TY - JOUR
T1 - Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression
T2 - Functional role of GC-box and CRE
AU - Tencomnao, Tewin
AU - Kapitonov, Dmitri
AU - Bieberich, Erhard
AU - Yu, Robert K.
N1 - Funding Information:
To whom correspondence should be addressed: Dr. Robert K. Yu, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, CA 1012, Augusta, GA 30912-2697, USA. Tel: 706-721-0699; Fax: 706-721-8727; E-mail: ryu@mail.mcg.edu ∗This work was supported by an NIH grant, NS11853-30, to RKY.
PY - 2003
Y1 - 2003
N2 - 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.
AB - 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.
KW - Galactocerebroside
KW - Transcription factors
KW - Translational regulation
KW - UDP-galactose: Ceramide galactosyltransferase
UR - http://www.scopus.com/inward/record.url?scp=3543051690&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3543051690&partnerID=8YFLogxK
U2 - 10.1023/B:GLYC.0000033630.58533.16
DO - 10.1023/B:GLYC.0000033630.58533.16
M3 - Article
C2 - 15229398
AN - SCOPUS:3543051690
SN - 0282-0080
VL - 20
SP - 339
EP - 351
JO - Glycoconjugate Journal
JF - Glycoconjugate Journal
IS - 5
ER -