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MafA is a key transcriptional activator of islet beta cells, and its exclusive expression within beta cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs -8118 to -7750 relative to the transcription start site), one of six conserved 5' cis domains of the MafA promoter, is capable of directing beta-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs -10428 to +230), and R1-6 lacking R3 (R1-6(DeltaR3)) were generated. Only the R1-6 transgene was active in MafA(+) insulin(+) cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6(DeltaR3) line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6(DeltaR3) lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the beta-cell-specific expression of MafA.

The MafA transcription factor is both critical to islet beta-cell function and has a unique pancreatic cell-type-specific expression pattern. To localize the potential transcriptional regulatory region(s) involved in directing expression to the beta cell, areas of identity within the 5' flanking region of the mouse, human, and rat mafA genes were found between nucleotides -9389 and -9194, -8426 and -8293, -8118 and -7750, -6622 and -6441, -6217 and -6031, and -250 and +56 relative to the transcription start site. The identity between species was greater than 75%, with the highest found between bp -8118 and -7750 ( approximately 94%, termed region 3). Region 3 was the only upstream mammalian conserved region found in chicken mafA (88% identity). In addition, region 3 uniquely displayed beta-cell-specific activity in cell-line-based reporter assays. Important regulators of beta-cell formation and function, PDX-1, FoxA2, and Nkx2.2, were shown to specifically bind to region 3 in vivo using the chromatin immunoprecipitation assay. Mutational and functional analyses demonstrated that FoxA2 (bp -7943 to -7910), Nkx2.2 (bp -7771 to -7746), and PDX-1 (bp -8087 to -8063) mediated region 3 activation. Consistent with a role in transcription, small interfering RNA-mediated knockdown of PDX-1 led to decreased mafA mRNA production in INS-1-derived beta-cell lines (832/13 and 832/3), while MafA expression was undetected in the pancreatic epithelium of Nkx2.2 null animals. These results suggest that beta-cell-type-specific mafA transcription is principally controlled by region 3-acting transcription factors that are essential in the formation of functional beta cells.

The C1/RIPE3b1 (-118/-107 bp) binding factor regulates pancreatic-beta-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse beta TC-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 beta TC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the beta-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 beta 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 beta-cell function.