Transcriptional Regulation Study Of The Human Farnesoid X Receptor Gene

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Bile acid is the physiological ligand(hence the name bile acid receptor). FXR is primarily expressed in liver, intestine, kidney and adrenal glands and also can be detected in heart and fat tissues. It regulates target gene expression by binding to FXR response elements (FXREs), after heterodimerization with the retinoid X receptor (RXR). FXR plays an essential role in feedback regulation of bile acid biosynthesis and maintaining cholesterol, glucose homeostasis.Although researchers have identified many FXR target genes and known about the protein function and structural characteristic, the mechanisms underlying the regulation of this gene in human tissues are still elusive. In fact, the FXR expression level is an important factor on lipid homeostasis. Most of genes which participated in the carbohydrate metabolism can be negatively or positively regulated by glucose or insulin. Studies have demonstrated that D-glucose positively regulated FXR gene expression in a dose and time dependent manner, probably by acting at the transcriptional level. To further elucidate FXR gene transcriptional regulation and to study the mechanism by which the high-dose of glucose enhances the expression of the FXR gene, we carried out this study, and the major results are summarized below:1. Bioinformatics analysis showed FXR gene promoter-like sequences located on the region of 1600bp upstream of the Exon ? . To analyze the transcription factor binding sites in the promoter region, the TRANSFAC Professional software in the Biobase biological database (http: //www. biobase. de /pages/products/databases.html#transfac) was used. The transcriptional start site was nucleotide A, 380bp nucleotides upstream of the ATG translation start site, 29 nt more 5′upstream from the 5′end of previous published longest FXR cDNAs (U68233 and NM005123) by 5′rapid amplification of cDNA ends (5′RACE)method. 2. To study the transcriptional regulation of FXR, the 1.8 kb 5′flanking region of FXR was amplified from genomic DNA of HepG2 cells and used as template to create different deletion constructs containing 5′or 3′deleted fragments fused upstream to a luciferase gene reporter vector. They were transient transfection into hepatic cell lines by Liposome. Functional assays showed that the–150/+29 nucleotides region from the first nucleotide of exon I is the minimal promoter of the human FXR gene. Point mutation analysis further demonstrated that–89/–84 is crucial for the promoter activity of the human FXR gene.3. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that hepatic nuclear factor 1α(HNF1α) interacted with the-89～-82 promoter region of the human FXR gene in vivo and in vitro.4. The results obtained from the co-transfection experiments, with the HNF1αexpression vector and the reporter plasmids carrying wild type HNF1-binding sites or the reporter constructs carrying mutated HNF1-binding sequences, indicated that HNF1αstimulated the human FXR promoter activity through the identified HNF1-binding site on FXR gene. Real time quantitative RT-PCR and Western blot further demonstrated that overexpression of HNF1αpositively regulates FXR expression in HepG2 cells.5. Having identified the promoter region and its regulation by HNF1a of the FXR gene, the possible mechanism(s) by which high dose of glucose stimulates the expression of FXR in HepG2 cells was further investigated in this study. Upon the treatment of high-dose glucose, the luciferase activity of the cells transfected with the FXR promoter constructs dramatically increased, compared to that of untreated transfeted cells. Our data further suggests that the intact HNF1a-binding site on the FXR promoter reporter construct is responsible for this glucose stimulatory effect. Furthermore, the results from RT-PCR and western blot showed that glucose up-regulated the expression of both HNF1αand FXR genes., suggesting that glucose stimulates the expresson of FXR may through its positive regulatory effect on HNF1a expression and subsequent promoter binding.In summary, after identified the transcriptional start site using 5′RACE approach, we have mapped the minimal promoter region of the human FXR gene by a combination of deletion mutation and transient transfetion analyses. There are two putative HNF1α-binding sites within this minimal promoter region. We provided several lines of evidences that HNF1αbinds to these HNF1-binding sites. The transcriptional activity of identified promoter of FXR was further found to be enhanced by overexpresison of HNF1αIn addition, we also investigated the mechanism of how glucose orchestrates the regulation of FXR gene activation and expression in transcriptional level. The study on the identification of FXR promoter and the transcriptional factors that binds to the promoter will provide useful information with regarding to the transcriptional regulation of FXR gene itself and the physiological and pathological roles FXR plays.