Figure 4: MYC mutants compromised in MIZ1 interaction do not disrupt the circadian clock. | Nature Communications

Figure 4: MYC mutants compromised in MIZ1 interaction do not disrupt the circadian clock.

From: MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation

Figure 4

(a) Transactivation assay in HEK293 cells showing that MYC:V5 WT, V394D and V393D equally induce 6xEbox-luc expression and compete with CLOCK/BMAL1 in a dominant negative manner (n=3). (b) Anti-V5 immunoprecipitation of MYC:V5 versions in HEK293 lysates showing that MYC:V5 V394D and V393D interact with MAX (upper panel) but not with MIZ1 (lower panel). Co-immunoprecipitation (Co-IP) of FLAG-tagged MAX and MIZ1 was detected with anti-FLAG antibodies. Refer to Supplementary Fig. 4a for inputs and reciprocal anti-FLAG co-IP’s. (c) Overexpressed MYC:V5 V394D and V393D are inefficient in repression of MIZ1 target genes. Expression of transiently transfected p15-luc, p21-luc and Bmal1-luc reporters in U2OS t-rex tetO-MYC:V5 cells expressing the indicated versions of MYC. Bioluminescence was quantified 18 h after MYC induction with doxycycline and normalized to PBS-treated samples (n=3). (d) Western blot analysis (left) and densitometric protein quantification (right) of CLOCK and BMAL1 in U2OS cells overexpressing MYC and MYC V393D 24 h after doxycycline induction (n=3). (e) Baseline-subtracted bioluminescent traces from U2OS t-rex tetO-MYC:V5 WT, V394D and V393D cells transiently transfected with Bmal1-luc (n=3). For the raw data refer to Supplementary Fig. 4d. (f) Total fluorescence and fluorescent objects quantified from synchronized U2OS t-rex Rev-VNP cells stably transfected with inducible MYC:V5 WT and V393D (n=1). Data are presented as mean±s.e.m. *P<0.05; one-way analysis of variance (ANOVA) with Bonferroni post-test.

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