Mechanism of Circadian Clock. the 2017 Nobel Prize in Physiology Or Medicine

Tom 67 2018 Numer 2 (319) Strony 245–249

Jadwiga M. Giebultowicz

Oregon State University Department of Integrative Biology 3029 Cordley Hall Corvallis, OR 97331 USA E-mail: [email protected]

MECHANISM OF CIRCADIAN CLOCK. THE 2017 NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE Most animals lead rhythmic lives; some ferent species suggested that the clock may are active at night and sleep during the have genetic basis, because the period of day, while others are diurnal, being active free-running rhythms could be inherited, or during daytime. Although these rhythms modulated by long term selection. Yet, the have a period of 24 hours matching the so- mechanism of circadian clocks remained a lar day, they are not merely a response to total mystery until researchers working with a daylight or darkness at night. When ani- fruit flies, Drosophila melanogaster, decided mals are placed in constant conditions such to test experimentally whether genes are in- as constant darkness and constant temper- volved in the clock function. Fruit flies have ature, they do not lose a sense of time but been used as a genetic model for over a maintain rhythmicity of rest and activity. hundred years; in the early 20th century, However, in these conditions, the period be- US biologist Thomas Morgan used fruit flies tween two sequential activity onsets is not to confirm that genes are located on chro- exactly 24 hours, but rather it is about or mosomes like beads on a string, and estab- “circa” 24 hours; therefore, these cycles are lished genetics as a modern science. Flies called circadian rhythms. Even humans iso- have a high reproduction rate, short life cy- lated from a solar day and left in artificial cle of 10 days from egg to adult, and there light to schedule their own activities, main- are well-established methods to induce mu- tain a clear circadian rhythm of sleep and tations and map them on fly chromosomes. wakefulness. These types of experiments Working at the California Institute of demonstrate that organisms have evolved Technology (CalTech), Dr. Seymour Ben- their own internal sense of time, which is zer and his graduate student Ron Konop- synchronized daily to a 24h solar cycle but ka decided to use rhythm of emergence of in constant conditions displays its endog- adult flies from their pupal cases to probe enous, free-running nature. The internal the mystery of the clock. Individual adult sense of time allows anticipation of cyclical flies tend to emerge in the morning while life events. For example, before we wake up, no emergence takes place in the afternoon, our internal clock orchestrates an increase and a free-running rhythm of adult emer- in blood pressure and in the levels of hor- gence persists in constant darkness. The mone cortisol to prepare us for activities of experimental approach was to mutate hun- the day. dreds of flies in hope of finding a few that The question of how animals and hu- would emerge at the “wrong” time. Indeed, mans can measure time has intrigued sci- the authors of this study isolated several of entists for many decades. Chronobiogists such flies and by analyzing their progeny named the internal mechanism the “circa- they discovered that a single genomic lo- dian clock” and depicted it as a black box cus, which they named period (per) carried when drawing models. Experiments in dif- three different mutations (Konopka and Ben-

