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

Abstract

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.

FIG. 1.

Analysis of the effects of 5′ flanking deletions spanning the conserved sequence domains of mammalian mafA on β-cell-line-selective expression. (A) Diagram of the human, mouse, and rat 5′ flanking region. The percent identity of the human and rat to mouse is indicated with bars below the locus diagram. Mouse regions 1 (bp −9389 to −9194), 2 (bp −8426 to −8293), 3 (bp −8118 to −7750), 4 (bp −6622 to −6441), 5 (bp −6217 to −6031), and 6 (bp −250 to +56) are numbered relative to the transcription start site determined in panel B. (B) Total RNA from βTC3 cells was hybridized with the mouse antisense mafA bp −35 to +106 probe and then treated with RNase. The arrow in the autoradiograph denotes the mafA RNase protected band, the exact size of which was determined using the M13mp18 plasmid sequencing ladder produced with the M13-40 primer. The same initiation site was determined independently using a bp −35 to +339 probe (data not shown). (C) The 5′ flanking deletion mutants were transfected into β (βTC3) and non-β cells (NIH 3T3). The normalized activity of each construct was presented relative to region 6:pSVOAPL2L ± the standard error of the mean.

FIG. 2.

Region 3 imparts β-cell-line-specific activation. (A) Mouse mafA:pTk constructs driven by regions 1 to 3 (bp −9940 to −6615), regions 4 and 5 (bp −6615 to −4217), region 6 (bp −510 to +239), and nonconserved (bp −5777 to −4217, bp −3785 to −3286, and bp −1779 to −1280) sequences were transfected into βTC3, MIN6, and NIH 3T3 cells. The ratio of the normalized activity of mafA:pTk to pTk and mafA:pSVOAPL2L to pSVOAPL2L were calculated for each cell line. Results are presented as the relative activity of each construct ± the standard error of the mean in β cells divided by the NIH 3T3 cell activity. (B) Mouse region 1, 2, 3, 4, 5, and 1 to 3 mafA:pTk constructs were transfected into βTC3, INS-1 (832/3), and NIH 3T3 cells. The ratio of the normalized activity of mafA:pTk to pTk was calculated for each cell line. Results are presented as the relative activity of each construct ± the standard error of the mean.

FIG. 3.

Sequence identity within region 3 between mouse, human, rat, and chicken mafA. Mouse, human, rat, and chicken mafA region 3 sequences were aligned in MacVector 7.0 using the ClustalW application. The shaded sequences are conserved between all species. The numbering is relative to the mouse mafA transcription start site. The potential Nkx6.1 (−8101 to −8094), PDX-1 (−8078 to −8071), BETA2 (−8036 to −8031), MafA and/or MafB (−8026 to −8008), Hnf-1α (−8000 to −7992), Hnf-4α (−7973 to −7955), FoxA2 (−7934 to −7920), Pax6 (−7818 to −7797), Pax4 (−7784 to −7764), and Nkx2.2 (−7762 to −7757) regulatory factor binding sites identified by TRANSFAC database analysis of mouse region 3 is shown, with the percent identity to the factor consensus binding site denoted. The identity to the Nkx2.2 consensus site was reduced in the human and chicken gene to 73% and 43%, respectively.

FIG. 4.

Identification of FoxA2, Nkx2.2 and PDX-1 binding sites in region 3. Sequences spanning −8087 to −8063 (PDX-1) (A), −7943 to −7910 (FoxA2) (B), and −7771 to −7746 (Nkx2.2) (C) were used in gel shift binding assays with βTC3 nuclear extract. The specificity of protein-DNA complex formation was determined by competition with a 5- to 50-fold molar excess of unlabeled wild-type competitor (R3 WT, lanes 2 to 5) or a 25- to 50-fold molar excess of binding mutant competitor (R3 MUT, lanes 6 and 7). In addition, preincubation of nuclear extract with anti-PDX-1 (A), anti-FoxA2 (B), or anti-Nkx2.2 (C) antibodies were found to influence the formation (anti-PDX-1 [αPDX-1]) or mobility (anti-FoxA2 [αFoxA2] and anti-Nkx2.2 [αNkx2.2]) of the factor-specific complex (lane 8). Arrowheads indicate the specific binding and supershifted complexes, with the asterisks denoting the nonspecific complexes.

FIG. 5.

FoxA2, Nkx2.2, and PDX-1 bind to region 3 of endogenous mafA in β cells. Formaldehyde cross-linked chromatin from βTC3 cells was incubated with antibodies specific to PDX-1 (lane 3) (A), FoxA2 (lane 3) (B), and Nkx2.2 (lane 3) (C). Immunoprecipitated DNA was analyzed by PCR using primers specific to region 3 (A to C) and PEPCK (D). As controls, PCRs were performed with total input DNA (1/100 dilution, all panels, lane 1), no DNA (all panels, lane 2), DNA immunoprecipitated with rabbit IgG (all panels, lane 4), and DNA that was precipitated in the absence of antibody (all panels, lane 5). Only the anti-FoxA2 (α-FoxA2) results are shown with PEPCK (D), although the same pattern was found with the anti-Nkx2.2 (α-Nkx2.2) and anti-PDX-1 (α-PDX-1) antibody precipitates (data not shown). Each experiment was repeated on three separate occasions.

FIG. 6.

FoxA2, Nkx2.2, and PDX-1 binding stimulate region 3 activity in β cell lines. βTC3 and INS-1 (832/13) cells were transfected with the wild type (WT) and PDX-1, FoxA2, or Nkx2.2 binding site mutant (MUT) versions of region 3:pTk. Normalized region 3:pTk activity ± standard error of the mean of each mutant is presented as a percentage of the region 3 WT activity. Asterisks denote that there was a statistically significant decrease between PDX-1, Nkx2.2, FoxA2, and the WT in a Student t test: *, P < 0.05; **, P < 0.005; ***, P < 0.001. The data were compiled from the results from at least six independently performed transfections.

FIG. 7.

mafA mRNA levels are decreased by siRNA-mediated knockdown of pdx-1. INS-1 (832/13) cells were treated with recombinant adenoviruses expressing siControl or siPDX-1. The effects on pdx-1 and mafA mRNA were analyzed by RT-PCR. The mafA and pdx-1 values are normalized to those for siControl-treated cells and presented as means ± standard errors of the means. Each experiment was performed in triplicate and repeated five times.

FIG. 8.

MafA is not present in Nkx2.2^−/− pancreata. Sections of E18.5 wild-type and Nkx2.2 mutant mice were stained with anti-MafA (green) and anti-ghrelin (red). MafA was not detected in the mutant nor in wild-type ghrelin⁺ cells. Nuclei were stained blue.