Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression: Functional role of GC-box and CRE

Tewin Tencomnao, Dmitri Kapitonov, Erhard Bieberich, Robert K. Yu

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)339-351
Number of pages13
JournalGlycoconjugate Journal
Volume20
Issue number5
DOIs
StatePublished - Dec 1 2003

Fingerprint

Ganglioside Galactosyltransferase
Gene expression
Electrophoretic mobility
Gene Expression
Genes
Local area networks
Assays
Local Area Networks
Oligodendroglioma
Electrophoretic Mobility Shift Assay
Cells
Myelin Sheath
Glycolipids
Transcription
Cell Line
Biosynthetic Pathways
Antibodies
Neuroblastoma
Enzymes

Keywords

  • Galactocerebroside
  • Transcription factors
  • Translational regulation
  • UDP-galactose: Ceramide galactosyltransferase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression : Functional role of GC-box and CRE. / Tencomnao, Tewin; Kapitonov, Dmitri; Bieberich, Erhard; Yu, Robert K.

In: Glycoconjugate Journal, Vol. 20, No. 5, 01.12.2003, p. 339-351.

Research output: Contribution to journalArticle

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abstract = "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.",
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