Figure 1: Overexpression of MYC attenuates the circadian clock.

(a) Overlap between native MYC (ref. 14) and BMAL1 (ref. 13) binding sites in U2OS cells. (b) PER2 and REV-ERBα loci with binding sites (BS) of BMAL1, CLOCK, native MYC and overexpressed MYC in U2OS cells (based on the data from refs 13, 14). (c) MYC and MAX do not substantially induce the BMAL1/CLOCK target genes REV-ERBα, PER2 and SCN5α. HEK293 cells were transfected with MYC, MAX, BMAL1 and CLOCK encoding plasmids (30 ng) together with the indicated circadian promoter-luc reporter plasmids. GAPDH-luc was transfected as a negative control (n=3). (d) MYC/MAX restricts stronger induction of 6xEbox-luc by CLOCK/BMAL1. HEK293 cells were transfected with 30 ng of each BMAL1 and CLOCK plasmids, and with the indicated amounts (in ng) of MYC and MAX vectors (n=3). ChIP-PCR analysis of (e) MYC:V5 and (f) BMAL1 binding to circadian E-boxes in PER2 and REV-ERBα promoters in synchronized and doxycycline-induced U2OS t-rex tetO-MYC:V5 cells (n=3). (g) Bioluminescence recorded from synchronized Bmal1-luc and PER2-luc U2OS t-rex tetO-MYC:V5 cells (n=3). (h) Quantitative PCR (qPCR) analysis of circadian expression profiles of PER2 and BMAL1 transcripts in synchronized U2OS t-rex tetO-MYC:V5 cells (n=3). Data are presented as mean±s.e.m. *P<0.05; one-way (c,d) and two-way (e,h) analysis of variance (ANOVA) with Bonferroni post-test.