Oscillating Molecules and Circadian Clock Output Mechanisms

Molecular Psychiatry (1998) 3, 381–385 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 NEWS & VIEWS Oscillating molecules and circadian clock output mechanisms The circadian clock system is crucial for the regulation of daily rhythms in processes ranging from gene expression to the mammalian sleep/wake cycle. This review will summarize evidence from studies in model systems for the involvement of transcriptional and post-transcriptional mechanisms in circadian clock output. Most neurobiologists, when told about circadian clock output pathways mediating physiological and genes and the seminal advances in understanding the behavioral rhythmicity. molecular mechanisms constituting the circadian clock, will ask: ‘Yes, but how does the clock do what Transcriptional mechanisms and clock output it does?’. This is to say, what are the mechanisms that dictate the accurate transmission of temporal infor- Many investigators in the field of circadian rhythms mation from the clock, located in a discrete region of would speculate that output mechanisms must, in the central nervous system, to the neuroeffector sys- some way, involve the action of transcription factors tems that generate defined circadian rhythms? In the and the regulation of ‘downstream’ genes that encode lingo of the field: what are the signaling or output path- clock output components. This aphorism stems in part ways that mediate the clock control of diverse circad- from several observations suggesting an involvement of ian rhythms? Questions about clock output, posed by transcriptional mechanisms in clock biology. As long such neurobiologists, are indicative of how little we ago as the late 1960s, an explicit model for the circad- really know about such output pathways. This mini- ian clock itself, was formulated that postulated an review will attempt to summarize recent molecular important role for transcriptional activity;7 this model genetic studies that have provided insights about cir- hypothesized that the timing of RNA synthesis from cadian clock output mechanisms. An understanding of a polycistronic complex of DNA (a so-called chronon) circadian clock output in any system may provide provided the temporal information required for circad- insights about the output mechanisms that control ian functions. This hypothesis was based on experi- complex mammalian behaviors. mental results which were interpreted as suggesting an involvement of nucleic acid metabolism in light- induced phase shifting of the clock.8 While the Biochemistry of the clock mechanism Chronon theory has been discounted in its purest form, The isolation and analysis of clock genes in a variety contemporary molecular analysis has established that of organisms, from fruitflies and fungi to mammals1–6 transcriptional mechanisms really are crucial for clock have generated tremendous knowledge about clock function. The best indication of this axiom is that cer- molecules and mechanisms, and have driven the for- tain transcription factors, of the bHLH/PAS class mulation of explicit models that purport to explain the (which contain a basic helix-loop-helix DNA-binding biochemistry of the circadian clock. The prevailing domain and a PAS dimerization domain) are molecular model of circadian clock function includes oscillating constituents of the circadian clock in both fungi and RNAs and proteins as state variables of the clock, and it mammals4–6,9 and a PAS-containing protein called Per- is couched in terms of an autoregulatory loop in which iod (the P in PAS) is known to be a component of the changes in one set of state variables (the proteins) nega- Drosophila clock mechanism.2,3 Furthermore, studies tively regulate the other. This model is supported prim- from several laboratories have demonstrated that acute arily by studies of clock genes1 in Neurospora (frq, wc- perturbations of transcription, using reversible inhibi- 1, wc-2) and Drosophila (per, tim). Currently, however, tors, can alter circadian period and reset the clock the oscillator is described as a closed molecular loop, mechanism.10,11 and it is not obvious how this loop is coupled to the In attempts to identify pacemaker or clock output components, circadian regulated RNAs, encoding transcription factors12–14 and other products15–17 have been Correspondence: FR Jackson, X Zhang and GP McNeil, Dept of described in several different organisms, and at least Neuroscience, Tufts University School of Medicine, Boston, MA some of these RNA oscillations are known to depend 02111, USA. E-mail: [email protected] on transcriptional mechanisms.18 The best charac- News & Views 382 terized of these RNAs, however, encode metabolic overexpression) do not affect all clock-driven RNA enzymes15,17 or structural proteins19 that are