Islet beta-cell-specific MafA transcription requires the 5'-flanking conserved region 3 control domain.

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 stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

MafA and MafB regulate Pdx1 transcription through the Area II control region in pancreatic beta cells.

Pancreatic-duodenal homeobox factor-1 (Pdx1) is highly enriched in islet beta cells and integral to proper cell development and adult function. Of the four conserved 5'-flanking sequence blocks that contribute to transcription in vivo, Area II (mouse base pairs -2153/-1923) represents the only mammalian specific control domain. Here we demonstrate that regulation of beta-cell-enriched Pdx1 expression by the MafA and MafB transcription factors is exclusively through Area II. Thus, these factors were found to specifically activate through Area II in cell line transfection-based assays, and MafA, which is uniquely expressed in adult islet beta cells was only bound to this region in quantitative chromatin immunoprecipitation studies. MafA and MafB are produced in beta cells during development and were both bound to Area II at embryonic day 18.5. Expression of a transgene driven by Pdx1 Areas I and II was also severely compromised during insulin+ cell formation in MafB(-/-) mice, consistent with the importance of this large Maf in beta-cell production and Pdx1 expression. These findings illustrate the significance of large Maf proteins to Pdx1 expression in beta cells, and in particular MafB during pancreatic development.

MafA regulates expression of genes important to islet beta-cell function.

Insulin transcription factor MafA is unique in being exclusively expressed at the secondary and principal phase of insulin-expressing cell production during pancreas organogenesis and is the only transcriptional activator present exclusively in islet beta-cells. Here we show that ectopic expression of MafA is sufficient to induce a small amount of endogenous insulin expression in a variety of non-beta-cell lines. Insulin mRNA and protein expression was induced to a much higher level when MafA was provided with two other key insulin activators, pancreatic and duodenal homeobox (PDX-1) and BETA2. Potentiation by PDX-1 and BETA2 was entirely dependent upon MafA, and MafA binding to the insulin enhancer region was increased by PDX-1 and BETA2. Treatment with activin A and hepatocyte growth factor induced even larger amounts of insulin in AR42J pancreatic acinar cells, compared with other non-beta endodermal cells. The combination of PDX-1, BETA2, and MafA also induced the expression of other important regulators of islet beta-cell activity. These results support a critical role of MafA in islet beta-cell function.

FoxA2, Nkx2.2, and PDX-1 regulate islet beta-cell-specific mafA expression through conserved sequences located between base pairs -8118 and -7750 upstream from the transcription start site.

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.

MafB: an activator of the glucagon gene expressed in developing islet alpha- and beta-cells.

The large Maf family of basic leucine-zipper-containing transcription factors are known regulators of key developmental and functional processes in various cell types, including pancreatic islets. Here, we demonstrate that within the adult pancreas, MafB is only expressed in islet alpha-cells and contributes to cell type-specific expression of the glucagon gene through activation of a conserved control element found between nucleotides -77 to -51. MafB was also shown to be expressed in developing alpha- and beta-cells as well as in proliferating hormone-negative cells during pancreatogenesis. In addition, MafB expression is maintained in the insulin(+) and glucagon(+) cells remaining in mice lacking either the Pax4 or Pax6 developmental regulators, implicating a potentially early role for MafB in gene regulation during islet cell development. These results indicate that MafB is not only important to islet alpha-cell function but may also be involved in regulating genes required in both endocrine alpha- and beta-cell differentiation.

Identification of a novel PDX-1 binding site in the human insulin gene enhancer.

