Clocks go forward: progress in the molecular genetic ... - Springer Link

Mamm Genome (2009) 20:67–70 DOI 10.1007/s00335-008-9166-1 COMMENTARY SERIES: FUTURE CHALLENGES AND OPPORTUNITIES IN MAMMALIAN FUNCTIONAL GENOMICS

Clocks go forward: progress in the molecular genetic analysis of rhythmic behaviour Patrick M. Nolan Æ Michael J. Parsons

Received: 21 November 2008 / Accepted: 8 December 2008 / Published online: 15 January 2009 Ó Springer Science+Business Media, LLC 2009

Abstract The molecular basis of circadian homeostasis has proven to be amenable to genetic dissection in many model organisms. Surprisingly, additional factors contributing to an organism’s ‘‘chronotype’’ continue to be identified using both forward and reverse genetics. As more factors are identified, the importance of rhythm regulation in all body systems is becoming apparent. Moreover, recent evidence confirms that the regulation of circadian homeostasis can be fine-tuned at a number of molecular levels. This not only ensures that biological rhythms are maintained at a robust level in all cells but also allows for the precise and rapid readjustment of rhythms in response to environmental factors.

Molecular processes regulating circadian rhythms, approximately 24-h oscillations in diverse biological parameters, provide a framework whereby an organism can anticipate and react to environmental change. It is now barely a decade since the first genes regulating mammalian circadian rhythms have been identified and characterised. Since then, additional insight gained through distinct experimental routes has attested to the complexity of the circadian regulatory mechanism in terms of how the internal pacemaker might respond to environmental cues, how clock genes interact in a tissue-specific manner, and how the core components of the system are regulated at multiple levels. The availability of molecular genetic tools—multiple cell-based reporter assays, sophisticated P. M. Nolan (&) M. J. Parsons MRC Harwell, Mammalian Genetics Unit, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK e-mail: [email protected]

protein interaction studies, analysis of compound mutants, conditional targeted mutagenesis—have given us the ability to investigate rhythms at a systematic level.

An expanding transcriptional oscillator The molecular components at the core of the oscillator are, by now, familiar to many (reviewed by Ko and Takahashi 2006). Clock and Bmal1 transcription factors drive the expression of Per and Cry genes, among others, through Ebox elements. Per and Cry negatively regulate Clock/ Bmal1-dependent transcription and events mediated through nuclear receptors Rora and Rev-erba (Nr1d1) maintain the cycle in an interlocking positive feedback loop. However, redundancy in this system is evident in that single Per and Cry mutations show mild rhythm phenotypes whereas double mutants are arrhythmic. Similarly, Npas2 is able to functionally substitute for Clock in mice to regulate rhythmicity (De Bruyne et al. 2007). Moreover, the Clock/Bmal1 complex is modifiable by a number of additional interacting/competing factors. In a yeast twohybrid screen, Zhao et al. (2007) have identified as many as 21 Clock-interacting proteins. One of these components, Clock-interacting protein, circadian (Cipc, mKIAA1737, 2310044G17Rik), is rhythmically expressed in multiple tissues and is a potent inhibitor of Clock/Bmal1 activity. Cipc has no invertebrate homologues, no similarity with any known protein, and no recognisable functional motifs or domains. Basic helix-loop-helix transcription factors Dec1 and Dec2 (Bhlhb2 and Bhlhb3) can repress Clock/ Bmal1-induced transactivation (Honma et al. 2002). Dec1/ Dec2 double mutants show a small but significant lengthening of the circadian cycle and an altered amplitude of circadian gene expression. One observation would suggest

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P. M. Nolan, M. J. Parsons: Molecular genetic analysis of rhythmic behaviour

that complex interactions can fine-tune E-box-mediated transcription in a tissue-specific fashion as this amplitude is higher in the suprachiasmatic nucleus (SCN, pacemaker) of double mutants whereas it is lower in cortex and liver (Rossner et al. 2008). Additional transcriptional oscillations may be modified independently of E-boxes. Reinke et al. (2008) used differential display of DNA-binding proteins to identify additional circadian transcription factors. They found that heat shock factor 1 (Hsf1) binds DNA in the dark phase and that mutant mice have a signifcantly longer wheel-running period although the effect size is small. This mechanism may be particularly relevant in perpheral circadian oscillators where homeostasis may be more reliant on temperature-dependent synchronisation, perhaps related to feeding and metabolic rate.

Histone modification and core protein acetylation Rhythmic oscillations in chromatin modification, including control of histone methylation and acetylation status, have been known to covary with circadian transcriptional rhythms (Etchegaray et al. 2003; Ripperger and Schibler 2006). The subtleties of this dynamic process are still under investigation, although this particular association is augmented by the fact that Clock can mediate histone acetylation (Doi et al. 2006), that both Bmal1 and Per2 can be acetylated, and that Per proteins can associate with a number of histone methylases (Brown et al. 2005; Etchegaray et al. 2006). Moreover, Clock-mediated acetylation can be counterbalanced by the action of Sirt1, an NAD?dependent deacetylase (Asher et al. 2008; Nakahata et al. 2008). Sirt1 itself is regulated in a circadian manner, can be recruited to the Clock/Bmal1 complex, and promotes the deacetylation of histone H3, Bmal1, and Per2. This action by Sirt1 potentially promotes Per2 degradation. Although Sirt1-/- mice have a poor postnatal survival rate, it is still possible to explore the consequence of this mutation in embryonic fibroblasts and in liver-specific conditional mutants. Molecular oscillations in core components are certainly affected although the nature of the effect is still not clear. These findings are particularly relevant as they provide another functional link between metabolic activity and clock homeostasis. Sirt1 could effectively be a conduit allowing metabolic events to modify the molecular clock and vice versa.

