were subsequently analysed by immunoblotting. the same buffer. 24. Kozyraki, R. et al. The human intrinsic factor-vitamin B12 receptor, cubilin: Complex formation and crystallization. IF–Cbl and CUB5–8 were mixed in a 2:1 molecular characterization and chromosomal mapping of the gene to 10p within molar ratio and incubated at 4 uC overnight with 50 mg Endo H. The complex the autosomal recessive megaloblastic anemia (MGA1) region. Blood 91, was purified by Source 15Q (GE Healthcare) ion-exchange chromatography, 3593–3600 (1998). running a gradient from 50 to 300 mM KCl in a buffer containing 20 mM 25. Kabsch,W. Automaticprocessing ofrotation diffraction data from crystalsofinitially Tris-HCl, pH 7.6, 2 mM CaCl2 and 1 mM MgCl2. Peak fractions were pooled unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993). and concentrated to 5 mg ml21 using a 500 ml Vivaspin cell. Crystals were 26. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, obtained using the sitting-drop vapour diffusion method mixing 2 ml protein 658–674 (2007). solution with 2 ml reservoir solution containing 18% PEG MME 2k, 12% glycerol, 27. Kleywegt, G. J. & Jones, T. A. xdlMAPMAN and xdlDATAMAN – programs for 40 mM MES, pH 6.2, and 160 mM KCl. reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr. D 52, 826–828 (1996). Data collection and structure determination. Before data collection, crystals 28. Adams, P. D. et al. PHENIX: building new software for automated crystallographic were equilibrated against reservoir solution containing 20% glycerol and then structure determination. Acta Crystallogr. D 58, 1948–1954 (2002). flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K on the 29. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for XO6SA beamline at the Swiss Light Source and the i911-3 beamline at MAX-lab. building protein models in electron density maps and the location of errors in Data were indexed, integrated and scaled with the XDS-package25. Initial phases these models. Acta Crystallogr. A 47, 110–119 (1991). were calculated by molecular replacement using the program PHASER26. The 30. Kleywegt, G. J. & Jones, T. A. Halloween... Masks and Bones Æhttp:// structures of IF–Cbl (PDB accession 2PMV) and human neuropilin-2 (PDB xray.bmc.uu.se/gerard/papers/halloween.htmlæ. accession 2QQL) were used as search models. Owing to the three-fold non- 31. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystallographic symmetry (NCS), a test set in thin shells was chosen with crystalline state. J. Mol. Biol. 372, 774–797 (2007). DATAMAN27. The model was refined using iterative cycles of refinement in 32. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK:a program to check the stereochemical quality of protein structures. J. Appl. PHENIX28 followed by model building in the program ‘O’29. Both B-factors Crystallogr. 26, 283–291 (1993). and atomic coordinates were restrained by tight three-fold NCS with one 33. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific) Æ http:// B-factor group per residue throughout the refinement procedure. Electron www.pymol.org/æ (2002). density maps are shown in Supplementary Figs 12 and 13. Three-fold real space 34. Bond, C. S. & Schuttelkopf, A. W. ALINE: a WYSIWYG protein-sequence averaging of electron density maps was done with AVE30. PISA31 and alignment editor for publication-quality alignments. Acta Crystallogr. D 65, PROCHECK32 were used in structure analysis, and validation and figures were 510–512 (2009).

BEAUTY CONTEST For those who don’t mind researching the tints, smells, sheens and softeners that contribute to everyday cosmetic products, the industry can offer a rewarding career path. Alaina Levine reports.

