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KMID : 0356620140290030379
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Journal of Korean Society of Endocrinology
2014 Volume.29 No. 3 p.379 ~ p.387
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Effect of Resveratrol, a SIRT1 Activator, on the Interactions of the CLOCK/BMAL1 Complex
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Park In-Sung
Lee Yool
Kim Hee-Dae
Kim Kyung-Jin
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Abstract
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Background: In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression.
Methods: To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression.
Results: BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes.
Conclusion: These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity.
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KEYWORD
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Circadian clocks, CLOCK/BMAL1 heterodimer, SIRT1, BiFC analysis, Resveratrol
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