Transcriptional regulation by epigenetic factors

描述(由申请人提供)：在发育过程中，基因表达模式的变化定义了细胞的身份。这些特定的基因表达模式是在早期胚胎阶段建立的，必须在细胞分裂后传递给子细胞，这一过程被称为表观遗传维持。其潜在的机制是通过高度保守的三胸基团(TrxG)和多梳基团(PcG)蛋白实现的，它们分别负责维持许多靶基因的转录激活和抑制。这些蛋白质在转录中的作用仍然不清楚，它们在细胞周期中传递表观遗传信息的方式也完全未知。我们发现trxG蛋白是转录延长所必需的。我们还发现，在DNA复制过程中，几个trxG和PcG蛋白在体内仍然稳定地与它们在DNA上的结合位置相关联。这项提议的目的是检验两个假设：(1)表观遗传维持的建立是通过trxG和PcG蛋白质之间的复杂相互作用实现的；以及(2)一旦基因表达状态被确定，这些蛋白质在细胞周期中作为表观遗传标记将信息传递给子细胞。为了验证第一个假设，我们开发了一种技术，允许在胚胎发育的胚泡发育早期对trxG激活的和PcG沉默的HOX靶基因的染色质组成进行生化分析。在这些阶段，表观遗传维持正在建立。利用这项技术，我们建议解决以下问题：(1)在体内建立和维持表观遗传转录状态的过程中，PcG蛋白在转录抑制中扮演什么角色？(Ii)在建立和维护阶段，活跃的UBX和静默的UBX之间的分子差异是什么？(Iii)相互对立的监管者集团是相互对抗还是独立行事？为了验证我们的第二个假设，我们开发了一种新的体内方法来检查DNA复制过程中染色体蛋白和组蛋白与特定染色质区域的关联。这一方法将被用来(Iv)检验这一假设，即trxG和PcG蛋白是在DNA复制后重新建立目标基因染色质结构域的必要的表观遗传标记。鉴于trxG和PcG蛋白在高等真核生物中功能的保守性，这些研究将极大地促进我们对转录调控和表观遗传的基本机制及其与癌症等疾病的相关性的了解。 与公共健康相关：决定身体特定部位基因表达状态的表观遗传学程序是在胚胎发育的很早就建立的，该程序中的任何干扰都可能导致死亡或癌症等疾病。一旦这个程序建立，每个细胞中的基因活动状态必须在子细胞中忠实地遗传。这个项目的目的是了解在胚胎发生中这些表观遗传程序的建立、维持和遗传所必需的调节事件的机制。

DESCRIPTION (provided by applicant): In development, changes in gene expression patterns define cell identity. These specific gene expression patterns are established at early embryonic stages, and must be passed on to daughter cells after cell division, a process termed epigenetic maintenance. The underlying mechanisms are achieved by the highly conserved trithorax-group (trxG) and Polycomb-group (PcG) proteins, which are responsible for the maintenance of transcriptional activation and repression, respectively, of numerous target genes. The roles of these proteins in transcription remain obscure, and the way they pass epigenetic information during the cell cycle is completely unknown. We found that trxG proteins are essential for transcriptional elongation. We also found that during DNA replication several trxG and PcG proteins remain stably associated with their binding sites on DNA in vivo. The goal of this proposal is to test two hypotheses: (1) that establishment of epigenetic maintenance is achieved by the complex interactions between trxG and PcG proteins; and (2) once the status of gene expression is determined, these proteins serve as epigenetic marks during the cell cycle to pass this information to daughter cells. To test the first hypothesis, we developed a technique that allows biochemical analysis of the chromatin composition of the trxG-activated and the PcG-silenced Hox target gene Ultrabithorax at very early blastoderm stages of embryo development. During these stages epigenetic maintenance is being established. Using this technique, we propose to address the following questions: (i) What is the role of PcG proteins in transcriptional repression during the establishment and maintenance of epigenetic transcription status in vivo? (ii) What are the molecular differences between active and silenced Ubx during establishment and maintenance phases? (iii) Do the opposing groups of regulators act antagonistically or independently? To test our second hypothesis, we developed a new in vivo approach to examine association of chromosomal proteins and histones with specific chromatin domains during DNA replication. This approach will be used to (iv) test the hypothesis that trxG and PcG proteins are essential epigenetic marks in re-establishing chromatin domains of the target gene following DNA replication. Given conservation of the function of trxG and PcG proteins in higher eukaryotes, these studies will greatly advance our knowledge of the basic mechanisms of transcriptional regulation and epigenetic inheritance and their relevance to diseases like cancer. PUBLIC HEALTH RELEVANCE: The epigenetic program that determines the status of gene expression in a particular part of the body is established very early during embryo development, and any perturbations in this program may lead to death or diseases, like cancer. Once this program is established, the state of gene activity in each cell has to be faithfully inherited in daughter cells. The aim of this project is to understand the mechanisms that underline the regulatory events that are essential for the establishment, maintenance and inheritance of these epigenetic programs in embryogenesis.