ost science-minded children He disagrees, suggesting that the technical & Henkel. “There’s a lot of don’t dream of a career challenges in, say, the chemistry of hair care innovation,” Conway says, noting studying shampoo or designing often resemble those in pharmaceuticals. the potential to make frequent Mdeodorant. But for many What are these challenges? In the hope discoveries that contribute to scientists who make the personal-care of repairing or maintaining hair health, new products. At P&G, for example, and cosmetics industry their intellectual scientists such as Shiel are researchers designed an playground, applying a knowledge of trying to understand the electrostatic spray to ensure molecules and cells to making people smell process by which hair grows, liquid make-up is applied and look good is rewarding as it is a way its three-dimensional protein uniformly. Another team, to craft products that the general public structure and its chemical and studying the chemistry and appreciates. Of course, there are downsides. physical properties. Cosmetic surface properties of mascara The push to release new products and the chemists also work on and lashes, helped develop tricky regulatory landscape can be onerous maintaining product stability. a plastic mascara brush that at times. And those hoping to return to “Most cosmetic products have delivers an even flow and limits academia one day may have difficulty. things in the bottle that don’t clumps. Conway says her work And then there are the sneers from other want to be together, such as oil with cosmetics stopped her researchers who don’t exactly have the utmost and water. By thermodynamics, leaving industrial chemistry respect for cosmetics research. “My peers they want to separate,” notes “We’re partnering altogether. She had been think that it’s a big joke that I do cosmetics,” Randall Wickett, director disillusioned with previous jobs says Robert Lochhead, professor of polymer of the cosmetic science more and more in which she had tested waste science in the school of polymers and high- programme at the University of with academics water and blood products. performance materials at the University of Cincinnati and vice-president on research.” Having become fascinated Southern Mississippi, and president of of the SCC. But a product — with the chemistry of altering the industry organization the Society such as shampoo, mascara — Steve Shiel viscosity and researching skin of Cosmetic Chemists (SCC). Often, or moisturizer — has to benefits, she enjoys developing he says, those critics speak out of remain stable inside its container without everyday products. “It’s never become boring ignorance, not understanding the real separating for up to five years and in to me,” Conway says. science that goes on. harsh environmental conditions, such as Steve Shiel, scientific director freezing weather. A foundation for make-up of global hair care and grooming at Such challenges suggest that Starting a research career in the personal- Proctor & Gamble (P&G) Beauty and cosmetic science is a science like care industry requires a bachelor’s degree Grooming, recalls how, early in his career, any other, says Mary Conway, in science at minimum, but a PhD or his colleagues in big pharma said he was R&D director of hair-care master’s will greatly aid progression within

wasting his scientific training on make-up. development for US Schwarzkopf companies, says Rob Snyder, owner of Aristo ULVE/CORBIS KIRSTEN BY ILLUSTRATIONS

4452-45352-453 JobsJobs FeatFeat - CosmeticsCosmetics MH.inddMH.indd 452452 111/3/101/3/10 117:50:517:50:51 NATURE|Vol 464|18 March 2010 CAREERS

