or the past 25 years, a pair of Dartmouth regular daily rhythms. The most obvious example is researchers has been trying to figure out its growth cycle. To study Neurospora’s develop- F how a fungus tells time. Along the way, ment, researchers in the Dunlap-Loros lab intro- Jay Dunlap, Ph.D., and Jennifer Loros, Ph.D., have duce it into thin, 15-inch-long glass containers helped to explain one of biology’s fundamental mys- called race tubes and provide it with enough food teries—how organisms, including humans, have to fuel growth. The fungus spends most of the day adapted to the 24-hour cycle that we call a day. spreading down the length of the tube, a few mil- During the time it takes for the Earth to rotate limeters per hour. But around midnight, it starts to on its axis once, the average person experiences a lay down cells that can produce spores. Over the host of physiological changes. Your body tempera- next several hours, as Neurospora continues to grow ture rises from a low of about 97 degrees to a high horizontally, these cells will simultaneously grow of about 99 degrees, then falls again. Your short- vertically, producing orange spores that rise above term memory peaks just after you wake up, while the mass of the fungus on the bottom of the race your athletic performance improves in the late af- tube. By mid-morning, the spores have matured and Clock-wisePhotographs by Jon Gilbert Fox Text by Amos Esty

ternoon. And after midday, as drowsy workers in natural conditions could be picked up by a This pair of Dartmouth around the world can attest, a siesta starts to seem breeze. At this point, Neurospora stops producing very appealing. spores, growing only horizontally until the clock researchers has become These changes, and many more, are regulated again strikes midnight. by an internal circadian clock (“circadian” comes If left for a week, Neurospora will continue this increasingly wise to the from the Latin words for “about a day”). This clock same pattern, spreading horizontally for most of the is powered by a series of biochemical reactions that day and then producing spores for several hours ear- ways of the circadian clocks take place within individual cells. In other words, ly each morning. At the end of the week, the race Dunlap says, “you wake up in the morning because tube will contain seven orange spots where the that control the daily of things that are going on inside single cells.” spores are clearly visible, each separated by an inch Long before the field of circadian biology exist- or so of horizontal growth. routines of virtually every ed, close observers of nature realized that many or- Like the heliotrope, Neurospora exhibits this ganisms exhibit daily rhythms. In 1729, French sci- cycle even when it’s kept in the dark. The length organism on Earth—from entist Jean Jacques d’Ortous de Mairan wondered of the cycle changes slightly—from 24 hours to why the leaves of a plant in his garden—a helio - about 22 hours—but the rhythm remains the human beings to the simple trope—opened during the day and closed at night. same. And that raises the question: how is this The obvious explanation was that the heliotrope simple organism able to tell time? fungus that they study. was responding to the sun. But when de Mairan dug up the plant and placed it in a dark room, he found unlap now expounds enthusiastically that it continued to follow the same regular pat- about this question, but circadian biolo- tern, despite the lack of light. So the sun couldn’t D gy was not a field that he originally in- be causing the changes. Charles Darwin, too, was tended to enter. As an undergraduate, he majored intrigued by leaf movement, a topic that he dis- in oceanography. He then enrolled in graduate cussed at length in an 1880 book titled The Power school at Harvard, planning to get a degree in bio- of Movement in Plants. chemistry and apply it to marine life. Early in his The fungus Neurospora crassa (pronounced nuh- study of biochemistry, he came across tiny creatures RAH-spuh-ruh CRASS-uh) is the focus of much of called dinoflagellates. Some species of these single- Dunlap and Loros pose in front of a the research carried out by Dunlap and Loros. As do celled organisms can produce flashes of light— genetic map of Neurospora crassa, humans and heliotropes, it exhibits a number of something like seafaring fireflies. Their biolumines- the fungus whose biological clock cence is the result of a reaction between a number they have studied for 25 years. Amos Esty is the managing editor of Dartmouth Medicine. of proteins, and these proteins are present in much

