Analysis of Transcriptional Repression and Silencing

转录抑制和沉默的分析

• 批准号: 7069033
• 负责人: Austin J Cooney
• 金额: $ 29.3万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7069033
• 关键词: DNA methylation; cell line; chromatin; gene induction /repression; genetic transcription; human embryonic stem cell line; methyltransferase; protein protein interaction; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): Repression of gene expression involves the binding of transcriptional repressors to promoter elements, which in turn recruit co regulators that are part of large complexes with various chromatin modifying activities. There are multiple epigenetic modifications of chromatin that contribute to transcriptional repression that include histone acetylation and methylation, and DNA methylation. Transcriptional repression can vary from decreases in expression levels to reversible silencing of a gene to irreversible silencing of a gene. Understanding how genes are repressed to different degrees will yield greater understanding of basic processes that underlie development, differentiation and homeostasis and will have implications for providing greater understanding of diseases that disrupt these central processes. The Oct4 gene represents an excellent model system to dissect transcriptional repression and silencing. Oct4 is expressed in embryonic stem (ES) cells in developing embryos and upon gastrulation its expression is repressed and eventually silenced in somatic cells. We identified the transcription factor that plays a pivotal in this process the orphan nuclear receptor Germ Cell Nuclear Factor (GCNF), which is a transcriptional repressor. In order to understand the mechanism of repression of Oct4 we used a yeast two hybrid screen to identify GCNF interacting factors. We identified the co-repressor NCoR, the methylated DNA binding factors (MBDs) and DNA Methyl transferases (DNMTs) as factors that specifically interact with GCNF. We propose to test the hypothesis that GCNF mediates repression and silencing of Oct4 expression by the sequential recruitment of multiple factors involved in chromatin modification by execution of the following specific aims: 1, Analysis of Oct4 repression and silencing in differentiating P19 and ES cells, focusing on chromatin modifications, factor recruitment and gene silencing. 2, Analysis of the role of NCoR and SMRT in Oct4 repression. 3, Analysis of the role of MBDs in Oct4 repression. 4 Analysis of the role of DNMTs in Oct4 repression. 5, Analysis of the regulation of Oct4 repression in human ES cells (NIH Code BG01) from Bresagen Inc. The culminating of these experiments will have profound implications for manipulation of human ES cells for therapeutic purposes and somatic cell nuclear transfer into oocytes, which relies on the re-expression of silenced Oct4 and cancers in which the Oct4 is mis-expressed.

描述（由申请人提供）：基因表达的抑制涉及转录抑制器与启动子元素的结合，而启动子元素又是招募CO调节器，这些调节器是具有各种染色质修饰活性的大型复合物的一部分。 染色质的多种表观遗传修饰有助于转录抑制，包括组蛋白乙酰化和甲基化以及DNA甲基化。转录抑制可以从表达水平降低到基因的可逆沉默到基因不可逆的沉默。了解基因如何被抑制到不同程度将对基本过程产生更深入的理解，这些过程是发展，分化和稳态的基础，并将对破坏这些中心过程的疾病提供更深入的理解。 OCT4基因代表了一个出色的模型系统，用于剖析转录抑制和沉默。 OCT4在发育的胚胎中在胚胎干细胞（ES）中表达，胃胃后其表达被抑制并最终在体细胞中沉默。我们确定了在此过程中起关键作用的转录因子孤儿核受体生殖细胞核因子（GCNF），该因子是转录抑制剂。 为了了解Oct4抑制的机制，我们使用了酵母两种混合筛选来识别GCNF相互作用因子。 我们确定了共抑制剂NCOR，甲基化的DNA结合因子（MBD）和DNA甲基转移酶（DNMT）是与GCNF特别相互作用的因素。我们建议测试以下假设：GCNF通过执行以下特定目的通过执行以下特定目的介导了OCT4表达的抑制和沉默，从而介导了OCT4表达的抑制和沉默：1，分析P19和ES细胞中OCT4抑制和沉默的分析，集中于染色质修饰，因子募集，基因累积和基因沉积。 2，分析NCOR和SMRT在OCT4抑制中的作用。 3，分析MBD在OCT4抑制中的作用。 4分析DNMT在OCT4抑制中的作用。 5，分析Bresagen Inc.人类ES细胞中OCT4抑制（NIH代码BG01）的调节。这些实验的最终形式对操纵人类ES细胞的治疗目的和体细胞核转移到卵母细胞中的操纵将具有深远的影响，这与ocycers in Oct4和Cancers inth pertect at pertect ect44有关。