Elimination of cholesterol from the body is largely by way of the bile both through bile salt synthesis and bile salt-dependent bile flow. Thus, the control of the rate of bile salt synthesis, which is determined by the activity of the enzyme, cholesterol 7a hydroxylase (CYP7), is of major importance in understanding how nutritional, genetic and hormonal factors affect whole body cholesterol homeostasis. In cultured hepatoma cells (HepG2), cholesterol 7a hydroxylase activity, and mRNA levels are regulated by bile salts, sterols, glucocorticoids, thyroid hormone and serum factors. We propose to identify and characterize the DNA sequences in the human cholesterol 7a hydroxylase gene responsible for its regulation by these various modulators and the transcription factors involved in these processes. To accomplish this, we will first search for the presence of DNaseI hypersensitive (DH) sites in candidate control regions. DH sites present in HepG2 cells but not in HeLa cells (that do not express the gene), will pinpoint critical liver-specific sequences required for expression. In a similar manner, DH sites may help in the identification of sequences responsive to dietary or hormonal changes in transcription. These studies will be followed by functional analysis of the candidate control sequences using transfection assays with wild type and mutant constructs harboring the sequences of interest. Binding of transcription factors to the control sequences will be assessed by gel retardation and DNaseI footprinting assays. We will also identify the DNA sequence of the CYP7 gene involved in anchoring the human gene to the nuclear matrix. This information will be utilized to test whether the elements identified in cell culture studies do indeed function in the regulation of CYP7 gene expression in vivo , in transgenic animals. Selected constructs comprising key liver-specific elements and boundary insulator sequences (to ensure protection from position effects at the site of integration) will be used to generate transgenic mice that express human CYP7 reporter genes in the liver in a copy number-dependent manner. These constructs will be used to identify the elements responsible for bile salt down regulation of this gene, as well as for induction by cholesterol by mutagenesis of candidate sequences identified in the cell culture experiments. These studies will reveal whether the elements required for bile salt and cholesterol regulation are the same or different, and whether the elements characterized in cell culture function in a similar manner to control expression of the CYP7 gene in transgenic mice. Furthermore, they will serve as the basis for future studies directed toward identifying mutations in these control regions that may help explain heterogeneity among individuals in response to various diets.