38 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2009 Fall 2009 online at dartmed.dartmouth.edu—Dartmouth Medicine 39 made available by the revolution taking place in “You could see detectable progress, and there were molecular biology—tools that made it easier to dis- ‘eureka’ moments.” sect the inner workings of cells. It was also while he Cloning frq allowed Dunlap and Loros to try to was a postdoc that he met Loros. nail down the gene’s exact role in Neurospora’s cir- Jennifer Loros was a graduate student at UC- cadian clock, which turned out to be a big step to- Santa Cruz, and, like Dunlap, she started her scien- ward answering the first question posed by Dun- tific career with a very different focus from circadi- lap—how the clock runs at the molecular level. an rhythms. “I’m a gardener,” she says. “That’s ac- tually how I got into science.” She’d planned to orking with postdoc Benjamin Aron- study hormones in plants, but, she says, “I ran into son, Ph.D., Dunlap and Loros discov- sort of a wall with my first Ph.D. project and end- W ered that the expression of frq is turned ed up changing laboratories to work in circadian up at some points during the day and down at oth- Above, these tubes show Neuros - rhythms. And I’ve been doing it ever since.” ers, following a regular 22-hour cycle. Simplified, pora’s growth over the course of a Early in 1984, Dunlap took a position as an as- there are two basic steps in gene expression: tran- week; the spots mark the few hours sistant professor at Dartmouth Medical School. scription, in which DNA is transcribed into RNA, each day when the fungus produces Grad student Chen-Hui “They were looking for someone who was doing and translation, in which RNA is translated into spores. At left, this illustration Chen is one of many something different,” he recalls. At the time, circa- proteins. The researchers found that frequency pro- from a book by Charles Darwin dian rhythms certainly fit that description; in fact, teins limit the production of frq RNA—in other students and postdocs shows early awareness of circadian some scientists still weren’t convinced of the mer- words, the translation part of the process feeds back who have worked in rhythms; the leaves of this plant, its of circadian biology and didn’t think studying a to regulate transcription. When frq expression did the Dunlap-Loros lab. fungus could lead to a better understanding of cir- not fluctuate—for example, when Neurospora was Cassia corymbosa, are open during cadian clocks in humans. There was another factor manipulated so that frq RNA levels remained con- the day (left) but curl up at night. in the decision, as well. “It was the only job I got,” stant—the fungus no longer followed the 22-hour The DMS researchers concluded that circadian clocks work as a negative Dunlap says. “That made the choice easy.” cycle of horizontal growth and spore production. In 1994, Dunlap and Loros published their results in another top journal, For a time, Loros remained in California to fin- Instead, it produced spores constantly. Clearly the feedback loop. An example often given of a negative feedback loop is a ish her dissertation. But that fall the two married, cycling of frq expression was central to the proper Science, noting similarities between their findings and the circadian clocks household thermostat. Too much heat causes the furnace to turn off, which and in January 1985 Loros joined Dunlap’s lab as his functioning of the clock. of fruit flies. Studying a fungus was by then looking like a very promising first postdoc. They have since shared lab space, as Based on their findings in Neurospora, and the allows the temperature to drop, which makes the furnace turn back on. well as authorship on most of their papers. Within work of other researchers in fruit flies, Dunlap and way to find out how circadian clocks work in more complex organisms. a few years, Loros became a fellow member of the Loros inferred that circadian clocks work as a neg- greater amounts at night than during the day, when faculty. Now, their offices lie on opposite ends of ative feedback loop. An example often given of a Over the past 15 years, Dunlap and Loros have the flashes of light would be overpowered by the the same large lab and they run lab meetings to- negative feedback loop is the regulation of house- continued to add more pieces to this puzzle, result- sun. Somehow, these dinoflagellates are able to gether, but they are both independently funded hold temperature. If a thermostat is set to 68 de- ing in a detailed understanding of Neurospora’s cir- schedule their use of bioluminescence to maximize principal investigators. grees, it will turn on the furnace when the temper- cadian clock. Along with frq, two other cellular its effectiveness. “The