not likely rhythms, it has been hypothesized that this negative to function as regulatory elements of a clock output feedback loop acts as a ‘slave’ oscillator, coupling the pathway. Nonetheless, based on models of the circad- clock to specific output pathways.25 At present, how- ian system,20 which postulate the existence of ‘master’ ever, the physiological functions of the presumed out- and ‘slave’ (or driven) oscillators, it is possible that cer- put pathways are unknown. tain oscillating RNAs might be important for clock out- Whereas oscillations in clock state variables (such as put functions. per, tim and frq products) are thought to be driven by RNAs encoding certain basic leucine zipper (bZip) changes in the abundances and/or activities of tran- transcription factors such as D-binding Protein (DBP), scription factors (based largely on the identity of cer- Thyroid Embryonic Factor (TEF), and Hepatocyte Leu- tain clock components as putative bHLH transcription kemia Factor (HLF) exhibit marked circadian fluctu- factors; see earlier discussion), the Atgrp7 rhythm ations in abundance (Ref. 21 and references therein),21 clearly involves the activity of a putative RNA-binding and it has been postulated that circadian changes in protein; ie, the product of the Atgrp7 gene itself. the activities of such factors account for the observed Although it is not yet known whether the Atgrp7 RNA- diurnal regulation of known target genes.22 Interest- binding protein influences transcriptional or post-tran- ingly, DBP mRNA abundance shows circadian oscil- scriptional events to regulate Atgrp7 RNA abundance, lations within the suprachiasmatic nuclei, the anatom- the identity of the protein is consistent with obser- ical location of the mammalian circadian pacemaker, vations in at least two other systems that RNA-binding and DBP-null mutant mice have slightly shorter circad- proteins might have roles as clock output components. ian periods as assayed by locomotor activity rhythms.21 However, the mutants also have lower levels of spon- Gonyaulax bioluminescence rhythms and RNA- taneous activity, and it is known that acute changes in binding proteins activity levels can phase shift the clock and/or alter The unicellular alga Gonyaulax polyedra exhibits well- circadian period.23 Thus, at present there is little evi- characterized circadian rhythms in bioluminescence,28 dence that DBP functions as a clock molecule, and per- which have been used as an assay of clock output by haps it is more likely that DBP plays a role as a clock Hastings and colleagues. Elegant studies by these output component.21 investigators (reviewed in Ref. 29) have demonstrated Perhaps not surprisingly, there is also evidence from that clock-driven translational regulatory mechanisms studies in other model systems that specific transcrip- contribute to the observed circadian fluctuations in the tion factors can mediate clock output functions. In the abundance of proteins which are known to participate cyanobacterium Synechococcus, for example, a sigma- in luminescence (such as luciferase-binding protein or like transcription factor is postulated to function as a LBP). That is to say, in contrast to clock RNAs such as clock output element to control a subset of rhythmi- per, tim,orfrq, lbp mRNA does not oscillate in abun- cally transcribed genes.24 Obviously, transcriptional dance; rather LBP protein synthesis changes in a cir- regulatory mechanisms or changes in RNA stability cadian manner.29 Furthermore, studies in Gonyaulax must be involved in the control of all rhythms in and the unrelated unicellular Chlamydomonas rhein- RNA abundance. hardii, suggest that RNA-binding proteins, with an affinity for a defined GU-rich element within lbp 3′ untranslated sequence, are responsible for the circad- Post-transcriptional mechanisms and clock output ian regulation of lbp mRNA translation.30 It is also of The Arabidopsis Atgrp7 RNA-binding protein and interest that these RNA-binding proteins, which so far clock output have been identified only as lbp-binding factors from The activities of certain oscillating molecules, in parti- extracts of Gonyaulax or Chlamydomonas, have cular the PAS domain-containing clock proteins, are characteristics of repressor proteins; ie, the apparent critical for the function of the circadian system; how- binding activities of these factors are highest at the cir- ever, many of the rhythmically transcribed RNAs more cadian time when LBP synthesis is lowest. likely represent very distal targets of the clock rather than key regulatory elements of a clock output path- Lark, a Drosophila RNA-binding protein mediating way. A clear exception to this statement is the Arabi- clock output dopsis thaliana Atgrp7 gene and its encoded product

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