Fig. 4

FUS is in the repressor complex and mediates the recruitment of PSF-HDAC1 to the promoters of E box-containing genes. a Co-immunoprecipitation of FLAG-FUS with PER2, BMAL1 and CLOCK in HEK293T cells. b, c Per2 promoter-luciferase reporter activity in synchronized Neuro-2a cells after transfection and siRNA silencing with indicated conditions. Cells were first transfected with indicated RNAi constructs at 0 hr, followed by expression plasmid transfection at 24 hr if needed. Cells were synchronized at the 48 hr point for 2 hrs, and harvested for analysis at the 72 hr time point (mean ± s.e.m.; N = 3 experiments in duplicates; One-way ANOVA followed by Newman-Keuls multiple comparisons test; *:P≤0.05, **:P≤0.01). d ChIP-qPCR showing the promoter loading of PSF relative to IgG control. Neuro-2a cells were harvested at indicated time points after serum shock synchronization (mean ± s.e.m.; N = 3-5 experiments; One-way ANOVA followed by Dunnett's multiple comparison test; *:P≤0.05). e, f ChIP-qPCR showing the effect of FUS silencing on the binding of endogenous PSF (e) or HDAC1 (f) onto the E box-containing promoters of circadian genes. Neuro-2a cells were harvested at 28 hrs after serum shock synchronization (IgG as the ChIP control, mean ± s.e.m.; N = 3-5 experiments; t-test; **:P≤0.01)