Fig. 2

REV-ERBα activates the circadian expression of FUS. a ChIP-seq analysis showing REV-ERBα binding signals on the Fus promoter. The black bar below indicates the Fus promoter region (WT-P in Fig. 2c) used in the Fus promoter-luciferase construct, while the grey area in the middle of black bar indicates the region harboring the REV-ERBα-binding site based on ChIP-seq data [32]. b ChIP-qPCR showing the binding of FLAG-REV-ERBα to the Fus promoter in Neuro-2a cells (Fus-1 and Fus-2 are two pairs of primers specific for regions located in the predicted REV-ERBα binding sites in Fig. 2a; FLAG-GFP was used as the control; mean ± s.e.m.; N = 4 experiments; t-test; *:P≤0.05). c Luciferase activity of the intact (WT-P) and REV-ERBα-binding site deleted (Del-P) Fus promoter-luciferase constructs in Neuro-2a cells after siRNA silencing (mean ± s.e.m.; N = 4 experiments; t-test; ***:P≤0.001). The right panel showed the knock-down efficiency of Nr1d1-targeting siRNA (mean ± s.e.m.; N = 4 experiments; t-test; ***:P≤0.001; Ctrl represents scrambled control siRNA; NS: non-significant). d FUS expression level in synchronized wild-type or Nr1d1 knock-out (KO) MEFs. Quantification result was shown in the bar graph (mean ± s.e.m.; three lines of wild-type MEFs and four lines of Nr1d1 KO MEFs were generated from two pregnant Nr1d1 heterozygous mice, t-test; **: P≤0.01). e FUS expression level in the liver of free-running wild-type and Nr1d1 knock-out mouse at indicated time point, quantification result was shown in the right bar graph (N = 3 pairs of littermates for CT-0 hr and N = 2 pairs for CT-12 hr; mean ± s.e.m.; two-way ANOVA with Sidak's multiple comparison test, *:P≤0.05, **:P≤0.01). f Activating/repressive functional prediction analysis based on the published REV-ERBα ChIP-seq data [32] and transcriptional profile in Nr1d1 knock-out mice [53]. Genes are cumulated by the rank on the basis of the regulatory potential score from high to low according REV-ERBα ChIP-seq data (x-axis). The red and purple lines represent the percentage of up-regulated (UP) or down-regulated (DOWN) genes that harbor REV-ERBα binding sites from Nr1d1 knock-out microarray data, respectively. The black dashed line indicates the non-differentially (NON) expressed genes among REV-ERBα-binding genes. P values that represent the significance of the UP or DOWN group distributions are compared with the NON group by the Kolmogorov-Smirnov test. The right panel is an example showing the fraction of up-regulated (red) or down-regulated genes (purple) that contain REV-ERBα binding sites when the top 2,000 peaks from the ChIP-seq data were included (gray dash line in the left panel). The cumulative fractions of genes that are down-regulated in Nr1d1 knock-out mice indicate that REV-ERBα could also act as an activator