Functional Analysis of Epigenetic Complexes

翻译后摘要：为了人类正常发育，受精卵必须分裂的方式， 生成一个在正确的地方有正确的器官和附属物的身体。这在很大程度 是由主调节基因的适当时间和空间调节决定的， 引导分化细胞形成器官和附属物。一个关键方面 这种发育过程的一个重要特征是能够在一个受抑制的环境中维持主调节基因， 它们的表达可能导致不适当的细胞行为。错误表达 即使是一个单一的主调控基因，如HOX基因座编码的基因，也可以导致细胞 以与其身体位置和组织类型不相容的方式表现。Polycomb- PcG组基因主要负责维持这种抑制，基于 在过去的70年里进行了深入的研究。保持基因的活跃表达模式是 对发展同样重要。一组维持活性的基因，叫做三胸- 组（trxG），通过它们抑制PcG突变的能力来分离。本申请 描述了我们对理解编码蛋白质功能的工作的继续 PcG和trxG基因。这些蛋白质形成几种复合物。PcG复合物PRC 1 PRC 2和PRC 2各自是含有几种PcG基因产物的大复合物。PRC 1是主要的 在Polycomb-组中抑制的“引擎”，并且已知与染色质相互作用， 在细胞核中包装基因的结构。染色质结构可以使 封闭的DNA激活因子无法接近。一个突出的假设是，压抑可以 通过产生高度包装的DNA而产生，该DNA不再能够被转录， 表达。我们描述了研究PRC 1使用的各种机制的方法， 产生包装的染色质。PRC 1与PRC 2一起工作以产生抑制。PRC2 甲基化核小体，染色质的初级包装单位，在残基H3 K27上， 在众多实验室和公司进行深入研究。我们将提供新颖的 通过探索与甲基化无关的PRC 2的抑制活性来获得信息。我们 还将表征参与基因调控的单独蛋白质的PRC 2甲基化， Elongin A.最后，我们将描述两个独立的核小体重塑活动编码 通过trxG基因，哺乳动物SWI/SNF（BAF）复合物和哺乳动物CHD 7。我们将 探索SWI/SNF亚基和粘着蛋白（一种染色质组织活性）之间的相互作用， 并确定这些相互作用如何影响染色质中的长程相互作用。我们将 研究称为HERVH的lncRNA在靶向CHD 7活性中的作用。

Abstract: For humans to develop properly, the fertilized egg must divide in a manner that generates a body that has the correct organs and appendages in the correct places. Much of this is determined by the proper temporal and spatial regulation of master regulatory genes that guide formation of the differentiated cells that make up organs and appendages. A key aspect of this developmental process is the ability to maintain master regulatory genes in a repressed state in cells where their expression might cause inappropriate cell behavior. Mis-expression of even a single master regulatory gene, such as those encoded in the HOX loci, can result in a cell behaving in a manner incompatible with its body location and tissue type. The Polycomb- Group (PcG) group of genes is largely responsible for maintaining this repression, based upon intensive study over the past 70 years. Maintaining active expression patterns for genes is equally critical for development. A set of genes that maintains activation, called the trithorax- Group (trxG), was isolated via their ability to suppress PcG mutations. This application describes the continuation of our work on understanding the function of the proteins encoded by the PcG and trxG genes. These proteins form several complexes. The PcG complexes PRC1 and PRC2 are each large complexes that contain several PcG gene products. PRC1 is the main `engine' of repression in the Polycomb-Group and is known to interact with chromatin, the structure that packages genes in the nucleus of cells. Chromatin structure can render the enclosed DNA inaccessible to activating factors. A prominent hypothesis is that repression can be generated by generating highly packaged DNA that is no longer able to be transcribed and expressed. We describe approaches to investigate the various mechanisms used by PRC1 to generate packaged chromatin. PRC1 works together with PRC2 to generate repression. PRC2 methylates nucleosomes, the primary packaging unit of chromatin, on residue H3K27, a topic of intensive study in numerous laboratories and companies. We intend to provide novel information by exploring repressive activities of PRC2 that are unrelated to methylation. We will also characterize methylation by PRC2 of a separate protein involved in gene regulation, ElonginA. Finally, we will characterize two separate nucleosome remodeling activities encoded by trxG genes, the mammalian SWI/SNF (BAF) complex and mammalian CHD7. We will explore interactions between SWI/SNF subunits and cohesin, a chromatin organizing activity, and determine how those interactions impact long range interactions in chromatin. We will examine the role for a lncRNA called HERVH in targeting CHD7 activity.