Atherosclerotic disease is the predominant underlying organic disorder leading to myocardial infarction, stroke, and peripheral vascular disease. Increased levels of serum cholesterol is a major risk factor for the development of atherosclerosis. The primary form of circulating cholesterol has been defined in recent years. Intracellular cholesterol content is regulated by integration of two pathways that govern the supply of extracellular and endogenous cholesterol. Cells depleted of cholesterol increase their LDL receptor numbers and increase the activity of cholesterol biosynthetic enzymes. As tholesterol builds up in the cell, LDL receptor number and enzyme activities of a number of cholesterol biosynthetic enzymes are downregulated. The decrease in LDL receptor number, and in activity of a rate-limiting cholesterol biosynthetic enzyme. HMG-CoAreductase, is controlled, in part, by repression of transcription of these genes. The experiments proposed are designed to elucidate the nature of the cellular factors which interact with specific DNA elements to repress transcription of these genes in states of cholesterol abundance. Using DNA fragments from previously identified sterol response elements, gel mobilitiy shift analysis will be performed in nuclear and whole cell extracts from sterol-starved cells and from cells grown in abundant sterols. Site directed mutagenesis of the sterol response element(s) will be performed to correlate the binding activity in vitro and activity as a repressor element in vivo. This will be correlated with in vivo occupancy of specific DNA binding sites by in vivo methylation protection. With rapid sensitive assays developed for detection of this trans-acting factor(s), it will be possible to characterize the biochemical nature of the protein, and the clone the cDNA encoding this protein(s). This will allow structural and functional analysis of the mechanism of action of this important transcription regulatory factor. The developmental control of the activity of this repressive factor will be examined. A search will be made for related transcriptional factors. An analysis of the steric constraints on action of the sterol repressive factor will be undertaken, in order to better understand the requirements for protein-protein interaction intranscriptional regulation. Mutant cell lines will derived in order to identify genes whose products participate in sterol mediated gene repression. Later studies will seek to examine the dynamics of sterol repression by an identified factor(s) in metabolic states leading to elevated serum cholesterol such as hyperthroidism and diabetes. Further insight developed from these experiments into the normal process of receptor regulation will allow the design of new strategies to reverse hypercholesterolemia and diabetes. Further insight developed from these experiments into the normal process of receptor regulation will allow the design of new strategies to reverse hypercholesterolemia and prevent athersclerosis.
| Stark, H C; Weinberger, O; Weinberger, J (1992) Common double- and single-stranded DNA binding factor for a sterol regulatory element. Proc Natl Acad Sci U S A 89:2180-4 |
