REV-ERBα and REV-ERBβ function as key factors regulating Mammalian Circadian Output

Abstract

The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERBα and REV-ERBβ are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erbα/Rev-erbβ double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the double knockout of both Rev-erbα and Rev-erbβ does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERBα/β deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study highlight the relevance of REV-ERBs as pivotal output mediators of the mammalian circadian clock.

Figure 1

CRISPR/Cas9-mediated targeting of Rev-erbα and Rev-erbβ in mES cells. (a) Schematic of Rev-erbα and Rev-erbβ target regions. Green and pink letters indicate the CRISPR-targeted sequence and the PAM sequence, respectively (see Methods). (b) Relative expression levels of Rev-erbα and Rev-erbβ mRNA including CRISPR-targeted exon were determined by quantitative PCR. The values were normalized to 18S rRNA and presented as means ± SD (n = 3; *p < 0.001).

Figure 2

REV-ERBα/β deficiency affects their target gene expression in differentiated mES cells. (a) Venn diagram of expressed genes in WT and KO cells. (b) Expression of specific differentiation markers of the three germ layers. RPKM values from 12 time points are shown in bee swarm box plots. (c) Expression of core clock genes (n = 12; *p < 0.01). (d) Expression of known target genes of REV-ERBα/β (n = 12; *p < 0.01).

Figure 3

Comprehensive analysis of circadian gene expression in Rev-erbα/β-deficient cells. (a) Venn diagram of cycling genes in WT and KO cells. (b) Heatmap view of cycling genes. Each gene is represented as a horizontal line ordered vertically by phase as determined by MetaCycle. (c) The phase distribution of cycling genes. (d) Homer known motif enrichment analysis reveals HNF1b and NF-Y binding motifs are enriched in promoter region of cycling genes in WT cells but not in KO cells. (e) Expression of circadian clock genes cycling in both WT and KO cells. mRNA expression levels in WT and KO cells are plotted with blue and red lines, respectively. (f) Expression levels of Bmal1 and Npas2 in WT and KO cells are plotted with blue and red lines, respectively. (g) Cyclic expression of Per2, Cry1, Bmal1, and Npas2 is analyzed by quantitative PCR. mRNA expression levels in WT and KO cells are plotted with black and red lines, respectively. The values were normalized to 18S rRNA and presented as means ± SD (n = 3).

Figure 4

PER2^Luc bioluminescence rhythms in Rev-erbα/β-deficient cells. (a) PER2^Luc bioluminescence rhythms were measured in differentiated mES cells. Raw and detrended data are shown in the left and right graphs, respectively. The mean traces ± SD are plotted (n = 11 (WT) and 12 (KO)). (b) The period length and the amplitude are calculated and plotted. The values are the means ± SD (n.s., not significant). (c) Schematics of REV-ERB-mediated circadian gene regulation. REV-ERBs regulate the secondary loop of circadian transcriptional/translational feedback loops via RRE and control various output gene expression rhythms in a context-dependent manner.