An insulin-responsive element in the pancreatic enhancer of the amylase gene.
We have identified a distinct insulin-responsive element (IRE) located within the pancreatic enhancer of the mouse amylase gene Amy-2.2. A 30-base pair (bp) fragment was previously shown to be sufficient to transfer insulin response to a heterologous promoter. The 30-bp fragment overlaps the tissue-specific pancreatic enhancer that binds the putative transcriptional activator PTF1. To determine whether enhancer and IRE activities could be separated, we introduced three 10-base pair substitutions into the 30-bp region. The mutated regulatory regions were cloned upstream of a heterologous promoter and transferred to transgenic mice. Mutants 1 and 2 retained PTF1 binding activity and insulin response. Mutant 3 retained PTF1 binding, but was defective in insulin response. The results indicate that the IRE is functionally distinct from the PTF1-binding site, although the two overlap physically. In contrast to the wild-type gene, mutant 3 is characterized by constitutive expression in diabetic animals, suggesting that a binding site for a repressor has been destroyed. A nuclear protein with affinity for the IRE was detected in normal and diabetic pancreas. This protein does not bind mutant 3, suggesting that it may be involved in negative regulation of amylase in diabetic pancreas.
Johnson
T M
TM
Department of Human Genetics, University of Michigan, Ann Arbor 48109-0618.
Rosenberg
M P
MP
Meisler
M H
MH
D000681:Amylases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D007328:Insulin; D007527:Isoenzymes; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; C062602:transcription factor PTF1; D000681:Amylases