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Home , Cycle (gene), Michael Rosbash, Seymour Benzer, Timeless (gene)

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Celebrating clocks and – 2017 for or Medicine

At a time when most ‘Nobel watchers’ single could yield sig- Around the same time, two adjacent expected the much coveted prize in nificantly large effects on rhythmic laboratories in nearby New England were physiology or medicine to go to CRISPR behaviour. Understandably, clocks were engaged in a similar quest to unravel the technology or even to immunotherapy, expected to be a more complex entity mysteries of . Piqued by the find- the announcement to award discoveries with built-in redundant machinery that ing that the period gene which has an made using fruit flies, in deciphering the was unlikely to be laid bare by mutations effect on daily rhythms also impacts high molecular underpinnings of the funda- in single . frequency rhythms in courtship songs, mental biological process of time- Michael W. Young, one of the the foundations of a highly fruitful col- keeping, is reassuring to scientists en- awardees, was a graduate student at the laboration between two professors, Jef- gaged in basic research across the world. University of Texas in 1971, when frey C. Hall and his friend and neighbour The humble has had its share of period mutants were described and hap- at the Department in Brandeis recognition from the Nobel Committee in pened to be studying the University, were laid. the past; the earliest with Thomas Hunt genome using classical cytogenetic Soon the duo was harnessing the power Morgan for the fundamental idea of the methods. He had fly lines with chromo- of molecular , that had just be- Chromosomal Theory of Inheritance somal rearrangements on the X chromo- come available, to isolate period gene (1933) followed by his student Hermann some, including one with a break in the and elucidate its properties. Nearly si- J. Muller for his discovery of mutagene- region predicted by the Benzer study, multaneously as the group from Rocke- sis by radiation (1946). Developmental which he would use only later. While a feller, the Brandeis labs published their biologists Edward B. Lewis, Christiane post-doc at Stanford, Young recalls hav- first collaborative paper on the molecular Nüsslein-Volhard and Eric F. Wieschaus ing heard a highly animated and persua- analysis of the period , and showed were recognized in 1995, Linda Buck sive talk by Seymour Benzer who evidence of the rescue of a null and for their discoveries of likened his genetic dissections of behav- by P-element mediated transformation. olfactory receptors (2004) were followed iour to those of surgical lesions that had These studies can be considered as the by Jules Hoffman for his discoveries in been used previously to localize the sites official start of a race that was some- innate immunity (2011). of brain functions. Incidentally, the times bitter, but in hindsight may have In the early 1980s, the three awardees , a structure in been the catalyst for the speed with for this year’s Nobel Prize ventured forth the mammalian brain had been localized which studies progressed in the direction in an attempt to uncover genes that may to be the site of the circadian pacemaker of understanding the biochemical and play central roles in circadian (~24 h in 1972 using surgical lesions. In 1978 cell biological basis for self-sustained, periodicity) rhythm generation, mainte- when Young had a faculty position at the near-24 h rhythm generation. Thus, within nance and synchronization to the geo- Rockefeller Centre in he a decade of isolating and mapping the physical day and night, at a time when was able to pursue the idea of locating period gene, all the other molecular giants in the field of or and mapping the period gene using a players involved in the study of biological rhythms, were chromosomal walk (a method of posi- generation were identified and character- skeptical of such an approach. However, tional cloning). With Thaddeaus Bar- ized. Today we celebrate what circadian another discovery made more than a dec- giello, a post-doctoral fellow at his new biologists now agree was a rivalry that ade before, by Seymour Benzer and laboratory, the precise location and size put forth a highly conserved mechanism Ronald Konopka1 was pivotal in steering of period was described, followed by the that has since been found in the vast ma- the efforts of the trio. Konopka and demonstration that P-element mediated jority of organisms examined. Benzer showed that a locus on the transformation of mutant arrhythmic flies In the early 1980s the question re- X- of the fly is critical for with the wildtype copy of the gene can mained as to how a single gene could rhythmic behaviours such as adult emer- restore both adult emergence as well as bring about rhythmic phenomenon? The gence from pupal cases in populations of activity rhythms. first breakthrough came in the form of a flies and the locomotor activity of indi- vidual flies. Allelic mutations at this locus either rendered flies arrhythmic in both behaviours or altered the periodicity of the rhythms in opposite directions (either lesser than or greater than 24 h), thus prompting them to name it period. This landmark study took the chronobi- ology community by storm, partly due to the remarkably clear and robust pheno- type obtained by a random screen. At that time, the field was domi- nated by scientists who did not think that Jeffrey C. Hall Michael Rosbash Michael W. Young

CURRENT SCIENCE, VOL. 113, NO. 8, 25 OCTOBER 2017 1497 NEWS discovery by Paul Hardin, a post- ments. In a primary screen of about 7000 and were shown to physically doctoral fellow with Rosbash who fly lines, a candidate was found on the interact with one another and bind to E- showed that period mRNA from fly second chromosome that did not show Box elements on the period and heads exhibited a circadian oscillation the normal preference for dawn adult genes, thus providing evidence of being and that the oscillation was affected by emergence and instead emerged more-or- members of the core-. Further stud- mutations in the gene. Subsequently the less equally throughout the day and ies showed that in fact CLOCK–CYCLE same group showed that the precursor night, which they named timeless4. Like activity was inhibited by PERIOD and and mature forms of period mRNA have the period null mutant, these flies also TIMELESS. In later years a second similar amplitude and are in-phase and showed arrhythmicity in locomotion. feedback loop comprising of vrille-pdp1 that the period gene promoter can pro- Strikingly, this mutation also affected the as transcriptional regulators was pro- duce oscillation even to heterologous oscillation of period mRNA, such that it posed by the laboratory of Justin Blau9. mRNA. Most importantly, they found was no longer rhythmic, suggesting an In the same year, a more complex model that the period oscillation is de- interaction between the period and time- of interlocked feed-back loops incorpo- layed in phase by 6 hours from the pe- less loci. Leslie Vosshall and colleagues rating both the loops (PERIOD– riod mRNA oscillation. Around that in the same lab showed that indeed the TIMELESS and VRILLE-PDP1) bound time, it became known that PERIOD timeless mutant has profound effects on by a common requirement of the shuttles between the cytoplasm and the the PERIOD protein, the latter’s ability CLOCK-CYCLE complex for transcrip- nucleus2 in a temporally restricted man- to enter the nucleus at a certain specific tional activation, was put forth by the ner in brain neurons (the proposed loca- time of day was contingent upon timeless studies of Paul Hardin’s laboratory10. tion of the fly clock) and that its amino function, leading to the hypothesis that While the above studies explained how acid sequence showed similarity with timeless and period are both part of one a self-sustained clock machinery with a two other proposed transcriptional regula- intracellular mechanism that generates a near 24 h period might be generated in tors (SINGLE MINDED and ARNT). self-sustained oscillation. Subsequently, the absence of external time cues, it was This led to the hypothesis that the lag in timeless mRNA was also shown to cycle, also necessary to explain how such PERIOD accumulation may be due to and PERIOD and TIMELESS clocks might synchronize with the exter- negative regulation of itself. For the first were shown to interact in order to enter nal environment – the phenomenon of time ever, there was reasonable evidence the nucleus. entrainment. Previously, the behavioural to propose that period mRNA and its In a flurry of reports that followed, yet attributes of entrainment had been the protein product influence each other3, other mutants that affect the nature of subject of intense study mostly using through a negative feedback mechanism the activity/rest rhythm via defects in the mammalian systems of a wide hue (series that had been proposed as the basis for underlying TTFL were isolated by the of papers by Pittendrigh and Daan, self-sustained oscillators. Although it Young and the Hall-Rosbash laborato- 1976). Light, a major entraining agent seems obvious today that this may well ries. Jeffery Price, Brian Kloss and col- (‘’ or time-giver in German) be the case for a bonafide leagues at the Young Lab demonstrated was shown to impact the fly circadian gene, many coincident factors were criti- that a protein affects the clock via TIMELESS degradation. The cal to establishing this idea. Amongst stability of PERIOD due to its role as a finding of a dedicated photoreceptor – them were, the previous discovery from kinase in of PERIOD for the clock in flies the Hall lab in 1988, that PERIOD pro- protein5. The phosphorylated form of the came once again from the Rosbash-Hall tein also oscillates with a day–night monomeric PERIOD protein being un- laboratories in 1999. Subsequently, stud- rhythm, the fact that the Rosbash lab was stable ensures that only the heteromeric ies from various laboratories showed that at the fore-front of molecular techniques and stable PERIOD–TIMELESS com- light via CRYPTOCHROME modifies to handle RNA samples, and the reason- plexes can enter the nucleus, thus build- TIMELESS to enable its proteasomal ing of Paul Hardin to assay mRNA in fly ing in a time delay between peaks degradation and thus reset the circadian heads rather than whole bodies. of period/timeless mRNA and protein clock11. Integral to the ability of TTFLs The race that was alluded to previ- levels. These studies and others from the to function as the core-clock mechanism ously, was following another parallel path three laboratories provided evidence for are several other processes that have in the Young lab at Rockefeller. Given the idea of a –translation been discovered over the past two the proposition that PERIOD is regulat- feedback loop (TTFL) as a molecu- decades. Among them are epigenetic ing its own transcription, it required lar/biochemical basis for generation of modifications that can change chromatin PERIOD to have a DNA-binding do- self-sustained rhythms. architecture, RNA-mediated post- main, yet its sequence did not reveal any. The discovery of two more new genes transcriptional modifications and micro- This led to the idea that perhaps PERIOD clock6 and cycle7 brought an essential RNA induced alternative splicing medi- may interact with other proteins bearing missing piece, namely transcriptional ated regulation of transcription. As also a DNA-binding domain. In this pursuit, a activators (positive elements) into the post-translational modifications such as post-doctoral fellow, along feedback loop. Interestingly for the first SUMOylation and O-GlcNAcylation, and with her colleagues Jeffery Price and time, the identification of a fly circadian ubiquitination, in addition to phosphory- Bernice Man took on the task of repeat- gene was done by working backwards lation have been demonstrated to alter ing the heroic approach of Konopka and from a mouse mutagenesis screen that clock output across mice and flies. So Benzer to uncover other mutations on gave the first mammalian circadian gene how prevalent and integral are the TTFLs autosomes, this time by a genetic method named Circadian locomotor output in sustaining rhythms across life forms? of mutagenesis using transposable ele- cycles kaput (Clock)8, Drosophila CLOCK Analogous systems have been found

1498 CURRENT SCIENCE, VOL. 113, NO. 8, 25 OCTOBER 2017 NEWS across phyla – cyanobacteria, Neurospora, activity around the lights-off transition, policy makers across the globe. While plants and , have TTFLs gov- and these cells also influenced the phase chronotherapeutics might still be in its erning their rhythms; however, the lack of the evening bout of activity via the infancy, a great deal of basic research of homology among the components of production of a neuropeptide, Pigment akin to the one conducted by the 2017 the TTFL suggests that such a mecha- Dispersing Factor that was proposed to Nobel laureates and several giants before nism has arisen independently and more actively delay the onset of evening activ- them remains to be conducted. It is not than once along evolutionary timescales. ity. Subsequently, other subsets among difficult to imagine that once again the More recently, non-TTFL based clocks the approximately 150 neurons that fruit fly will be at the forefront of this have also been discovered. Prominently, express TIMELESS/PERIOD have been endeavour. The Nobel Prize this year is a a protein-phosphorylation dependent sys- shown to be critical for various aspects definite shot-in-the arm for clockwatch- tem has been found in cyanobacteria, of rhythmic behaviour including the eve- ers and fly biologists alike! where a self-sustained, near-24 h rhythm ning activity under light cycles, or tem- can be generated in the absence of tran- perature cycles. The wealth of genetic scriptional machinery by a series of tools in flies along with the somewhat 1. Konopka, R. J. and Benzer, S., PNAS, sequential phosphorylation steps12,13. distributed anatomical localization of the 1971, 68, 2112–2116. 