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 catalized by cholesterol 7 alpha-hydroxylase, a microsomal cytochrome P450. 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, and cholesterol, the substract of the enzyme, exerts a positive control. Both regulations occur at the level of RNA accumulation, presumably and by analogy with other genes involved in cholesterol homeostasis, 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 7alpha-hydroxylase expression. We have cloned the cholesterol 7alpha-hydroxylase gene, characterized its transcriptional initiation site, and sequenced 1.6 kb of its 5'-flanking region. By transfection experiments we have demonstrated that this fragment contains the DNA elements necessary for the promotion of its transcription in a liver-specific manner. However, we have been unable to demonstrate either cholesterol or bile acid-dependent regulation of gene transcription mediated by the 5'-flanking region of the 7alpha-hydroxylase gene. Because that regulation seems to require the complex interaction of whole organ systems, we will develop transgenic mice that harbor fusion genes containing the 7alpha-hydroxylase promoter. Once we obtain a promoter construct that shows regulated expression in transgenic mice, we will use combined in vivo and in vitro approaches to identify and characterize the DNA elements involved in cholesterol and bile acid- mediated regulation of 7alpha-hydroxylase expression. We will do this by trimming down the 5'-flanking region of the 7alpha-hydroxylase gene, creating deletion mutants that will be analyzed in an in vitro transcription system, prior to introducing them into mice. Protein/DNA-binding studies will allow us to localize the promoter region(s) most likely to contain regulatory elements. These region(s) will be mutated by oligonucleotide-mutagenesis techniques and studied by the in vivo and in vitro approaches mentioned above. These studies should provide a better understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.