original plan was not necessarily to work ature drops below that point, producing heat. Once components form the heart of the negative feed- together the rest of our lives,” Loros says. “But I the heat raises the temperature above 68, the ther- back loop—the proteins white collar-1 and white unlap wondered how these organisms helped set the lab up while he was writing grants.” mostat turns off the furnace, until the temperature collar-2 (named for the effect that mutations in could possibly know to expend energy to In those grant proposals, Dunlap outlined three again drops below 68. With help from the furnace these genes have on Neurospora’s appearance). To- D generate light only at night. No one he bold lines of research. He wanted to know how cir- and the thermostat, heat regulates itself—too much gether, white collar-1 and white collar-2 form a talked to had an explanation. “It was clear this was cadian clocks work at the molecular level, how heat causes the furnace to turn off, which allows transcription factor for frq, meaning that when they a problem where no one had any clue as to how it clocks are affected by external cues (such as light), the temperature to drop, which makes the furnace bind to that gene, they turn it on, leading to the worked,” Dunlap says. “And, as far as I could tell, and how the clock shapes Neurospora’s behavior by turn back on to produce more heat, and so on. The production of frq RNA. they weren’t even pursuing it in the ways in which regulating gene expression. expression of the frq gene plays a role in Neurospo- you’d have to pursue it in order to get an answer. So A second postdoc, C. Robertson McClung, ra’s circadian clock akin to that of heat in keeping n the hours after midnight, this transcription I saw it as a great problem.” Ph.D., soon joined the lab. “When I came to Jay’s a house at the right temperature. factor gets to work, prompting the expression Dunlap decided to take on circadian rhythms, lab, I wasn’t necessarily thinking that I would be- In 1994, Dunlap and Loros published the results I of frq. By mid-morning, there’s an abundance but in Neurospora, an organism that is widely used come a chronobiologist,” says McClung, who is of this research in another top journal, Science, out- of frq RNA available in Neurospora’s cells. Eventu- for genetic studies. He went on to a postdoctoral now a Dartmouth College professor of biology. lining the basic steps of the negative feedback loop. ally, the frq RNA is translated into frequency pro- position in the lab of Jerry Feldman, Ph.D., at the “But the problem, as Jay explained it to me, was They also pointed out similarities between their teins. These proteins provide negative feedback by University of California at Santa Cruz. Feldman just beautiful.” findings and the work of scientists who were study- blocking the transcription factor from binding to had earlier identified a Neurospora gene called fre- Working together, Dunlap and McClung suc- ing the circadian clocks of fruit flies. The similari- the frq gene. As a result of this obstruction, the pro- quency (frq). Feldman found that strains of Neu- ceeded in cloning the frq gene, opening up new ties, they wrote, suggested that negative feedback duction of frq RNA slows down in the late morn- rospora with a mutated form of frq display abnormal ways of investigating the role of the gene in the cir- loops “may be a universal feature of circadian oscil- ing and early afternoon. The frq RNA that has al- circadian rhythms, indicating that the gene must cadian clock. The breakthrough earned them a spot lators.” Studying a fungus was by then looking like ready been produced continues to be translated, play some role in the organism’s clock. in the prestigious journal Nature. a very promising way to find out exactly how circa- which means that protein levels reach their peak in As a postdoc, Dunlap encountered new tools “It was tremendously exciting,” McClung says. dian clocks work in more complex organisms. the mid-afternoon or early evening—hours after

