Functional analysis of promoter activity of murine beta-1,6-N-acetylglucosaminyltransferase

In the mouse, beta-1,6-N-acetylglucosaminyltransferase (IGnT) forms branches in poly-N-acetyllactosamines, which are good scaffolds for oncodevelopmental cell-surface antigens and recognition markers. There are two isoforms of IGnT, IGnT A and B, which are produced by alternative splicing of the IGnT gene; the unique portion is encoded by exon 1 and common portion is encoded by exons 2 and 3. Thus, the expression of each isoform is controlled by a different promoter. Here, we identified the regulatory regions of the mouse IGnT A and B genes. The promoter regions for IGnT A and B did not contain putative TATA or CAAT boxes, but each contained GT boxes. The upstream regulatory region of each gene was examined by transient luciferase reporter gene transfection experiments and gel mobility shift assay. Promoter activity for each gene was detected in F9 embryonal carcinoma cells, which express IGnT A and B, but not in N2a cells, which do not express the gene. Deletion analysis demonstrated that the regions 308 bp upstream from the transcriptional initiation site of IGnT A and 430 bp upstream from the transcriptional initiation site of IGnT B showed minimal promoter activity. Mutation of the single GT box in IGnT A and two GT boxes in IGnT B caused marked reduction of the promoter activity. These findings provided strong evidence that the GT boxes play crucial roles in transcriptional regulation of the genes.