Abstract
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.
Highlights
Gene ablation studies have revealed that each of the isletenriched transcriptional activators of the insulin gene mediate critical steps involved in both endocrine and exocrine pancreas formation [11, 12]
The bp Ϫ144 mutants resulted in 3– 4-fold activation over wild type (Fig. 1B). These results suggested that human insulin nucleotides Ϫ145 to Ϫ141 are part of the functional core of the GG2 element
Islet  cell-selective and glucose-mediated expression of the insulin gene is mediated by sequences within 350 bp of the transcription start site [1, 2]
Summary
Gene ablation studies have revealed that each of the isletenriched transcriptional activators of the insulin gene mediate critical steps involved in both endocrine and exocrine pancreas formation [11, 12]. The human GG2 vates rodent insulin C2 (Ϫ329/Ϫ307 bp)-driven expression [17], activator binding factor(s) was shown to be ϳ38 – 40 kDa is essential for the generation of islet glucagon hormone-proand distinct from other size-matched islet-enriched ducing ␣ cells [17, 18]. 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. Tissue-specific expression of the insulin gene has been shown in transgenic animals in vivo to be metostatin (PDX-1 [33] and PAX-6 [17])) These observations link essential insulin gene transcription factors with broader functions associated with  cell formation and physiology.
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