標題: Analysis of human UDP-glucose dehydrogenase gene promoter: Identification of an Sp1 binding site crucial for the expression of the large transcript
作者: Vatsyayan, J
Peng, HL
Chang, HY
生物科技學系
Department of Biological Science and Technology
關鍵字: EMSA;promoter analysis;Sp-1;transcription start site
公開日期: 1-六月-2005
摘要: 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.
URI: http://dx.doi.org/10.1093/jb/mvi082
http://hdl.handle.net/11536/13663
ISSN: 0021-924X
DOI: 10.1093/jb/mvi082
期刊: JOURNAL OF BIOCHEMISTRY
Volume: 137
Issue: 6
起始頁: 703
結束頁: 709
顯示於類別:期刊論文


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  1. 000230308900008.pdf