Rev-erbα and Rev-erbβ coordinately protect the circadian clock and normal metabolic function

Abstract

The nuclear receptor Rev-erbα regulates circadian rhythm and metabolism, but its effects are modest and it has been considered to be a secondary regulator of the cell-autonomous clock. Here we report that depletion of Rev-erbα together with closely related Rev-erbβ has dramatic effects on the cell-autonomous clock as well as hepatic lipid metabolism. Mouse embryonic fibroblasts were rendered arrhythmic by depletion of both Rev-erbs. In mouse livers, Rev-erbβ mRNA and protein levels oscillate with a diurnal pattern similar to that of Rev-erbα, and both Rev-erbs are recruited to a remarkably similar set of binding sites across the genome, enriched near metabolic genes. Depletion of both Rev-erbs in liver synergistically derepresses several metabolic genes as well as genes that control the positive limb of the molecular clock. Moreover, deficiency of both Rev-erbs causes marked hepatic steatosis, in contrast to relatively subtle changes upon loss of either subtype alone. These findings establish the two Rev-erbs as major regulators of both clock function and metabolism, displaying a level of subtype collaboration that is unusual among nuclear receptors but common among core clock proteins, protecting the organism from major perturbations in circadian and metabolic physiology.

Figure 1.

Loss of both Rev-erb subtypes renders MEFs arrhythmic. Gene expression over a 40-h period in synchronized wild-type (WT) or Rev-erbα knockout (αKO) MEFs transduced with adenovirus encoding a shRNA targeting Rev-erbβ (Revβ) or βgal as control. The experiment was performed in triplicate, and SEM is indicated by vertical bars. All values have been normalized to Arbp mRNA expression and the 8-h time point in the wild-type control. (A) Rev-erbα mRNA levels in wild-type MEFs infected with control virus (WT/shβgal) or Rev-erbβ knockdown virus (WT/shRevβ) and Rev-erbα-null MEFs infected with control virus (αKO/shβgal) or Rev-erbβ knockdown virus (αKO/shRevβ). (B) Rev-erbβ mRNA levels in the cells described in A. (C) Bmal1 mRNA levels in WT/shRevβ and αKO/shβgal cells. (D) Bmal1 mRNA levels in WT/shβgal and αKO/shRevβ cells. (E) Cry1 mRNA levels in WT/shRevβ and αKO/shβgal cells. (F) Cry1 mRNA levels in WT/shβgal and αKO/shRevβ cells.

Figure 2.

Diurnal rhythm of Rev-erbβ expression and genomic binding in mouse livers. (A) Rev-erb mRNA levels in the livers of 12-wk-old wild-type male mice euthanized at the indicated time points. n = 4 or 5; SEM is indicated by vertical bars. (B) Rev-erb protein levels. Western blot of liver extracts from 12-wk-old wild-type male mice euthanized at the indicated time points. The same extract was used on both blots. Ras-related nuclear protein (RAN) was used as a loading control. (C) Rev-erbβ ChIP on the liver samples described in B; Arbp intron 3 is a negative control region; n = 3 or 4; SEM is indicated by vertical bars. (D) Rev-erbα ChIP on the samples described in B and C. Arbp intron 3 is a negative control region; n = 3 or 4; SEM is indicated by vertical bars.

Figure 3.

Extensive correlation of liver Rev-erbβ and Rev-erbα cistromes. (A) Overlap of the liver Rev-erbβ cistromes generated at 5:00 a.m. and 5:00 p.m. Binding sites were considered overlapping when the peak calls overlapped by at least 1 bp. Numbers indicate the total number of binding sites in each cistrome and the number of binding sites in each group. (B) Quantitative analysis of Rev-erb ChIP-seq signal. Scatter plot of the maximum stack height at each Rev-erbα and Rev-erbβ site not detected by Rev-erbα ChIP in the Rev-erbα-null mouse. Sites are classified as subtype-specific if the difference in binding profiles is statistically significant (Fisher's exact test, Benjamini-Hochberg-corrected P-value < 0.05) and indicates at least a twofold difference in binding strength. For clarity, 16 common and 23 Rev-erbβ-specific outliers were omitted from the plot. r² and the slope of the best-fit line are shown for common sites. (C) Gene ontology analysis (PANTHER) on genes with a Rev-erb-binding site within 10 kb of the TSS (Galaxy/Cistrome). Shown are the top five enriched GO terms.

Figure 4.

