Serial VECTOR Clock Pro 2.2.rar
Dortha Chuang
Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism. Validating BMAL1 and REV-ERBα targets using luciferase assays and in vivo rescue, we demonstrate how a major role of the muscle clock is to promote diurnal cycles of neutral lipid storage while coordinately inhibiting lipid and protein catabolism prior to awakening. This occurs by BMAL1-dependent activation of Dgat2 and REV-ERBα-dependent repression of major targets involved in lipid metabolism and protein turnover (MuRF-1, Atrogin-1). Accordingly, muscle-specific loss of BMAL1 is associated with metabolic inefficiency, impaired muscle triglyceride biosynthesis, and accumulation of bioactive lipids and amino acids. Taken together, our data provide a comprehensive overview of how genomic binding of BMAL1 and REV-ERBα is related to temporal changes in gene expression and metabolite fluctuations.
Circadian clocks are known to regulate local and systemic homeostasis by anticipating rhythmic changes in behavior and nutritional state and by compartmentalizing incompatible metabolic pathways within precise temporal and spatial windows. Yet a precise mechanistic understanding of how the circadian clock in skeletal muscle controls homeostasis is just beginning to come to light. Here, we investigated how the muscle clock directs 24-hr metabolic rhythms. We compared genome-wide binding of clock transcription factors brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα with 24-hr transcriptional and metabolic effects after their loss of function specifically in muscles. We found that the muscle clock plays a major role anticipating the transition from fasting to feeding. This occurs by direct activation of transcriptional programs promoting lipid storage, insulin sensitivity, and glucose metabolism, with coordinated repression of programs controlling lipid oxidation and protein catabolism. Importantly, these gene expression changes occur in the hours prior to systemic metabolic and hormonal cues that arise upon awakening. As such, we find that the muscle clock tips the scales in favor of glucose metabolism, whereas loss of function of the clock transcription factor BMAL1 is associated with persistent lipid metabolism, protein catabolism, and metabolic inefficiency.
Copyright: 2018 Dyar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: ChIP-seq data are available at Gene Expression Omnibus, accession number GSE108650. Microarray data are available at Gene Expression Omnibus, accession number GSE43071. All other relevant data are within the paper and its Supporting Information files.
Funding: Emmy Noether grant (DFG) (grant number UH275/1-1). NHU. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. European Commission (Integrated Project MYOAGE) (grant number 223576). SS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Telethon Italy Grant (grant number GGP13222). LS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fondazione CARIPARO, Padova. LS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Circadian rhythms of metabolism are endogenously generated and maintained by tissue-specific gene networks under the transcriptional control of molecular clocks [1]. While chronic misalignment/disruption of circadian clocks has consistently been linked to metabolic disorders and diseases [2], precise pathogenic mechanisms and their relation to tissue-specific clock function remain largely undefined.
Global and tissue-specific conditional or inducible loss-of-function mouse models targeting clock genes have begun to unravel such relationships in skeletal muscle [3], a multifaceted and highly dynamic tissue and a major player in whole-body energy homeostasis. Depending on fluctuations in energy supply and demand, skeletal muscle plays various essential metabolic roles in the uptake, storage, utilization, and release of oxidative substrates. Experimental efforts have established muscle as the main site for insulin-stimulated glucose disposal [4] and a major consumer of lipoprotein-triacylglycerol-derived fatty acid and plasma free fatty acids (FFAs) [5]. Importantly, muscle tissue serves a highly dynamic role as the main destination for circulating amino acids in the fed state and the main source of circulating amino acids during starvation and insulin deficiency [6].
To understand how the muscle clock can transcriptionally direct rhythmic metabolism, we mapped in vivo genome-wide chromatin occupancy (cistromes) of the positive master clock regulator BMAL1 and the dominant repressor REV-ERBα in adult mouse gastrocnemius muscles. Accordingly, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) on tissues collected during maximum diurnal protein expression for BMAL1 and REV-ERBα at Zeitgeber time (ZT) 4 and 8, respectively (S1A Fig).
