Regulation of Chromosomal DNA Replication Dynamics in S. Cerevisiae

描述(由申请人提供)： 复制起始期的时间调控是表观遗传调控的一种形式，其意义和机制尚不清楚。起源计时程序被认为在发育过程中与基因表达程序相连，可能是为了确保转录状态的稳定遗传。差异起源时间也被认为在基因组稳定性中发挥作用。染色质结构与调控复制时间有关，但其确切机制仍不明确。我们现在已经确定了Forkhead转录因子Fkh1和Fkh2(Fkh1/2)，通过确定酵母基因组中许多最有效的、早期激发的起始点的激活，在建立基因组复制模式中发挥主要作用。没有其他因素被确定为在水平和广度上对基因组复制模式具有如此显著的影响。Fkh1/2在起源调控中的作用独立于它们在转录调控中的作用。相反，我们的结果 表明Fkh1/2是早期起源聚集或亚核定位所必需的，以及它们与启动因子CDc45的关联，两者都处于G1期。这些结果表明，总体假设，叉头蛋白在起源调控中发挥关键作用，将起始点捆绑成簇，将成为S阶段的第一个复制工厂。我们认为起源聚集是由一种新的Fkh1/2二聚化机制和/或与ORC的相互作用所介导的，这是我们最近证明的。这些发现为理解基因组组织和表观遗传调控机制开辟了令人兴奋的新途径。这项提案将研究叉头转录因子在复制中的新功能机制，这将使我们了解它们在转录调控中的作用，以及我们新假设的在高级基因组组织中的作用。我们的具体目标是确定Fkh1和Fkh2用于调节染色体复制和结构的机制：1)开发分子工具来分析和操纵起源聚集动力学2)阐明Fkh1/2结合在起源调控机制中的作用3)对Fkh1和Fkh2进行结构-功能分析4)表征Fkh1和Fkh2介导的长程染色质相互作用

DESCRIPTION (provided by applicant): The temporal regulation of replication origin initiation timing is a form of epigenetic regulation whose significance and mechanism remain poorly understood. The origin timing program is thought to be connected with the gene expression program during development, perhaps to ensure the stable inheritance of transcriptional states. Differential origin timing is also propose to play a role in genome stability. Chromatin structure has been implicated in modulating replication timing but a precise mechanism remains vague. We have now determined that Forkhead transcription factors, Fkh1 and Fkh2 (Fkh1/2), play a major role in establishing genomic replication patterns by determining the activation of many of the most efficient, early-firing origins in the yeast genome. No other factor has been identified as having such a significant impact, in level and breadth, on genomic replication patterns. The role of Fkh1/2 in origin regulation is independent of their roles in transcriptional regulation. Instead, our results show that Fkh1/2 are required for early origin clustering or subnuclear localization and their association with the initiation factor Cdc45, both in G1-phase. These results suggest the overall hypothesis that Forkhead proteins play a key role in origin regulation by tethering origins into clusters that will become the first replication factories in S-phase. We propose that origin clustering is mediated by a novel mechanism of Fkh1/2 dimerization and/or by interactions with ORC, which we recently demonstrated. These findings have opened exciting new avenues toward understanding genome organization and mechanisms of epigenetic regulation. This proposal will examine new mechanisms of function for Forkhead transcription factors in replication that will inform our understanding of their roles in transcription regulation, and our newly hypothesized role in higher-order genome organization. Our Specific Aims to determine the mechanisms that Fkh1 and Fkh2 use to regulate chromosome replication and structure are: 1) Develop molecular tools to analyze and manipulate origin clustering dynamics 2) Elucidate the role of Fkh1/2 binding in the mechanism of origin regulation 3) Perform structure-function analysis of Fkh1 and Fkh2 4) Characterize the long-range chromatin interactions mediated by Fkh1 and Fkh2