The Rev-erb subtypes bind simultaneously to genomic sites. (A) At 5:00 p.m., hepatic Rev-erbβ, Rev-erbα, NCoR, and HDAC3 bind together at two neighboring sites in the Bmal1 promoter. Genome browser tracks of stack height profiles from ChIP-seq experiments for HDAC3 (Feng et al. 2011), NCoR (Feng et al. 2011), Rev-erbβ (Revβ), and Rev-erbα (Revα) in wild-type mice (Feng et al. 2011), and Rev-erbα in Rev-erbα knockout mice (αKO). Peak height is represented in reads per million. (B) Top de novo motif under the shared Rev-erb-binding sites (HOMER, P-value = 1 × 10⁻⁷⁰¹) with the core NR-binding motif boxed by a dashed outline, and the significantly enriched de novo motif similar to the published RORE (HOMER, P-value = 1 × 10⁻³¹³). (C) The Rev-erb subtypes are present simultaneously at the Bmal1 and Npas2 genes in the liver. Sequential ChIP of Rev-erbα followed by either Rev-erbβ or IgG ChIP in mouse livers harvested at 5:00 p.m. n = 4; SEM is indicated by vertical bars. (D) The distance to the nearest adjacent region was computed for each binding region in the common Rev-erb cistrome or randomly matched control sites and grouped in bins of 200 bp. (*) P-value < 1 × 10⁻⁷ (Fisher's exact test). (E) Identification of two or more binding motifs under Rev-erb peaks. All regions in the common Rev-erb cistrome were scanned for the core NR motif identified by de novo motif analysis (dashed outline; Fig. 3B) and compared with randomly selected control regions with a similar distribution of distances from the nearest gene TSS. (*) P-value < 1 × 10⁻¹⁶ (Fisher's exact test).

Figure 5.

Rev-erbβ protects the regulation of clock and metabolic genes from loss of Rev-erbα. (A) shRNA-mediated knockdown of Rev-erbβ. Hepatic Rev-erb mRNA levels at 5:00 p.m., 7 d after tail vein injection of adenovirus encoding a shRNA targeting Rev-erbβ (Revβ) or βgal as control in 12-wk-old wild-type or Rev-erbα knockout (αKO) male mice. n = 4–6; SEM is indicated by vertical bars. (B) Western blots showing the Rev-erb protein levels in the livers of the mice described in A. The same extracts were loaded twice on separate gels and probed for either Rev-erbβ or Rev-erbα with Ras-related nuclear protein (RAN) or heat-shock protein 90 (Hsp90) as loading control, respectively. (C) Hepatic mRNA levels of circadian and metabolic genes in the mice described in A. (*) P-value ≤ 0.05 versus the αKO/shβgal condition as determined by Student's t-test.

Figure 6.

Complete Rev-erb deficiency disrupts NCoR and HDAC3 recruitment at 5:00 p.m. (A) NCoR ChIP in liver at 5:00 p.m., 7 d after tail vein injection of adenovirus encoding a shRNA targeting Rev-erbβ (Revβ) or βgal as control in 12-wk-old wild-type or Rev-erbα-null (αKO) male mice. Arbp intron 3 is a negative control region; n = 3; SEM is indicated by vertical bars. (*) P-value ≤ 0.05 relative to WT/shβgal; (§) P-value ≤ 0.05 relative to all other treatments, as determined by a Student's t-test. (B) HDAC3 ChIP on liver extracts from the mice described in A. Arbp intron 3 is a negative control region; n = 3; SEM is indicated by vertical bars. (*) P-value ≤ 0.05 relative to WT/shβgal; (§) P-value ≤ 0.05 versus all other treatments, as determined by Student's t-test.

Figure 7.

Total hepatic Rev-erb deficiency leads to exacerbated steatosis. (A) Hepatic triglyceride levels in two pooled experiments of 12-wk-old wild-type or Rev-erbα knockout (αKO) mice 7 d after injection of adenovirus encoding shRNA targeting Rev-erbβ (Revβ) or βgal as control. n = 5–7; SEM is indicated by vertical bars. (*) P-value = 0.0272 as determined by Student's t-test. (B) Oil red O stains of liver sections from the mice described in A at a magnification of 40× Bar, 50 μm.

Figure 8.

Rev-erbα and Rev-erbβ cooperate in regulating core clock function and mediating the interplay between circadian rhythm and metabolism. The Rev-erbs are central repressors of both the positive (Bmal1) and the negative (Cry1 and Rev-erbα) limb of the core clock. In addition, the Rev-erbs mediate the interplay between the cellular clock and metabolism together with BMAL1 and CLOCK/NPAS2. Note that other core clock components (shown as PER, CRY, and BMAL in the model for simplicity) all have homologs (PER1–3, CRY1/2, and BMAL1/2) that, like the two Rev-erb subtypes, function as a backup system.