40 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2009 Fall 2009 online at dartmed.dartmouth.edu—Dartmouth Medicine 41 possible for a single cell to contain all the compo- set—if it consistently followed a 22-hour growth 100 frq RNA nents of a circadian clock. (In fact, there are sever- cycle? At first, it might not make much difference. frequency proteins al other pieces that add still more complexity to the But within about a week, the fungus would produce picture.) But this explanation does not account for spores at noon, when the sun is most intense. The one of the fundamental properties of circadian spores are vulnerable to ultraviolet rays and so less white collar-1 clocks: when an organism is kept in constant dark- likely to survive if left to bake in the sun, which ness, its circadian clock does not stick to a strict would reduce Neurospora’s ability to spread. 24-hour cycle. In Neurospora, the cycle reverts to 22 Producing spores costs Neurospora in energy ex- hours; in humans, it’s between 24 and 25 hours. A penditure, but the spores are an essential part of its related issue is the fact that, given a few days, it’s life cycle. So it makes sense that, over evolutionary SPORE PRODUCTION possible to adjust to very different day-night cycles, time, Neurospora has developed a rhythm that en- RELATIVE LEVELS STARTS such as when traveling across an ocean. hances the odds that spores survive. Dunlap cau- Dunlap, at left, is a member of the The second challenge posed by Dunlap in his tions that this explanation for Neurospora’s devel- National Academy of Sciences, the early grant proposals—how circadian clocks are af- opment pattern hasn’t been proven experimental- premier U.S. scientific body. He and 0 fected by external factors—targets these questions. ly. But he notes that studies in other organisms, in- Loros—who is pictured above with 0 HRS 6 HRS 12 HRS 18 HRS 24/0 HRS 6 HRS Again, he and Loros deserve much of the credit for cluding plants and cyanobacteria, have shown that their dog, Fiona—collaborate 24/7. (dawn) (noon) (dusk) (midnight) (dawn) (noon) finding the answers. adhering to a cycle in tune with the Earth’s rotation They were married in ’84, she TIME Progress in this area resulted from the work of does provide fitness advantages. became a postdoc in his lab in ’85, This graph shows fluctuations in Neurospora’s relative levels of frq RNA, frequency Susan Crosthwaite, Ph.D., a postdoc in Dunlap and and they’ve shared lab space ever proteins, and white collar-1 proteins over the course of a little more than one circadian Loros’s lab, and Allan Froehlich, Ph.D., who his leads to Dunlap’s third challenge—fig- since, though they’re both now cycle; day and night are indicated by the white and black portions of the bottom bar. worked in the lab as both a graduate student and a uring out which parts of Neurospora’s postdoc. Crosthwaite’s research examined how light T physiology are regulated by the circadian independently funded investigators. affects the expression of frq, and Froehlich built on clock and how the clock exerts this control. Those Say a Neurospora cell catches a morning flight from Paris to New York. her findings by outlining the “nuts and bolts,” as he are questions Loros has devoted much of her career There are now more than 180 Neurospora genes confirmed to be under puts it, of how light resets Neurospora’s circadian to investigating, ever since she was a postdoc. When it arrives in mid-morning, the cell feels as if it’s mid-afternoon; if it clock at the molecular level. While Dunlap and McClung worked to clone the control of the clock, but there are still many questions about exactly had stayed in Paris, its frequency proteins would be starting to decline. Essentially, white collar-1 plays the role in Neu- frq, Loros tried to identify genes that are controlled how the clock regulates genes. But whatever the exact mechanism, rospora’s clock that eyes play for humans—it recog- by the clock. She used both a normal strain of Neu- But the extra hours of daylight in New York adjust its circadian clock. nizes light. When that happens, the white collar rospora and a mutated form—called frq7—that has it has become clear that circadian clocks affect a wide range of behaviors. proteins become more active as a transcription fac- a 29-hour cycle. She measured the amount of RNA the amount of frq RNA goes into decline. Over tor, triggering the production of frq RNA, regard- present in Neurospora cells at different times of day confirmed to be under the control of the clock, but time, frequency proteins are broken down by en- less of the time of day. and found two genes whose expression follows the there are still many questions about exactly how zymes, diminishing their numbers. And with no re- As an example, say a Neurospora cell catches a same rhythm as Neurospora’s internal clock. Unlike the clock regulates genes. In some cases, Loros says, maining reservoir of frq RNA waiting to be trans- morning flight from Paris to New York. When it ar- the frq gene, these newly identified genes were not the white collar proteins might directly determine lated, protein levels continue to decline. rives, it will be mid-morning in New York, but the part of the clock itself; they could be knocked out whether another gene is expressed. In other in- cell—still on Paris time—will