Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.
UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.
Tencomnao
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Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Commonwealth University, Richmond 23298-0614, USA.
Yu
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RK
Kapitonov
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D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics*; D051147:Ganglioside Galactosyltransferase; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D012313:RNA; D008156:Luciferases; D005700:Galactosyltransferases; C511211:UGT8 protein, human; D051147:Ganglioside Galactosyltransferase