Analysis of Transcriptional Repression and Silencing

描述（由申请人提供）：基因表达的抑制涉及转录抑制因子与启动子元件的结合，启动子元件反过来招募作为具有各种染色质修饰活性的大型复合物的一部分的共同调节因子。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的降低到基因的可逆沉默到基因的不可逆沉默。了解基因是如何在不同程度上受到抑制的，将有助于更好地理解发育、分化和体内平衡的基本过程，并有助于更好地理解破坏这些中心过程的疾病。Oct4基因代表了一个很好的模型系统来解剖转录抑制和沉默。Oct4在发育中的胚胎干细胞中表达，在原肠胚形成时，其表达被抑制并最终在体细胞中沉默。我们发现在这一过程中起关键作用的转录因子是孤儿核受体生殖细胞核因子（GCNF），这是一种转录抑制因子。为了了解Oct4的抑制机制，我们使用酵母双杂交筛选来鉴定GCNF的相互作用因子。我们确定了协同抑制因子NCoR、甲基化DNA结合因子（MBDs）和DNA甲基转移酶（dnmt）是特异性与GCNF相互作用的因子。为了验证GCNF通过序贯募集参与染色质修饰的多个因子介导Oct4表达的抑制和沉默的假说，我们提出以下具体目标：1、分析P19和ES细胞分化过程中Oct4的抑制和沉默，重点关注染色质修饰、因子募集和基因沉默。2、NCoR和SMRT在Oct4抑制中的作用分析。3、mbd在Oct4抑制中的作用分析。4 dnmt在Oct4抑制中的作用分析。5、Bresagen公司的人类胚胎干细胞（NIH代码BG01） Oct4抑制调控分析。这些实验的最终结果将对用于治疗目的的人类胚胎干细胞的操纵和体细胞核移植到卵母细胞中具有深远的意义，这依赖于沉默的Oct4的重新表达和Oct4错误表达的癌症。

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.