Key words: biological clock, circadian rhythms, clock genes, Drosophila melanogaster, Nobel Prize 246 Jadwiga M. Giebultowicz zer 1971). One mutant completely lost the emergence rhythm (per0), another mutation shortened the free-running rhythm from circa 24h to 19h (pershort), and the third mutation produced long-period rhythms of 29h (perlong) of adult emergence. Excitingly, the same mutations caused corresponding changes in the period of the free-running rhythm of locomotor activity in individual flies, indicating that the period gene is part of the clock controlling different behavioral rhythms. This 1971 discovery of the gene period was the first milestone on the way to understanding biological clocks. However, the sequence and function of period remained unknown until the mid-80s, when three Americans, Drs. Jeffrey Hall and Michael Rosbash at Brandeis University and Mi- chael Young at Rockefeller University, used newly developed genetic and molecular tools to sequence period DNA. The Brandeis and Rockefeller teams independently demonstrated that the introduction of period genomic fragments into an arrhythmic per01 mutant caused rescue of both adult emergence rhythm and locomotor activity rhythm (Bar- giello and Young 1984, Reddy et al. 1984). Further studies in the labs of J. Hall and M. Rosbash showed that PER protein (Si- Fig. 1. Schematic depiction of the negative feed- wicki et al. 1988) and per mRNA (Hardin back loop that forms the core mechanism of the et al. 1990) undergo daily oscillations and Drosophila clock. suggested that clock may consist of a negative feedback loop with the PER protein act- At night (upper panel) the CLK/CYC heterodimers bind ing as a repressor of transcription (Hardin to E-box sequences in per and tim promoters and ac- et al. 1990). Meanwhile, another mutant tivate transcription of these genes. Resulting PER and that abolished circadian rhythms in flies TIM proteins form heterodimers, enter the nucleus and bind to CLK/CYC repressing further transcription of per was uncovered in the lab of M. Young (Se- and tim. Morning light activates the CRY protein (low- hgal et al. 1994). This second clock gene er panel) which binds to TIM causing its degradation. was named timeless (tim) and the TIM pro- PER, which is stabilized by TIM, also degrades, ending tein turned out to be a partner of PER, the repressive phase of the clock and allowing posi- necessary for its stability and nuclear entry tive arm of the clock to restart. Many clock-controlled (Gekakis et al. 1995, Vosshall et al. 1994) genes (CCGs) also contain E-boxes in their promoters Although it was evident that PER and and their transcription is directly stimulated by CLK/ TIM proteins somehow affected transcrip- CYC. Some of these CCGs encode transcription factors, tion of their own genes, the mechanism was which indirectly generate rhythmic transcription of ad- not clear owing to the lack of DNA-binding ditional CCGs (modified from Giebultowicz 2017). domains in both proteins. Fortunately, a search for more arrhythmic mutants in the feedback loop is at the core of the circadian labs of J. Hall and M. Rosbash revealed clock in both flies and mammals. A model two genes Clock (Clk) and cycle (cyc) en- of the core feedback loop in Drosophila was coding transcription factors (Allada et al. reviewed recently (Giebultowicz 2017) and 1998, Rutila et al. 1998) that activate per is shown in Fig. 1. Two transcription fac- and tim mRNA transcription. Interestingly, tors encoded by the genes Clock (Clk) and the Clock gene was first identified as part of cycle (cyc) act as the positive clock factors, the mammalian timing mechanism (Vitater- whereby CLK and CYC proteins form com- na et al. 1994), and communication between plexes, which bind to the E-box sequenc- fly and mouse researchers greatly facilitated es in the promoters of per and tim genes, the progress in the understanding of the stimulating their transcription in the early circadian clock mechanism. night. After translation, PER and TIM pro- By the turn of the century, it was clear teins act as the negative limb of the clock that the transcription-translation negative when they accumulate in the cell nuclei Mechanism of circadian clock 247

