Circadian regulation of c-MYC in mice

Abstract

The circadian clock is a global regulatory mechanism that controls the expression of 50 to 80% of transcripts in mammals. Some of the genes controlled by the circadian clock are oncogenes or tumor suppressors. Among these Myc has been the focus of several studies which have investigated the effect of clock genes and proteins on Myc transcription and MYC protein stability. Other studies have focused on effects of Myc mutation or overproduction on the circadian clock in comparison to their effects on cell cycle progression and tumorigenesis. Here we have used mice with mutations in the essential clock genes Bmal1, Cry1, and Cry2 to gain further insight into the effect of the circadian clock on this important oncogene/oncoprotein and tumorigenesis. We find that mutation of both Cry1 and Cry2, which abolishes the negative arm of the clock transcription-translation feedback loop (TTFL), causes down-regulation of c-MYC, and mutation of Bmal1, which abolishes the positive arm of TTFL, causes up-regulation of the c-MYC protein level in mouse spleen. These findings must be taken into account in models of the clock disruption-cancer connection.

Keywords: BMAL1; c-MYC; circadian clock; cryptochromes; transcription regulation.

Fig. 1.

Bmal1 deletion increases c-MYC expression in vivo. (A) β-Catenin and c-MYC expression were measured by Western blot of spleen extracts from WT and Bmal1 KO mice using GAPDH and Ponceau S as loading controls. Samples were collected at the indicated time points (ZT = Zeitgeber time). (B) Quantification of c-MYC and β-catenin protein levels. For each genotype and time point, three mice were used for quantification. White and black bars indicate lights on and lights off, respectively. Error bars correspond to SEM (standard error of the mean). For c-MYC, data were normalized to a value of 1 for WT at ZT0; for β-catenin, data were normalized to a value of 1 for WT at ZT16. N.S, not significant, *P < 0.05, **P < 0.01, as determined by t test. (C) c-MYC target gene mRNA levels in spleen of WT and Bmal1 KO mice. For each genotype and time point, three mice were used for quantification after normalizing to gapdh RNA. White and black bars indicate lights on and lights off, respectively. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT0. N.S, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, as determined by t test.

Fig. 2.

BMAL1 regulates c-Myc at the transcriptional level. (A) Expression of genes that regulate c-MYC degradation was measured by reverse transcription-qPCR using spleens of WT and Bmal1 KO mice and using gapdh as an internal control. Samples were collected at the indicated time points. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT0. (B) c-Myc mRNA levels in spleen samples detected by reverse transcription-qPCR. Three biological repeats were used for quantification. White and black bars indicate lights on and lights off, respectively. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT0. N.S, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, as determined by t test.

Fig. 3.

Cry1/2 double knockout decreases c-MYC expression in vivo. (A) Endogenous proteins c-MYC and β-catenin were detected by WB (Western blotting) in spleens of WT and three Cry mutants using GAPDH and Ponceau S as loading controls. Samples were collected at the indicated time points. (B–D) Quantification of c-MYC and β-catenin protein levels in the indicated genotypes. For each genotype and time point, three mice were used for quantification. White and black bars indicate lights on and lights off, respectively. Error bars correspond to SEM. For c-MYC, data were normalized to a value of 1 for WT at ZT8; for β-catenin, data were normalized to a value of 1 for WT at ZT4. N.S, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, as determined by t test.

Fig. 4.

CRYs modulate c-MYC expression through transcriptional regulation. (A) c-Myc levels in the spleen samples of WT and Cry1/2 KO mice detected by reverse transcription-qPCR. Three biological repeats were used for quantification. White and black bars indicate lights on and lights off, respectively. (B) mRNA levels of c-MYC target genes in the spleens of WT and Cry1/2 KO. For each genotype and time point, four mice were used for quantification. (C) Expression of genes that regulate c-MYC degradation were measured by reverse transcription-qPCR in spleens of WT and Cry1/2 KO mice using gapdh as an internal control. Samples were collected at the indicated time points. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT0. n.s, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, as determined by the t test.

Fig. 5.

Transcription of Ctnnb1 is controlled by circadian clock proteins. (A) Sequence of the BMAL1 binding site in the Ctnnb1 intron region is in blue (10, 12, 38). Nucleotides conserved in the E-box-like (CANNTG) sequences are colored in yellow. Nucleotides targeted by qPCR primers are marked by bold font. (B–D) Levels of BMAL1 binding to the Nr1d1 E-box, Dbp E-box, and Ctnnb1 intron region in the spleens of WT and Cry1/2 KO mice. For each genotype and time point, at least three mice were used for quantification. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT8. *P < 0.05, **P < 0.01, as determined by the t test. (E and F) Ctnnb1 mRNA levels in spleen samples from WT, Bmal1 KO, and Cry1/2 KO mice detected by reverse transcription-qPCR. Three biological repeats were used for quantification. White and black bars indicate lights on and lights off, respectively. Error bars correspond to SEM. Data were normalized to a value of 1 for WT at ZT0. N.S/n.s, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, as determined by the t test.

Fig. 6.

BMAL1 and CRY control of c-MYC expression in mouse spleen (model of circadian regulation of c-MYC). (A) BMAL1 deficiency induces the transcription of Ctnnb1 (β-catenin), which binds the TCF/LEF family of transcription factors resulting in high expression of c-Myc. (B) In the Cry1/2 KO, CLOCK-BMAL1 at its binding site in the Ctnnb1 intron suppresses the transcription of Ctnnb1 (β-catenin), which results in low expression of c-Myc.