feel as if it’s mid-af- without affecting the functioning of the clock. stances, they might be the start of a longer cascade, ere’s where it gets complicated: in addi- ternoon. If it had stayed in Paris, the cell’s level of In 1989, Loros published this research in Science, triggering the expression of one gene that in turn tion to blocking the expression of the frequency proteins would be starting to decline, coining the now widely used term “clock-controlled causes the expression of another. But whatever the H frq gene, frequency proteins also pro- reaching a low point about eight hours later. gene” (ccg) to describe such genes. In subsequent exact mechanism, it has become clear that circadi- mote the translation of white collar-1 RNA into Instead, the cell is exposed to several extra hours years, she worked with postdoc Deborah Bell-Ped- an clocks affect a wide range of behaviors. “In Neu- white collar-1 proteins. (White collar-2 levels re- of daylight in New York, and its circadian clock ersen, Ph.D., to try to figure out what those genes rospora, like in people, the clock controls an awful main constant through the day, but white collar-1 starts to adjust. White collar-1 senses light, prompt- do. They found that ccg-2 produces a protein that lot of the physiology of the cell,” Dunlap says. fluctuates.) The more frequency proteins there are, ing the white collar proteins to continue producing coats the outside of spores, making them repel wa- the more white collar-1 proteins are produced. But frq RNA. As a result, frequency proteins are pro- ter. In moist environments, this protein helps spores ome of those effects have implications for when frequency protein levels are high, it’s hard for duced for several hours more than would have been survive. In strains with an inactive ccg-2 gene, the human health. In 2006, António Preguei - white collar proteins to act as a transcription factor, the case had the cell remained in Paris. spores clump together, making it harder for them to S ro, Ph.D., then a graduate student, cloned leaving all that white collar-1 and white collar-2 If a Neurospora cell travels in the opposite di- be dispersed by the wind. a mutation in the Neurospora gene period-4, which sitting around waiting for something to do. rection, leaving New York at night and arriving in Loros and Bell-Pedersen also identified a num- is a homologue of checkpoint kinase-2, a tumor sup- Late at night, this process reaches a tipping Paris at dawn, the exposure to light earlier than ex- ber of additional clock-controlled genes not relat- pressor gene in humans. Checkpoint kinase-2 stops point. There are no longer enough frequency pro- pected will cause the white collar proteins to ramp ed to Neurospora’s growth cycle. Bell-Pedersen says the cell cycle when DNA damage occurs. teins to block the white collar proteins from bind- up their activity several hours ahead of schedule, that these findings added to the evidence for the Pregueiro’s finding linked circadian clocks to the ing to the frq gene, which means frq RNA will again again resetting the clock. This same process hap- importance of circadian rhythms. “We’d known for cell cycle, showing that damage to a Neurospora be produced, leading to the production of frequen- pens on a small scale every day, lengthening Neu- a long time that the clock regulates development, cell’s DNA can reset that cell’s circadian clock— cy proteins. The cycle starts all over again. rospora’s innate circadian rhythm from 22 to 24 but it wasn’t clear what other processes in the cell just as changes in light exposure can. Many cells By identifying these pieces and putting them to- hours to keep it in line with the Earth’s rotation. were affected by the clock, if any,” she says. divide only at a specific time of day. If DNA dam- gether, Dunlap and Loros proved that it is indeed But what if Neurospora’s clock couldn’t be re- There are now more than 180 Neurospora genes continued on page 62

42 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2009 Fall 2009 online at dartmed.dartmouth.edu—Dartmouth Medicine 43 Clock-wise Neurospora at the absolute cutting edge of clock research.” ROGER CLARKSON REALTORS continued from page 43 Today the study of circadian rhythms no age occurs, resetting the clock might keep a longer qualifies as “something different,” in WillowBrook Prosthetics & Orthotics Serving the Hanover area with sales, investments, and rentals since 1975 cell from dividing at a time when a mutation part due to all the researchers who have is likely to result, thereby preventing muta- passed through the Dunlap-Loros lab. Bell- tions that could lead to tumor growth. Earli- Pedersen, now a professor of biology