Characterization of the human UDP-galactose : ceramide galactosyltransferase gene promoter

UDP-galactose :ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key e nzyme in the biosynthesis of galactocerebroside, the most abundant glycosph ingolipid in the myelin sheath. An 8 kb fragment upstream from the transcri ption initiation site of CGT gene was isolated from a human genomic DNA lib rary. Primer extension analysis revealed a single transcription initiation site 379 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start sit e: however, this region contains a high GC content and multiple putative re gulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated a nd 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-spe cific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential bind ing 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 a s 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, N F1-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 t he 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. (C) 2001 Elsevier Science B.V. All rights reserved.