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