Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.epigenomics | systems biology T he molecular clockwork consists of well-characterized transcriptional components [including Clock, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like (Bmal) 1, Period, Cryptochrome, and nuclear receptor subfamily 1, group D, member 1/2 (Rev-erbα/β)], as well as more recently discovered posttranscriptional processes (1-4). Despite progress in understanding the make up of circadian transcriptomes and proteomes, which are thought to encompass more than 10% of known genes and proteins, the systems-level mechanisms driving their rhythmic abundance have remained unclear (5-7).Based on observations that a few specific clock-regulated genomic loci undergo changes in chromatin state over the circadian cycle (8-10), we hypothesized that this might be more generally applicable at other clock-controlled gene (CCG) loci. We investigated this by performing chromatin immunoprecipitation (ChiP) and high-throughput sequencing (ChIP-seq) on mouse liver tissue collected over the circadian cycle to delineate global changes in the epigenome over a 24-h time frame. Results and DiscussionWe focused mostly on the activation mark, H3K4me3 (histone H3 trimethylated at lysine 4) (11), which exhibited a clear circadian profile at thousands of genomic loci ( Fig. 1 A and B). This was in stark contrast to H3K9me3 (histone H3 trimethylated at lysine 9), which is associated with transcriptional inhibition (12), and unmodified histone H3, the binding of which changed only at relatively few loci (Fig. 1A). Therefore, genomic scale changes in the evolutionarily conserved activation mark H3K4me3 are regulated in a circadian manner at thousands ...

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

Circadian clock genes regulate 10-15% of the transcriptome, and might function as tumor suppressor genes. Relatively little is known about the circadian clock in tumors and its effect on surrounding healthy tissues. Therefore, we compared the 24-hr expression levels of key circadian clock genes in liver and kidney of healthy control mice with those of mice bearing C26 colorectal tumor metastases in the liver. Metastases were induced by injection of C26 colorectal carcinoma cells into the spleen. Subsequently, tumor, liver and kidney tissue was collected around the clock to compare circadian rhythmicity. Expression levels of five clock genes (Rev-Erba, Per1, Per2, Bmal1 and Cry1) and three clock-controlled genes (CCGs; Dbp, p21 and Wee1) were determined by qRT-PCR. Liver and kidney tissue of healthy control mice showed normal 24-hr oscillations of all clock genes and CCGs, consistent with normal circadian rhythmicity. In colorectal liver metastases, however, 24-hr oscillations were completely absent for all clock genes and CCGs except Cry1. Liver and kidney tissue of tumor-bearing mice showed a shift in clock periodicity relative to control mice. In the liver we observed a phase advance, whereas in the kidney the phase was delayed. These data show that hepatic metastases of C26 colon carcinoma with a disrupted circadian rhythm phase shift liver and kidney tissue clocks, which strongly suggests a systemic effect on peripheral clocks. The possibility that tumors may modify peripheral clocks to escape from ordinary circadian rhythms and in this way contribute to fatigue and sleep disorders in cancer patients is discussed.The circadian timing system plays an essential role in the development of cancer. Cancer cells carry a similar machinery of increased proliferation rate, reduced apoptotic sensitivity and escaping cell-cycle control. These parameters are controlled by the circadian clock. [1][2][3][4] Circadian rhythms are generated by a molecular oscillator composed of a set of clock genes that act in a cellautonomous way and are present in all cell types. These clocks are coordinated by a master clock in the neurons of the suprachiasmatic nuclei (SCN), which is located in the anterior hypothalamus. 5 Peripheral clocks are regulated by the SCN through both the autonomic nervous system and neuroendocrine systems. Clocks in the SCN neurons and peripheral cells make use of the same set of clock genes. 6 The positive branch of the mammalian clock machinery consists of CLOCK (Circadian Locomotor Output Cycles Kaput) and BMAL1 (Brain-Muscle Arnt-Like protein 1). CLOCK and BMAL1 are transcription factors that heterodimerize and activate transcription of the Cryptochrome (Cry1 and Cry2) and Period (Per1 and Per2) genes by binding to E-box elements in their promoters. After being synthesized in the cytoplasm, CRY and PER proteins heterodimerize and enter the nucleus where they inhibit CLOCK-BMAL1-mediated transcription and accordingly close the negative feedback loop. 7 The core mechanism is more complex than a single autor...

SummaryThe circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

The tau mutation in Syrian hamsters (Mesocricetus auratus) is phenotypically expressed in a period of the circadian rhythm of about 20 h in homozygotes (SS) and about 22 h in heterozygotes (S+). The authors investigate whether this well-defined model for variation in circadian period exhibits associated changes in energy metabolism. In hamsters of the three genotypes (SS, S+, and wild type [WT]), oxygen consumption measurements were performed at 28 degrees C (thermoneutral), 18 degrees C, and (after acclimatization) 10 degrees C. After correction for body mass, SS tau mutant hamsters had a higher overall metabolic rate (average oxygen consumption per hour over 24 h) and a higher resting metabolic rate (the lowest 30-min oxygen consumption in the subjective day) than did WT hamsters at all ambient temperatures. S+ hamsters were intermediate in both after taking body mass into account. The differences in metabolism among the three genotypes indicate that the increase in metabolic rate was statistically indistinguishable from a proportional increase in circadian frequency. The oxygen consumption totals per circadian cycle (24 h for WT, 22 h for S+, and 20 h for SS mutants) were not statistically different among the genotypes after correcting for body mass. The possible roles of pleiotropic effects, of linkage to genes involved in growth and metabolism, and of early ontogenetic influences are briefly discussed.