Defining chromatin architecture and maturation at sites of DNA replication in the Drosophila melanogaster genome

 描述(由申请人提供)：DNA复制是遗传和表观遗传中的一个基本过程。在每个细胞周期中，整个基因组必须在S期复制，DNA复制从多个染色体位置开始，称为起源，以起源识别复合体为标志。此外，表观遗传状态(例如，核小体定位、转录因子结合和翻译后组蛋白修饰)也必须在复制分叉后面重新建立。毫不奇怪，DNA复制和染色质环境是相互依赖的。越来越多的数据表明，染色质结构是起源选择和激活的主要决定因素，染色质组装与DNA复制叉的复制体和通道紧密相连。放松对DNA复制程序或染色质组装的调控可能会导致基因组不稳定和表观遗传状态的丧失--这两者都是肿瘤发生的标志。我建议在核苷酸分辨率上评估果蝇复制起源的染色质结构。我将应用最近开发的一种基于微球菌核酸酶(MNase)的分析方法来‘足迹’蛋白质-DNA在整个果蝇基因组中的占据。这种方法能够以不依赖于染色质免疫沉淀的因子不可知的方式，在核苷酸分辨率上全面解析核小体、转录因子、复制因子和染色质重构体。我希望确定特定的染色质特征，这些特征定义了ORC结合和起始激活的细胞类型特定模式。我将使用突变的依赖于三磷酸腺苷的染色质重构体来测试这一假设，即主动染色质重塑对于确定DNA复制的起始位置是重要的。此外，我将开发一种新的方法来表征 通过结合基于MNase的染色质分析和掺入的核苷酸类似物的免疫沉淀，在复制叉子后面的染色质成熟的时空动力学，这将使我能够区分新生的和成熟的染色质。我希望确定染色质成熟中的基因座和因子的特定差异，为每个细胞周期如何遗传表观遗传状态提供新的机制见解。

 DESCRIPTION (provided by applicant): DNA replication is an essential process involved in both genetic and epigenetic inheritance. In every cell cycle, the entire genome must be duplicated in S-phase with DNA replication starting at multiple chromosomal locations, termed origins, marked by the origin recognition complex (ORC). In addition, the epigenetic state (e.g. nucleosome positioning, transcription factor binding, and post-translational histone modifications) must also be re-established behind the replication fork. Not surprisingly, DNA replication and the chromatin environment are mutually dependent. Increasing data suggests that chromatin structure is a primary determinant for origin selection and activation, and that chromatin assembly is tightly coupled to the replisome and passage of the DNA replication fork. Deregulation of either the DNA replication program or chromatin assembly may result in genomic instability and loss of the epigenetic state -- both of which are hallmarks of tumorigenesis. I propose to assess the chromatin architecture of Drosophila replication origins at nucleotide resolution. I will apply a recently developed micrococcal nuclease (MNase) based assay to 'footprint' protein-DNA occupancy throughout the Drosophila genome. This assay is capable of comprehensively resolving nucleosomes, transcription factors, replication factors, and chromatin remodelers at nucleotide resolution in factor agnostic manner that is independent of chromatin immunoprecipitation. I expect to identify specific chromatin signatures that define cell type specific patterns of ORC binding and origin activation. I will use mutant ATP-dependent chromatin remodelers to test the hypothesis that active chromatin remodeling is important to define start sites of DNA replication. In addition, I will develop a novel approach to characterize the spatiotemporal dynamics of chromatin maturation behind the replication fork by coupling the MNase based chromatin assay with the immunoprecipitation of an incorporated nucleotide analog, which will allow me to discriminate between nascent and mature chromatin. I expect to identify locus and factor specific differences in chromatin maturation providing new mechanistic insights into how the epigenetic state is inherited every cell cycle.