Fig. 2. The poster depicting Nobel Prize winners, from left to right: Jeffrey C. Hall, Michael Rosbash and Michael W. Young. Copyright © The Nobel Assembly at Karolinska Institutet, source, https:// www.nobelprize.org/. late at night and repress CLK-CYC activity. Michael Rosbash and Michael W. Young, This results in the suppression of per and all three of them doing basic research in tim transcription until the repressive PER Drosophila melanogaster. It was not the first and TIM are degraded. Degradation of TIM time that the tiny fruit fly was “honored” in is initiated by light via the photoreceptive this way. At least five other groups have re- CRY protein encoded by the cryptochrome ceived Nobel Prize for their work using fruit (cry) gene characterized in Drosophila by J. flies to decipher the secrets of human phys- Hall and M. Rosbash (Emery et al. 1998, iology and disease. Sequencing of human Stanewsky et al. 1998). Upon activation by and Drosophila genomes revealed that about light, CRY binds to TIM protein leading to 75% of known human disease genes have its degradation. Because TIM stabilizes PER, a functional match in fruit flies, including the latter is also degraded within few hours genes involved in Down’s syndrome, Alzhei- of lights-on. Mammalian clocks operate by mer’s disease, autism, diabetes, cancer and the same mechanisms and contain mostly others. homologous genes as Drosophila clocks. A Based on early observations of behavio- major difference between fly and mamma- ral rhythms in sleep/activity, feeding, and lian clocks is the use of CRY, rather than cognitive functions, it was assumed that TIM, as the PER binding partner. Mamma- the clock would reside in specialized neu- lian CRY lost light sensitivity and gained a rons. Indeed, the circadian clocks regulating function as the circadian repressor. behavioral functions are located in specific The research that led to the understand- brain neurons of mammals and insects; ing of the circadian clock mechanism earned this was investigated using perturbation of their discoverers the 2017 Nobel Prize locomotor activity rhythms as a readout of in Physiology or Medicine. The Prize was clock function. However, it is now well es- awarded jointly to (Fig. 2): Jeffrey C. Hall, tablished that animals possess multi-oscilla- 248 Jadwiga M. Giebultowicz tory circadian systems. In addition to cen- always evident at the meetings of the So- tral or master clocks residing in the central ciety for Research on Biological Rhythms, nervous system, there are peripheral clocks which brings together researchers working in cells forming most other tissues, and on clocks in bacteria, plants, and animals their molecular mechanism is very similar. as well as medical doctors dealing with tim- The existence of peripheral clocks that can ing disorders in humans. They can learn function independently of the brain was from each other because most molecular first demonstrated in mothsG ( iebultowicz pathways are conserved in evolution and et al. 1989), then in Drosophila (Hege et al. human cells function and divide by the 1997) and finally in mammals (Balsalobre same mechanisms as in flies. The Nobel et al. 1998). Clocks that exist in cells mak- Prize for the three fly scientists highlights ing up most body organs in flies and mam- the unity of fundamental life processes and mals provide the temporal framework to or- underscores the value of basic research on ganize activity of different tissues, allowing simple model organisms for the understand- synchronization of compatible and separa- ing of our own physiology and for making tion of incompatible metabolic processes. progress in preventing and treating various The molecular rhythms generated by the human diseases. tissue-specific clocks contribute to rhythmic physiology such as daily fluctuations in the ACKNOWLEDGEMENTS levels of hormones, enzymes, and various The author thanks Eileen Chow for help metabolites. In fact, nearly all aspects of with the figure and the manuscript. Polish- metabolism vary with time of day, at both American Fulbright Commission is acknowl- cellular and systemic levels (Brown 2016). edged for financial support granted to the These rhythms are synchronized with daily author to teach and do research in Poland. cycles of food intake, digestion, motor, and Summary cognitive activities which are followed by a period of sleep, which is associated with Since 1901, the Nobel Prize has been awarded to fasting and cellular repair. Increasingly, scientists who have made the most important discover- modern humans tend to disrupt these cy- ies for the benefit of humanity. The 2017 Nobel Prize in Physiology or Medicine was awarded jointly to Jef- cles by shift work, irregular eating habits, frey C. Hall, Michael Rosbash and Michael W. Young prolonged exposure to artificial light, and “for their discoveries of molecular mechanisms control- travel across time zones and these disrup- ling the circadian rhythm.” It may be surprising to learn tions increase risk of several diseases. that those three scientists dedicated their entire careers Studies in model organisms including to research on the fruit fly, Drosophila melanogaster. Drosophila were first to suggest that disrup- However, as their studies progressed, it became increas- tion of circadian rhythms may have patho- ingly clear that the mechanism of the biological clock logical consequences. Laboratory mammals that they discovered in Drosophila is very similar to a with genetically engineered defects in their timekeeping mechanism present in mammals, including circadian clocks show many pathologies humans. Through interdisciplinary work between scientists performing basic research on model organisms including obesity, diabetes, steatosis, car- and medical doctors, we have learned over time that diomyopathy, and atherosclerosis (Brown daily rhythms support human health while disruption of 2016). There is also accumulating evidence these rhythms is associated with a range of pathological that age-related disruptions of normal cir- disorders such as cardiovascular problems, metabolic, cadian rhythms and sleep cycles can affect neurological, and many other diseases. This short re- neuronal health and contribute to pathogen- view highlights critical milestones on the way to under- esis of neurodegenerative diseases, such as standing biological clocks, focusing on the roles played Alzheimer’s disease (Musiek and Holtzman by the three Nobel Prize winners. 2016). 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Emery P., So V., Kaneko M., Hall J. C., Ros- Reddy P., Zehring W. A., Wheeler D. A., Pirrot- bash M., 1998. CRY, a Drosophila clock and ta V., Hadfield C., Hall J. C., Rosbash M., light-regulated cryptochrome, is a major con- 1984. Molecular analysis of the period locus tributor to circadian rhythm resetting and pho- in Drosophila melanogaster and identification tosensitivity. Cell 95, 669-679. of a transcript involved in biological rhythms. Gekakis N., Saez L., Sehgal A., Young M., Weitz Cell 38, 701-710. C., 1995. Isolation of timeless by PER protein Rutila J. E., Suri V., Le M., So V., Rosbash interaction: defective interaction between time- M., Hall J. C., 1998. CYCLE is a second less protein and long-period mutant PER. Sci- bHLH-PAS Clock protein essential for circadian ence 270, 811-815. rhythmicity and transcription of Drosophila pe- Giebultowicz J. M., 2017. The circadian sys- riod and timeless. Cell 93, 805-814. tem and aging of Drosophila. [In:] Circadian Sehgal A., Price J., Man B., Youngs M., 1994. Rhythms and Their Impact on Aging. Jazwin- Loss of circadian behavioral rhythms and per ski S., Belancio V., Hill S. (eds.). Cham, RNA oscillations in the Drosophila mutant Switzerland: Springer International Publishing timeless. Science 263, 1603-1606. AG, 129-145. Siwicki K. K., Eastman C., Petersen G., Rosbash Giebultowicz J. M., Riemann J. G., Raina A. K., M., Hall J. C., 1988. Antibodies to the period Ridgway R. L., 1989. Circadian system con- gene product of Drosophila reveal diverse tis- trolling release of sperm in the insect testes. sue distribution and rhythmic changes in the Science 245, 1098-1100. visual system. Neuron 1, 141-150. Hardin P. E., Hall J. C., Rosbash M., 1990. Stanewsky R., Kaneko M., Emery P., Beretta Feedback of the Drosophila period gene prod- B., Wager-Smith K., Kay S. A., Rosbash M., uct on circadian cycling of its messenger RNA Hall J. C., 1998. The cryb mutation identifies levels. Nature 343, 536-540. cryptochrome as a circadian photoreceptor in Hege D. M., Stanewsky R., Hall J. C., Giebul- Drosophila. Cell 95, 681-692. towicz J. M., 1997. Rhythmic expression of Vitaterna M. H., King D. P., Chang A. M., Korn- a PER-reporter in the Malpighian tubules of hauser J. M., Lowrey P. L., McDonald J. D., decapitated Drosophila: evidence for a brain- Dove W. F., Pinto L. H., Turek F. W., Taka- independent circadian clock. J. Biol. Rhythms hashi J. S., 1994. Mutagenesis and mapping 12, 300-308. of a mouse gene, Clock, essential for circadian Konopka R. J., Benzer S., 1971. Clock mutants behavior. Science 264, 719-725. of Drosophila melanogaster. Proc. Natl. Acad. Vosshall L., Price J., Sehgal A., Saez L., Young Sci. USA 68, 2112-2116. M., 1994. Block in nuclear localization of pe- Musiek E. S., Holtzman D. M., 2016. Mecha- riod protein by a second clock mutation, time- nisms linking circadian clocks, sleep, and neu- less. Science 263, 1606-1609. rodegeneration. Science 354, 1004-1008.

