Role of Chromatin Remodeling in Circadian Regulation

描述（由申请人提供）：粗神经孢子虫的昼夜节律调节部分由负反馈回路控制，其中频率（FRQ）蛋白通过与白领复合体（WCC）的直接相互作用抑制其自身的转录。目前还没有关于频率（frq）基因启动子染色质组织的信息，以及在昼夜周期中发生的任何类型的染色质重塑。我建议通过三个特定的目的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反应（LM-PCR）绘制frq启动子核小体在昼夜周期和光诱导下的位置。发生的核小体位置的相对变化将通过dna酶I保护和限制性位点可及性测定来证实。其次，我计划使用染色质免疫沉淀（ChiP）测定在组蛋白的氨基末端尾部区域上发生的共价修饰。最后，我将命令发生在frq位点的组蛋白修饰，以开发昼夜节律调节的“组蛋白代码”。这些目标的完成将进一步推动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.