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J. Biol. Chem., Vol. 283, Issue 13, 8164-8172, March 28, 2008

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Pdx1 and BETA2/NeuroD1 Participate in a Transcriptional Complex That Mediates Short-range DNA Looping at the Insulin Gene^*

Daniella A. Babu, Swarup K. Chakrabarti, James C. Garmey¹, and Raghavendra G. Mirmira²

From the Department of Pharmacology and the Department of Medicine and the Diabetes Center, University of Virginia, Charlottesville, Virginia 22908

The activity of the insulin gene, Ins, in islet β cells is thought to arise in part from the synergistic action of the transcription factors Pdx1 and BETA2/NeuroD1. We asked how the binding of these factors to A and E elements many tens or hundreds of base pairs upstream of the start site could influence activity of transcriptional machinery. We therefore tested the hypothesis that the complex of Pdx1 and BETA2/NeuroD1 maintains a DNA conformation such that distal regions of the gene are brought into proximity of the promoter and coding region. We show by coimmunoprecipitation that Pdx1 and BETA2/NeuroD1 exist within a complex and that the two physically interact with one another in this complex as assessed by fluorescence resonance energy transfer. Consistent with this interaction, we found that the two factors simultaneously occupy the same fragment of the Ins gene in β cell lines using the chromatin immunoprecipitation/re-chromatin immunoprecipitation assay. Using a modification of the chromosome conformation capture assay in vitro and in β cells, we observed that Pdx1 and BETA2/NeuroD1 mediate looping of a segment of Ins that brings EcoRI sites located at –623 and +761 bp (relative to the transcriptional start site) in proximity to one another. This looping appears to be dependent in vitro upon an intact A3 binding element, but not upon the E2 element. Based on our findings, we propose a model whereby Pdx1 and BETA2/NeuroD1 physically interact to form a nucleoprotein complex on the Ins gene that mediates formation of a short DNA loop. Our results suggest that such short loop conformations may be a general mechanism to permit interactions between transcription factors and basal transcriptional machinery.

Received for publication, January 14, 2008 , and in revised form, February 1, 2008.

^* This work was supported by National Institutes of Health Grants RO1 DK60581 (to R. G. M.) and T32 GM007055 (to D. A. B.) and by a Thomas R. Lee Career Development Award from the American Diabetes Association (to R. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202.

² To whom correspondence should be addressed: Indiana University School of Medicine, 635 Barnhill Dr., Rm. 2031B, Indianapolis, IN 46202. Tel.: 317-274-3889; Fax: 317-274-3882; E-mail: rmirmira{at}iupui.edu.

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