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.

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.

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.

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 (), NCoR (), Rev-erbβ (Revβ), and Rev-erbα (Revα) in wild-type mice (), 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; ) 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).

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.

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.

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.

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.