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.