Comparing our muscle cistromes to published mouse liver cistrome data for BMAL1 [14] and REV-ERBα [15], we found only 46 common BMAL1 peaks between tissues, associated with 42 common genes, and 264 common REV-ERBα peaks associated with 252 common genes (S1B Fig and S1 Table). Common muscle and liver targets were mainly core circadian clock and known clock-dependent output genes. Interestingly, common muscle and liver REV-ERBα targets were additionally enriched for p53 signaling components, chromatin modifiers, as well as macroautophagy and mitophagy mediators. However, the vast majority of sites (98% of BMAL1 peaks and 80% of REV-ERBα peaks) were muscle specific. This implies highly tissue-specific roles for BMAL1 and REV-ERBα beyond their common regulation of core clock genes.
To provide a functional context for associations between BMAL1 and REV-ERBα binding sites and target gene regulation, we focused on transcriptional changes of key targets and coordinately regulated gene networks after mKO and loss of REV-ERBα [7,17]. REACTOME pathway enrichment analysis [20] performed on 931 differentially expressed genes in muscles from mKO mice [7] uncovered changes in canonical clock-related pathways and general perturbations in fatty acid, triglyceride (TG), and phospholipid metabolism (Fig 2A).
REV-ERBα/β are known to regulate lipid metabolism in peripheral tissues [12,15,27], including skeletal muscle [28,29], but a comprehensive understanding of their direct muscle targets, their cross-talk with other nuclear hormone receptors, and the effects of muscle-specific loss of function remain largely unknown. REV-ERBα is known to directly compete with and inhibit TR/RXR binding at target sites during muscle differentiation [30], and REV-ERBα and PPAR signaling pathways are likewise known to converge [31]. PPARs are nuclear hormone receptors that mediate adaptive metabolic responses, including increased lipid oxidation and amino acid catabolism in muscle [32] following their activation by endogenous or dietary lipids or lipid derivatives [33]. REV-ERBα/β is known to repress transactivation of some PPAR targets by competitively binding to nearby genomic sites in a concentration-dependent manner [34].
Consistent with motif enrichment of BMAL1 and REV-ERBα peaks (Fig 1D and S2E Fig), PPAR signaling was also among the top-ranking pathways impacted by mKO (Fig 2A). Furthermore, mKO muscles showed an altered gene expression profile reminiscent of acute pharmacological activation using the PPARδ receptor agonist GW501516 [35]. In particular, we noticed significantly increased expression of major regulatory genes involved in mobilizing intracellular lipid stores (Atgl/Pnpla2) and channeling fatty acids from lipid droplets to the mitochondria for oxidation (Plin5) (Fig 2E). Additionally, mKO muscles showed significantly increased expression of genes involved in fatty acid transport (Fatp-1/Slc27a1), activation of fatty acids to corresponding acyl-CoAs (Acsl1), and finally breakdown (Acadm) and oxidation (Hadha) of fatty acids. However, we observed no major changes in diurnal expression of PPARs in fast or slow muscles (S3B Fig), suggesting that increased PPAR target expression in mKO muscles results from increased presence of endogenous ligands and/or PPAR activation. Highlighting potential sites for cross-talk/competition between REV-ERBα and PPARs, we noted REV-ERBα peaks near known PPAR regulatory elements (PPREs) in promoters of Plin5 and Acsl1 [36,37].
To gain more mechanistic insight into how BMAL1 and REV-ERBα regulate muscle targets, and their functional roles, we further stratified our cistrome data according to direction of expression changes in mKO muscles (Fig 2G and S1 Table). Hundreds of BMAL1 and REV-ERBα targets showed differential regulation, suggesting a direct link between loss of BMAL1 and REV-ERBα and gene expression changes in mKO muscles. According to pathway enrichment analysis [20], down-regulated BMAL1 and REV-ERBα targets reflected common regulation of circadian clock genes and transforming growth factor beta (TGFβ) signaling components. Down-regulated BMAL1 targets were further associated with mitogen-activated protein kinase (MAPK) and other stress response pathways, whereas down-regulated REV-ERBα targets were associated with nuclear factor kappa B (NFκB) activation. Interestingly, up-regulated target genes showed less cooperation between BMAL1 and REV-ERBα and suggested more specific regulation of calcium and Wnt signaling by BMAL1, while REV-ERBα showed specificity for the GR signaling pathway and heme biosynthesis (Fig 2G). We further corroborated REV-ERBα-specific association with GR targets by visually inspecting prominent REV-ERBα peaks with high enrichment scores near selected GR-regulated genes (S2F Fig), including transcriptional regulators like Fos and Trp53 [38].
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