Circadian Transcriptional Regulation Research Articles

Temperature-driven coordination of circadian transcriptional regulation.

The circadian clock is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce circadian oscillations in thousands of genes in a tissue-specific manner, orchestrating myriad biological processes. While previous studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome: genes are cycling in a temperature-specific manner, and the distributions of their phases also differ between the two conditions. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low temperatures and synchronization of rhythmic genes that are network neighbors. We report that the phase differences between cycling genes increase as a function of geodesic distance in the low temperature condition, suggesting increased coordination of cycling on the gene regulatory network. Our results suggest a potential mechanism whereby the circadian clock mediates the fly's response to seasonal changes in temperature.

Acute Deletion of the Glucocorticoid Receptor in Hepatocytes Disrupts Postprandial Lipid Metabolism in Male Mice.

Hepatic lipid metabolism is highly dynamic, and disruption of several circadian transcriptional regulators results in hepatic steatosis. This includes genetic disruption of the glucocorticoid receptor (GR) as the liver develops. To address the functional role of GR in the adult liver, we used an acute hepatocyte-specific GR knockout model to study temporal hepatic lipid metabolism governed by GR at several preprandial and postprandial circadian timepoints. Lipidomics analysis revealed significant temporal lipid metabolism, where GR disruption results in impaired regulation of specific triglycerides, nonesterified fatty acids, and sphingolipids. This correlates with increased number and size of lipid droplets and mildly reduced mitochondrial respiration, most noticeably in the postprandial phase. Proteomics and transcriptomics analyses suggest that dysregulated lipid metabolism originates from pronounced induced expression of enzymes involved in fatty acid synthesis, β-oxidation, and sphingolipid metabolism. Integration of GR cistromic data suggests that induced gene expression is a result of regulatory actions secondary to direct GR effects on gene transcription.

Circadian mechanisms in adipose tissue bioenergetics and plasticity.

The circadian clock plays an essential role in coordinating feeding and metabolic rhythms with the light/dark cycle. Disruption of clocks is associated with increased adiposity and metabolic disorders, whereas aligning feeding time with cell-autonomous rhythms in metabolism improves health. Here, we provide a comprehensive overview of recent literature in adipose tissue biology as well as our understanding of molecular mechanisms underlying the circadian regulation of transcription, metabolism, and inflammation in adipose tissue. We highlight recent efforts to uncover the mechanistic links between clocks and adipocyte metabolism, as well as its application to dietary and behavioral interventions to improve health and mitigate obesity.

Circadian clock protein BMAL1 broadly influences autophagy and endolysosomal function in astrocytes

An emerging role for the circadian clock in autophagy and lysosome function has opened new avenues for exploration in the field of neurodegeneration. The daily rhythms of circadian clock proteins may coordinate gene expression programs involved not only in daily rhythms but in many cellular processes. In the brain, astrocytes are critical for sensing and responding to extracellular cues to support neurons. The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function but also leads to a unique cell-autonomous activation phenotype. We report here that astrocyte-specific deletion of Bmal1 influences endolysosome function, autophagy, and protein degradation dynamics. In vitro, Bmal1-deficient astrocytes exhibit increased endocytosis, lysosome-dependent protein cleavage, and accumulation of LAMP1- and RAB7-positive organelles. In vivo, astrocyte-specific Bmal1 knockout (aKO) brains show accumulation of autophagosome-like structures within astrocytes by electron microscopy. Transcriptional analysis of isolated astrocytes from young and aged Bmal1 aKO mice indicates broad dysregulation of pathways involved in lysosome function which occur independently of TFEB activation. Since a clear link has been established between neurodegeneration and endolysosome dysfunction over the course of aging, this work implicates BMAL1 as a key regulator of these crucial astrocyte functions in health and disease.

Butt-seq: a new method for facile profiling of transcription.

A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, Butt-seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that Butt-seq is a simple and powerful technique to analyze transcription at a high level of resolution.

558 An aging-susceptible circadian rhythm controls cutaneous antiviral immunity

Aged skin is prone to viral infections, but the mechanisms responsible for this are unclear. We hypothesize that an aging circadian rhythm compromises cutaneous innate antiviral immunity. In support of this hypothesis, we observed that aged mammalian skin exhibits significant reduction in transcriptional levels of antiviral proteins (AVPs) and circadian transcriptional regulators including BMAL1 and CLOCK, in vivo and in vitro. Using BMAL1 and CLOCK knockout mouse models, we found that loss of circadian factors significantly reduces AVP expression in the skin.

