Coactivation of the CLOCK-BMAL1 complex by CBP mediates resetting of the circadian clock
Coactivation of the CLOCK-BMAL1 complex by CBP mediates resetting of the circadian clock
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DOI：
10.1242/jcs.070300
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发表时间：
2010-10-15
影响因子：
4
通讯作者：
Kim, Kyungjin
中科院分区：
文献类型：
--
作者：
Lee, Yool;Lee, Jiwon;Kim, Kyungjin
转录因子CLOCK-BMAL 1是驱动哺乳动物昼夜节律基因表达和生理的分子时钟机制的核心组成部分。最近，我们报道了这种异二聚体转录因子通过Ca（2+）依赖的蛋白激酶C途径作为信号分子应答重置刺激。在这里，我们证明了CREB结合蛋白（CBP）在快速激活CLOCK-BMAL 1异源二聚体中起着关键作用，导致生物钟的相位重置。在生理条件下，双分子荧光互补（BiFC）测定显示，CLOCK和BMAL 1在细胞质中二聚化，随后易位到细胞核中，以响应血清刺激（平均持续时间为29.2分钟，平均速度为0.7 μ m/分钟）。同时，BMAL 1在Per 1启动子E-box上迅速募集CBP，但在离散的核灶中不募集p300（CBP的功能类似物）。然而，cAMP/Ca（2+）反应元件结合（CREB）蛋白在CRE上的募集在给予重置刺激后并没有显著增加。此外，CBP的过表达大大增强了CLOCK-BMAL 1介导的Per 1转录，这种效果被E-box元件的定点突变完全消除，但不能被Per 1启动子中CRE的突变所消除。此外，CBP的分子敲低严重抑制了由重置刺激触发的时钟基因表达的昼夜节律振荡。这些研究结果表明，CBP招聘BMAL 1介导急性反式激活的时钟BMAL 1，从而诱导即时早期Per 1转录和相位重置的生物钟。
The transcription factor CLOCK-BMAL1 is a core component of the molecular clock machinery that drives circadian gene expression and physiology in mammals. Recently, we reported that this heterodimeric transcription factor functions as a signaling molecule in response to the resetting stimuli via the Ca(2+)-dependent protein kinase C pathway. Here, we demonstrate that the CREB-binding protein (CBP) plays a key role in rapid activation of the CLOCK-BMAL1 heterodimer that leads to phase resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation (BiFC) assay revealed that CLOCK and BMAL1 dimerize in the cytoplasm and subsequently translocate into the nucleus in response to serum stimuli (mean time duration was 29.2 minutes and mean velocity 0.7 mu m/minute). Concomitantly, BMAL1 rapidly recruited CBP on Per1 promoter E-box, but not p300 (a functional analog of CBP), in the discrete nuclear foci. However, recruitment of CBP by cAMP/Ca(2+) response element-binding (CREB) protein on CRE was not markedly increased upon delivery of the resetting stimuli. Furthermore, overexpression of CBP greatly potentiated the CLOCK-BMAL1-mediated Per1 transcription, and this effect was completely abolished by site-directed mutation of E-box elements, but not by the mutation of CRE in the Per1 promoter. Furthermore, molecular knockdown of CBP severely dampened circadian oscillation of clock gene expression triggered by the resetting stimuli. These findings suggest that CBP recruitment by BMAL1 mediates acute transactivation of CLOCK-BMAL1, thereby inducing immediate-early Per1 transcription and phase resetting of the circadian clock.