Identification of a Novel Cryptochrome Differentiating Domain Required for Feedback Repression in Circadian Clock Function
Identification of a Novel Cryptochrome Differentiating Domain Required for Feedback Repression in Circadian Clock Function
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DOI：
10.1074/jbc.m112.368001
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发表时间：
2012-07-27
影响因子：
4.8
通讯作者：
Liu, Andrew C.
中科院分区：
文献类型：
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作者：
Khan, Sanjoy K.;Xu, Haiyan;Liu, Andrew C.
哺乳动物的昼夜节律时钟基于负反馈环，其中隐花色素 CRY1 和 CRY2 的转录抑制是该机制的核心。尽管它们在序列、结构域结构和生化活性方面相似，但它们在时钟功能中发挥着不同的作用。然而，详细的生化研究并不简单，并且尚未使用动力学测量在真实的时钟细胞中检查 Cry 功能。在这项研究中，我们通过基于细胞的遗传互补和实时分子记录证明，单独的 Cry1 能够维持细胞自主的昼夜节律，而 Cry2 则不能。利用这种新颖的功能测定，我们在 CRY1 的光裂合酶同源区 (PHR) 内鉴定了一个隐花色素区分 α 螺旋结构域，称为 CRY1-PHR(313-426)，它是时钟功能所必需的，并将 CRY1 与 CRY2 区分开来。与推测相反，发散的羧基末端尾结构域（CTD）是可有可无的，但可以调节节律幅度和周期长度。最后，我们确定了它们独特功能的生化基础； CRY1 是比 CRY2 更有效的转录抑制因子，各种形式的 CRY 蛋白的抑制强度与节律振幅显着相关。综上所述，我们的结果表明 CRY1-PHR(313-426)，而不是发散的 CTD，对时钟功能至关重要。这些发现为黄素蛋白光裂合酶/隐色素家族的多种功能的进化提供了新的见解，并为 CRY 功能的机制研究提供了新的机会。
Circadian clocks in mammals are based on a negative feedback loop in which transcriptional repression by the cryptochromes, CRY1 and CRY2, lies at the heart of the mechanism. Despite similarities in sequence, domain structure, and biochemical activity, they play distinct roles in clock function. However, detailed biochemical studies have not been straightforward and Cry function has not been examined in real clock cells using kinetic measurements. In this study, we demonstrate, through cell-based genetic complementation and real-time molecular recording, that Cry1 alone is able to maintain cell-autonomous circadian rhythms, whereas Cry2 cannot. Using this novel functional assay, we identify a cryptochrome differentiating alpha-helical domain within the photolyase homology region (PHR) of CRY1, designated as CRY1-PHR(313-426), that is required for clock function and distinguishes CRY1 from CRY2. Contrary to speculation, the divergent carboxyl-terminal tail domain (CTD) is dispensable, but serves to modulate rhythm amplitude and period length. Finally, we identify the biochemical basis of their distinct function; CRY1 is a much more potent transcriptional repressor than CRY2, and the strength of repression by various forms of CRY proteins significantly correlates with rhythm amplitude. Taken together, our results demonstrate that CRY1-PHR(313-426), not the divergent CTD, is critical for clock function. These findings provide novel insights into the evolution of the diverse functions of the photolyase/cryptochrome family of flavoproteins and offer new opportunities for mechanistic studies of CRY function.