Post-transcriptional processing A number of reports implicate the regulation of RNA stability as an additional mechanism contributing to both the robustness and flexibility to the clock. Many of these

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mechanisms could lead to the design of efficient therapeutic agents to target disturbed circadian homeostatic and/ or metabolic processes. Post-transcriptional regulation of circadian gene expression through microRNAs (miRNAs) is evident both at a level of homeostasis and as a response to light signals from the environment (Cheng et al. 2007). Although their specific targets have yet to be identified, two miRNAs, miR-132 and miR-219, have a rhythmical expression in wild-type SCN that is disrupted in circadian mutants. Reduction in the expression of miR-219 results in lengthening of the circadian cycle in mice, while reduced miR-132 expression amplifies the response to nocturnal light on the cycle. Clock gene mRNA can also be regulated through 30 -untranslated region (UTR)-mediated mRNA decay. Recently, a polyA ribonuclease, nocturnin (Noc), with known circadian expression patterns in mouse (Wang et al. 2001), has been shown to play a crucial role as a downstream regulator of the circadian system by causing the post-transcriptional regulation of metabolic genes (Green et al. 2007). Circadian oscillations in Per2 mRNA may also be regulated by 30 -UTR-mediated decay. Depletion of polypyrimidine tract-binding protein (PTB) by RNAi results in Per2 mRNA stabilisation, whereas oscillations in Per2 mRNA show a reciprocal expression profile to that of cytoplasmic PTB (Woo et al. 2008).

Protein phosphorylation Phosphorylation state plays an important role in regulation of the circadian machinery (for a review, see Gallego and Virshup 2007). The phosphorylation state of a clock protein may influence its ability to form heterodimers or protein complexes, its subcellular localisation, or its stability. Although phosphorylation state is implicated for positive regulators such as the calcium-dependent, protein kinase C-mediated phosphorylation of Clock (Shim et al. 2007), much of the recent focus has been on the phosphorylation of negative regulators. In mammalian systems, GSK3b can mediate its circadian effects by stabilising a number of clock proteins (Iitaka et al. 2005; Yin et al. 2006), whereas lithium treatment can lead to the rapid proteasomal degradation of the clock protein Rev-erba and subsequent activation of the clock gene Bmal1 in culture. Per2 is the focus of the majority of clock phosphorylation studies. It can be heavily phosphorylated by casein kinase Ie (CKIe), leading to b-TrCP protein-mediated proteasomal degradation. Conversely, protein phosphatase 1 (PP1) has been shown to shift Per2 to the dephosphorylated state. Not surprisingly, both a dominant negative form of PP1 and PP1 inhibitors can accelerate the degradation of Per2 (Gallego et al. 2006). Mutations in humans and mice affecting Per2 phosphorylation by casein kinases (Xu et al.


2005, 2007) can dramatically shorten the clock cycle in mice and underlie familial advanced sleep phase syndrome in humans.

Ubiquitination, sumoylation, and protein degradation Regulation of clock elements at the level of protein stability provides a further means of fine-tuning daily oscillations. Recent studies have shown that the transactivation and subsequent degradation of the Clock/Bmal1 complex is dependent on the dual sumoylation, by SUMO2/3, and ubiquitination of Bmal1 (Lee et al. 2008). A great deal of recent work has shown that a number of genes from the F-box protein family of ubiquitin E3 ligases differentially affect the degradation of the negative regulators of the mammalian circadian cycle. Both b-TrCP1 and b-TrCP2 have been shown to bind phosphorylated Per1 (Shirogane et al. 2005), and Per2 (Ohsaki et al. 2008). While these two genes are involved in the in vitro regulation of circadian oscillations, b-TrCP1-deficient mice have a normal period length, again suggesting redundancy in the system (Ohsaki et al. 2008). Mutations in Fbxl3, whose protein product is ubiquitously expressed, results in lengthened behavioural and molecular rhythms due to the failure to mark Cry1 and Cry2 for proteasomal degradation (Godinho et al. 2007). Another F-box protein with rhythmic SCN expression, Fbxl21, similarly binds to Cry1 preventing its negative regulatory effects on the Per1 promoter (Dardente et al. 2008). Interestingly, Per and Cry protein families are marked for degradation by distinct Fbox proteins, suggesting that there are differences in temporal or spatial degradation patterns of these two classes of negative regulators.

Perspective Over the past decade, defining the mammalian circadian oscillator has progressed at a dramatic rate. Given the multiple levels at which the system is regulated, the tissuespecificity of its components, and their probable redundancy, the investigation of the circadian system will continue to be a challenge. Nevertheless, progress in the development of a number of molecular genetic tools should facilitate future studies. Analysis of tissue-specific and inducible targeted mutants will help in defining the spatiotemporal complexities of clock homeostasis, its environmental responsiveness, and its reciprocal interaction with other homeostatic mechanisms throughout the organism. Careful selection of compound mutants that ‘‘hit’’ the homeostatic mechanism at multiple levels will facilitate behavioural and molecular analysis of rhythms.

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Finally, the development of new cellular reporter assays will allow one to investigate an individual factor’s effect on multiple components of the system in parallel. Acknowledgment This work was supported by the MRC and by the 6th Framework Project EUCLOCK (No. 018741).

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