Recruitment of transcriptional machinery following DNA replication

DNA 复制后转录机制的募集

• 批准号: 9767525
• 负责人: Tyler Kennedy Fenstermaker
• 金额: $ 4.55万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767525
• 关键词: Address; Architecture; Belief; Biological; Biological Assay; Biological Process; Cell Cycle; Cell Cycle Stage; Cell Differentiation process; Cell Lineage; Cell division; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Conflict (Psychology); Cultured Cells; DNA; DNA Polymerase II; DNA biosynthesis; DNA replication fork; DNA-Directed RNA Polymerase; Data; Data Analyses; Daughter; Development; Disease; Drosophila genus; Embryo; Enzymes; Epigenetic Process; Excision; Experimental Designs; Foundations; Generations; Genes; Genetic Transcription; Goals; Histone Code; Histones; Interruption; Investigation; Knowledge; Label; Ligation; Maintenance; Memory; Microscopy; Mitosis; Modeling; Normal Cell; Nuclear; Pattern; Peptide Initiation Factors; Phase; Physical condensation; Physiologic pulse; Process; Proteins; RNA; RNA replication; Resolution; S Phase; Techniques; Transcript; Transcriptional Activation; Transcriptional Elongation Factors; Work; base; chromatin immunoprecipitation; design; gene repression; genomic locus; histone modification; novel; preservation; programs; promoter; recruit; restoration; theories; transcription factor

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.

项目总结/摘要 在细胞分裂期间维持转录程序是表观遗传学的关键作用。让细胞传输 由于在分裂过程中存在转录记忆，因此认为某些蛋白质标志着活性和抑制的基因组记忆， 的位点在大多数情况下，这些因素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，从而记住转录模式。两 由于转录中断，细胞周期的主要阶段是S期和M期 DNA复制和染色质凝聚。表观遗传标记的典型候选者是 修饰的组蛋白，被认为是转移到新生的DNA，然后招募染色体 蛋白质到两个女儿链。然而，最近的研究表明，许多与染色质相关的 在复制过程中，蛋白质不会从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。这就提出了两个问题：转录蛋白是否也 可能不受复制机器的影响，如果不是，它们在新的载体上重组的速度有多快？ 合成DNA？这个项目的目标是研究转录蛋白的命运，包括RNA 聚合酶II（Pol II）和相关因素，在S期，特别是了解他们的招聘 新生的DNA，因为转录程序的维护是表观遗传学的首要功能。到 为了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型免疫荧光测定法， 染色质组装测定（CAA）。使用CAA的力量在于它可以进行脉冲追踪工作 流来跟踪蛋白质募集到培养细胞中的DNA。为了从机制上理解这些 蛋白质被招募，CAA将与超分辨率显微镜相结合，研究这些蛋白质是如何被招募的。 相互作用在空间上关于复制和转录工厂发生。此外，我将使用 连续染色质免疫沉淀（re-ChIP）试验，旨在研究蛋白质募集， 在特定基因组位点的新生DNA。这些检测将有助于验证CAA的发现，并进一步 在基因特异性水平上询问这些初始结果。此外，基因特异性测定的能力将 允许在不同类型的基因上进一步查询转录蛋白的募集和/或保留，包括 活跃、停顿和平静的基因。这些分析将提供一个更好的理解表观遗传学如何 功能，以维持转录程序，并将在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。