Cheng Ping, Yang Yuhong, Wang Lixin, He Qiyang, Liu Yi
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75390, USA.
J Biol Chem. 2003 Feb 7;278(6):3801-8. doi: 10.1074/jbc.M209592200. Epub 2002 Nov 25.
WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression of wc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series of wc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains.
白领蛋白1（WC-1）和WC-2这两种含PAS结构域的转录因子，是粗糙脉孢菌昼夜节律反馈环中的正向元件。此外，这两种蛋白质都是各种蓝光反应光输入的必需成分，包括生物钟的光诱导。最近，我们确定WC-1是负责这些光反应的蓝光光感受器。在本研究中，我们表明体内FRQ-WC复合物的形成是关闭昼夜节律负反馈环的关键步骤，这需要WC-1。此外，我们表明WC-1在转录水平上负调控wc-2的表达，形成另一个相互作用环。在wc-1突变体中，我们证明存在WC-1的替代蛋白起始，并且WC-1对frq和其他基因的光诱导需求显著不同，这表明细胞中存在不同的WC复合物。与此解释一致，我们的结果表明体内至少存在两种不同类型的WC-1/WC-2复合物，并且较大的WC-1/WC-2复合物包含不止一个WC-1分子。使用一系列wc-1突变体，我们表明WC-1的PASC结构域及其C末端区域对于WC-1/WC-2复合物的形成至关重要。功能分析表明，WC-1的DNA结合结构域仅在黑暗中激活frq时需要，而对于该蛋白的光功能则不需要，这证实WC-1是一种具有可分离蛋白结构域的多功能蛋白。
