PROFILE

Profile of Steve Kay linking mustard plants and glow- Rockefeller University (New York) lab of ing fruit flies are just a few of the Nam-Hai Chua, who is a leader in the clever tricks that Steve Kay de- study of light-dependent gene expression vised to explore the molecular in plants. B ‘‘We were collaborating closely with genetic basis of circadian clocks in plants, flies, and mammals. Monsanto on constructing some of the Kay, who was elected into the National first vectors for making transgenic plants, Academy of Science in 2008, has spent mostly tobacco and petunias,’’ he said. ‘‘It two decades identifying the photorecep- was very, very exciting.’’ tors, genes, and complex networks that Kay and another postdoc named Ferenc make these internal clocks tick. Nagy began studying the light-activated Kay’s work has revealed applications chlorophyll a/b binding (CAB) gene. They from agriculture to disease. For exam- were trying to discover how plants convey ple, by tweaking one of the clock genes, light signals to the nucleus where they researchers can engineer crops to alter rapidly alter CAB expression level. their growth patterns, ultimately increas- While recording CAB mRNA levels, ing yields and possibly expanding the the two noticed that their results con- geographical range where the plants can flicted. To resolve the discrepancy, they thrive. Circadian clocks are also impor- began conducting around-the-clock experi- tant for how plants deal with stresses ments for several days. such as drought and salinity. ‘‘We discovered that our results were Kay’s work linking the clock to human different because I was doing my experi- health suggests that chemicals targeting Steve Kay ments in the morning, because I’m an the body’s timing system may lead to new early bird, and Ferenc was a night owl and doing his experiments in the treatments for bipolar disorder, diabetes, evening,’’ said Kay. and cardiovascular disease. Although his parents had not pursued college, they were phenomenally support- The finding implied that a circadian Although Kay, dean of biological sci- ive of his interest in science. clock regulated the CAB gene; some ences at the University of California, San ‘‘When you come from generations of mechanism switched it on in the morning Diego, is best known for his work on cir- fishermen,” Kay said, “they realize that and off in the late afternoon. cadian rhythms, his inaugural article (1) is there are better things to do than being ‘‘That [3] was my first clear glimpse of a divergence from his mainstream work frozen in the middle of the ocean. The something called a circadian rhythm,’’ said on circadian networks. North Sea is not the most inviting place Kay. It was 1985 and no one had de- The article is a tribute to his mother, Ͼ in the world, however thrilling!’’ scribed circadian rhythms acting at the who died 2 years ago of a progressive molecular level in any organism. Re- neurodegenerative disease. The article Planted in the Beginning searchers had only recently cloned the identifies a novel E3 ubiquitin ligase as Kay began his studies at the University of period (PER) gene in flies, which con- the culprit behind some types of motor Bristol in the United Kingdom, where he trolled the insects’ 24-h body clock, and and sensory neuron degeneration in a mu- earned a bachelor’s degree in biochemis- researchers had not yet identified such a tant mouse model. try. There he met a Welshman named gene in plants. Seduced by Nature’s Rhythms Trevor Griffiths who would become his Ph.D. supervisor and introduce him to the Blinking Mustard Plants Another of nature’s rhythms inspired world of plants. To examine the idea of a circadian clock Steve Kay’s interest in biology: the ex- Griffiths had observed that plants grow in plants, Kay developed transgenic plants traordinary tides that sculpted his birth- differently in the dark than they do in in which he fused a light-dependent pro- place and childhood home on Jersey, the light. Previous studies had found that moter to the reporter gene LAC Z. This southernmost of the Channel Islands lo- plants stop producing chlorophyll, the key meant that the LAC Z gene was turned cated between England and France. to transforming light into energy, when off and on in a circadian manner. Ͼ On those shores, the sea receded 3 grown in darkness. Kay’s doctoral project When Kay presented his work at the miles at some low tides, he recalled, leav- was to identify and characterize the en- annual meeting of the Society for Re- ing beaches pockmarked with rock pools zyme that catalyzes the light-dependent search on Biological Rhythms in 1988 ‘‘ev- of the English Channel. In this environ- step of chlorophyll synthesis. erybody went absolutely nuts,’’ he said. ment, he discovered ‘‘amazing creatures’’ Kay noticed that the concentration of ‘‘We showed we had a promoter element and other oddities of marine life, and his the enzyme seemed to rise and fall regulated by the clock.’’ interest in science was born. throughout a day–night cycle. Kay’s results were among the first ob- When he was Ϸ9 years old, his life Using molecular biology techniques that servations to suggest that clocks were not changed when a teacher from mainland were just being developed in England and just clusters of neurons in the brain that England brought a microscope to his the United States, he discovered, to his synchronized each other and regulated small elementary school. surprise, that light regulated the expres- sleep/wake cycles. Instead, his findings ‘‘That absolutely blew me away,’’ he sion of the gene that produced the en- showed that an individual cell could dis- recalled. ‘‘I’d never known what was in zyme for chlorophyll synthesis (2). pond water or what the edges of a torn To further pursue this research, Grif- piece of paper looked like.’’ By his early fiths advised Kay to study in the United This is a Biography of a recently elected member of the National Academy of Sciences to accompany the member’s teens he knew he wanted to study for a States. After completing his thesis, Kay Inaugural Article on pages 2097–2103 in issue 7 of volume Ph.D. secured a postdoctoral fellowship at The 106.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910583106 PNAS ͉ October 27, 2009 ͉ vol. 106 ͉ no. 43 ͉ 18051–18053 Downloaded by guest on October 1, 2021 play circadian rhythms and that clocks ing used by the whole circadian field,’’ photoreceptor while screening their mu- could regulate intracellular processes such said Kay. tant flies (7), a discovery Science hailed as as gene expression. one of the top 10 breakthroughs of 1997. The circadian field reacted to Kay’s Flashing Flies Also in 1997, Joe Takahashi, then at work with enthusiasm and excitement. At In 1996, Kay moved to the Center for Bi- Northwestern University (Evanston, IL) the time, he understood the importance of ological Timing at the University of Vir- found the mouse circadian clock gene and his findings and decided to shift his focus ginia (Charlottesville, VA). Along with wanted to find its counterpart in the fly. to plant circadian rhythms. trying to identify all parts of the plant He and Kay teamed up. Later, while still at Rockefeller, Kay clock, he wanted to find more fruit fly Kay and Takahashi’s teams worked to- collaborated with geneticist Mike Young clock genes. To this end, he engineered gether and identified not only the fly to use fly clock genes to search for homo- flies with the luciferase gene. When he CLOCK gene, but the fly partner of logues in plants. When that approach fed these transgenic flies luciferin they CLOCK, a gene called BMAL1. failed, he realized that they had to de- started glowing. ‘‘It is a complete reversal of what nor- velop a method to ‘‘visualize the circadian ‘‘And so we had these Drosophila run- mally happens in biology,’’ said Kay, rhythms in plants’’ and developed trans- ning around that were ‘fire-fruit’ flies,’’ he meaning that in this case a mouse forward genic Arabidopsis plants. said. genetics screen led to the identification of Kay and graduate student Andrew Mil- In collaboration with Jeff Hall at new genes in flies. lar took photos of the Arabidopsis plants Brandeis University (Waltham, MA), Kay continued to tease apart to ulti- every few hours with a sensitive camera. Kay used these flashing flies to search mately define a molecular feedback loop. ‘‘We were just shocked and elated to dis- for clock genes. Kay and Hall discov- The two then demonstrated that CLOCK cover we could see the plants glowing on ered fly mutants that could not be en- and BMAL1 acted to turn on the PER and off in a circadian fashion. That was trained by light; these flies were glowing gene and a gene called TIM. They later definitely a ‘eureka!’ moment,’’ said Kay. all over. This finding led the pair and showed that PER and TIM proteins re- Because the CAB promoter is light- their collaborators to discover a fly ver- pressed their own expression by antago- dependent, the plants glowed early in the sion of a gene called cryptochrome that nizing CLOCK and BMAL1. This was the morning. But when he mutated these Kay’s lab had been working on in plants. first demonstration of a molecular feed- glowing plants, he identified individuals Kay recalled being perplexed by the back loop defining the core mechanism of that glowed out of sync, for example, once glowing flies. Hall and Kay held a the circadian clock (8). every 21, rather than 24, h. ‘‘cranio-centric view,’’ which suggested This work was part of an increasingly The first gene Kay and Millar identified that that the circadian clock rested in the competitive circadian rhythms field. from this screen was TOC1, the first clock brain where it controlled behavior. How- ‘‘There was this amazing meeting in gene in plants (4). It took Kay another 12 ever, when Kay dissected his glowing flies May in 1998, a clock