Creative Consultants, a long-time the major players in a company, but also the having their scientists reformulate products recruiter for personal-care, areas of science where the firm has focused to remove formaldehyde. They feared a cosmetics and drug companies. its resources, notes Jeni Thomas, a senior consumer backlash, whether warranted or Graduates with degrees in scientist at P&G Beauty & Grooming. not. The removal of ingredients because of chemistry and chemical engineering, There are also opportunities what Lochhead calls ‘misinformation myths’ the largest group, tend to focus on areas for scientists on the marketing creates “busy work for scientists” that can such as toxicology, formulation, and communications side of be frustrating. “More than annoying, it’s an clinical trials, quality, beauty care. Shiel, for example, economic waste of intellectual resources,” claims, regulatory affairs circles the planet translating he says. Industry regulatory hurdles and and process development and P&G’s science into user- numerous lawsuits over patent rights can manufacturing. Biologists work friendly product materials for also be frustrating: short product lifecycles in areas such as skin care. More different audiences, mean little time to file patents and more companies are looking from Walmart customers to before a product goes to for expertise in genomics, bioinformatics dermatologists to hair stylists. market, which increases the and biotechnology, as these fields become As part of his efforts to reach chances that ideas will be embedded in cosmetics product design. a global audience, he recently stolen. This does, however, Physics-trained professionals are sought after appeared as the main guest on a create company positions in in specialist areas such as shaving, where an Pakistani TV talk show during intellectual property, either understanding of friction is key. which he spent 45 minutes as patent attorneys or as And it’s not just the big consumer fielding questions about the scientists supporting the firm’s corporations that hire scientists to research causes of dandruff. There are technology transfer. rouge. Supplier firms such as BASF and communication challenges, he Some scientists worry that Dow Chemical value chemists, physicists says, pertaining to how different a career in cosmetics research and chemical engineers to do chemical cultures perceive, and therefore is a one-way ticket, making formulations, test, find ingredients from all treat, the flaky white stuff. As a “There’s a lot of a move back to academia all over the world and perform regulatory and scientist-communicator, he tries innovation. It’s but impossible. Lochhead, intellectual-property work. Those with a to be aware of cultural nuances never become who joined the University of background in environmental science may associated with beauty care so Southern potentially apply their knowledge to the areas that he can effectively educate boring to me.” Mississippi of regulation and safety. the public about product — Mary Conway after more Backgrounds certainly vary. Bridget benefits. than 20 years in the Newman made a transition from Aveda’s Newman loves industry, planned his return researching snails’ reproductive habits as the quick pace of well in advance. He recommends an undergraduate to studying skin care her job. “If you staying engaged with academic colleagues, at Aveda, where she works in the claims screw up, you gotta attending non-industry science conferences and performance laboratory. In her lab, a fix it right away,” she says. The rapid velocity and giving talks on topics outside the machine holds a comb up to a tress of hair, at which the sector moves may be a surprise cosmetics arena. Demonstrable teaching mechanically stroking it to test for inter-fibre to some academics. “If you’re not someone skills, a good publishing record and a friction. Newman, who earned her bachelor’s who can jump from question to question, penchant for garnering grants will all help degree in biology in 2008 from St Catherine then industrial science is not for you,” says Jay with a potential transition. Both Lochhead University in St Paul, Minnesota, works to Tiesman, principal scientist and genomics and Wickett taught courses at the University substantiate claims associated with finished group leader in P&G’s biotechnology division. of Cincinnati while toiling in industry. products, prototypes and raw materials Shiel, though, believes that the academic — backing up, for example, a company Cosmetic caveats world has begun to appreciate the rigour of claim that texturizing cream increases hair But that pace has a downside. Because cosmetic science. “We’re partnering smoothness by 50%. She wasn’t sure of her product lifecycles are so fast — often more and more with academics on next step after college. But after Newman moving from concept to consumer research,” he says, noting a recent posted her résumé on an online employment within six weeks to three months — collaborative clinical study site a temp agency contacted her about a scientific inquiry may be sacrificed with university scientists position. “You just don’t realize how much in order to deliver a product to the that compared the benefits science is in everyday products,” says marketplace quickly, says Lochhead. The of two skin regimens on Newman. hardcore scientists who truly love their craft wrinkle reduction. But many jobs are not advertised. usually won’t have time to study a product’s Even in the recession, “It’s who you know, not what you underlying mechanisms. If a product doesn’t cosmetic companies are still hiring, albeit in know,” says Snyder, who advises work, they swap ingredients for others that smaller numbers, says Snyder — although attending meetings of are known to work, rather than conducting he admits that 2009 was his industry’s worst the SCC and finding an a detailed examination. “This impinges on year ever for placing job seekers. The hiring internship, possibly via your creativity,” says Lochhead. process is slower, but the industry is bouncing listings on the websites Sometimes ingredient swapping is a back, Snyder suggests. “The industry tends of consumer product reaction to a health scare, which can be to weather bad economic times better than companies and suppliers. frustrating. For example, a news report most,” reckons John Bailey, executive vice- Internships at large companies once indicated that formaldehyde can president of science and chief scientist for such as P&G or Johnson & Johnson can cause cancer — and very small amounts of the Personal Care Products Council, a trade be lucrative, says Lochhead, and usually formaldehyde are used as a preservative in group for companies. “People always want to last about 10 weeks in the summer. Job many products, including shampoo and look their best.” ■ seekers may also want to peruse a company’s various cosmetics. Once the public heard Alaina Levine is a freelance writer based in publication record. One can learn not only the report, companies quickly responded by Tucson, Arizona.

4452-45352-453 JobsJobs FeatFeat - CosmeticsCosmetics MH.inddMH.indd 453453 111/3/101/3/10 117:50:557:50:55 FUTURES NATURE|Vol 464|18 March 2010 Expectancy theory Wishful thinking.

