Transcriptional regulation by epigenetic factors

描述（由申请方提供）：在开发过程中，基因表达模式的变化定义了细胞身份。这些特定的基因表达模式在早期胚胎阶段建立，并且必须在细胞分裂后传递给子细胞，这一过程称为表观遗传维持。其基本机制是通过高度保守的三胸组（trxG）和多梳组（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蛋白是必不可少的转录延伸。我们还发现，在DNA复制过程中，几个trxG和PcG蛋白保持稳定的DNA上的结合位点在体内。该提案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蛋白之间的复杂相互作用实现的;（2）一旦确定了基因表达的状态，这些蛋白质在细胞周期中充当表观遗传标记，将此信息传递给子细胞。为了验证第一个假设，我们开发了一种技术，允许在胚胎发育的早期胚盘阶段对trxG激活和PcG沉默的Hox靶基因Ultrabithorax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。使用这种技术，我们建议解决以下问题：（一）什么是PcG蛋白在转录抑制过程中的作用，建立和维持表观遗传转录状态在体内？(ii)在建立和维持阶段，活性和沉默的Ubx之间的分子差异是什么？(iii)对立的监管机构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研究DNA复制过程中染色体蛋白和组蛋白与特定染色质结构域的关联。这种方法将用于（iv）检验以下假设：trxG和PcG蛋白是在DNA复制后重建靶基因的染色质结构域中必不可少的表观遗传标记。考虑到trxG和PcG蛋白在高等真核生物中的功能保守性，这些研究将极大地推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认识。 公共卫生相关性：决定身体特定部位基因表达状态的表观遗传程序在胚胎发育的早期就建立了，这个程序中的任何扰动都可能导致死亡或疾病，如癌症。一旦这个程序建立起来，每个细胞中的基因活性状态必须忠实地遗传到子细胞中。该项目的目的是了解强调调控事件的机制，这些调控事件对于胚胎发生中这些表观遗传程序的建立、维持和遗传至关重要。

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.