Analysis of Transcriptional Repression and Silencing

描述(申请人提供)：基因表达抑制涉及转录抑制物与启动子元件的结合，而启动子元件又招募作为具有各种染色质修饰活性的大复合体的一部分的协同调节器。染色质有多种表观遗传修饰，导致转录抑制，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，再到基因的不可逆沉默。了解基因如何在不同程度上受到抑制，将有助于更好地理解构成发育、分化和动态平衡的基本过程，并将有助于更好地了解扰乱这些中央过程的疾病。Oct4基因代表了分析转录抑制和沉默的一个很好的模型系统。Oct4在胚胎发育中的胚胎干细胞中表达，原肠形成后在体细胞中的表达被抑制并最终沉默。我们鉴定了在这一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)，它是一种转录抑制因子。为了了解Oct4的抑制机制，我们利用酵母双杂交筛选方法对GCNF相互作用因子进行了鉴定。我们确定共抑制因子NCoR、甲基化DNA结合因子(MBDS)和DNA甲基转移酶(DNMT)是与GCNF特异相互作用的因子。我们提出的假设是，GCNF通过顺序招募参与染色质修饰的多个因子来验证这一假说：1，分析P19和ES细胞分化过程中Oct4的抑制和沉默，重点研究染色质修饰、因子招募和基因沉默。2、分析NCoR和SMRT在Oct4抑制中的作用。3、Mbds在10月4日压制中的作用分析。4分析DNMT在Oct4抑制中的作用。5，分析Oct4在人类ES细胞中的抑制作用(NIH代码BG01)。这些实验的最终结果将对操纵人类ES细胞用于治疗目的和体细胞核移植到卵母细胞具有深远的意义，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.