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The Transcriptional Repressor Nkx6.1 Also Functions as a Deoxyribonucleic Acid Context-Dependent Transcriptional Activator during Pancreatic ß-Cell Differentiation: Evidence for Feedback Activation of the nkx6.1 Gene by Nkx6.1

Department of Internal Medicine and the Diabetes Center (T.I., S.M.Z., J.C.G., R.G.M.) and Department of Pharmacology (D.G.T., R.G.M.), University of Virginia, Charlottesville, Virginia 22903; and Department of Medicine (H.W.), Metabolism, and Endocrinology, Jutendo University School of Medicine, Tokyo 113-8421, Japan

Address all correspondence and requests for reprints to: Raghavendra Mirmira, University of Virginia Health System, 450 Ray C. Hunt Drive, Box 801407, Charlottesville, Virginia 22903. E-mail: mirmira{at}virginia.edu.

In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of ß-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, we identify a novel ß-cell-specific enhancer element in the nkx6.1 gene between –157 and –30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element. We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. We show by EMSAs that Nkx6.1 binds to the ß-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in ßTC3 cells. We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during ß-cell differentiation while simultaneously effecting broader gene repression events.

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