Interrogation of functional aspects of transcription at heterochromatic loci

描述（由申请人提供）：异染色质对于真核生物基因组内的DNA组织是必需的，并且在保护基因组完整性、着丝粒形成和核组织中起重要作用。传统上，人们认为异染色质的更密集的结构通过阻止RNA pol II机制访问DNA来阻止基因组异染色质区域内的转录。从那时起，它已被证明，异染色质区域内的转录起着重要的作用，在建立和维持异染色质在三个主要位点，即近着丝粒，交配型和亚端粒。然而，它仍然没有很好地理解转录是如何容纳在异染色质区域。此外，异染色质维持机制的差异已经在上述三个主要异染色质区域中的每一个区域被发现。在这个建议中提出的目的是寻求扩大目前的异染色质基因座内的转录知识。首先，目标1将集中在生物化学纯化的RNA转录本产生的三个主要异染色质区的S。使用MS2外壳蛋白选择性地分离含有编码的MS2茎环的RNA转录物。鉴定与RNA转录物相关的蛋白质将提供一个全面的分析，参与沉默异染色质的因素。 基因座，并暴露沉默发生在三个主要异染色质基因座的每一个的机制可能存在的差异。其次，对于目标2，将对异染色质基因座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的异染色质基因座。这将是特别重要的，因为RNA聚合酶II机器在浓缩和拥挤的异染色质区域的挑战。两者合计，这些目标将寻求发现以前不受重视的方面转录异染色质基因座，并将提供一个理想的机会，在一个新的生物学研究领域的培训。

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.