Bridge-1, a novel PDZ-domain coactivator of E2A-mediated regulation of insulin gene transcription

Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neu rogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancrea tic beta cell, E2A proteins, in combination with tissue-specific transcript ion factors, regulate expression of the insulin gene and other genes critic al for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-I) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator o f gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Brid ge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E 2A proteins is further demonstrated by coimmunoprecipitation of in vitro-tr anslated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy te rminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12 . An E12 mutant (E12 Delta C) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivat ional activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein tra nsactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienha ncer promoter-reporter construct in transient-transfection experiments. Sub stitution of the mutant E12 Delta C for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge- 1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA dim inishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-lik e domain to interact with E12, may pro vide a new mechanism for the coactiv ation and regulation of transcription of the insulin gene.