Interrogation of functional aspects of transcription at heterochromatic loci

描述（由申请人提供）：异染色质是真核生物基因组中DNA组织所必需的，在保护基因组完整性、着丝粒形成和细胞核组织方面起着重要作用。传统上，人们认为异染色质的更浓缩的结构通过阻止RNA pol II机制接近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（即周中心、交配型和亚端粒）的异染色质的建立和维持中起着重要作用。然而，转录是如何在异染色质区调节的，目前还不清楚。此外，在上述三个主要的异染色质区域中，已经发现了维持异染色质的机制差异。在本提案中提出的目的是寻求扩大目前对异染色质位点内转录的了解。首先，目的1将着重于利用MS2外壳蛋白选择性地分离含有编码MS2茎环的RNA转录物，从S. pombe基因组的三个主要异色区域产生RNA转录物的生化纯化。鉴定与RNA转录相关的蛋白质将提供对异染色质沉默相关因素的全面分析

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.