Study of the human apolipoprotein CIII (apoCIII) gene and the human apolipoprotein E (apoE) protein

In this study, the ability of ligand-dependent nuclear receptors to bind and modulate the human apoCIII promoter activity was investigated. DNA binding and competition assays showed that the proximal element CIII-B (-87/-72) binds heterodimers of RXR a with RAR a , T3R b or PPAR a . Element CIII-G (-669/-648) binds heterodimers of RXR a with either RAR a or T3R b and element CIII-I4 (-732/-712) binds ARP-1 and EAR-3, as well as RXR a /RAR a and RXR a /T3R b heterodimers. To determine the core motifs within these elements, methylation interference experiments were performed that identified the protein-DNA interactions between nuclear receptors and the respective HREs on the apoCIII promoter. RXR a /RAR a heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements CIII-B and CIII-I4, respectively. RXR a /T3R b heterodimers and ARP-I bind to DR-5 and DR-0 motifs respectively on element CIII-G. Co-transfection experiments in HepG2 cells showed that RXR a , a combination of RXR a with RAR a or T3R b increased apoCIII promoter activity approximately 2-fold in the presence of 9-cis or all-trans RA. Mutations in the HREs of elements CIII-B, CIII-G or CIII-I4 in the SP1 binding site of element CIII-H, abolished the binding of nuclear receptors and SP1 to their cognate sites, reduced promoter strength and resulted in different responses to the ligand-dependent nuclear receptors. These experiments indicate that CIII-B is the most important element in mediating the effect of nuclear receptors. They also suggest that modulation of the apoCIII promoter activity by nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly, other factors bound to the enhancer and the proximal promoter region.; In the second part of this thesis, stable cell lines that express wild type and mutant apoE forms were generated and new procedures for purification of apoE from the culture medium of cells were developed. The purified apoE variants will be utilized for in vitro functional studies. The wild type and mutant apoE forms have also been cloned in adenovirus vectors. The recombinant adenoviruses will be utilized in future studies for functional analyses of apoE variants in apoE-deficient mice.