(Taken directly from the application). We propose to pursue our studies of factors which bind both far upstream and downstream to the PEPCK gene chromatin. The biological role of these factors particularly HS-A, and the regions to which they bind, has been identified in part through a combination of transient transfection studies and the generation of transgenic mice. Additional experiments are planned to test the hypothesis that the factor which binds to the HS- E site is involved in insulating adjacent domains against the activation of the PEPCK gene domain. Thus these factors appear to be playing a key role in the establishment of the overall chromatin domain of the PEPCK gene, and its activation in specific tissues. We plan to continue the purification and sequencing of the factors which bind to these regions and to clone and express the genes encoding them. Since HS-A site consists of binding sequences for cyclic AMP response element-binding protein (CREB)/ATF as well as PepA we will ask what role methylation of the underlying sequences plays in regulating CREB binding in tissues which do not express PEPCK; and what role the PepA protein plays in modifying methylation to permit CREB binding to its cognate site. We will assay if the binding of protein factors to the upstream sequences facilities a communication with proteins bound to the basal promoter. Additionally we will ask if expression of the cloned PepA gene is sufficient to induce an opening of the PEPCK chromatin in a non- expressing cell line. We plan to clone the mouse PEPCK gene along with substantial amounts of flanking sequence (ca. 35 kb both upstream and downstream). This will permit us to look for additional HS sites; however the primary goal will be to generate a knockout mouse in which the upstream HS sites have been ablated. This will permit us to assay the role of these sites within the immediate context of the PEPCK chromatin, rather than as additions into random sites in the chromatin. We will assay for the effects on the development of PEPCK gene expression in the liver, the kidney and the adipose tissue.
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