| Summary: | 博士 === 國立清華大學 === 生命科學系 === 94 === The prominence of UDP-Glc Dehydrogenase (UGDH, EC1.1.1.22) is in its unique and pivotal role in catalyzing the oxidation of UDP-Glc to UDP-GlcA accompanied with the reduction of two molecules of NAD. UDP-GlcA plays many important physiological roles in liver by conjugating with a variety of xeno- and endobiotic compounds to aid in their solubilization and excretion through the action of phase II drug detoxifying enzymes. UDP-GlcA serves as a critical component of the glycosaminoglycans, proteoglycans, hyaluronan, chondroitan sulfate and heparan sulfate.
Analysis of a human EST database, as well as the results of a 5’-RACE experiment revealed the presence of two transcription start sites approximately 160 bp apart in the human UGDH gene confirming previous Northern hybridization results. An extensive bioinformatics analysis of the gene sequence of the UGDH 5’-flanking region revealed many interesting putative transcription factor-binding sites. The most unique fact was the prediction of fourteen putative Sp1 sites upto –632 bp region of the promoter, besides the presence of an atypical TATA box. To delineate the regions in the UGDH promoter required for regulating the expression of the gene, in particular the synthesis of the large transcript, serial deletions of the 2.1-kb UGDH promoter region were constructed and their activities determined by the firefly luciferase reporter gene assay. Our results indicated that the region from nucleotide position –486 to –632 relative to the start of the small transcript contains positive regulatory elements that contribute to gene expression. Mithramycin A, an inhibitor of transcription factor Sp1, abrogated the promoter activity, suggesting the involvement of this specific protein in UGDH expression. By using site-directed mutagenesis, we analyzed the functional contribution of three putative Sp1 binding elements within this region. A mutation at position –564 demonstrated that this site serves as an enhancing element in both HepG2 and HeLa cells. The complex formation pattern revealed by an electrophoretic mobility shift assay as well as an anti-Sp1 antibody-mediated supershift assay confirmed the identity of this GC box as an Sp1 binding motif. Our results thus identified an alternative transcription start site on the UGDH promoter, and located the cis-element that greatly enhances the basal transcriptional activity of UGDH gene.
Besides defining the 714 bp 5’-flanking region of the UGDH gene as the core promoter, in another study, we delineated the region from nucleotide positions –1057 to –957 on the UGDH promoter to be responsible for the repression of the promoter activity. A mutation at nucleotide –1003, which is contained within a motif predicted as the response element for peroxisome proliferator receptor a�n (PPARa), abolished the suppression effect. The proteins interacting with the PPRE-like repressor motif were purified by biotin labeled DNA affinity purification with streptavidin-coated beads. Subsequently MALDI-TOF identified the purified proteins as a 62-kDa zinc finger and a 42-kDa b-actin protein.
In addition, it is not known whether xenobiotics can modulate the UGDH gene expression. An attempt to answer this question was made by treating the human hepatoma cells HepG2 with several medicinal compounds and the UGDH gene expression was analyzed by using Real time-PCR. Through promoter-reporter gene assays, we found that rifampicin showed multiple folds activation of a 1.23-kb UGDH promoter construct, and the region likely to respond to rifampicin treatment is located within the region –632 to –1050. A bioinformatics search for xenobiotic response element in this region predicted a binding motif for the PPAR at position –1003. A mutation at the predicted PPAR recognizing motif eliminated normal suppression as well as the rifampicin activation effect on the UGDH promoter activity. Cotransfection with the PPARa and RXRa expression vectors and subsequent treatment with the PPARa agonist led to the suppression of the UGDH promoter activity either in the presence or absence of rifampicin. Our study for the first time showed the UGDH gene to be under xenobiotic regulation and delineated a motif responsible for rifampicin response and transcriptional repression of the UGDH gene.
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