DNA methylation of the BMAL1 promoter.

We previously analyzed transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system and found that the BMAL1 gene is expressed with circadian oscillation and that its regulatory region is located in hypomethylated CpG islands with an open chromatin structure. Here, we found that the BMAL1 gene is not expressed with circadian oscillation in CPT-K cells because the CpG islands located in the BMAL1 promoter are hypermethylated and that 5-aza-2'-deoxycytidine (aza-dC) recovered BMAL1 expression. In contrast, CpG islands in the PER2 promoter were hypomethylated, the PER2 gene was expressed and aza-dC enhanced PER2 gene expression in CPT-K cells. Reporter gene assays showed that intracellular transcriptional machinery for the BMAL1 gene is active, suggesting that BMAL1 inactivation is caused by DNA methylation and not by malfunctional promoter activity. Incubating CPT-K cells with aza-dC also increased CRY1 expression, whereas CLOCK expression was not altered and the CRY1 promoter was unmethylated. These results suggest that aza-dC induces BMAL1 expression via DNA demethylation in the BMAL1 promoter and enhances PER2 and CRY1 transcription. Finally, aza-dC recovered the circadian oscillation of BMAL1 transcription. These results suggest that DNA methylation of the BMAL1 gene is critical for interfering with circadian rhythms.