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

The 5'-flanking region of the mouse glucagon receptor has been previously c loned and two promoter regions were characterized. Functional analysis of t he proximal promoter was now performed to characterize cis-acting element(s ) regulating basal gene expression. Promoter analysis using deletion constr ucts in a rat cell line (CA-77) expressing the glucagon receptor, showed th at 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 de monstrated to be functional as a cis-acting enhancer. Constructs including several copies of this motif strongly increased activity of the heterologou s thymidine kinase promoter. Gel mobility shift assays performed with diffe rent DNA fragments spanning this region confirmed that it specifically boun d nuclear protein(s) from CA-77 cells, mouse MIN-6 cells or mouse liver. Mu tations in the core sequence of this site impaired both reporter gene activ ity and nuclear protein binding. The palindrome is a novel DNA sequence wit h 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 constitut ive expression of the gene. (C) 2001 Elsevier Science B.V. All rights reser ved.