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock‐controlled protein expression, driven by daily cycles of CRYPTOCHROME‐dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild‐type and CRY‐deficient fibroblasts under constant conditions. In CRY‐deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild‐type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY‐deficient cells. This proteome imbalance in CRY‐deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide‐ranging phenotypes of CRY‐deficient mice. Rather than generating large‐scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post‐translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Dysfunction of the circadian clock in the kidney tubule leads to enhanced kidney gluconeogenesis and exacerbated hyperglycemia in diabetes

The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes.

Reversible dysregulation of renal circadian rhythm in lupus nephritis

BackgroundWe have found disruption of expression of major transcriptional regulators of circadian rhythm in the kidneys of several mouse models of lupus nephritis. Here we define the consequence of this disturbance with respect to circadian gene expression and renal homeostatic function in a mouse model of lupus nephritis.MethodsMolecular profiling of kidneys from 47 young and 41 nephritic female NZB/W F1 mice was performed at 4 hourly intervals over a 24 h period. Disruption of major circadian transcriptional regulators was confirmed by qPCR. Molecular data was normalized and analyzed for rhythmicity using RAIN analysis. Serum aldosterone and glucose and urine sodium and potassium were measured at 4 hourly intervals in pre-nephritic and nephritic mice and blood pressure was measured every 4 h. Analyses were repeated after induction of complete remission of nephritis using combination cyclophosphamide and costimulatory blockade.ResultsWe show a profound alteration of renal circadian rhythms in mice with lupus nephritis affecting multiple renal pathways. Using Cosinor analysis we identified consequent alterations of renal homeostasis and metabolism as well as blood pressure dipper status. This circadian dysregulation was partially reversed by remission induction therapy.ConclusionsOur studies indicate the role of inflammation in causing the circadian disruption and suggest that screening for loss of normal blood pressure dipping should be incorporated into LN management. The data also suggest a potential role for circadian agonists in the treatment of lupus nephritis.

N6-methyladenosine demethylase ALKBH5 suppresses malignancy of esophageal cancer by regulating microRNA biogenesis and RAI1 expression.

N6-Methyladenosine (m6A) is the most prevalent epigenetic RNA modification and is vital in regulating malignancies. The roles of m6A modifiers on noncoding RNAs have not been fully investigated in esophageal cancer. By screening all m6A modifiers, ALKBH5 was the most potent member related to patient outcomes and suppressing esophageal cancer malignancy in cell and animal models. It demethylated pri-miR-194-2 and inhibited miR-194-2 biogenesis through an m6A/DGCR8-dependent manner. RAI1, previously considered as a circadian clock transcriptional regulator, was the main target of miR-194-2. It enhanced transcription of Hippo pathway upstream genes by binding to their 3'UTR and suppressed YAP/TAZ nuclear translocation. The ALKBH5/miR-194-2/RAI1 axis was also validated in clinical samples. In addition, the increased malignancy by low ALKBH5 was abolished by the YAP inhibitor verteporfin. Our findings uncover a critical role of ALKBH5 in miRNAs biogenesis and provide novel insight for developing treatment strategies in esophageal cancer.

Expression Signatures of microRNAs and Their Targeted Pathways in the Adipose Tissue of Chickens during the Transition from Embryonic to Post-Hatch Development.

As the chick transitions from embryonic to post-hatching life, its metabolism must quickly undergo a dramatic switch in its major energy source. The chick embryo derives most of its energy from the yolk, a lipid-rich/carbohydrate-poor source. Upon hatching, the chick’s metabolism must then be able to utilize a lipid-poor/carbohydrate-rich source (feed) as its main form of energy. We recently found that a number of hepatically-expressed microRNAs (miRNAs) help facilitate this shift in metabolic processes in the chick liver, the main site of lipogenesis. While adipose tissue was initially thought to mainly serve as a lipid storage site, it is now known to carry many metabolic, endocrine, and immunological functions. Therefore, it would be expected that adipose tissue is also an important factor in the metabolic switch. To that end, we used next generation sequencing (NGS) and real-time quantitative PCR (RT-qPCR) to generate miRNome and transcriptome signatures of the adipose tissue during the transition from late embryonic to early post-hatch development. As adipose tissue is well known to produce inflammatory and other immune factors, we used SPF white leghorns to generate the initial miRNome and transcriptome signatures to minimize complications from external factors (e.g., pathogenic infections) and ensure the identification of bona fide switch-associated miRNAs and transcripts. We then examined their expression signatures in the adipose tissue of broilers (Ross 708). Using E18 embryos as representative of pre-switching metabolism and D3 chicks as a representative of post-switching metabolism, we identified a group of miRNAs which work concordantly to regulate a diverse but interconnected group of developmental, immune and metabolic processes in the adipose tissue during the metabolic switch. Network mapping suggests that during the first days post-hatch, despite the consumption of feed, the chick is still heavily reliant upon adipose tissue lipid stores for energy production, and is not yet efficiently using their new energy source for de novo lipid storage. A number of core master regulatory pathways including, circadian rhythm transcriptional regulation and growth hormone (GH) signaling, likely work in concert with miRNAs to maintain an essential balance between adipogenic, lipolytic, developmental, and immunological processes in the adipose tissue during the metabolic switch.

