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 neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic beta cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated 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 terminus 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 (E12DeltaC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12DeltaC 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 diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.
Thomas
M K
MK
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts 02114, USA.
Yao
K M
KM
Tenser
M S
MS
Wong
G G
GG
Habener
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007328:Insulin / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D051820:TCF Transcription Factors; D014018:Tissue Distribution; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007328:Insulin; D009419:Nerve Tissue Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C402482:Psmd9 protein, rat; C544912:TCF7L1 protein, human; C553060:Tcf7l1 protein, rat; C093564:Neurogenic differentiation factor 1