Characterization of the promoter and the transcription factors for the mouse UDP-Gal:betaGlcNAc beta1,3-galactosyltransferase gene.
Galbeta1-3Gal-NAcbeta1-4Gal(3-2alphaNeuAc)beta1-4Glcbeta1-1Cer (GM1) is one of the most extensively investigated gangliosides that plays critical roles in the development and functions of the nervous system. UDP-Gal:betaGlcNAc beta1,3-galactosyltransferase (Gal-T-II) is responsible for synthesis of ganglioside GM1 in the ganglioside biosynthetic pathway. To understand the transcriptional regulation of Gal-T-II gene expression, we cloned a 1448 bp 5'-flanking fragment from the mouse Gal-T-II gene. The transcriptional activity of the fragment was demonstrated in mouse Neuro-2a cells by a luciferase assay. The proximal 550 bp fragment showed the highest transcriptional activity as determined by a series of 5'-truncated constructs of the promoter. One negative regulatory region was also identified. Primer extension assay revealed a transcription initiation site approximately 242 bp upstream from the ATG translation start codon. Analysis of the promoter sequence revealed a number of potential binding sites for known transcription factors. To determine which transcription factors bind to the promoter, we carried out a systematic search for the binding proteins using the 1142 bp Gal-T-II promoter fragment containing both positive and negative regulatory regions in a combination of DNA pull-down assay and transcription factor array analysis. Twenty-seven transcription factors bound to consensus sites in the promoter region. In addition, four other factors without consensus binding sites in this region were also recruited, possibly as components of transcription factor complexes. These data indicate that the basal regulation of Gal-T-II gene transcription involves multiple transcription factors, some of which may be present in complexes.
Xia
Tian
T
Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA.
Gao
Luoyi
L
Yu
Robert K
RK
Zeng
Guichao
G
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D005700:Galactosyltransferases; C470633:UDP-galactose N-acetylgalactosamine peptide beta1,3-galactosyltransferase