Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation - Interaction with BMAL1
Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation - Interaction with BMAL1
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DOI：
10.1111/j.1432-1033.2004.04379.x
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发表时间：
2004-11-01
期刊：
影响因子：
--
通讯作者：
Kato, Y
中科院分区：
文献类型：
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作者：
Sato, F;Kawamoto, T;Kato, Y
碱性螺旋-环-螺旋转录因子DEC1在视交叉上核以昼夜节律的方式表达，它似乎通过抑制Clock/BMAL1激活的启动子来调节哺乳动物的昼夜节律。DEC1与BMAL1的相互作用被认为是抑制BMAL1的分子机制之一[Honma，S.，Kawamoto，T.，Takagi，Y.，Fujimoto，K.，Sato，F.，Noshio，M.，Kato，Y.&Honma，K.(2002)Nature419,841-844]。DEC1的缺失分析表明，其N末端区域包括基本螺旋-环-螺旋结构域，对于抑制活性和与BMAL1的相互作用都是必不可少的，因为缺少碱性区域的DEC1不显示任何抑制或相互作用。此外，我们还发现B组碱性螺旋-环-螺旋蛋白中保守的碱性区域的Arg65负责抑制BMAL1的表达，并与BMAL1相互作用，并与CACGTG E-box结合。然而，His57取代ALA显著降低了DEC1的E-box结合活性，尽管它不影响与BMAL1的相互作用或抑制CLOCK/BMAL1诱导的转录。另一方面，碱性区域缺失的DEC1对DEC1活性起显性-负性作用，表明碱性区域不是形成DEC1同源二聚体所必需的。此外，突变体DEC1还以显性-负性方式对抗DEC2介导的抑制活性。通过下拉实验证实了DEC1和DEC2的异源二聚体的形成。这些发现表明，DEC1的碱性区域通过与BMAL1的蛋白质相互作用和与E-box的DNA结合而参与转录调控。
The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature419, 841-844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loop-helix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein-protein interaction with BMAL1 and DNA binding to the E-box.