Abstract
The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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Animals
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Base Sequence
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Binding Sites
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CHO Cells
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COS Cells
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Cell Line
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Chlorocebus aethiops
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Cricetinae
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DNA Footprinting
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DNA-Binding Proteins / metabolism*
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Enhancer Elements, Genetic*
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Gene Expression Regulation*
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Genes, Reporter
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Hepatocyte Nuclear Factor 1
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Hepatocyte Nuclear Factor 1-alpha
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Hepatocyte Nuclear Factor 1-beta
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Hepatocyte Nuclear Factor 3-beta
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Homeodomain Proteins / genetics
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Humans
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Islets of Langerhans / metabolism*
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Luciferases / genetics
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Mice
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Molecular Sequence Data
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Nuclear Proteins / metabolism*
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Podophyllin / analogs & derivatives*
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Podophyllin / metabolism*
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Podophyllotoxin / analogs & derivatives
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Recombinant Proteins / metabolism
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Sequence Deletion
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Trans-Activators / genetics*
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Transcription Factors / metabolism*
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Transfection
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Tumor Cells, Cultured
Substances
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DNA-Binding Proteins
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FOXA2 protein, human
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Foxa2 protein, mouse
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HNF1A protein, human
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HNF1B protein, human
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Hepatocyte Nuclear Factor 1-alpha
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Hnf1a protein, mouse
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Hnf1b protein, mouse
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Homeodomain Proteins
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Nuclear Proteins
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Recombinant Proteins
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Trans-Activators
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Transcription Factors
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pancreatic and duodenal homeobox 1 protein
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Hepatocyte Nuclear Factor 1
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Hepatocyte Nuclear Factor 3-beta
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Hepatocyte Nuclear Factor 1-beta
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mitopodozide
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Podophyllin
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Luciferases
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Podophyllotoxin