2. Liu, X., Zwiebel, L. J., Hinton, D., Another non-TTFL based clock has been pacemakers have enabled fly circadian Benzer, S., Hall, J. C. and Rosbash, M., discovered, first in anucleate cells such biologists to dissect the neuronal circuit J. Neruosci., 1992, 12, 2735–2744. as erythrocytes from many organisms, to an unprecedented level, possibly more 3. Hardin, P. E., Hall, J. C. and Rosbash, including . Here the ratio of over- than for any other behavioural pheno- M., Nature, 1990, 343, 536–540. and hyperoxidized form of peroxiredoxin type. Now we know that both in flies and 4. Sehgal, A., Price, J. L., Man, B. and was found to show a free running cir- in mammals, clocks exist in a variety of Young, M. W., Science, 1994, 263, cadian rhythm along with the ability to tissues throughout the body. So-called 1603–1606. entrain to temperature cycles as well as peripheral clocks are located outside the 5. Price, J. L., Blau, J., Rothenfluh, A., robust temperature compensation, all of brain and even in non-neural tissues, no- Abodeely, M., Kloss, B. and Young, M. which are considered as characteristic tably in the liver, kidneys, adipocytes W., Cell, 1998, 94, 83–95. 6. Allada, R., White, N. E., So, W. V., Hall, features of endogenous circadian clocks14. and stomach in mammals; in the fat- J. C. and Rosbash, M., Cell, 1998, 93, Remarkably such a mechanism has hence body, Malpighian tubules and even 791–804. been discovered in distantly related organ- antennae in flies. It is well accepted that 7. Rutila, J. E., Suri, V., Le, M., So, W. V., isms such as the alga Ostreococcus tauri, proper synchrony among these peripheral Rosbash, M. and Hall, J. C., Cell, 1998, the archaeon Halobacterium salinarum, clocks is integral to well being, based on 93, 805–814. cyanobacterium Synechoccus and yeast empirical evidence from multiple ex- 8. Vitaterna, M. H. et al., Science, 1994, Saccharomyces cerevisae. Not withstand- perimental models and paradigms. 264, 719–725. ing the fact that the last decade or so has Indeed, the field is ripe with questions 9. Cyran, S. A. et al., Cell, 2003, 112, 329– revealed many non-TTFL based clocks, about how circadian clocks and timing 341. one cannot but acknowledge the fact that impinge upon metabolism and physio- 10. Glossop, N. R., Houl, J. H., Zheng, H., Ng, F. S., Dudek, S. M. and Hardin, P. widespread interest was fuelled by the logy. Ironically, rhythm research has E., Neuron, 2003, 37, 249–261. early breakthroughs achieved via eluci- come a full circle as mutations in genes 11. Ceriani, M. F., Darlington, T. K., dating the TTFLs, in what some may go that were first identified as core clock Staknis, D., Mas, P., Petti, A. A., Weitz, so far as to say, excruciating detail. members, have also now been found to C. J. and Kay, S. A., Science, 1999, 285, Coming back to flies, it is important to be linked with several /wake disor- 553–556. note here that while the intracellular ders in humans such as familial advanced 12. Nakajima, M. et al., Science, 2005, 308, mechanisms were being investigated, sleep-phase syndrome (Per2), delayed 414–415. there were also studies asking the ques- sleep-phase syndrome (Per3), bipolar 13. Tomita, J., Nakajima, M., Kondo, T. and tion of where in the brain these clocks disorder (Timeless and Per3) and seaso- Iwasaki, H., Science, 2005, 307, 251– lay. In 1998, in parallel with the ‘clock- nal affective disorder (Npas2). Addition- 254. 14. O’Neill, J. S. and Reddy, A. B., Nature, work explosion’ in molecular terms, ally, sleep disturbances occurring due to 2011, 469, 498–503. Charlotte Helfrich-Forster from Tubin- shiftwork and jetlag result in metabolic gen, Germany continuing on a long tradi- and hormonal imbalances, thereby giving tion of neurobiologists interested in an impetus to chronotherapeutic treat- ACKNOWLEDGEMENTS. I thank my stu- circadian clocks showed by very elegant ments of lifestyle and related disorders. dents Abhilash, Arijit, Sheetal and Viveka for means that in fact, a set of about 8 cells Chronotherapy has proceeded to an ex- suggesting several improvements to an earlier in the fly’s brain are sufficient for the tent of appropriately timed drug delivery version of this article. self-sustained rhythm in locomotion. and post-surgical treatments for optimum These cells were shown to be integral recovery. The impact of clocks on health also for generation of the morning bout and well-being are being recognized by Sheeba Vasu, Unit, Jawa- of activity in the presence of light/dark medical practitioners, personnel man- harlal Nehru Centre for Advanced Scien- cycles earning the sobriquet ‘M’ cells. agement boards of business establish- tific Research, Bengaluru 560 064, India. However, flies exhibit a second peak of ments, defence organizations, as well as e-mail: [email protected]

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