Interrogation of functional aspects of transcription at heterochromatic loci

异染色质位点转录功能方面的询问

• 批准号: 8313422
• 负责人: Keith Michael Connolly
• 金额: $ 4.92万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313422
• 关键词: Adopted; Affect; Base Pairing; Biochemical; Biological; Biological Assay; Cataloging; Catalogs; Cell physiology; Centromere; Chromosome Segregation; Code; Complex; Crowding; DNA; DNA Polymerase II; Elements; Euchromatin; Event; Factor Analysis; Fission Yeast; Gene Silencing; Genetic; Genetic Recombination; Genetic Transcription; Genome; Heterochromatin; Knowledge; MS2 coat protein; Maintenance; Mating Types; Mediating; Modeling; Northern Blotting; Nuclear; Play; Prevention; Primer Extension; Process; Promoter Regions; Property; Proteins; RNA; RNA Interference; RNA Processing; RNA purification; Recruitment Activity; Repetitive Sequence; Reporter Genes; Repression; Role; Structure; Training; Transcript; Transcription Initiation; prevent; promoter; research study; stem

DESCRIPTION (provided by applicant): Heterochromatin is essential for the organization of DNA within eukaryotic genomes and plays an important role in protecting genome integrity, centromere formation and nuclear organization. Traditionally, it was assumed the more condensed structure of heterochromatin prevented transcription within heterochromatic regions of the genome by preventing RNA pol II machinery from accessing DNA. Since then, it has been shown that transcription within heterochromatic regions plays an important role in the establishment and maintenance of heterochromatin at three major loci, namely pericentromic, mating-type and subtelomeric. However, it is still not well understood how transcription is accommodated in heterochromatic regions. Furthermore, differences in the mechanisms by which heterochromatin is maintained have been uncovered at each of the three major heterochromatic regions mentioned above. The aims presented in this proposal seek to expand current knowledge of transcription within heterochromatic loci. First, aim 1 will focus on the biochemical purification of RNA transcripts arising from each of the three major heterochromatic regions of the S. pombe genome using the MS2 coat protein to selectively isolate RNA transcripts containing coded MS2 stem loops. Identification of proteins associated with RNA transcripts will offer a comprehensive analysis of factors involved in silencing of heterochromatic loci and expose possible differences in the mechanisms by which silencing occurs at each of the three major heterochromatic loci. Second, for aim 2, an interrogation of upstream elements at heterochromatic loci will be undertaken to analyze the properties of transcription initiation at heterochromatic loci by direct replacement of promoter elements. This will be particularly important given the challenges placed on the RNA pol II machinery at condensed and crowded heterochromatic regions. Taken together, these aims will seek to uncover previously unappreciated aspects of transcription at heterochromatic loci, and will provide an ideal opportunity for training in a new field of biological study. PUBLIC HEALTH RELEVANCE: Establishment and maintenance of heterochromatin is important for a number of cellular processes including proper segregation of chromosomes and prevention of potentially deleterious recombination events [1]. Thus, heterochromatin protects the integrity of the genome. This proposal seeks to investigate mechanistic details of transcription within heterochromatic domains that are important for maintenance of heterochromatin structure.

描述（由申请人提供）：异染色质对于在真核基因组中的DNA组织至关重要，并且在保护基因组完整性，Centromere形成和核组织中起着重要作用。传统上，假定异染色质的更凝结结构可以通过防止RNA POL II机械访问DNA来阻止基因组异质区域内的转录。从那以后，已经表明，异质区域内的转录在三个主要基因座的异染色质建立和维持中起着重要作用，即丁香膜，交配型和亚端子体。但是，仍然不太了解在异性区域如何适应转录。此外，在上述三个主要的异晶区中的每个区域中，都发现了保持异染色质的机制的差异。该提案中提出的目的旨在扩大异质基因座中的当前转录知识。首先，AIM 1将使用MS2涂层蛋白选择性地分离包含编码的MS2茎回路的RNA转录本，将其由S. pombe基因组的三个主要杂色区域中的每个区域中的每一个引起的RNA转录物的生化纯化。与RNA转录本相关的蛋白质的鉴定将对杂质沉默涉及的因素进行全面分析 基因座并暴露了可能在三个主要的异质基因座中每一个发生沉默的机制上可能存在差异。其次，对于AIM 2，将对异性层的上游元素进行审问，以分析在 通过直接替换启动子元件的直接替代位点。考虑到在RNA Pol II机械上的挑战凝结和拥挤的异性区域，这将是尤其重要的。综上所述，这些目标将寻求在异色症基因座上发现以前未批准的转录方面，并将为在新的生物学研究领域提供理想的培训机会。 公共卫生相关性：异染色质的建立和维护对于许多细胞过程至关重要，包括适当的染色体隔离和预防潜在有害的重组事件[1]。因此，异染色质保护基因组的完整性。该提案旨在研究异质域中转录的机理细节，这对于维持异染色质结构很重要。