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.
Ben-Shushan
E
E
Department of Endocrinology and Metabolism, Hebrew University Hadassah Medical Center, 91120 Jerusalem, Israel.
Marshak
S
S
Shoshkes
M
M
Cerasi
E
E
Melloul
D
D
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