Characterization of an enhancer element in the proximal promoter of the mouse glucagon receptor gene

Abstract

The 5′-flanking region of the mouse glucagon receptor has been previously cloned and two promoter regions were characterized. Functional analysis of the proximal promoter was now performed to characterize cis-acting element(s) regulating basal gene expression. Promoter analysis using deletion constructs in a rat cell line (CA-77) expressing the glucagon receptor, showed that the region from −64 to +127 relative to the proximal transcription start site was sufficient for maximal proximal promoter activity. A DNA sequence spanning the −28 to −16 region organized as an imperfect palindrome was demonstrated to be functional as a cis-acting enhancer. Constructs including several copies of this motif strongly increased activity of the heterologous thymidine kinase promoter. Gel mobility shift assays performed with different DNA fragments spanning this region confirmed that it specifically bound nuclear protein(s) from CA-77 cells, mouse MIN-6 cells or mouse liver. Mutations in the core sequence of this site impaired both reporter gene activity and nuclear protein binding. The palindrome is a novel DNA sequence with no homology to existing transcription factor binding site database. This is the first characterization of a functional cis-acting sequence into the proximal promoter of the mouse glucagon receptor that may support constitutive expression of the gene.