Apolipoprotein B is the sole protein component of LDL and mediates the catabolism of LDL via the LDL receptor. Their is strong evidence that alterations in the structure of regulation of synthesis of this protein play a major role in the pathogenesis of atherosclerosis. Thus, high or low levels of apoB or LDL are associated with increases or decreased risk, respectively, of coronary artery disease. The goals of the application are to study the mechanism of transcriptional regulation of the apoB gene.
The specific aims are: 1) to identify the recognition sequences of nuclear factors involved in the transcriptional activation of the human apoB gene. These recognition sequences will be identified by: a) site-directed mutagenesis; b) competition footprinting analysis; c) fractionation of nuclear extracts on a heparin sepharose column and analysis of the various fractions by DNAse I footprinting and gel retardation assays. 2) to identify the different positive or negative transcriptional factors which regulate the hepatic expression of human apoB gene. The factors will be identified by: a) gel retardation competition experiments; b) biochemical purification by DNA affinity chromatography. The purified factor will be used to obtain limited protein sequence information; b) isolation and characterization of cDNA and genomic clones. Cloning will involve initial screening of a liver cDNA library with synthetic oligonucleotides corresponding to the amino acid sequence or screening an expression library with the oligonucleotide recognition sequence. The genomic sequence will be determined by screening a genomic library with the cDNA probe; c) identification of the properties of this factor from the cRNAs produced in vitro. To fully understand the apoB gene regulation will eventually require the purification of all transcriptional factors involved and the identification of the means which they interact or their activity is modulated by events of cell differentiation. Understanding the regulation of expression of apoB may provide in the near future new approaches toward controlling our plasma LDL and apoB levels.
