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

Abstract

The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.

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.

Figure 2. MYC represses BMAL1 independent of REV-ERBα.

(a) Quantitative PCR (qPCR) analysis of circadian expression profiles of REV-ERBα and REV-ERBβ transcripts in synchronized U2OS t-rex tetO-MYC:V5 cells (n=3). (b) Total fluorescence and fluorescent objects quantified from synchronized U2OS t-rex Rev-VNP tetO-MYC:V5 cells (n=1). Inset: normalized number of fluorescent objects (24–96 h integration; n=2). (c) qPCR analysis of REV-ERBα and BMAL1 transcripts in siRNA-transfected U2OS t-rex tetO-MYC:V5 cells (n=3). Unsynchronized cells were collected 24 h after doxycycline induction. (d) Integrated bioluminescence of Bmal1-luc transfected U2OS t-rex tetO-MYC:V5 cells. Cells were transfected with indicated siRNAs 2 days before synchronization. Average expression levels (area under the curve) were determined over 72 h (n=3). Data are presented as mean±s.e.m. *P<0.05; two-way analysis of variance (ANOVA) with Bonferroni post-test.

Figure 3. MYC directly represses clock genes via MIZ1.

(a) Kinetics of MYC-dependent repression of p21-luc, p15-luc and Bmal1-luc and activation of 6xEbox-luc (n=3). Unsynchronized U2OS t-rex tetO-MYC:V5 cells were transiently transfected with the indicated reporter plasmids. At time point 0 MYC:V5 expression was either induced with doxycycline (MYC ox) or not induced with PBS (Ctrl). (b) Left panel: schematic of MIZ1 binding sites in NPAS2, CLOCK and BMAL1 based on data from Walz et al.. Black triangles indicate regions amplified in ChIP-PCR analysis. Right panel: ChIP-PCR analysis showing recruitment of induced MYC:V5 to MIZ1 binding sites in NPAS2, CLOCK and BMAL1 at 24 and 36 h after synchronization of U2OS t-rex tetO-MYC:V5 cells (n=3). (c) Downregulation of MIZ1 with siRNA reduces expression of NPAS2, CLOCK and BMAL1 in U2OS cells (n=3). Transcript levels of the indicated genes were determined by quantitative PCR (qPCR). (d) MYC-induced attenuation of the circadian clock is rescued by downregulation of MIZ1 (n=3). Synchronized U2OS t-rex tetO-MYC:V5 Bmal1-luc cells were transfected with MIZ1 or control siRNAs. Note that downregulation of MIZ1 attenuates the circadian rhythm of Bmal1-luc and lengthens the period (Supplementary Fig. 3a,b). Data are presented as mean±s.e.m. *P<0.05; Student's t-test.

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

(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.

Figure 5. MYC inversely regulates the circadian clock and proliferation.

(a) MYC:V5 WT and V393D U2OS cells were stained with propidium iodide 48 h after induction and DNA content was quantified by FACS (n=3). Values indicate difference (in %) to PBS-treated cells in the respective cell cycle phase. (b) Left panel: fluorescence microscopy of U2OS t-rex tetO-MYC:V5 cells stably expressing mCherry-Cdt1 (FUCCI-Red G1 marker) 48 h after treatment with doxycycline to induce MYC:V5 (MYC ox) or PBS (Ctrl). Scale bar, 300 μm. Right panel: quantification of cells in G0 or G1 phase by mCherry-Cdt1 expression (n=3) (c) FACS analysis of U2OS cells stained with propidium iodide 48 h after transfection with MYC siRNA (n=3). Values indicate difference to cells transfected with negative siRNA. (d) Growth curve of U2OS cells transfected with MYC siRNA and negative siRNA (n=3). (e) Western blot analysis (left) and densitometric protein quantification (right) showing that MYC depletion by siRNA supports increased BMAL1 and CLOCK expression in U2OS cells (n=3). (f) Relative amplitudes (ChronoStar software) of circadian luciferase rhythms of indicated U2OS reporter cell lines (n=3). The cells were transfected with MYC siRNA and negative siRNA as indicated. Data are presented as mean±s.e.m. *P<0.05; Student's t-test (e,f), one-way (b,c) and two-way (a,d) analysis of variance (ANOVA) with Bonferroni post-test.

Figure 6. MYC inversely correlates with BMAL1 expression in human lymphomas.

(a) Scatter plots of expression levels of MYC versus the indicated clock genes in 102 human lymphoma samples of the ICGC MMML-Seq project (RPKM: log2 sequence reads per kilobase transcript per million reads38). Expression of MYC versus its established target NUCLEOLIN (NCL) is shown as a positive control. (b) Data were binned according to the indicated MYC expression levels. Clock gene expression levels are shown by Box-plots. Significant differences (P<0.05; Student's t-test) of expression levels relative to box 1 (0–50) and box 2 (50–100) are indicated by asterisks (*) and number signs (#), respectively. (c) Model of the coordinating function of MYC. Left panel: high levels of MYC suppress the circadian clock by MIZ1-dependent downregulation of BMAL1/CLOCK (see text), which results in low amplitude expression rhythms of clock-controlled genes. On the other hand, high MYC levels support cell growth and proliferation (for example, by inhibition of p15 and p21, see text). Right panel: at low levels of MYC, the circadian clock is not inhibited and supports high amplitude expression rhythms of clock-controlled genes. Low levels of MYC do not support cell growth and proliferation.