Identification of a novel PDX-1 binding site in the human insulin gene enhancer.
Islet beta cell type-specific transcription of the insulin gene is regulated by a number of cis-acting elements found within the proximal 5'-flanking region. The control sequences conserved between mammalian insulin genes are acted upon by transcription factors, like PDX-1 and BETA-2, that are also involved in islet beta cell function and formation. In the current study, we investigated the contribution to human insulin expression of the GG2 motif found between nucleotides -145 and -140 relative to the transcription start site. Site-specific mutants were generated within GG2 that displayed a parallel increase (i.e. -144 base pair) or decrease (i.e. -141 base pair) in insulin enhancer-driven reporter and gel shift binding activity in beta cells consistent with human GG2 being under positive regulatory control. In contrast, the corresponding site in the rodent insulin gene, which only differs from the human at nucleotides -144 and -141, is negatively regulated by the Nkx2.2 transcription factor (Cissell, M. A., Zhao, L., Sussel, L., Henderson, E., and Stein, R. (2003) J. Biol. Chem. 278, 751-756). Human GG2 activator binding activity was present in nuclear extracts prepared from human islets and enriched in those from rodent beta cell lines. The human GG2 activator binding factor(s) was shown to be approximately 38-40 kDa and distinct from other size-matched islet-enriched transcription factors, including Nkx2.2, Pax-4, Cdx2/3, and Isl-1. Combined DNA chromatographic purification and mass spectrometry analysis revealed that the GG2 activator was PDX-1. These results demonstrate that the GG2 element, despite its divergence from the core homeodomain consensus binding motif, is a site for PDX-1 activation in the human insulin gene.
Le Lay
John
J
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37215, USA.
Matsuoka
Taka-Aki
TA
Henderson
Eva
E
Stein
Roland
R
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002845:Chromatography; D017124:Conserved Sequence; D006224:Cricetinae; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D015534:Trans-Activators / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry; D014162:Transfection; D029961:Zebrafish Proteins
D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; D009687:Nuclear Proteins; D010455:Peptides; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; C000715630:nkx2.2b protein, zebrafish; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA