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: 8635377
• 负责人: Keith Michael Connolly
• 金额: $ 5.51万
• 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/8635377
• 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.

描述（由申请人提供）：异染色质对于真核基因组内 DNA 的组织至关重要，并且在保护基因组完整性、着丝粒形成和核组织方面发挥着重要作用。传统上，人们认为异染色质的更浓缩结构通过阻止 RNA pol II 机器接触 DNA 来阻止基因组异染色质区域内的转录。从那时起，研究表明，异染色质区域内的转录在三个主要位点（即着丝粒周围、交配型和亚端粒位点）异染色质的建立和维持中发挥着重要作用。然而，目前尚不清楚转录如何适应异染色质区域。此外，在上述三个主要异染色质区域中，已发现维持异染色质的机制存在差异。该提案提出的目标旨在扩展当前异染色质基因座内转录的知识。首先，目标 1 将重点关注使用 MS2 外壳蛋白选择性分离含有编码 MS2 茎环的 RNA 转录物，对来自粟酒裂殖酵母基因组的三个主要异染色区域中的每一个区域的 RNA 转录物进行生化纯化。与 RNA 转录物相关的蛋白质的鉴定将为异染色质沉默所涉及的因素提供全面的分析 基因座并揭示三个主要异染色质基因座中每一个发生沉默的机制可能存在的差异。其次，对于目标 2，将询问异染色质位点的上游元件，以分析转录起始的特性 通过直接替换启动子元件产生异染色质位点。考虑到 RNA pol II 机器在浓缩和拥挤的异染色质区域面临的挑战，这一点尤其重要。总而言之，这些目标将寻求揭示异染色质位点转录以前未被重视的方面，并将为新的生物学研究领域的培训提供理想的机会。