Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer.

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.

[1]  W. Rutter,et al.  Pan: a transcriptional regulator that binds chymotrypsin, insulin, and AP-4 enhancer motifs. , 1990, Genes & development.

[2]  K. Docherty,et al.  Positive and negative regulation of the human insulin gene by multiple trans-acting factors. , 1990, The Journal of biological chemistry.

[3]  Harold Weintraub,et al.  The protein Id: A negative regulator of helix-loop-helix DNA binding proteins , 1990, Cell.

[4]  R. Stein,et al.  Identification of a pancreatic beta-cell insulin gene transcription factor that binds to and appears to activate cell-type-specific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence , 1990, Molecular and cellular biology.

[5]  A. Aronheim,et al.  A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene. , 1990, Nucleic acids research.

[6]  Y. Jan,et al.  Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence , 1989, Cell.

[7]  R. Stein,et al.  Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements , 1989, Molecular and cellular biology.

[8]  M. Tsai,et al.  Mutagenesis of the rat insulin II 5'-flanking region defines sequences important for expression in HIT cells , 1989, Molecular and cellular biology.

[9]  David Baltimore,et al.  A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins , 1989, Cell.

[10]  S. McKnight,et al.  In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. , 1988, Genes & development.

[11]  S. McKnight,et al.  The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. , 1988, Science.

[12]  T. Edlund,et al.  A beta-cell-specific protein binds to the two major regulatory sequences of the insulin gene enhancer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W. Rutter,et al.  Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators , 1988, Molecular and cellular biology.

[14]  W. Rutter,et al.  A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Roeder,et al.  Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region , 1985, Cell.

[16]  D. Hanahan,et al.  Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. , 1985, Nature.

[17]  G. Church,et al.  B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo. , 1985, Science.

[18]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[19]  D. Crothers,et al.  Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. , 1981, Nucleic acids research.

[20]  M. M. Garner,et al.  A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system , 1981, Nucleic Acids Res..

[21]  G. Church,et al.  Cell-type-specific contacts to immunoglobulin enhancers in nuclei , 1985, Nature.

[22]  D. Steiner,et al.  Structure and evolution of the insulin gene. , 1985, Annual review of genetics.

[23]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.