Identification of a novel PDX-1 binding site in the human insulin gene enhancer

Abstract

Islet β 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 β 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 β 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 nuelcotides -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 β cell lines. The human GG2 activator binding factor(s) was shown to be ∼38-40 kDa and distinct from other size-matched islet-enriched transcription factors, including Nkx2.2, Pax-4, Cdx2/3, and IsI-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.