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

Abstract

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-α and REV-ERB-β 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 the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-β function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-β 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.

Figure 1. Cistromic analyses of REV-ERBα and REV-ERBβ in liver

a, De novo HOMER motif analysis of in vivo REV-ERBα and REV-ERBβ binding. b, Venn diagram depicting the unique and common REV- ERBα and REV-ERBβ bound peaks. c, Commonly bound REV-ERBα and REV-ERBβ peaks are enriched for genes involved in lipid metabolism and associated with PPARs. d, REV-ERBα, REV-ERBβ and BMAL1binding at canonical circadian clock genes. Left axis indicates tag counts. e, BMAL1 cistrome significantly overlaps with REV-ERBα and REV-ERBβ. Examples of Clock related genes in overlap are listed and selected peaks shown in Supplementary Figure 4.

Figure 2. Circadian gene expression of many canonical core clock genes and output genes are disrupted in Livers of Rev-erbα^lox/lox Rev-erbβ^lox/lox Albumin-Cre (L-DKO) mice

The expression levels of a, Rev-erbα, b, Rev-erbβ, c-f, canonical core clock genes (Cry1, Clock, Bmal1 and Per2) g-i, presumed output genes (PoR, PPARα and Sco2) in livers from L-DKO (Albumin-Cre positive, red labels) and wildtype (Albumin-Cre negative, black labels) mice. Livers (n=3) were harvested at each indicated ZT under 12-hour light:dark cycle. QPCR was performed in technical triplicates. Relative Unit (RU) normalized with 36B4. Error bars indicate standard error of the mean, statistical significance determined by Student t-test (* p<0.05, ** p<0.01, *** p<0.001).

Figure 3. Broad disruption of circadian transcriptome in the absence of Rev-erbα and Rev-erbβ

a, Heatmap of genes with circadian expression in wild-type (left panel) and L-DKO (right panel) livers. 1227 unique accession numbers were selected based on fdr < 0.05. b, Genes expressed in a circadian manner that lose rhythm are highly associated with circadian and energy homeostasis functions as assessed by KEGG Pathway analysis.

Figure 4. Loss of both Rev-erbα and Rev-erbβ results in disrupted circadian wheel-running behavior and metabolic shift

a-c, Voluntary locomotor activity of wildtype, Rev-erbα^−/−, Rev-erbβ^−/−, and Rev-erbα^−/−Rev-erbβ^−/− (iDKO) mice. a, Actograms showing wheel-running activity in constant darkness after prior entrainment in light/dark. b, Activity profiles during light dark cycles. c, Chi-square periodogram of the initial 20 days in constant darkness. (n=5–9 for each mutant strain, n= 5–6 littermate controls). Representative actograms from individual mice are shown. d, Triglyceride (n=6), fasting glucose (n=6) and free fatty acid (n=6) levels in iDKO and wildtype mice. e, Respiratory exchange ratio (RER) for wildtype (black) and iDKO (red) mice (n=4). f, Model depicting the activating (Clock/BMAL1) and repressive (REV-ERBα/REV-ERBβ) transcriptional complexes whose coordinate actions generate rhythmic gene expression.