KOSMOS Vol. 67, 2, 245–249, 2018

Jadwiga M. Giebultowicz

Oregon State University, Department of Integrative Biology, 3029 Cordley Hall, Corvallis, OR 97331 USA, E-mail: [email protected]

MECHANIZM ZEGARA BIOLOGICZNEGO. NAGRODA NOBLA 2017 W DZIEDZINIE FIZJOLOGII LUB MEDYCYNY

Streszczenie Od roku 1901 Nagroda Nobla jest przyznawana naukowcom za najważniejsze odkrycia służące dobru ludzkosci. Nagrodę Nobla w dziedzinie fizjologii lub medycyny w 2017 roku otrzymali trzej amerykańscy uczeni Jeffrey C. Hall, Michael Rosbash i Michael W. Young „za odkrycie mechanizmu molekularnego, który kontroluje rytmy okołodobowe”. Może się to wydać zaskakujące, ale ci trzej nobliści poświęcili swoje kariery naukowe badaniom nad muszką owoco- wą, Drosophila melanogaster. Jednak w miarę postępu ich badań stawało się coraz bardziej oczywiste, że mechanizm zegara biologicznego, odkryty u muszki Drosophila, jest bardzo podobny do zegara, który posiadają ssaki, łącznie z człowiekiem. Interdyscyplinarna współpraca między naukowcami prowadzącymi badania podstawowe na organizmach modelowych i lekarzami prowadzącymi badania kliniczne ujawniła istotną rolę rytmów dobowych w utrzymaniu zdro- wia człowieka. Dlugotrwałe zakłócenie tych rytmów stanowi czynnik ryzyka wielu patologii, takich jak choroby serca, cukrzyca, otyłość czy choroby układu nerwowego. Artykuł krótko podsumowuje odkrycia, stanowiące kamienie milo- we na drodze poznania mechanizmu zegara biologicznego, ze szczególnym uwzględnieniem roli trzech noblistów 2017 w tym procesie.

Słowa kluczowe: Drosophila melanogaster, geny zegarowe, nagroda Nobla, rytmy okołodobowe, zegar biologiczny