Islet beta cell type-specific transcription of the insulin gene is regulated by a number of cis-acting elements found within the proximal 5'-flanking region. The control sequences conserved between mammalian insulin genes are acted upon by transcription factors, like PDX-1 and BETA-2, that are also involved in islet beta cell function and formation. In the current study, we investigated the contribution to human insulin expression of the GG2 motif found between nucleotides -145 and -140 relative to the transcription start site. Site-specific mutants were generated within GG2 that displayed a parallel increase (i.e. -144 base pair) or decrease (i.e. -141 base pair) in insulin enhancer-driven reporter and gel shift binding activity in beta cells consistent with human GG2 being under positive regulatory control. In contrast, the corresponding site in the rodent insulin gene, which only differs from the human at nucleotides -144 and -141, is negatively regulated by the Nkx2.2 transcription factor (Cissell, M. A., Zhao, L., Sussel, L., Henderson, E., and Stein, R. (2003) J. Biol. Chem. 278, 751-756). Human GG2 activator binding activity was present in nuclear extracts prepared from human islets and enriched in those from rodent beta cell lines. The human GG2 activator binding factor(s) was shown to be approximately 38-40 kDa and distinct from other size-matched islet-enriched transcription factors, including Nkx2.2, Pax-4, Cdx2/3, and Isl-1. Combined DNA chromatographic purification and mass spectrometry analysis revealed that the GG2 activator was PDX-1. These results demonstrate that the GG2 element, despite its divergence from the core homeodomain consensus binding motif, is a site for PDX-1 activation in the human insulin gene.

The MafA transcription factor appears to be responsible for tissue-specific expression of insulin.

Insulin gene expression is regulated by several islet-enriched transcription factors. However, MafA is the only beta cell-specific activator. Here, we show that MafA selectively induces endogenous insulin transcription in non-beta cells. MafA was also first detected in the insulin-producing cells formed during the second and predominant phase of beta cell differentiation, and absent in the few insulin-positive cells found in Nkx6.1(-/-) pancreata, which lack the majority of second-phase beta cells. These results demonstrate that MafA is a potent insulin activator that is likely to function downstream of Nkx6.1 during islet insulin-producing cell development.

The islet beta cell-enriched RIPE3b1/Maf transcription factor regulates pdx-1 expression.

Pancreatic duodenal homeobox factor-1, PDX-1, is required for pancreas development, islet cell differentiation, and the maintenance of beta cell function. Selective expression in the pancreas appears to be principally regulated by Area II, one of four conserved regulatory sequence domains found within the 5'-flanking region of the pdx-1 gene. Detailed mutagenesis studies have identified potential sites of interaction for both positive- and negative-acting factors within the conserved sequence blocks of Area II. The islet beta cell-enriched RIPE3b1 transcription factor, the activator of insulin C1 element-driven expression, was shown here to also stimulate Area II by binding to sequence blocks 4 and 5 (termed B4/5). Accordingly, B4/5 DNA-binding protein's molecular mass (i.e. 46 kDa), binding specificity, and islet beta cell-enriched distribution were identical to RIPE3b1. Area II-mediated activation was also unaffected upon replacing B4/5 with the insulin C1/RIPE3b1 binding site. In addition, the chromatin immunoprecipitation assay showed that the Area II region of the endogenous pdx-1 gene was precipitated by an antiserum that recognizes the large Maf protein that comprises the RIPE3b1 transcription factor. These results strongly suggest that RIPE3b1/Maf has an important role in generating and maintaining physiologically functional beta cells.

Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived beta TC-3 cells revealed that multiple promoter regions, located between -306 and -97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans -acting factor-binding sites in the IGRP promoter that were identified in beta TC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans -acting factor-binding sites located between -97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the -306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 (neurogenic differentiation/beta-cell E box transactivator 2) and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx2.2.

Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. This raises the possibility that factors that are capable of binding to a cis-acting element in vitro and regulating transcription from a transiently transfected plasmid, which would not have higher order chromatin structure, may not occupy this site within the endogenous gene. Closed chromatin structure and competition from another DNA-binding protein with similar nucleotide specificity are two possible mechanisms by which a transcription factor may be excluded from a potential binding site in vivo. Multiple transcription factors, including Pdx-1, BETA-2, and Pax6, have been implicated in expression of the insulin gene in pancreatic beta cells. In this study, the chromatin immunoprecipitation assay has been used to show that these factors do, in fact, bind to insulin control region sequences in intact beta cells. In addition, another key islet-enriched transcription factor, Nkx2.2, was found to occupy this region using the chromatin immunoprecipitation assay. In vitro DNA-binding and transient transfection assays defined how Nkx2.2 affected insulin gene expression. Pdx-1 was also shown to bind within a region of the endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in vitro. Because Pdx-1 does not regulate gene transcription in isolation, these sequences were examined for occupancy by the other insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and pax-4 control sequences in vivo. These studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and Nkx2.2 transcription factors in regulating expression of genes selectively expressed in islet beta cells.