High-density lipoprotein (HDL) and its major protein component, apolipoprotein AI (apoAI), play a major role in cholesterol homeostasis. Knowledge of the molecular mechanisms that regulate apoAI gene expression is clinically important because reduced plasma levels of apoAI and HDL are associated with premature atherosclerosis and coronary heart disease. Our long term objective is to study the transcriptional regulation of the apoAI gene and to identify novel signals that modulate apoAI synthesis in liver and intestine. Towards this goal, we have found that regulatory elements in the apoAI promoter interact with four nuclear receptors with unknown ligands, ARP-1, EAR-2, EAR-3, and HNF-4, and with heterodimers formed between the 9-cis retinoic acid receptor alpha (RXRalpha) and either ARP-1, EAR-2, EAR-3, all -trans retinoic acid receptor alpha (RARalpha), vitamin D receptor (VDR), or peroxisome proliferator-activated receptor (PPAR). ApoAI gene expression is repressed by ARP-1 and activated by HNF-4 and RXRalpha, however, the effects of the other nuclear receptors on apoAI expression are not known.
The specific aims are: 1) To determine the functional effects of the nuclear receptors that bind to the apoAI promoter on the expression of this gene in hepatic and intestinal cells. 2) To map the functional domains of HNF-4 and ARP-1, and to identify the basal transcription factors which are targets for these nuclear receptors. 3) To identify the factors that regulate the DNA-binding activity of ARP-1, EAR-2, and EAR-3. The experimental design and methods for achieving these aims are; 1) Cotransfections of plasmids expressing the above nuclear receptors with constructs containing the CAT reporter gene under the control of the apoAI promoter region in human hepatoma (HepG2) and human colon carcinoma (Caco- 2) cells, using the calcium phosphate coprecipitation method. Transfections with RXRalpha, RARalpha, VDR, and PPAR will include posttransfection treatments with 9-cis and all-trans retinoic acid, vitamin D, and clofibric or linoleic acid, respectively. 2) Deletion mutagenesis of HNF-4 and ARP-1 in combination with cotransfection experiments will be employed to map the activation and repression domains of these nuclear receptors. Protein-protein interaction assays will be used to study the interactions of HNF-4 and ARP-1 with basal transcription factors., including the TATA-binding protein (TBP), TFIIB, and other TBP-associated factors (TAFs), an to identify the domains involved in these interactions. Furthermore, interaction cloning, will be used to clone tissue-specific TAFs that may interact with HNF-4 or ARP-1. 3) A biochemical assay and High performance liquid Chromatography will be employed to purify and characterize the factor that regulates the DNA-binding activity of ARP-1. These studies will enhance our understanding of the molecular mechanisms of apoAI transcriptional regulation by nuclear receptors and will reveal novel signal transduction pathways that modulate apoAI synthesis and contribute to cholesterol homeostasis. Furthermore, this information may provide a basis for the development of novel pharmacological strategies for monitoring common lipid disorders.
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