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Role of Chromatin Accessibility in the Occupancy and Transcription of the Insulin Gene by the Pancreatic and Duodenal Homeobox Factor 1

Department of Pharmacology (J.F., D.A.B., T.G.D., D.G.T., R.G.M.) and Department of Medicine and the Diabetes Center (S.K.C., J.C.G., C.E.-M., R.G.M.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Raghavendra G. Mirmira, University of Virginia Health System, 450 Ray C. Hunt Drive, Box 801407, Charlottesville, Virginia 22908. E-mail: mirmira{at}virginia.edu.

The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20–40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in ß-cells (ßTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in ßTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp –126 to –296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the ß-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp –126 to –296, thereby permitting occupancy by transactivators such as Pdx-1.

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