Fig. 3: CHRONO differentially regulates the expression of REV-ERBα via cis-binding to the TRE box.

A The UCSC genome browser view of CHRONO occupancy at the REV-ERBα locus in both U2OS-WT and U2OS-CHR cells. The binding peak that contains the TRE box was labeled. Notice there are three binding peaks for CHRONO. B The UCSC genome browser view of CHRONO occupancy at the REV-ERBβ locus in both U2OS-WT and U2OS-CHR cells. The only binding peak at the REV-ERBβ locus contains an E-box. C The UBC locus contains only the TRE element but not the E-box. ChIP-PCR confirmed that CHRONO can bind to the TRE box on the UBC locus. D CHRONO still binds to UBC and REV-ERBα -B3, but not PER2, REV-ERBβ loci when BMAL1 was knocked out. ChIP-PCR confirmed that CHRONO binds to the UBC locus. E JUN/FOS was present in the immuno-precipitation product when CHRONO was precipitated. “*” labels the non-specific bands of FOS proteins. F Co-IP confirmed the interaction between CHRONO and the JUN/FOS complex. G CHRONO suppressed the luciferase activity which is under the control of a UBC promoter containing only the TRE element. H CHRONO suppressed the luciferase activity which is under the control of a REV-ERBα promoter containing a TRE element. I JUN/FOS did not activate the expression when the TRE element was mutated. Data are presented as the mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).