Interactions between areas I and II direct pdx-1 expression specifically to islet cell types of the mature and developing pancreas.
PDX-1 regulates transcription of genes involved in islet beta cell function and pancreas development. Islet-specific expression is controlled by 5'-flanking sequences from base pair (bp) -2917 to -1918 in transgenic experiments, which encompasses both conserved (i.e. Area I (bp -2761/-2457), Area II (bp -2153/-1923)) and non-conserved pdx-1 sequences. However, only an Area II-driven transgene is independently active in vivo, albeit in only a fraction of islet PDX-1-producing cells. Our objective was to identify the sequences within the -2917/-1918-bp region that act in conjunction with Area II to allow comprehensive expression in islet PDX-1(+) cells. In cell line-based transfection assays, only Area I effectively potentiated Area II activity. Both Area I and Area II functioned in an orientation-independent manner, whereas synergistic, enhancer-like activation was uniquely found with duplicated Area II. Chimeras of Area II and the generally active SV40 enhancer or the beta cell-specific insulin enhancer suggested that islet cell-enriched activators were necessary for Area I activation, because Area II-mediated stimulation was reduced by the SV40 enhancer and activated by the insulin enhancer. Several conserved sites within Area I were important in Area I/Area II activation, with binding at bp -2614/-2609 specifically controlled by Nkx2.2, an insulin gene regulator that is required for terminal beta cell differentiation. The ability of Area I to modulate Area II activation was also observed in vivo, as an Area I/Area II-driven transgene recapitulated the endogenous pdx-1 expression pattern in developing and adult islet cells. These results suggest that Area II is a central pdx-1 control region, whose islet cell activity is uniquely modified by Area I regulatory factors.
Van Velkinburgh
Jennifer C
JC
Department of Molecular Physiology and Biophysics, Vanderbilt Medical Center, Nashville, Tennessee 37232, USA.
Samaras
Susan E
SE
Gerrish
Kevin
K
Artner
Isabella
I
Stein
Roland
R
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic; D005697:Galactosides / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000378:metabolism; D007328:Insulin / Q000235:genetics; D007340:Insulinoma / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D007763:Lac Operon; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins; D010179:Pancreas / Q000196:embryology* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D029961:Zebrafish Proteins
D002843:Chromatin; D005697:Galactosides; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007211:Indoles; D007328:Insulin; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; C000715630:nkx2.2b protein, zebrafish; C084010:pancreatic and duodenal homeobox 1 protein; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside