Role of Chromatin Remodeling in Circadian Regulation

描述(申请人提供)：粗糙脉孢菌的昼夜节律部分由负反馈环控制，在负反馈环中，频率(FRQ)蛋白通过与白领复合体(WCC)直接相互作用抑制自己的转录。目前还没有关于染色质频率组织(FRQ)基因启动子的信息，以及在昼夜节律周期中发生了什么类型的染色质重塑。我建议通过三个具体目标来研究染色质重塑在昼夜节律调节中的作用。首先，我计划利用连接介导的聚合酶链式反应(LM-PCR)来定位FRQ启动子上的核小体在昼夜节律周期中的位置，并响应光诱导。核小体位置的相对变化将通过DNase I保护和限制位点可及性分析来确认。其次，我计划使用染色质免疫沉淀(CHIP)分析来确定组蛋白氨基末端尾部结构域上发生的共价修饰。最后，我将对FRQ基因座上发生的组蛋白修饰进行排序，以形成一个昼夜节律调节的组蛋白密码。这些目标的完成将促进昼夜节律生物学和染色质重塑领域的发展。

DESCRIPTION (provided by applicant): Circadian regulation in Neurospora crassa is controlled in part by a negative feedback loop where the Frequency (FRQ) protein inhibits its own transcription through a direct interaction with the White Collar complex (WCC). Currently there is no information on chromatin organization at the frequency (frq) gene promoter and what, if any type of chromatin remodeling occurs during the circadian cycle. I propose to examine the role of chromatin remodeling in circadian regulation through three specific aims. First, I plan to map the nucleosome positions at the frq promoter over the circadian cycle and in response to light induction using ligation-mediated polymerase chain reaction (LM-PCR). Relative changes in nucleosome positions that occur will be confirmed by DNase I protection and restriction site accessibility assays. Secondly, I plan to determine the covalent modifications that occur on the amino-terminal tail domains of the histones using the chromatin immunoprecipitation (ChiP) assays. Finally, I will order the histone modifications that occur at the frq locus to develop a circadian regulated 'histone code'. The completion of these aims will further both the circadian biology and chromatin remodeling fields.