Regulation of Chromosomal DNA Replication Dynamics in S. Cerevisiae

描述（由申请人提供）： 复制起点起始时间的时间调节是表观遗传调节的一种形式，其意义和机制仍然知之甚少。起源时间程序被认为与发育过程中的基因表达程序有关，可能是为了确保转录状态的稳定遗传。不同起源时间也被认为在基因组稳定性中起作用。染色质结构已被牵连在调制复制时间，但精确的机制仍然模糊。我们现在已经确定，叉头转录因子，Fkh 1和Fkh 2（Fkh 1/2），通过确定酵母基因组中许多最有效的，早期点火起点的激活，在建立基因组复制模式中发挥重要作用。没有其他因素已被确定为具有如此显着的影响，在水平和广度，基因组复制模式。Fkh 1/2在起源调节中的作用独立于它们在转录调节中的作用。相反，我们的结果 表明Fkh 1/2是早期起源聚集或亚核定位所必需，以及它们与起始因子Cdc 45的关联，两者都在G1期。这些结果表明，叉头蛋白发挥了关键作用，在起源调节拴成集群，将成为第一个复制工厂在S期的起源。我们建议，起源集群介导的Fkh 1/2二聚化和/或通过与ORC的相互作用，我们最近证明了一种新的机制。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。该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阶基因组组织中的作用。本研究的主要目的是：1）开发分析和调控Fkh 1/2结合的分子工具; 2）阐明Fkh 1/2结合在染色体起源调控机制中的作用; 3）对Fkh 1和Fkh 2进行结构-功能分析; 4）表征Fkh 1和Fkh 2介导的长距离染色质相互作用

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