at Texas er this year, postdoc Joshua Gamsby, Ph.D., A&M, says Dunlap and Loros take a strong reported similar results in mouse cells, prov- interest in promoting the careers of their grad ing that in mice, as in Neurospora, DNA students and postdocs. They “gave us a lot of damage can reset the clock. freedom to really test ourselves and make These findings add to the growing recog- sure that we were cut out for this field,” she nition of the relationship between the circa- says. “It was a great experience.” dian clock and cancer. “We are, as a field, Froehlich, a senior scientist at a biotech now understanding that the clock is very im- company, also credits Dunlap and Loros for Mountaintop Hideaway • Minutes to Dartmouth-Hitchcock Medical Center portant in a number of different cell-cycle helping develop new generations of scien- events,” says Loros, “and that misregulation tists. “The training in that lab set me up well With 57 acres of privacy, views, and southern exposure. in clocks often results in cancer.” for what I’m doing now,” he says. Priced below appraised value at $765,000. In one study, mice lacking the gene Per2, Giles Duffield, Ph.D., spent time in their A smaller full-service agency representing buyers a key clock component in mammals, were lab as a postdoc before leaving to join the bi- more likely to develop tumors. Another ology faculty at Notre Dame. “When people and sellers of Upper Valley properties. study, done in Denmark, found that women leave the lab, they know how to do good sci- We love helping people, and we love referrals from clients and friends! who work at night had a greater risk of breast ence,” he says. Of course, he adds, “many Robert Diebold, C.O., Orthotist • David Loney, C.P., Prosthetist cancer than woman who work day shifts. people don’t want to leave, because it’s such CONTACT US AT 603-643-6004 OR VISIT www.rogerclarkson.com And a number of studies have shown that a nice environment.” 190 Hanover Street • Lebanon, NH • 603/448-0070 38 South Main Street, Hanover, NH 03755 timing chemotherapy to take advantage of Providing the researchers in their lab with circadian cycles can improve outcomes. the freedom to develop their own ideas has been a conscious decision for Dunlap and he complexity and importance of Loros. “We give people an enormous amount the circadian clock explains why of independence,” Loros says. T it’s held Dunlap and Loros’s inter- “We rarely tell people what to do,” Dun- est for so many years. “Although I’ve worked lap agrees. “We’ll suggest projects when they on clocks my whole life, I feel like I’m con- get here if they don’t come in with some- stantly being thrown into other areas, com- thing they want to do, but almost always pletely new areas of science, because clocks within some months they have either devel- affect such a broad spectrum of an organism’s oped entirely their own ideas or developed biology,” says Loros. additional ideas that are their own.” “Most of physiology is at least indirectly Loros points out that much of their own under clock control,” Dunlap adds. “It bears success is due to the hard work of these up- on all of biology, and you work on it at the and-coming researchers. “I feel very grateful molecular level. So it’s the perfect mix.” for all the people I’ve worked with over Dunlap, who chairs DMS’s Department time,” she says. “Good science is very often of Genetics, has earned numerous honors a collaborative effort.” over the years. He was recently elected to the Of course, each one’s most important co - National Academy of Sciences, the country’s worker has always been the other. “In sci- premier scientific body. He’s also received ence, people constantly talk about how it’s many awards, including the Honma Prize, an important to have colleagues at the institute, international award for biological clock re- somebody that you can talk about your work search, and the George W. Beadle Medal of with,” Loros says. “Well, Jay and I just have the Genetics Society of America. a built-in colleague.” Together, they have put McClung says the field owes much to together the puzzle of Neurospora’s circadian Dunlap and Loros. “For a long time, only the clock, constantly learning more about how work of Jay and Jennifer was what was push- significant circadian rhythms are to the well- ing ahead Neurospora as a molecular genetic being of virtually every organism on Earth. model for clockwork,” he says. “They pretty Given their track record, more findings are much have been the key players in keeping sure to come. It’s only a matter of time.

62 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2009 Fall 2009 online at dartmed.dartmouth.edu—Dartmouth Medicine 63