Our long-term goal is to identify the molecular interactions involved in pancreas-specific gene expression and transcriptional regulation by insulin. Expression of pancreatic amylase is repressed in diabetic animals, reducing absorption of dietary carbohydrate, and re-expressed after treatment with insulin. We have identified a 63 bp DNA segment from the 5' flanking region of a murine pancreatic amylase gene which contains an insulin-responsive element and a pancreas-specific enhancer. Several proteins from pancreatic nuclei bind specifically to this fragment. We will localize the nucleotides essential for insulin response and enhancer activity by functional assays of synthetic mutant enhancer elements with 3 bp substitutions. Band shift assays with each mutant will localize the binding sites for each of the nuclear proteins. By correlating loss of binding with loss of function, we will identify putative transcriptional regulatory proteins. These proteins will be cloned by screening cDNA libraries with oligonucleotide probes. The structure and developmental expression of positive clones will be studied. We will select mutants of the AR42J pancreatic acinar cell line which are defective individual transcription factors. These lines can by used to test the function of cloned proteins. We will evaluate the activity of a glucocorticoid receptor binding site located at position-17 to +1 of the amylase gene. Interaction between two conserved pancreas consensus elements by insulin will be tested in adipocytes and hepatocytes. The experiments proposed here will contribute to understanding the mechanism of insulin action, and will extend our knowledge of pancreas-specific regulation of mammalian genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036089-09
Application #
2139722
Study Section
Endocrinology Study Section (END)
Project Start
1985-08-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1995-07-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Genetics
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Wu, K J; Wilson, D R; Shih, C et al. (1994) The transcription factor HNF1 acts with C/EBP alpha to synergistically activate the human albumin promoter through a novel domain. J Biol Chem 269:1177-82
Ting, C N; Burgess, D L; Chamberlain, J S et al. (1993) Phosphoenolpyruvate carboxykinase (GTP): characterization of the human PCK1 gene and localization distal to MODY on chromosome 20. Genomics 16:698-706
Meisler, M H; Ting, C N (1993) The remarkable evolutionary history of the human amylase genes. Crit Rev Oral Biol Med 4:503-9
Johnson, T M; Rosenberg, M P; Meisler, M H (1993) An insulin-responsive element in the pancreatic enhancer of the amylase gene. J Biol Chem 268:464-8
Yu, H; Thun, R; Chandrasekharappa, S et al. (1993) Human PCK1 encoding phosphoenolpyruvate carboxykinase is located on chromosome 20q13.2. Genomics 15:219-21
Ting, C N; Rosenberg, M P; Snow, C M et al. (1992) Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene. Genes Dev 6:1457-65
Wu, K J; Samuelson, L C; Howard, G et al. (1991) Transactivation of pancreas-specific gene sequences in somatic cell hybrids. Mol Cell Biol 11:4423-30
Keller, S A; Rosenberg, M P; Johnson, T M et al. (1990) Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer. Genes Dev 4:1316-21
Johnson, T M; Meisler, M H; Bennett, M I et al. (1990) Vanadate induction of pancreatic amylase mRNA in diabetic rats. Diabetes 39:757-9
Meisler, M H; Howard, G (1989) Effects of insulin on gene transcription. Annu Rev Physiol 51:701-14

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