Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism

Abstract

The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα^-/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.

Keywords: NR1D1; circadian clock; energy metabolism; liver; nuclear hormone receptor.

Fig. 1.

Antibody-independent ChIP-seq maps the liver REVERBα cistrome. (A) Targeting design for CRISPR-mediated knockin, with CRISPR target site in exon 1 of Reverbα, and homology flanked HaloTag long ssDNA donor Below. Homology-directed repair (HDR) results in the integration of HaloTag in frame with exon 1 of Reverbα. (B) Gene expression profiles of core circadian clock genes in liver tissue collected from WT (black) or HaloReverbα (red) mice confirming the maintenance of robust molecular rhythms with normal phase and amplitude in the HaloReverbα mice. Plot shows mean normalized gene expression ± SEM, n = 4 to 6 mice per time point. (C) HaloREVERBα ChIP-seq peaks were called across ZT8 HaloReverbα samples (n = 3) with ZT8 wild-type (n = 2) or ZT20 (n = 2) HaloReverbα samples serving as background; 8,660 peaks were common to both strategies. (D) Heatmap shows aggregated signal at these 8,660 sites (peak center ± 5 kbp). (E) Piechart of annotated peak locations shows that the majority of sites lie within a gene body or regulatory domain. (F) Tracks showing aggregated signal at sites associated with Bmal1, Rora, Cry1, and Npas2.

Fig. 2.

The HaloREVERBα cistrome is enriched for RORE and RevDR2 motifs, and sites of corepressor complex binding, but shows limited overlap with antibody datasets. (A) Motif analysis at HaloREVERBα-binding sites. Plots show enrichment of known motifs; below plot is top de novo motif identified. Analysis using random background shown on Left, and using a background of ZT6 to ZT10 open chromatin (DNase sites) shown on Right. Point size proportional to percentage of target sites containing motif. For clarity, only motifs with more than twofold enrichment are labeled; full results are in SI Appendix, Tables S1–S4. A RORE-like motif is the top motif identified de novo against either random or DNase background. (B) GIGGLE identifies transcription factors whose cistromes (top 1-k peaks, from a database of published ChIP-seq) overlap with HaloREVERBα sites. Ten factors with highest mean GIGGLE scores are plotted; full results are in SI Appendix, Table S5. Points indicate individual datasets, line indicates mean score. (C) Venn diagram shows overlap of HaloREVERBα cistrome with published HDAC3/NCOR ChIP-seq. (9). (D) Venn diagrams show overlap of HaloREVERBα cistrome with published antibody REVERBα ChIP-seq (14, 21). Numbers of sites are shown in bold. Left Venn is shaded to show enrichment of the RORE motif (over DNase background) in each group of sites; Right Venn is shaded to show RevDR2 enrichment. Fold-enrichment values are shown in italics. “Not enriched” = HOMER did not find that motif to be significantly enriched on standard known motif analysis. (E) Tracks showing aggregated ChIP-seq signal from this study and two published REVERBα studies, including REVERBα ChIP-seq performed in global REVERBα^−/− mice (“Kim et al. KO ZT10”; track shown in red) (21). Locations of putative RevDR2 motifs indicated with black arrows, putative RORE motifs with gray.

Fig. 3.

