TY - JOUR
T1 - Characterization of the human UDP-galactose
T2 - Ceramide galactosyltransferase gene promoter
AU - Tencomnao, Tewin
AU - Yu, Robert K.
AU - Kapitonov, Dmitri
N1 - Funding Information:
This work was supported by USPHS grant NS11853 and NMSS grant RG 2690A6. TT was also supported by a Royal Thai Government’s Scholarship. The authors gratefully acknowledge the generous gift of the HOG cell line from Dr. Glyn Dawson. We thank Dr. Carmen Sato-Bigbee for her helpful discussion, and Dr. Stacey A. Kraemer for her critical comments on this manuscript.
Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 2001/2/16
Y1 - 2001/2/16
N2 - 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 γ-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.
AB - 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 γ-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.
KW - Gene expression
KW - Myelination
KW - Promoter
KW - Transcription
KW - Uridine diphosphate-galactose:ceramide galactosyltransferase
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U2 - 10.1016/S0167-4781(00)00283-9
DO - 10.1016/S0167-4781(00)00283-9
M3 - Article
C2 - 11342220
AN - SCOPUS:0035895812
SN - 0167-4781
VL - 1517
SP - 416
EP - 423
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 3
ER -