Our long-term objective is to understand the mechanism of transcriptional regulation of human apolipoprotein genes. It is our hypothesis that this regulation is controlled by protein-protein interactions of tissue-specific and ubiquitous nuclear proteins which recognize the apolipoprotein promoter. To understand this regulation we propose to identify the factors which bind to three apolipoprotein promoters (apoA-I, apoCIII, and apoA-II), to purify and clone unique factors, and to study in detail the mechanism of transcriptional regulation of human apoA-I.
The specific aims are: 1) To determine the significance of the promoter elements for the transcription of the human apoA-I, apoCIII, and apoA-II genes by identifying the ubiquitous and tissue-specific transcription factors which bind to these elements and which regulate hepatic transcription. This will be accomplished by a) DNA binding gel electrophoresis, DNase I footprinting and methylation interference with normal and mutated promoter regions, and b) promoter (CAT) assays and in vitro transcription; 2) To purify and characterize new factors, the binding of which significantly affects the transcription of the human apoA-I, apoCIII, and apoA-II genes. Purification from nuclear extracts of rat liver will be accomplished by conventional procedures, followed by DNA sequence specific affinity chromatography. The DNA binding and specificity of the factors will be further tested by DNA binding, DNA protection, and in vitro transcription assays; 3) To clone and sequence cDNAs encoding new factors important for hepatic transcription of the human apoA-I, apoCIII, and apoA-II genes. This will be accomplished by a) screening of human liver cDNA libraries with synthetic oligonucleo- tides corresponding to the protein sequence of the factor, and b) screening expression libraries with the synthetic oligonucleotides corresponding to the recognition sequence of the factor and, 4) To express cDNAs encoding new transcription factors and to study in detail the mechanism of transcriptional regulation of the human apoA-I gene. Normal and mutated full length cDNAs encoding factors will be placed under the control of strong heterologous promoters and will be overexpressed in eukaryotic or bacterial cells in order to study the structure/function relationship of the corresponding factor. Co-trans- fection experiments will determine the importance of a factor for the activation or repression of transcription of target genes. The information which will emerge may then provide rational means of controlling plasma lipoprotein levels in ways that are protective against atherosclerosis.
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