Characterization of the 5′-Flanking Region of Human α-N-acetylglucosaminyltransferase Gene

ZHANG Mu-Xia, ZHANG Yao, ZHAO Meng, GUO Xiao-Hua, GU Wei-Wei

Chinese Journal of Biochemistry and Molecular Biology ›› 2010, Vol. 26 ›› Issue (10) : 953-961.

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Chinese Journal of Biochemistry and Molecular Biology ›› 2010, Vol. 26 ›› Issue (10) : 953-961.
Research Papers

Characterization of the 5′-Flanking Region of Human α-N-acetylglucosaminyltransferase Gene

  • ZHANG Mu-Xia, ZHANG Yao, ZHAO Meng, GUO Xiao-Hua, GU Wei-Wei
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Abstract

To investigate the regulation of human α1,4-N-acetylglucosaminyltransferase (A4GNT) gene transcription, 5′RACE (rapid amplification of cDNA ends) and primer extension assays were deployed to obtain the A4GNT transcription start site. A series of deletions and site-directed mutantions were constructed into the luciferase reporters based on bioinformatics analyses, and then transiently transfected into gastric cancer cells MKN45 and AGS. The luciferase assays showed that the core promoter of A4GNT gene was from -141~+116 bp, which contained multiple CCAAT boxes and putative binding sites for transcription factor Sp1 and ETS-1, but lacked the consensus TATA box. The Sp1 binding site(-136~-131 bp)and the forward CCAAT(-93~-89 bp) appeared to be critical for the transcriptional activity of A4GNT as shown by mutation analyses. Electrophoretic mobility shift assays (EMSA) have demonstrated the specific binding of Sp1 and NF-Y to these elements. Our additional data also suggested that the region of -1 464~-771 bp might be responsible for the cell-specific gene expression of A4GNT.

Key words

&alpha / 1,4-N-acetylglucosaminyltransferase; promoter; luciferase reporter gene; electrophoretic mobility shift assay; transcription factor

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ZHANG Mu-Xia, ZHANG Yao, ZHAO Meng, GUO Xiao-Hua, GU Wei-Wei. Characterization of the 5′-Flanking Region of Human α-N-acetylglucosaminyltransferase Gene[J]. Chinese Journal of Biochemistry and Molecular Biology, 2010, 26(10): 953-961

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Funding

Supported by Natural Science Foundation of Hebei Province (No.C2006000917) and Science and Technology Foundation from Department of Health of Hebei Province(No.08-144)

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