ApoB, the only protein of low density lipoprotein (LDL) is synthesized primarily in human liver. Plasma levels of LDL cholesterol and apoB correlate directly with atherosclerosis susceptibility and premature heart disease. Moderately decreased levels of LDL cholesterol and hypobetalipoproteinemia are generally associated with decreased risk of coronary artery disease. Regulation of apoB gene transcription is critically important for assembly and secretion of very low density lipoprotein (VLDL), and therefore, may ultimately determine plasma LDL cholesterol. However, we do not yet understand the precise regulatory mechanisms controlling the transcription of the human apoB gene, and in the absence of that information it has been difficult to develop effective therapy directed toward transcriptional regulation of the apoB gene. The experiments proposed in this application will elucidate the regulatory mechanisms of apoB gene transcription. A secondary aim of this proposal tests the hypothesis that trans-acting factors that regulate apoB gene transcription may also control plasma LDL cholesterol level. Since trans-acting factors mediate apoB gene transcription by interacting with cis-acting DNA elements as well as with specific co-activators, it is necessary to clone genes encoding these trans-acting factors and their co-activators to elucidate the mechanism of apoB gene transcription in the liver. The proposed specific aims are to (1) clone trans-acting factor BRF-4; (2) map its DNA-binding and trans-activation domains; (3) elucidate the mechanism of interaction between HNF-4 and BRF-4 as well as HNF-4 and BRF-2, and their roles in apoB secretion; (4) identify co-activators of BRF-4 and elucidate their roles in apoB transcription and secretion. BRF-4 will be purified from rat liver by DNA-specific affinity column and characterized by SDS/PAGE, gel mobility shift, DNase I footprinting, and in vitro transcription assay. RACE PCR will be used to clone the gene encoding BRF-4. DNA-binding and trans-activation domains of BRF- 4 will be determined by expressing mutated or truncated BRF-4 in a recombinant baculovirus expression system. Co-activators will be identified by a yeast two-hybrid system. Understanding the mechanism of apoB transcription may someday lead to the control of apoB expression and consequently to the control of LDL cholesterol levels.