Liver-targeted Reverbα deletion and HaloREVERBα ChIP-seq define direct REVERBα targets. (A) No genotype differences in body weight, lean, and fat mass were observed between Reverbα^Flox2-6Alb^CreERT2 mice and Reverbα^Flox2-6 littermate controls (12- to 14-wk-old males, n = 6 to 13 per group, 15 d after the first tamoxifen injection [10 d after the last]). (B and C) Reverbα^Flox2-6Alb^CreERT2 mice do not accumulate liver lipid. Quantification of total liver triacylglyceride (TAG) (B) and fatty acid composition analysis (C) show diminished TAG levels in Reverbα^Flox2-6Alb^CreERT2 liver (compared to controls) and no differences in fatty acid desaturation (reflected by C16:1n-7/C16:0 ratio) or de novo lipogenesis (reflected by C16:0/C18:2n ratio). (n = 6 to 8 per group, 12- to 14-wk-old males and females, 15 d after the first tamoxifen injection). (D and E) Liver-specific deletion of REVERBα has some effect on carbohydrate metabolism, as shown by increased liver glycogen in the fed state in Reverbα^Flox2-6Alb^CreERT2 mice (D), but no differences in blood glucose levels (E) (n = 7 to 12 per group, 12- to 14-wk-old males and females, 15 d after the first tamoxifen injection). (F) Mean difference (MD) plot of liver RNA-seq from Reverbα^Flox2-6Alb^CreERT2 male mice (relative to littermate controls); genes with significant differential expression (FDR < 0.05) highlighted in color (up-regulated in blue, down-regulated in red). (G) Circadian clock, but not lipid metabolism genes are differentially expressed (DE) in the livers of Reverbα^Flox2-6Alb^CreERT2 mice compared to controls (fold change [FC] and FDR of selected clock and lipid metabolism genes from F). (H) Reactome pathway analysis of significantly DE genes does not show enrichment of lipid metabolism pathways. Four pathways with lowest Padj values are shown. Full results are in SI Appendix, Table S6. (I) Phase plots of rhythmic, differentially expressed genes identified in Reverbα^Flox2-6Alb^CreERT2 livers (orange), with all rhythmic genes in liver shown in gray. Gene rhythmicity and peak phase (acrophase) were taken from ref. . Introns are in darker bars, exons in lighter bars. (J) Proximity of differentially expressed genes to HaloREVERBα peaks. (J, Top) Proportion of each gene set lying within a given distance of peaks. (J, Bottom) Significance of the enrichment of each gene set compared to all genes in the genome at each distance. Individual data points are shown, line at mean, *P < 0.05, **P < 0.01, unpaired t test (A–C). Two-way ANOVA with Tukey’s multiple comparisons tests (D and E).

Fig. 4.

The broader repressive effects of REVERBα become evident under conditions of metabolic perturbation. (A) MD plot of liver RNA-seq from Reverbα^−/− male mice (relative to littermate controls); genes with significant differential expression (FDR < 0.05) highlighted in color (up-regulated in blue, down-regulated in red). (B) Reactome pathway analysis of significantly DE genes shows enrichment of metabolic pathways. Four pathways with lowest Padj are shown. Full results are in SI Appendix, Table S7. (C) Lipid metabolism genes are differentially expressed in the livers of Reverbα^−/− mice compared to controls (fold change [FC] and FDR of selected clock and lipid metabolism genes from A). (D) Overlap of significantly DE genes in each mouse model. (E) Proximity of DE genes to HaloREVERBα peaks in both liver-specific and global deletion of REVERBα. (E, Top) Proportion of each gene set lying within a given distance of peaks. (E, Bottom) Significance of the enrichment of each gene set compared to all genes in the genome at each distance. (F) Cistrome-transcriptome associations respect TAD/subTAD boundaries. Table lists percentage of DE genes from each RNA-seq cluster which are found within the same TAD/subTAD as HaloREVERBα sites. Percentage of all genes is shown for comparison. Depth of shading indicates significance of enrichment (compared to all genes). (G) IPA identifies regulators putatively upstream of genes which are (i) only up-regulated (derepressed) or (ii) only down-regulated in Reverbα^−/− livers, and which are within 100 kbp of HaloREVERBα sites. (H) Mistimed feeding causes significant increase in expression in Reverbα^Flox2-6Alb^CreERT2 liver (Right) at HaloREVERBα-associated genes, which are not derepressed under basal conditions (Left). *P < 0.05, **P < 0.01 Welch’s t tests, n = 8 to 14 per group.