Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression: functional role of GC-box and CRE.
UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthetic pathway of galactocerebroside (GalC), the most abundant glycolipid in myelin. Using a GalC expressing cell line, human oligodendroglioma (HOG), one which does not express GalC, human neuroblastoma (LAN-5), we previously demonstrated that the human CGT (hCGT) gene promoter functions in a cell-specific manner. Because the proximal (-292/-256) and distal (-747/-688) positive domains were shown to be critically involved in regulating the expression of several myelin-specific genes, we further investigated the functional roles of these two motifs in hCGT expression. Mutation analysis confirmed that a GC-box (-267/-259) and a CRE (-697/-690) were critical for hCGT expression. Electrophoretic mobility shift assay (EMSA) demonstrated that these motifs specifically bound to nuclear extracts from both cell lines. Using antibodies to Sp1, Sp3, pCREB-1, and ATF-1, these proteins were shown to be components of the EMSA complexes. However, the only difference between the HOG and LAN-5 cells was found in the EMSA profile of the CRE complexes. This difference may account for the differential transcription of the hCGT gene in the two cell types. Furthermore, the expression levels of ATF-1 detected were much higher in HOG cells than in LAN-5 cells. Thus, our data suggest that the GC-box and CRE function cooperatively, and that the CRE regulates the cell-specific expression of the hCGT gene.
Tencomnao
Tewin
T
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2697, USA.
Kapitonov
Dmitri
D
Bieberich
Erhard
E
Yu
Robert K
RK
D051697:Activating Transcription Factor 1; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism*; D051147:Ganglioside Galactosyltransferase; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
D051697:Activating Transcription Factor 1; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D005700:Galactosyltransferases; C511211:UGT8 protein, human; D051147:Ganglioside Galactosyltransferase