Recruitment of transcriptional machinery following DNA replication

项目摘要/摘要 细胞分裂过程中转录程序的维持是表观遗传学的关键作用。让细胞进行传输 转录记忆在分裂过程中，人们认为某些蛋白质标志着基因组的活跃和抑制 精神错乱。在大多数情况下，这些因子包括染色质相关蛋白，其功能是保持稳定 在整个细胞周期中与DNA相关，从而使转录模式被记住。两个人 由于转录中断，细胞周期的主要阶段是S期和M期 分别通过DNA复制和染色质缩合。表观遗传标记的典型候选者是 被修饰的组蛋白，据信被转移到新生的DNA，然后招募染色体 蛋白质连接到两条子链。然而，最近的研究表明，许多与染色质相关的 蛋白质在复制过程中不会从DNA中被取代，这挑战了长期以来认为复制是 固有地破坏染色质结构。这就引出了两个问题：转录蛋白是否也 潜在地不受复制机制的影响，如果不是，它们在新的 人工合成的DNA？这个项目的目标是研究包括rna在内的转录蛋白的命运。 聚合酶II(POL II)及其相关因素，在S阶段，特别是了解他们的招募 对于新生的DNA来说，因为转录程序的维持是表观遗传学的首要功能。至 追踪这些蛋白质的募集我将使用我们实验室开发的一种新的免疫荧光分析方法，称为 染色质组装试验(CAA)。使用CAA的强大之处在于它适合于脉冲追逐的工作 Flow跟踪培养细胞中蛋白质向DNA的募集。为了从机械上理解这些 蛋白质正在被招募，CAA将与超级分辨率显微镜相结合，以研究这些 相互作用在空间上发生在复制和转录工厂方面。此外，我将使用 顺序染色质免疫沉淀(Re-ChIP)分析旨在研究蛋白质在 新生的DNA在特定的基因组位置。这些分析将有助于验证CAA的结果，并进一步 在基因特定的水平上询问这些初步结果。此外，基因特异性分析的力量将 允许在不同类型的基因上进一步询问、招募和/或保留转录蛋白，包括 活跃、停顿和稳定的基因。这些分析将更好地理解表观遗传学是如何 维持转录程序的功能，对于了解细胞如何改变这些程序至关重要 在细胞分化和疾病发展过程中。

Project Summary/Abstract Maintenance of transcriptional programs during cell division is the key role of epigenetics. For cells to transmit transcriptional memory during division, it is believed that certain proteins mark active and repressed genomic loci. For the most part, these factors include chromatin-associated proteins whose function is to remain stably associated with DNA throughout the cell cycle such that transcriptional patterns are remembered. The two main phases of the cell cycle where this is critical are S-phase and M-phase, due to transcriptional interruption by DNA duplication and chromatin condensation, respectively. The typical candidates for epigenetic marks are modified histones, which are believed to be transferred to nascent DNA and to then recruit chromosomal proteins to the two daughter strands. Recently, however, it was shown that many chromatin-associated proteins are not displaced from DNA during replication, challenging the long held belief that replication is inherently disruptive to chromatin structure. This raises two questions: are transcriptional proteins also potentially impervious to replication machinery, and if not, how quickly do they reassemble on newly synthesized DNA? The goal of this project is to investigate the fate of transcriptional proteins, including RNA polymerase II (Pol II) and associated factors, during S-phase, and in particular to understand their recruitment to nascent DNA, since maintenance of transcriptional programs is the paramount function of epigenetics. To track the recruitment of these proteins I will use a novel immunofluorescent assay developed by our lab, called the Chromatin Assembly Assay (CAA). The power of using CAA is that it is amenable to a pulse-chase work flow to track protein recruitment to DNA in cultured cells. In order to understand mechanistically how these proteins are being recruited, CAA will be combined with super resolution microscopy, to study how these interactions spatially occur with regard to replication and transcription factories. Additionally, I will use sequential chromatin immunoprecipitation (re-ChIP) assays designed to investigate protein recruitment to nascent DNA at specific genomic loci. These assays will help to validate the CAA findings, and to further interrogate these initial results at the gene specific level. Furthermore, the power of gene-specific assay will allow further inquiry recruitment and/or retention of transcriptional proteins at different types of genes, including active, paused, and poised genes. These analyses will provide a better understanding of how epigenetics functions to maintain transcriptional programs, and will be vital in understanding how cells alter these programs during cellular differentiation and the development of disease.