5. Assays of Bmal1 transcription modulators

The circadian clock controls the daily oscillations of gene expression and physiological function at the cellular level, indicating that the control of circadian rhythms at the cellular level is important for human health. After we elucidated the transcriptional mechanism of the nonredundant essential unique clock gene, Bmal1, we developed a circadian functional assay system that consists of luminescent reporter cells and the application of Bmal1 findings. We found that

the minimal essential region of the Bmal1 promoter for circadian transcription is embedded in an open chromatin structure, suggesting that this region can remain functional even when inserted into a reporter plasmid [39]. We then established stable reporter cell lines with which to analyse circadian clock function [83] and the effects of DNA methylation on circadian clock function [43]. Figure 3 shows the application of these systems to further dissection of the molecular mechanisms underlying the mammalian circadian clock [46, 84, 85].

Another important finding was the epigenetic inactivation or DNA methylation of the Bmal1 promoter. The methylation status of the Bmal1 promoter is critical for the circadian system. Because the Bmal1 gene is inactivated by the DNA hypermethylation of its promoter, the circadian oscillation of Bmal1 transcription was absent in the haematological malignant cells. The demethylating agent aza-dC restored circadian oscillation, whereas continuous Bmal1 expression did not. Because BMAL1 protein has distinct tissue-specific regulation and functions [87], tissue-specific regulation of BMAL1 expression might be required, and this can be introduced endogenously by aza-dC to establish the negative feedback loop system and restore circadian oscillation. Because the Bmal1 promoter is basically hypomethylated, the methyltransferases DNMT3a and DNMT3b might be mainly responsible for introducing cytosine methylation de novo at unmethylated CpG sites in the promoter [40]. The methylation of DNA contributes to the expression of clock genes [45] in addition to Bmal1, a key player in the

Epigenetic Modulation of Circadian Rhythms: *Bmal1* Gene Regulation

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Circadian rhythms control all aspects of physiology. When they are disrupted by changes in clock gene expression, various critical intracellular physiological processes become dysregulated, and this can lead to diseases that are induced partly by epigenetic effects including DNA methyation. The pathologies that are closely associated with disrupted circadian rhythms include cancer [88], dementia [89], Parkinson's disease [90] and obesity [91]. Among the clock genes, Bmal1 is unique because the loss of BMAL1 protein in mice results in immediate and complete loss of circadian rhythmicity [33], indicating the importance of a specific amount of BMAL1 expression for circadian rhythms. In addition, DNA methylation of the Bmal1 promoter disrupts the circadian system even when the Per and Cry gene promoters are unmethylated, indicating that the Bmal1 gene is functionally important [43]. Epigenetic regulation, especially DNA methylation status, is specific to DNA sites and gene functions. Therefore, the finding that the epigenetic transcriptional regulation of Bmal1 is functionally important for adaptation to environmental changes provides novel insights into clock gene functions that should affect the clinical diagnosis and treatment of

diseases. Therefore, modulators of Bmal1 transcription are needed for the human health.

This work was supported by a KAKENHI grant [number 23592756].

The authors have no conflicts of interest to declare.

disruption of circadian rhythms.

6. Conclusion

Acknowledgements

Conflict of interest

One of the most important findings was that altering the DNA configuration of the Bmal1 promoter causes the epigenetic regulation of Bmal1 circadian transcription [86]. Topoisomerase I (TOP1) is located at an intermediate region between two ROREs that are critical cis-elements of circadian transcription, which is required for transcriptional suppression in cooperation with the distal RORE. The DNA fragment between the ROREs, where the TOP1-binding site is located, behaved like a right-handed superhelical twist, and the modulation of TOP1 activity by the TOP1 inhibitor, camptothecin and Top1 siRNA altered the footprint, indicating the modulation of the chromatin structure. These findings indicated that TOP1 modulates the chromatin structure of the Bmal1 promoter, regulates the Bmal1 transcription and influences the circadian period.

Figure 3. Monitoring cellular circadian clock system using stable reporter cell line. (A) Monitoring method. Promoter region (202 to +27) of Bmal1 was inserted into pGL3-dluc and used to create cell lines with stable, real-time reporter gene to evaluate cellular circadian clock system. (B) Circadian oscillation monitored using host NIH3T3 cells. (C) Circadian oscillation monitored using host CPT-K cells with hypermethylated Bmal1 promoter region. Cells with stable gene expression derived from CPT-K cells were incubated with 2.5 μM aza-dC for 2 days, stimulated with 50% FBS for 2 h and then bioluminescence was measured. Detrended fit curves are representative of at least three independent experiments (control, grey; aza-dC, black). Dots, raw values; lines, fit curve data.

Another important finding was the epigenetic inactivation or DNA methylation of the Bmal1 promoter. The methylation status of the Bmal1 promoter is critical for the circadian system. Because the Bmal1 gene is inactivated by the DNA hypermethylation of its promoter, the circadian oscillation of Bmal1 transcription was absent in the haematological malignant cells. The demethylating agent aza-dC restored circadian oscillation, whereas continuous Bmal1 expression did not. Because BMAL1 protein has distinct tissue-specific regulation and functions [87], tissue-specific regulation of BMAL1 expression might be required, and this can be introduced endogenously by aza-dC to establish the negative feedback loop system and restore circadian oscillation. Because the Bmal1 promoter is basically hypomethylated, the methyltransferases DNMT3a and DNMT3b might be mainly responsible for introducing cytosine methylation de novo at unmethylated CpG sites in the promoter [40]. The methylation of DNA contributes to the expression of clock genes [45] in addition to Bmal1, a key player in the disruption of circadian rhythms.
