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(申请人提供)：异染色质对于真核基因组中DNA的组织是必不可少的，并在保护基因组完整性、着丝粒形成和核组织方面发挥重要作用。传统上，人们认为异染色质更紧密的结构通过阻止RNA PolII机制访问DNA来阻止基因组异染色质区域内的转录。自那以后，异染色质区域内的转录在三个主要的位点，即着丝粒周围、交配型和亚端粒的异染色质的建立和维持中起着重要的作用。然而，目前还不清楚转录是如何在异染色质区域中被调节的。此外，在上面提到的三个主要异染色质区域中的每一个都发现了维持异染色质的机制的差异。在这项建议中提出的目标是寻求在异染色质基因座内扩展现有的转录知识。首先，目标1将专注于使用MS2外壳蛋白对来自庞氏链霉菌基因组三个主要异染色区的RNA转录本进行生化纯化，以选择性地分离包含编码MS2茎环的RNA转录本。鉴定与RNA转录本相关的蛋白质将提供对参与异染色质沉默的因素的全面分析 并揭示了在三个主要的异染色质基因座上发生沉默的机制可能存在的差异。第二，对于目标2，将对异染色质位点的上游元件进行询问，以分析转录起始的特性 通过直接替换启动子元件而产生的异染色位点。考虑到在浓缩和拥挤的异染色区对RNA PolII机制提出的挑战，这一点尤其重要。综上所述，这些目标将寻求揭示以前不为人知的异染色位点转录方面，并将提供一个理想的机会，在一个新的生物学研究领域进行培训。