Regulation of human UDP‐galactose: ceramide β‐galactosyltransferase (CGT) gene expression

Abstract

Myelination is a highly coordinated event during nervous system development, which involves the synthesis of many myelin specific components. The most abundant glycolipid in myelin is galactocerebroside (GC), whose synthesis is catalysed by CGT. To elucidate the transcriptional control of the hCGT gene, we sequenced 2.3 kb of the 5′‐flanking region of this gene. Transient transfection of the human oligodendroglioma (HOG; GC+) and neuroblastoma (LAN‐5; GC–) cell lines using a series of luciferase reporter plasmids containing deletion fragments of the hCGT 5′‐flanking region showed that this promoter functions in a cell‐specific manner. Three positive regulatory regions were identified: a region at −292/−256 containing an Sp1 site, a region at −747/−688 containing sites for TGGCA‐BP, CRE, and ER half‐site, a distal region at −1325/−1279 containing a nitrogen regulatory site. A negative regulatory domain (−1594/−1326) was also identified. Using mutation analyses and cotransfection experiments in HOG cells, we confirmed that the Sp1 motif (−267/−259) and the CRE motif (−697/−690) were critical for hCGT expression. Gel shift assays showed that these motifs are bound by nuclear extracts from both cell lines and mouse brains. Using antibodies to Sp1, Sp3, and CREB, these proteins were shown to be components of the complexes. However, the CRE complexes were the only notable differences between HOG and LAN‐5 cells, which may be responsible for the differential transcription of this gene. Thus, the hCGT promoter contains both positive and negative regulatory regions for the cell‐specific expression, and these regions function cooperatively.Acknowledgements: Supported by NIH NS11853‐27.