The goal of the proposed work is to understand how different genes that are regulated by intracellular cholesterol levels are controlled coordinately and independently. By identifying the important cis acting DNA controlling elements and proteins that regulated gene activity through the cis-elements, a more thorough understanding of how diverse genes are regulated will be obtained. The first specific aim in the experiments of the current proposal are aimed at characterizing the roles for the YY1 (Red25) and SREBP proteins in regulation of the gene encoding HMG CoA reductase which is the rate controlling enzyme of cholesterol biosynthesis. YY1 and SREBP bind to an overlapping site that is required for feedback regulation by cholesterol in the reductase promoter. The studies described will evaluate the role for YY1 and SREBP through expression analyses of wild type and mutant forms of each protein in cultured cells along with cell free methods. The experiments will determine how YY1 and SREBP function (together or antagonistically) and will identify the functional domains of YY1 involved. In addition, the cell free DNA binding and protein-protein interaction studies will help uncover a mechanism for how each of the two proteins influences the function of the other. In the second specific aim, the role for the SREBP protein in the stimulation of the promoter for the LDL receptor, the main protein involved in cholesterol uptake from outside the cell, will be evaluated. The exact experiments will determine which specific features of the SREBP molecule are required to stimulate the DNA binding of its dependent co-regulatory Sp1. Previous studies have shown that the stimulation of Sp1 binding by SREBP is an essential part of the activation process of the LDL receptor in response to cholesterol levels. In the third specific aim, the DNA binding specificity of the different SREBP proteins SREBP-1 and -2 will be evaluated to help determine how the two proteins may function differently inside the cell to maintain cholesterol homeostasis. These studies focus on the exact mechanism for how intracellular cholesterol metabolism is regulated to maintain an adequate supply for this essential nutrient. By understanding the mechanism for regulation of intracellular cholesterol metabolism at the molecular level the principal investigator will be able to use this information in the future to determine why individuals in the human population have trouble maintaining cholesterol balance even when they significantly alter its intake through dietary restriction.
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