Analysis of human UDP-glucose dehydrogenase gene promoter: identification of an Sp1 binding site crucial for the expression of the large transcript.
UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is required in liver for the excretion of toxic compounds, and for the biosynthesis of complex carbohydrates, such as hyaluronan, in many cell types. Analysis of a human EST database, as well as the results of a 5'-RACE experiment, have revealed the presence of two transcription start sites approximately 160 bp apart in the human UGDH gene confirming previous Northern hybridization results. 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 indicate 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, abrogates 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 identify an alternative transcription start site on the UGDH promoter, and locate the cis-element that greatly enhances the basal transcriptional activity of UGDH gene.
Vatsyayan
Jaya
J
Institute of Molecular Medicine, National Tsing Hua University, Hsin Chu 300, Taiwan..
Peng
Hwei-Ling
HL
Chang
Hwan-You
HY
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008156:Luciferases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000502:physiology*; D024363:Transcription Initiation Site; D014162:Transfection; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics*
D016329:Sp1 Transcription Factor; D014533:Uridine Diphosphate Glucose Dehydrogenase; D008156:Luciferases