Transcription factors NF‐κB and Sp1 are major determinants of the basal promoter activity of the rat GD3‐synthase gene

Abstract

GD3‐synthase is one of the key sialyltransferases responsible for synthesis of ganglioside GD3, the substrate for initiation of the ‘b’ and ‘c’ series ganglioside synthesis. We have previously cloned the rat GD3‐synthase gene promoter, and preliminary characterization has identified a minimal 0.5‐kb region that has a strong basal promoter activity, and is GC‐rich and has no CAAT or TATA boxes. In this study, we showed that the Sp1 and NF‐κB sites in this region significantly contributed to basal GD3‐synthase promoter activity. When either the Sp1 or NF‐κB sites were deleted, a 50% decrease in promoter activity was observed. The same results were obtained by a decoy strategy using oligonucleotides containing the Sp1 or NF‐κB sites. The binding to the Sp1 and NF‐κB sites was confirmed by electrophoretic mobility shift assay (EMSA), competition and supershift EMSA. In addition, cell‐type specific activation of the promoter was also determined. The promoter was highly activated in the GD3‐expressing F‐11 cells while repressed in NG‐108 cells in which GD3 is almost undetectable. An additional band of NF‐κB family was identified only in the F‐11 nuclear extract using the NF‐κB consensus probe by EMSA. DNA pull‐down assays were further carried out to screen proteins that bound to the promoter including the basal region and the potential negative‐regulatory region between −526 and −769. More than 10 major binding proteins were pulled down, some of which were present only in the F‐11 or NG‐108 nuclear extracts. Our data demonstrate that NF‐κB and Sp1 are the major determinants for the basal promoter activity and some factors such as NF‐κB may be involved in cell type‐specific expression of the gene.