We propose to study proteins which bind to the PEPCK gene chromatin in order to ask how these interactions affect transcriptional modulation by insulin and glucocorticoids. In addition, we will probe the role of these proteins in generating transcriptionally competent PEPCK chromatin. We will perform high resolution footprinting over the proximal PEPCK promoter to assay the effects of hormonal modulation. We will attempt to answer the question of why ubiquitous transcription factors can bind to the PEPCK gene promoter in cells in which the gene is transcribed; but although they are still present, they are unable to bind in cells in which the gene is quiescent. As a part of this analysis we will do genomic sequencing to determine the degree of CpG methylation in tissues which contain promoter- binding factors which are not able to interact with the PEPCK promoter. As an additional part of this analysis, a protein factor, pep, which binds 4800 bp upstream of the PEPCK start site, will be purified. Experiments will be performed to assay if this factor (and its cognate binding site) function to facilitate protein binding to the PEPCK gene promoter. These experiments will include a test of DCR function using transgenic mice as well as transient transfection assays to test if this protein can trigger HS site formation over the promoter region. The effect of mutation of the upstream cognate binding domain on both pep binding in vitro and on hypersensitive site formation in vivo, it will be assayed. The role of this protein will be studied in the loss of PEPCK gene activity during the extinction process in hybrid cells.
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