Structural Organization Of Human Nuclear Receptor Hb1f(nr5a2) Gene And Its Transcriptional Regulation

Human nuclear receptor hB1F (human B1-fragment binding Factor, nomenclatured as NR5A2) is a member of the FTZ-F1(NR5A) subfamily of nuclear receptor superfamily, which was firstly cloned as a novel transcription factor in our previous study of the enhancer II (ENII) of hepatitis B virus (HBV). It was revealed that hB1F(NR5A2) could regulate the transcriptions of a number of target genes as well as those of several critical transcription factor genes. Moreover, it played essential roles in the regulation of cholesterol homeostasis and various physiological pathways. This dissertation emphasizes on the characterization of hb1f(nr5a2) gene's structural organization and its transcriptional regulation mechanisms.The first chapter of this dissertation elaborated the process of obtaining the whole genomic sequence of hb1f(nr5a2) gene via shotgun approach. Characterization of the structural organization of hb1f(nr5a2) gene showed that this gene was nearly 150 kb in length, comprising of 8 exons and 7 introns. Three isoforms of hb1f(nr5a2) gene arise from alternative splicing, while 3'-RACE revealed that hb1f(nr5a2) gene could utilize two polyadenylation signals. Bioinformatic investigation of hb1f(nr5a2) genomic sequence implied that there might be other coding regions hidden in the two largest introns.We studied the transcriptional regulation of hb1f(nr5a2) gene in the secondchapter. We determined the transcription start site (TSS) of hb1f(nr5a2) gene via RT-PCR and primer extension and then identified the promoter of hb1f(nr5a2) gene, which exhibited high hepatocyte specific activity. Co-expression assays revealed that HNF1 (Hepatocyte Nuclear Factor 1) and HNF3β (Hepatocyte Nuclear Factor 3β) could activate the promoter of hb1f(nr5a2) gene. Further studies proved that HNF1 could bind to a canonical HNF1 site upstream the TATA box of hb1f(nr5a2) promoter and activate its transcription. Elaborate examination of the core promoter of hb1f(nr5a2) gene revealed a strong cis-element located in a 50bp-region downstream the TSS, which harbored a conserved E-box motif (CATGTG). Mutation of this E-box almost annulled the activity of hb1f (nr5a2) promoter, futhermore, EMSA assay indicated that a ubiquitous expressed factor could bind the E-box specifically. Although SREBPs (Sterol Response Element Binding Proteins) could bind this E-box in vitro, alteration of the sterol condition of the culture medium exerted little effect on hb1f(nr5a2) promoter activity, suggesting that SREBPs might not regulate the transcription of hb1f(nr5a2) gene directly. Supershift assay validated that the protein factor binding to the E-box was USFs (Upstream Stimulatory Factors), implying that USFs would play an important role in the regulation of hb1f(nr5a2) gene transcription.Characterization of the genomic sequence, structure organization and promoter of hb1f(nr5a2) gene in this dissertation could promote the study of its transcriptional regulation, and as well facilitate portraying the physiological roles of human nuclear receptor hB1F(NR5A2) in the process of various complicated physiological pathways.