Genome-wide Expression Analysis of Mouse Liver Reveals CLOCK-regulated Circadian Output Genes

Teikyo University, Edo, Tōkyō, Japan
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2003; 278(42):41519-27. DOI: 10.1074/jbc.M304564200
Source: PubMed


CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5'-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues.

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    • "This is notable, because among the hundreds of clock-controlled transcripts, only a small minority cycles in multiple locations. The circadian rhythm of Usp2 is blunted in Clock mutant and Bmal1 KO mice (Oishi et al., 2003; Molusky et al., 2012b), and the Usp2 promoter is activated by CLOCK/BMAL1 (Molusky et al., 2012b), indicating that Usp2 is a direct target of these transcription factors. In addition to its circadian regulation , Usp2 expression is also induced by starvation and it was therefore proposed that USP2 integrates nutritional and circadian timing cues (Molusky et al., 2012b). "
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    ABSTRACT: Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.
    Full-text · Article · Aug 2014 · Frontiers in Molecular Neuroscience
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    • "The Clock-Bmal1 heterodimer binds directly to the E-box element of the clock controlled genes (CCGs) [19], [20]. Occludin and Claudin-1 possess E-box elements in the promoter regions of the genes [21], [22]. "
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    Full-text · Article · May 2014 · PLoS ONE
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    • "Evidence suggests that circadian gene expression is achieved through a transcriptional cascade in which the expression of other TFs responsible for the majority of rhythmic tissue-specific expression are primarily driven directly by the main clock transcription factor complex CLOCK:BMAL1 [8]. Recently, this hypothesis has been reinforced by experiments showing that TFs whose expression patterns exhibit daily oscillation are direct targets of the CLOCK:BMAL1 complex [9,10]. However, those studies only explored the transcriptome with one main clock transcription factor mutated and thus the entire picture of how the system behaves or how target genes are controlled is still a question. "
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    ABSTRACT: The circadian clock is a critical regulator of biological functions controlling behavioral, physiological and biochemical processes. Because the liver is the primary regulator of metabolites within the mammalian body and the disruption of circadian rhythms in liver is associated with severe illness, circadian regulators would play a strong role in maintaining liver function. However, the regulatory structure that governs circadian dynamics within the liver at a transcriptional level remains unknown. To explore this aspect, we analyzed hepatic transcriptional dynamics in Sprague-Dawley rats over a period of 24 hours to assess the genome-wide responses. Using an unsupervised consensus clustering method, we identified four major gene expression clusters, corresponding to central carbon and nitrogen metabolism, membrane integrity, immune function, and DNA repair, all of which have dynamics which suggest regulation in a circadian manner. With the assumption that transcription factors (TFs) that are differentially expressed and contain CLOCK:BMAL1 binding sites on their proximal promoters are likely to be clock-controlled TFs, we were able to use promoter analysis to putatively identify additional clock-controlled TFs besides PARF and RORA families. These TFs are both functionally and temporally related to the clusters they regulate. Furthermore, we also identified significant sets of clock TFs that are potentially transcriptional regulators of gene clusters. All together, we were able to propose a regulatory structure for circadian regulation which represents alternative paths for circadian control of different functions within the liver. Our prediction has been affirmed by functional and temporal analyses which are able to extend for similar studies.
    Full-text · Article · Mar 2014 · BMC Bioinformatics
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