The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has widespread implications because the rate of elimination of cholesterol is a very important factor in diseases such as atherosclerosis, gallstone disease, and some lipid storage diseases. The initial and rate limiting step in this pathway is the hydroxylation of cholesterol at position 7 and is catalyzed by cholesterol 7alpha- hydroxylase. This enzyme is subject to a feedback control, whereby high levels of bile acids returning to the liver via the enterohepatic circulation suppress its activity. Cholesterol, the substrate of the enzyme, exerts a positive control. Both regulations occur at the level of the transcription of its gene. The overall goal of this proposal is to understand the molecular mechanisms involved in the cholesterol- and bile acid-mediated regulation of cholesterol 70:-hydroxylase transcription. We have recently demonstrated that bile acids and cholesterol mediate transcriptional regulation of 7alpha-hydroxylase promoter activity in transgenic mice, and that the DNA element(s) that mediates that regulation is located in the first 342 bp of the promoter region. We have also quantitatively reproduced the bile acid- and cholesterol-mediated regulation of the 7alpha-hydroxylase promoter activity in an established tissue culture system. We will use this tissue culture system to localize, at the nucleotide level, the DNA element(s) involved in the bile acid- and cholesterol-mediated 'regulation of 7alpha-hydroxylase transcription. Deletion mutants of the 7alpha- hydroxylase promoter and heterologous promoters will be created and tested to delineate those sequences. Important fusion genes that provide critical information in our tissue culture experiments will be tested in transgenic mice to confirm the obtained data. Once the DNA elements that mediate the bile acid and cholesterol regulation of 7alpha-hydroxylase transcription are known, we will establish a protein-DNA binding assay that show a precise correlation between 7alpha-hydroxylase promoter activity and binding to specific oligonucleotides that we will use as probes. This assay will be used to purify the transacting factors that bind to the DNA regulatory elements using standard and affinity chromatography techniques. This purification will lead to the preparation of antibodies and DNA probes essential for the study of the molecular mechanisms involved in that regulation. These studies should provide a better understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.
