A pancreatic beta -cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3beta (HNF-3beta ), HNF-1alpha, and SPs transcription factors.
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.
Department of Endocrinology and Metabolism, Hebrew University Hadassah Medical Center, 91120 Jerusalem, Israel.
E
E
Ben-Shushan
S
S
Marshak
M
M
Shoshkes
E
E
Cerasi
D
D
Melloul
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051542:Hepatocyte Nuclear Factor 3-beta; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011033:Podophyllin / Q000031:analogs & derivatives* / Q000378:metabolism*; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496503:Foxa2 protein, mouse; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C084010:pancreatic and duodenal homeobox 1 protein; D051537:Hepatocyte Nuclear Factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051539:Hepatocyte Nuclear Factor 1-beta; C003170:mitopodozide; D011033:Podophyllin; D008156:Luciferases; D011034:Podophyllotoxin