meeting, where Mi- years to figure out what the gene does; it into body parts (heads, thoraxes, and ab- chael Rosbash’s lab, our lab, Takahashi’s is a transcriptional regulator in Arabidop- domens), each part glowed independently. lab, and Chuck Weitz’s lab all got up and sis and is recruited to the promoter of When he grew these insect body parts in described in various versions in mammals other clock genes (5). culture, he could use light to reset the and flies this feedback loop at the molec- The screening method and the discov- rhythmic flashing. ular level, and people were just stunned,’’ ery of TOC1 led to back-to-back papers in These findings showed that clocks were Kay recalled. ‘‘That was incredibly satisfy- Science in 1995 (4, 6). all over the body and predicted that there ing to participate in.’’ ‘‘The luciferase tool became very, very should be a widely distributed local photo- In 1998, Science again cited Kay’s work powerful and, of course, it ended up be- receptor. Kay and Hall identified that as one of the breakthroughs of the year and the circadian rhythms field was work- ing overtime, with discoveries about clock genes appearing almost weekly. By then, Kay had moved his lab from Virginia to the Scripps Research Institute (La Jolla, CA), and his team generated a steady stream of fundamental breakthroughs re- vealing the inner workings of clocks in plants and flies. ‘‘It was really exciting,’’ he said. ‘‘You have to look back at it and say, wow, what a great mix of incredible colleagues, luck, and occasional good judgment.’’ From Flies to Mice In 1999, Kay turned his attention to mam- mals where understanding of the circadian clock was still relatively rudimentary. To facilitate this shift, he established a sec- ond laboratory at the Genomics Institute of the Novartis Research Foundation (GNF) (San Diego). At GNF, Kay and postdoctoral fellow John Hogenesch took advantage of auto- mation and large-scale genomics technol- Kay examining transgenic Arabidopsis plants. Image courtesy of Kim McDonald, University of California, ogy to identify new clock genes. He and San Deigo. his colleagues quickly realized that the

18052 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910583106 Trivedi Downloaded by guest on October 1, 2021 mammalian clock was far more complex appreciate the real impact of a clock most] satisfying work that I’ve ever done and made of multiple loops, not just one mutant, researchers must not only look in my life,’’ Kay said. feedback loop. This discovery turned at behavior, but also at single cell out to be universal in all organisms. phenotypes. A Tribute The technology at GNF also enabled This is because when the cells in the When it comes to Kay’s inaugural arti- Kay and his colleagues to characterize the suprachiasmatic nucleus (SCN), a section cle, he notes, ‘‘It’s one-off, opportunis- novel photoreceptor melanopsin. In 2002, of the brain’s hypothalamus known to tic...essentially a nod to my mum.’’ Kay’s group showed that melanopsin in contain a ‘‘master clock’’ that controls In 2006 his mother died of a very ag- retinal ganglion cells detects light and sig- behavior, are coupled into a network, they gressive motor neuron disease called pro- nals the master circadian oscillator in the are quite resistant to Clock gene muta- gressive bulbar palsy. The disease closely resembled a disease that Kay and a large hypothalamus, which then signals the rest tions. However, if intercellular communi- team of collaborators from Phenomix of the body. This photoreceptor can reset cation is disrupted, such as in dissociated identified and characterized in a mouse the clock (9). neurons or cultured cells, the Clock gene mutant. Kay followed this work in a second col- knockouts generally show dramatic ar- So, with the mouse model, the team laborative article (10) where he and his rhythmic phenotypes (12). proceeded to clone the defective gene, an colleagues showed that the visual photore- E3 ubiquitin ligase named Listerin that is ceptors and melanopsin work indepen- involved in motor and sensory neuron dently to entrain the clock gene. The ‘‘Understanding how degeneration when mutated. article showed that even blind mice lack- ‘‘I had a strong personal motivation to ing rods and cones can detect light, which circadian rhythms follow up on this mouse mutant, inspired then sets their body clock. But when both by the suffering that I saw my mum go the visual photoreceptors and melanopsin interact with other through,’’ he said. ‘‘And, I think, she was are eliminated, the animals become com- very keen that I would contribute some- pletely blind to light/dark cycles. networks could provide thing in this field.” In 2002 Science, once again, cited Meanwhile, his mammalian clock re- Kay’s melanopsin findings in the Top 10 new opportunities for search is yielding discoveries ripe for use breakthroughs. in medicine. Agricultural biotechnology ‘‘Those years are, I think, they are treatments.’’ companies are keen to collaborate and almost unbeatable, right?’’ said Kay. translate Kay’s discoveries in Arabidopsis ‘‘There were just so many amazing dis- to crop plants to improve yield. coveries and, it was a product of in- Kay is applying this finding by using cell Research has shown that circadian rhythms influence cancer treatment and tense, but reasonably civil competition, lines to screen for compounds that affect Kay believes they also play a role in meta- the pace or strength of the circadian as well as collaboration.’’ bolic diseases. He has been investigating clock. Those candidates that affect the Clock Disorders and Therapeutics the clock’s role in the liver and how it circadian clock could be useful tools for regulates hepatic glucose output, which By 2002, his clock work in animals had probing the clock pathways and might may reveal a connection between the shifted exclusively to mammals. In one serve as drug candidates for manipulating clock and diabetes. study (a collaboration with Takahashi and the clock for therapeutic purposes (13). In a recent study (14) Kay’s laboratory the University of California, San Diego’s Throughout Kay’s pursuit of the clock together with John Hogenesch have per- David Welsh) Kay’s team used the lucif- in the fly and mouse, he never abandoned formed a genomewide siRNA screen for erase screen to study CLOCK gene ex- his studies in his favorite model plant, clock modulators in human cells. pression in individual mammalian cells in Arabidopsis. ‘‘This work has revealed that the clock culture. Kay said it is now very clear that the is functionally interconnected with many They (11) found that the clock ticks clock network regulates plant growth by fundamental signaling modules in cells’’ along in liver cells, fibroblasts, and many issuing the wake-up call that triggers a said Kay. Of relevance is their discovery other cell types. However, the circadian growth spurt. of tight links between clock function and rhythm seems to ‘‘run down in whole tis- ‘‘I think it is going to reveal ways insulin signaling. sue samples in culture,’’ said Kay. ‘‘Each where, through a circadian rationale, ‘‘Understanding how circadian rhythms individual cell expresses a clear rhythm, we’re going to be able to influence yield interact with these other networks could just slightly different in pace from its in the fields [5],’’ he said. provide new opportunities for treat- neighbors.’’ He has also described an ‘‘exquisite’’ ments,’’ said Kay. The clock is ticking and he is not missing a beat. Kay and his team extended those find- mechanism whereby a 24-h clock is used ings in 2007 when they showed that to as a seasonal timer. ‘‘It is [some of the Bijal Trivedi, Freelance science writer

1. Chu J, et al. (2009) A mouse forward genetics screen iden- the Arabidopsis circadian clock. Science 323:1481–1485. image-forming photic responses in blind mice. Science tifies LISTERIN as an E3 ubiquitin ligase involved in neuro- 6. Millar AJ, Straume M, Chory J, Chua, N-H., Kay SA (1995) 301:525–527. degeneration. Proc Natl Acad Sci USA 106:2097–2103. Regulation of circadian period by phototransduction 11. Welsh DK, Yoo SH, Liu AC, Takahashi JA, Kay SA (2004) Bioluminescence imaging of individual fibroblasts reveals 2. Griffiths WT, Kay SA, Oliver RP (1985) The presence and pathways in Arabidopsis. Science 267:1163–1166. persistent, independently phased circadian rhythms of photoregulation of protochlorophyllide reductase in 7. Plautz J, Kaneko M, Hall J, Kay SA (1997) Independent clock gene expression. Curr Biol 14:2289–2295. green tissues. Plant Mol Biol 4:13–22. photoreceptive circadian clocks throughout Drosoph- 12. Liu A, et al. (2007) Intercellular coupling confers robust- 3. Nagy F, Kay SA, Chua N-H (1988) A circadian clock regulates ila. Science 278:1632–1635. ness against mutations in the SCN circadian clock net- transcription of the wheat Cab-1 gene. Genes Dev 2:376– 8. Darlington T, et al. (1998) Closing the circadian loop: work. Cell 129:605–616. 13. Hirota T, et al. (2008) A chemical biology approach 382. CLOCK-induced transcription of its own inhibitors per reveals period shortening of the mammalian circadian 4. Millar AJ, Carre I, Strayer C, Chua, N-H, Kay SA (1995) and tim. Science 280:1599–1603. clock by specific inhibition of GSK-3␤. Proc Natl Acad Sci Circadian clock mutants in Arabidopsis identified by 9. Panda S, et al. (2002) Melanopsin (Opn4) requirement USA 105:20746–20751. luciferase imaging. Science 267:1161–1163. for normal light-induced circadian phase shifting. Sci- 14. Zhang EE, et al. (2009) A genomewide RNAi screen for 5. Pruneda-Paz JL, Breton G, Para A, Kay SA (2009) A func- ence 298:2213–2216. modifiers of the circadian clock in human cells. Cell,in tional genomics approach reveals CHE as a component of 10. Panda S, et al. (2003) Melanopsin is required for non- press.

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