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Temperature Compensation and Temperature Sensation in the Circadian Clock

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Temperature Compensation and Temperature Sensation in the Circadian Clock Temperature compensation and temperature sensation in the circadian clock Philip B. Kidda,b, Michael W. Younga, and Eric D. Siggiab,1 aLaboratory of Genetics, Rockefeller University, New York, NY 10065; and bCenter for Physics and Biology, Rockefeller University, New York, NY 10065 Edited by Susan S. Golden, University of California, San Diego, La Jolla, CA, and approved October 8, 2015 (received for review June 8, 2015) All known circadian clocks have an endogenous period that is that set the 24-h circadian period are not fully known. Many remarkably insensitive to temperature, a property known as temper- transcriptional regulatory programs are carried out in a matter of ature compensation, while at the same time being readily entrained minutes (see, for example, refs. 16 and 17), so generating a 24-h by a diurnal temperature oscillation. Although temperature com- circadian rhythm requires extensive regulation to generate long pensation and entrainment are defining features of circadian clocks, time delays. In wild-type (WT) Drosophila, the translocation of their mechanisms remain poorly understood. Most models presume the PER-TIM dimer into the nucleus takes about 6–8 h (18, 19) that multiple steps in the circadian cycle are temperature-depen- and relies on a complex set of protein interactions and chemical dent, thus facilitating temperature entrainment, but then insist that modifications. The delay between peak expression of per and tim the effect of changes around the cycle sums to zero to enforce mRNA and protein is also long, about 6 h in constant darkness in temperature compensation. An alternative theory proposes that the WT (see Fig. 2 and Fig. S1), and mRNA stability and processing circadian oscillator evolved from an adaptive temperature sensor: a are also subject to circadian regulation (20). Although many gene circuit that responds only to temperature changes. This theory details about these processes are known (21), the full set of re- implies that temperature changes should linearly rescale the ampli- actions setting the associated time scales is not. Additionally, the tudes of clock component oscillations but leave phase relationships 12- to 14-h length of the combined transcriptional/translational timeless and shapes unchanged. We show using luciferase reporter delay and nuclear translocation time does not fully account for measurements and Western blots against TIMELESS protein that this the 24-h period of the clock. Drosophila prediction is satisfied by the circadian clock. We also re- Not knowing the full set of chemical reactions that contribute view evidence for pathways that couple temperature to the circadian to determining the circadian period has made research into the clock, and show previously unidentified evidence for coupling be- Drosophila temperature compensation difficult. However, some period- tween the clock and the heat-shock pathway. determining reactions are known, and experiments on those reactions have provided some insight, albeit fragmentary, into circadian clock | temperature compensation | mathematical models the nature of temperature compensation. Nuclear translocation of PER and TIM relies on many complicated chemical modi- ircadian rhythms are daily oscillations in gene expression and fications, in particular, daily rhythms of phosphorylation (22, 23) Cprotein concentration that regulate sleep (1, 2), metabolism involving the enzymes casein kinase 2 (CK2) (24, 25), DOUBLE- – (3, 4), and a host of other biological processes (5 8). Circadian TIME [a casein kinase 1 (CK1) homolog] (26–28), SHAGGY oscillations are known to be present in nearly all animals and [a glycogen synthase kinase (GSK-3) homolog] (23), and plants, as well as some fungi and bacteria (9). In all organisms in others (21). An experiment that altered gene dosages in Neu- which circadian oscillations have been observed, circadian os- rospora has shown that the activity of CK2 has a strong influ- cillations satisfy three defining properties (10). First, circadian ence on the period of the circadian rhythm (29). Additionally, oscillations are self-sustained and spontaneously maintain a pe- riod of about 24 h. Second, the circadian rhythm is sensitive to Significance light and temperature and can be synchronized to external os- cillations in the quantity of either. Third, the period of the cir- cadian oscillation is “temperature-compensated”;thatis,the Circadian clocks in animals, plants, and fungi possess the re- endogenous period of the oscillation is relatively insensitive to markable property of temperature compensation: the clock has temperature. a temperature-insensitive period, while retaining the ability to The genetic basis of circadian clocks has been elucidated in synchronize to temperature cycles. The conservation of tem- considerable detail over the past two decades, particularly in the perature compensation across clades means it is likely critical fruitfly Drosophila melanogaster, the mouse Mus musculus,andthe to the function of circadian clocks. Temperature compensation bread mold Neurospora crassa (11). In Drosophila, a self-sustained also places the circadian clock in contrast to other biological circadian oscillation in neurons is generated when a pair of proteins, pathways, which generally have temperature-sensitive time PERIOD (PER) and TIMELESS (TIM), dimerize and, after some scales. However, the mechanism of temperature compensation delay, translocate into the nucleus, where the proteins repress their remains unknown. We present a general scheme by which a own transcription by inactivating a transcription factor dimer com- circadian oscillator can be temperature-compensated while still posed of the proteins CLOCK and CYCLE. Light sensitivity is synchronizing to environmental temperature cycles. In partic- ular, we give experimental evidence that the circadian clock mediated by the blue light photoreceptor CRYPTOCHROME consists of a temperature-insensitive core oscillator coupled to (CRY), which upon activation binds TIMELESS and promotes a specific adaptive temperature signaling pathway. TIMELESS degradation. A remarkably similar mechanism under- lies circadian clocks in mouse and Neurospora. The circadian clock Author contributions: P.B.K., M.W.Y., and E.D.S. designed research; P.B.K. performed re- of cyanobacteria has also been studied in detail but has a signifi- search; P.B.K., M.W.Y., and E.D.S. analyzed data; and P.B.K., M.W.Y., and E.D.S. wrote cantly different structure (12, 13). the paper. Much is therefore known about the first two defining prop- The authors declare no conflict of interest. erties of circadian clocks. Temperature compensation, however, This article is a PNAS Direct Submission. remains poorly understood despite having been a key problem in 1To whom correspondence should be addressed. Email: [email protected]. chronobiology for nearly the entire history of the field (14, 15). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. In part, this lack of understanding is because the mechanisms 1073/pnas.1511215112/-/DCSupplemental. E6284–E6292 | PNAS | Published online November 2, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1511215112 Downloaded by guest on October 1, 2021 increasing CK2 levels several-fold by gene duplication leads to rate at high temperatures. The relationship between these models PNAS PLUS a decreasing period at higher temperature, whereas decreasing CK2 is examined further in the Discussion. levels leads to an increasing period at higher temperature. Another In this paper, we present compelling evidence against the net- experiment in mammalian cell culture used a chemical screen to work model and in favor of the pathway model for temperature identify CK1 as a key period-determining enzyme (30). Knockdown compensation. We first argue for the generality of the scaling and of CK1 leads to a loss of temperature compensation, and, most in- pathway predictions using a simple mathematical model. We then terestingly, the phosphorylation rate of CK1 was independently present experimental evidence from quantitative Western blot and temperature-compensated in an in vitro assay. It should be pointed luciferase reporter experiments demonstrating that the simple out, however, that the substrate in this assay was an artificial peptide, scaling prediction is satisfied by the circadian clock in Drosophila. not a physiologically relevant CK1 target, making the in vivo impli- We provide support for the prediction of specific signaling path- cations of the experiment unclear. In the flowering plant Arabidopsis, ways by showing that knockouts in the heat-shock pathway affect it has been shown that temperature-dependent changes in the con- both circadian temperature phase shifting and temperature regu- centration of CK2 and CCA1 are required for temperature com- lation of sleep behavior. Finally, we discuss the implications of pensation (31). In Drosophila, the equilibrium level of PER/TIM these results and suggest some ideas for continuing work on cir- binding is temperature-compensated (32). These experiments pro- cadian temperature compensation. vide interesting hints into how temperature compensation is achieved at the biochemical level, but no clear picture has emerged. Results Temperature compensation of the circadian period does not Temperature Scaling in Some Simple Examples. We begin by devel- imply that the circadian clock simply ignores temperature, and it oping a simple mathematical example of the pathway model. The is
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