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Molecular and Cellular Biology, September 2003, p. 6049-6062, Vol. 23, No. 17
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.17.6049-6062.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Members of the Large Maf Transcription Family Regulate Insulin Gene Transcription in Islet ß Cells

Taka-aki Matsuoka,1 Li Zhao,1 Isabella Artner,1 Harry W. Jarrett,2 David Friedman,3 Anna Means,4 and Roland Stein1*

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center,1 Department of Biochemistry,3 Department of Surgical Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,4 Department of Biochemistry, University of Tennessee, Memphis, Tennessee 381632

Received 13 February 2003/ Returned for modification 24 March 2003/ Accepted 28 May 2003

The C1/RIPE3b1 (-118/-107 bp) binding factor regulates pancreatic-ß-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse ßTC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N- and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (-340/-91 bp) of the endogenous insulin gene in ßTC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the ß-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet ß cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet ß-cell function.

* Corresponding author. Mailing address: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232. Phone: (615) 322-7026. Fax: (615) 322-7236. E-mail: Roland.Stein{at}vanderbilt.edu.

Molecular and Cellular Biology, September 2003, p. 6049-6062, Vol. 23, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.17.6049-6062.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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