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J. Biol. Chem., Vol. 280, Issue 17, 16798-16807, April 29, 2005

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Mechanism of insulin Gene Regulation by the Pancreatic Transcription Factor Pdx-1

APPLICATION OF PRE-mRNA ANALYSIS AND CHROMATIN IMMUNOPRECIPITATION TO ASSESS FORMATION OF FUNCTIONAL TRANSCRIPTIONAL COMPLEXES^*

Tessy Iype, Joshua Francis¶, James C. Garmey, Jonathan C. Schisler||, Rafael Nesher**, Gordon C. Weir**, Thomas C. Becker||, Christopher B. Newgard||, Steven C. Griffen, and Raghavendra G. Mirmira¶

From the Department of Internal Medicine and the Diabetes Center and ^¶Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, the ^||Sarah W. Stedman Nutrition and Metabolism Center, and Departments of Pharmacology and Cancer Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27704, the ^**Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, and the Department of Internal Medicine, University of California at Davis, Sacramento, California 95817

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

Received for publication, December 21, 2004 , and in revised form, February 28, 2005.

^* This work was supported by National Institutes of Health Grants R01 DK60581 (to R. G. M.), U01 DK56047 (to C. B. N. and T. C. B.), U19 DK61251 (to G. C. W.), and K08 DK002619 (to S. C. G.) and a 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.

Both authors contributed equally to this work.

To whom correspondence should be addressed: University of Virginia Health System, 450 Ray C. Hunt Dr., Box 801407, Charlottesville, VA 22908. Tel.: 434-243-5036; Fax: 434-982-3796; E-mail: mirmira{at}virginia.edu.

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