Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β

The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK–BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-a and REV-ERB-b have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with themaster circadian regulator BMAL1. Although REV-ERB-a has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-a and REV-ERB-b genomic regulatory circuits than was previously suspected. Geneswithin the intersection of the BMAL1, REV-ERB-a and REV-ERB-b cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-a and Rev-erb-b function by creating doubleknockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheelrunning behaviour and deregulated lipid metabolism. These data now unite REV-ERB-a and REV-ERB-b with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism. The circadian clock is a transcriptional mechanism that coordinates both behavioural and physiological processes such as the sleep–wake cycle and food intake. The existing model for the mammalian core molecular clock involves a transcriptional negative-feedback loop in which the transactivation of E-box-containing genes by CLOCK and BMAL1 is inhibitedby the expressionofPer1 andCry1, themselvesunder transcriptional control ofE boxes.As functional redundancy is common for core clock components, deletion of multiple paralogues in mice is often required to uncover relevant phenotypes such as perturbations in circadian gene expression, metabolism and wheel-running behaviour. As REV-ERB-a, REV-ERB-b, ROR-a, ROR-b and ROR-c bind to a common response element (the RORE), their intrinsic repressive and inductive activities, respectively, are believed to establish the rhythmic expressionof target genes suchasBmal1.However, thepartial penetrance and mild period phenotype of Rev-erb-a mice (essentially intact circadian wheel-running behaviour) and the modest rhythmic phenotype upon partial Rev-erb-b depletion of Rev-erb-a cultured cells, suggested that they are not required for principal core clock function. Rather, REV-ERBs are proposed to form an accessory feedback loop that stabilizes the clock and has a role in receiving input signals or transmitting output pathways. To clarify the regulatory potential of REV-ERB-a and REV-ERB-b in circadian regulation, we generated isoform-specific antibodies (Supplementary Methods and Supplementary Fig. 1) and determined their genome-wide cis-acting targets (cistromes) in the liver at Zeitgeber time (ZT) 8, the peak of their protein expression (data not shown and ref. 9).De novomotif analysis (Fig. 1a) revealed that in vivo, in addition to the classic REV-ERB direct repeat 2 (DR2) motifs, other nuclear receptor binding sites (particularly DR1) are predominant in the DNA elements bound by both REV-ERB-a and REV-ERB-b. Although not surprising, extensive overlap was observed between the REV-ERB cistromes, with commonly bound peaks accounting for 54.8% and 60.7% of the total REV-ERB-a and REV-ERB-b peaks, respectively (Fig. 1b). The limited overlap of our REV-ERB-a cistrome with one that has been published previously (,50%) can most likely be attributed to differences in the antibody specificities (Supplementary Figs 2 and 3). Pathway analyses of our REV-ERB-a and REVERB-b overlapping peaks revealed an enrichment in lipidmetabolism genes (Fig. 1c), consistent with the hyperlipidaemic phenotype previously observed in Rev-erb-a null mice. Notably, loci encoding circadian clock genes (Clk, Bmal1, Cry1, Cry2, Per1, Per2; see Fig. 1d) were also enriched in the REV-ERB-a/b cistromic overlap, suggesting that the coordinated actions of both REV-ERB isoforms are directly linked with clock function. A comparison of the REV-ERB-a/b cistrome with published BMAL1 binding sites revealed that 28% of BMAL1 peaks (at ZT6 and ZT10) were shared with the REV-ERB-a/b (FT8) cistrome and 68% of these peaks (781) were occupied by all three transcription factors. Clear binding sites for each of these transcription factorswere foundon ‘core clock’ gene loci aswell as onmany clock-controlled target genes (Rev-erb-a, Rev-erb-b, Ror,Dbp,Hlf, Tef, Nfil3; see Fig. 1d, e). In addition to circadian annotated loci, the BMAL1, REV-ERB-a, REV-ERB-b triple intersection is highly enriched for genes in the receptor tyrosine kinase signalling pathway as well as those known for energy homeostasis (Supplementary Table 1). Their confluence at hundreds of clock and clock output genes indicates that beyond a simple ‘binary relationship’, REVERB-a/b and BMAL1 cooperate to regulate clock and clock output genes coordinately. To test the aboveproposal,weusedCre/lox recombination to generate three genetically modified mouse lines in C57BL/6 backgrounds harbouring global, tissue-specific, or conditional knockouts of Reverb-a andRev-erb-b loci. GlobalRev-erb-a andRev-erb-b single knockout mice were generated using a CMV-cre transgenic allele (detailed in Supplementary Methods). Homozygous deletion of Reverb-b did not cause overt gross abnormalities, lethality or infertility, whereas Rev-erb-a mice, although viable, show frequent postnatal lethality (before 2weeks of age) (Supplementary Table 2), with survivors exhibiting diminished fertility in both sexes. In contrast,