A circadian clock regulates efflux by the blood-brain barrier in mice and human cells

The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY‐deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY‐less oscillations are variable in their expression and have shorter periods than wild‐type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY‐mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post‐translational.

Sleep Disturbance Induces Increased Cholesterol Level by NR1D1 Mediated CYP7A1 Inhibition.

Disturbed sleep is closely associated with an increased risk of metabolic diseases. However, the underlying mechanisms of circadian clock genes linking sleep and lipid profile abnormalities have not been fully elucidated. This study aimed to explore the important role of the circadian clock in regulating impaired cholesterol metabolism at an early stage of sleep deprivation (SD). Sleep disturbance was conducted using an SD instrument. Our results showed that SD increased the serum cholesterol levels. Concentrations of serum leptin and resistin were much lower after SD, but other metabolic hormone concentrations (adiponectin, glucagon, insulin, thyroxine, norepinephrine, and epinephrine) were unchanged before and after SD. Warning signs of cardiovascular diseases [decreased high density lipoprotein (HDL)-cholesterol and increased corticosterone and 8-hydroxyguanosine levels] and hepatic cholestasis (elevated total bile acids and bilirubin levels) were observed after SD. Cholesterol accumulation was also observed in the liver after SD. The expression levels of HMGCR, the critical enzyme for cholesterol synthesis, remained unchanged in the liver. However, the expression levels of liver CYP7A1, the enzyme responsible for the conversion of cholesterol into bile acids, significantly reduced after SD. Furthermore, expression of NR1D1, a circadian oscillator and transcriptional regulator of CYP7A1, strikingly decreased after SD. Moreover, NR1D1 deficiency decreased liver CYP7A1 levels, and SD could exacerbate the reduction of CYP7A1 expression in NR1D1−/− mouse livers. Additionally, NR1D1 deficiency could further increase serum cholesterol levels under SD. These results suggest that sleep disturbance can induce increased serum cholesterol levels and liver cholesterol accumulation by NR1D1 mediated CYP7A1 inhibition.

Abstract PR05: Epigenetic disruption of vitamin D receptor signaling in African American prostate cancer alters circadian signaling networks

Abstract The current study aimed to define the genomic functions of the vitamin D receptor (VDR) in African American (AA) prostate cancer (PCa) compared to European American (EA) counterparts. VDR-dependent ChIP-Seq and RNA-Seq gene was undertaken in EA (non-malignant HPr1AR and malignant LNCaP) and AA (non-malignant RC43N and malignant RC43T) prostate models, combined with analyses of three PCa cohorts. In AA prostate models the VDR is highly expressed, binds more frequently, and is enriched in active and poised enhancers. Motif analyses revealed selective enrichment, including ZBTB33/KAISO in AA cells and ERG family members in EA cells and, similarly, GIGGLE analyses revealed AA VDR cistromes were significantly overlapped with core circadian rhythm transcription factors (e.g. NONO). Combining VDR-dependent ChIP-Seq and RNA-Seq established that AA cells displayed a significantly stronger transcriptional response, compared to EA cells, and was most responsive in non-malignant RC43N. For example, RC43N transcriptional responses were enriched for circadian rhythm (NES 2.7) and inflammation, whereas in RC43T the same gene networks were repressed. To reveal how VDR/1,25(OH)2D3 signaling is corrupted in AA PCa, we mined TCGA data and revealed that the BAZ1A/SMARCA5 chromatin remodeling complex was uniquely altered in TMPRSS2:ERG fusion negative AA PCa. We are currently examining the impact of BAZ1A on the 1,25(OH)2D3 responsiveness. We also identified miRNA associated with progression from HGPIN to PCa in AA men, and that ~30% were bound by VDR and regulated by 1,25(OH)2D3, although ~5% of EA progression miRNA were VDR-responsive. For example, VDR binds to miR-199b, is uniquely 1,25(OH)2D3 up-regulated in RC43N but repressed in RC43T, and associates with AA progression from HGPIN to PCa. MiR-199b regulates expression of NPAS2, a core circadian transcription factor. Finally, leveraging a previously analyzed cohort of 1,25(OH)2D3-treated PCa patients revealed that AA tumors were intrinsically more 1,25(OH)2D3-responsive than EA counterparts, reflecting the cell line analyses. 1,25(OH)2D3 regulated circadian transcriptional regulators (e.g. NOCT and MYBBP1A) and inflammatory signals. Together, these data suggest VDR transcriptional control in AA men is more dynamic than in EA men, and is primed to govern inflammatory and circadian rhythm pathways. This is frequently disrupted, including by altered BAZ1A/SMARCA5 expression and/or reduced environmental-regulated serum vitamin D3 levels, and leads to altered regulation of circadian rhythm process, and inflammatory signals. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes that contributes to PCa health disparities. Citation Format: Manjunath Siddappa, Sajad D. Wani, Jaimie S. Gray, Mark D. Long, Hedieh Jafari, Hsuchang Wu, Honhe Wang, Rebecca Morgan, Gary Hardiman, James Marshall, Chanita Hughes-Halbert, Lara E. Sucheston-Campbell, Clayton L. Yates, Moray J. Campbell. Epigenetic disruption of vitamin D receptor signaling in African American prostate cancer alters circadian signaling networks [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR05.

Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila.

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W1118 and a mutant of the key circadian transcriptional regulator Clock (Clkjrk ). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clkjrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.

Boolean function network analysis of time course liver transcriptome data to reveal novel circadian transcriptional regulators in mammals.

Many biological processes in mammals are subject to circadian control at the molecular level. Disruption of circadian rhythms has been demonstrated to be associated with a wide range of diseases, such as diabetes mellitus, mental disorders, and cancer. Although the core circadian genes are well established, there are multiple reports of novel peripheral circadian regulators. The goal of this study was to provide a comprehensive computational analysis to identify novel potential circadian transcriptional regulators. To fulfill the aforementioned goal, we applied a Boolean function network method to analyze the microarray time course mouse and rat liver datasets available in the literature. The inferred direct pairwise relations were further investigated using the functional annotation tool. This approach generated a list of transcription factors (TFs) and cofactors, which were associated with significantly enriched circadian gene ontology (GO) categories. As a result, we identified 93 transcriptional circadian regulators in mouse and 95 transcriptional circadian regulators in rat. Of these, 19 regulators in mouse and 21 regulators in rat were known, whereas the rest were novel. Furthermore, we validated novel circadian TFs with bioinformatics databases, previous large-scale circadian studies, and related small-scale studies. Moreover, according to predictions inferred from ChIP-Seq experiments reported in the database, 40 of our candidate circadian regulators were confirmed to have circadian genes as direct regulatory targets. In addition, we annotated candidate circadian regulators with disorders that were often associated with disruptions of circadian rhythm in the literature. In summary, our computational analysis, which was followed by an extensive verification by means of a literature review, can contribute to translational study from endocrinology to cancer research and provide insights for future investigation.

Caloric restriction effects on liver mTOR signaling are time-of-day dependent.

The regulation of mechanistic target of rapamycin (mTOR) signaling contributes to the metabolic effects of a calorie restriction (CR) diet. We assayed the effect of CR on the activity of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) in the liver of mice at six different times across the day. CR effects on mTORC1 and mTORC2 activities were time-of-day dependent. CR induced mTORC1 activity at one time, reduced at two times and has no effect during other times. CR induced mTORC2 activity at one time of the day and has no effects at other times. Circadian clocks are implemented in the regulation of mTOR signaling in mammals and mechanisms of CR. We assayed the effect of CR on mTOR signaling in the liver of mice deficient for circadian transcriptional regulators BMAL1 and CRYs. The CR induced suppression of mTORC1 activity was observed in both clock mutants, while up regulation of mTORC2 was observed in the liver of CRY deficient but not in the liver of BMAL1 deficient mice. Our finding revealed that CR has different time dependent effect on the activity of mTOR complexes 1 and 2 and suggest that circadian clock protein BMAL1 is involved in the up regulation of mTORC2 upon CR in mammals.

A HAD family phosphatase CSP-6 regulates the circadian output pathway in Neurospora crassa.

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.

CREBH Maintains Circadian Glucose Homeostasis by Regulating Hepatic Glycogenolysis and Gluconeogenesis.

Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.