Ananyo Bhattacharya ourselves as hairless monkeys desperately

trying to cope with a somewhat oversized JACEY I conceived then that, irrespective of the brutal brain, and as a result we also abandoned history of our species and the multifarious the field of sociobiology, with its less than dark, disturbing truths revealed to us in the flattering findings about human nature. natural sciences by studies of the human In their place, we have adopted a more mind and instincts, the world was perfectible. edifying view: that we are beings designed Not through a long, desperate and tenacious by an all-powerful intelligence. As a result struggle against our own fell natures but of certain shortcomings of the human simply because a large enough number of brain, which we are trying very hard to well-intentioned folk wished it to be. wish away at the moment, the nature of — Jacques Monad, Journals Vol. III this entity remains somewhat nebulous. (2003–2006) We, as a people, aided by the exception- ally wishful thinking of a few radical femi- Expectancy theory, the scientific hypothesis nists, also went on to free ourselves of all that ended science and permanently gender differences. We no longer have sex- changed the lives of every member of the ual organs, choosing to divide whenever human race, grew out of a single line of we feel the urge to reproduce. The umbili- mathematics scribbled down hastily in the cus, a vestigial reminder of our undigni- journals of the former sociologist Jacques fied past, became our chief pleasure-giving Monad. organ. We can now attain orgasm at any At the time of his groundbreaking work, theory’s publication history time of the day or night simply by prod- Monad was a 43-year-old tax accountant is itself notorious. Rejected by ding ourselves in the navel. living in Basingstoke, but he claimed he Nature as “a cock-and-bull story that could In fact, in the light of Monad’s equations, had drawn the rule out of the surveys he only have been dreamed up by a madman”, we had to revise the history of science com- carried out as a PhD student at the Univer- it was swiftly accepted the following week pletely, as space and time had warped to fit sity of Liverpool. Although he spent four by Science, which in an editorial referred to the hypotheses of physicists. It seemed that years conducting research, his doctoral it as “a landmark”. science had been a social construct in a way thesis — ‘Study of the effects of alcohol The theory is simple enough but many that not even the most devoted advocates consumption on the behaviour of single fail to grasp it immediately because of its of science studies had comprehended. The females: an examination of contemporary counter-intuitive implications. The expect- Universe began to run more like clock- Merseyside mating rituals’ — was never ancy index e, in particular, requires further work after Newton wrote down his equa- completed. Monad was thrown out of the explanation. It is a measure of the effect tions of motion. In retrospect, expectancy university amid claims, always denied by a human mind can have on the course of theory provides the only explanation of him but, oddly, never contested formally, natural events by wishing them to be a cer- why Einstein’s preposterous ideas came to of scientific misconduct. tain way. In most situations it is, of course, be borne out by experiments. After all, he The simple law he formulated — the law negligibly small. It was found, however, had remarkable charisma. of modified probability — still lies at the that in certain circumstances, if enough Recently, we have abandoned the old heart of even the most complex papers of people wish for something to be true, the systems of governance, which at their most expectancy theory. It can be stated math- probability that it becomes true rises sig- absurd led to a confederacy of unelected oil ematically as follows: nificantly. This is because e is a cumulative barons ruling the richest and most power- quantity. Thus if three people, A, B and C, ful nation on Earth. Instead we operate by a

P(x) + eP(x) = Pr(x) (1) wish for the same event, the total expect- new principle: we, the people, get what we ancy index eT is a sum of their individual want. We are proud of this new arrange- where P(x) is the probability of an event x expectancy indices: ment and have named it ‘democracy’. occurring in the absence of anyone hoping And what has become of Jacques

that it will happen, e is the expectancy index eT = eA + eB + eC (2) Monad? He has joined the ranks of the and Pr(x) is the actual probability of the immortals. Quite literally, as we all now event occurring once the full expectation When the total expectancy is a positive live forever. Monad has wished into exist- of event x has been taken into account. value, Equation 1 becomes: ence a replica of the mansion built by the It is possible that Monad himself did not late-twentieth-century media mogul Hugh

perceive the true significance of what he Pr(x) >> P(x) (3) Hefner. It is complete in every detail. Puz- wrote that night. In an interview shortly zlingly, he still chooses to reproduce in the after his theory was published, Monad said This astonishing result has profoundly old-fashioned way. ■ that the law came to him after he had had changed the way we think about the world Ananyo Bhattacharya is Nature’s online “a few too many one evening”. A few months and ourselves. Science has largely been dis- news editor, where he wishes that great later, Monad seemed to have changed his carded as the cheerless product of unim- stories would arrive perfectly formed mind. “It was divine inspiration,” he said. aginative minds. The theory of evolution, a every day. “I was truly touched by the hand of God.” A particularly diseased example of the genre, Join the discussion of Futures in Nature at statement he was to repeat many times. The was trashed first. Now we no longer regard go.nature.com/QMAm2a FUTURES 456 © 2010 Macmillan Publishers Limited. All rights reserved

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