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• W2078561941 abstract "Pancreatic duodenal homeobox factor-1, PDX-1, is required for pancreas development, islet cell differentiation, and the maintenance of ॆ 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 thepdx-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 ॆ 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 ॆ 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 ॆ cells. Pancreatic duodenal homeobox factor-1, PDX-1, is required for pancreas development, islet cell differentiation, and the maintenance of ॆ 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 thepdx-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 ॆ 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 ॆ 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 ॆ cells. pancreatic duodenal homeobox factor-1 chloramphenicol acetyltransferase sequence blocks 4 and 5 phosphoenolpyruvate carboxykinase thymidine kinase insulin C1 polyvinylidene difluoride calf intestinal alkaline phosphatase baby hamster kidney Madin-Darby canine kidney Targeting of the pancreatic duodenal homeobox factor-1 (pdx-1)1 gene in mice has established that expression in a common progenitor cell population is essential for the development of both the endocrine and exocrine compartments of the pancreas. PDX-1 acts by stimulating proliferation, branching, and differentiation of the pancreatic epithelium (1Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Google Scholar, 2Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Google Scholar, 3Offield M.F. Jetton T.L. Labosky P.A. Ray M. Stein R.W. Magnuson M.A. Hogan B.L. Wright C.V. Development. 1996; 122: 983-995Google Scholar). In contrast, all other characterized islet endocrine- (e.g. PAX6 (4Sander M. Neubuser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Google Scholar, 5St-Onge L. Sosa-Pineda B. Chowdhury K. Mansouri A. Gruss P. Nature. 1997; 387: 406-409Google Scholar), Ngn3 (6Gradwohl G. Dierich A. LeMeur M. Guillemot F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1607-1611Google Scholar), BETA2 (7Naya F.J. Huang H.P. Qiu Y. Mutoh H. DeMayo F.J. Leiter A.B. Tsai M.J. Genes Dev. 1997; 11: 2323-2334Google Scholar), and exocrine (PTF1-p48 (8Krapp A. Knofler M. Ledermann B. Burki K. Berney C. Zoerkler N. Hagenbuchle O. Wellauer P.K. Genes Dev. 1998; 12: 3752-3763Google Scholar, 9Kawaguchi Y. Cooper B. Gannon M. Ray M. MacDonald R.J. Wright C.V. Nat. Genet. 2002; 32: 128-134Google Scholar)-enriched transcription factors act downstream of PDX-1 and are principally involved in islet or exocrine cell differentiation. Selective elimination of PDX-1 in mouse ॆ cellsin vivo also results in a reduction in both insulin secretion and islet ॆ cell numbers (1Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Google Scholar). These animals become glucose intolerant and diabetic, largely because of their inability to synthesize appropriate amounts of PDX-1-regulated gene products that are involved in maintaining glucose homeostasis (1Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Google Scholar) (e.g.insulin (10Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Google Scholar, 11Peshavaria M. Gamer L. Henderson E. Teitelman G. Wright C.V. Stein R. Mol. Endocrinol. 1994; 8: 806-816Google Scholar), GLUT2 (12Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Google Scholar), and glucokinase (13Watada H. Kajimoto Y. Umayahara Y. Matsuoka T. Kaneto H. Fujitani Y. Kamada T. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1478-1488Google Scholar)). Moreover, mutations in pdx-1 cause pancreatic agenesis (2Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Google Scholar, 3Offield M.F. Jetton T.L. Labosky P.A. Ray M. Stein R.W. Magnuson M.A. Hogan B.L. Wright C.V. Development. 1996; 122: 983-995Google Scholar) and a form of maturity onset diabetes of the young in humans (14Stoffers D.A. Stanojevic V. Habener J.F. J. Clin. Invest. 1998; 102: 232-241Google Scholar, 15Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Nat. Genet. 1997; 17: 138-139Google Scholar). These data have established an essential role for PDX-1 in islet ॆ cell development and function. The recent success in reversing type 1 diabetes by islet transplantation has led to renewed optimism for this form of treatment (16Shapiro A.M. Lakey J.R. Ryan E.A. Korbutt G.S. Toth E. Warnock G.L. Kneteman N.M. Rajotte R.V. N. Engl. J. Med. 2000; 343: 230-238Google Scholar). However, the availability of human islets is limited and will never be sufficient to treat all patients. Because islet-enriched transcription factors are essential for islet cell development, information valuable for generating transplantable cells will likely be gained by understanding how their expression is regulated. Therefore, efforts have recently focused on characterizing the transcriptional control regions in genes necessary for islet cell formation, includingpdx-1 (17Ben-Shushan E. Marshak S. Shoshkes M. Cerasi E. Melloul D. J. Biol. Chem. 2001; 276: 17533-17540Google Scholar, 18Gannon M. Gamer L.W. Wright C.V. Dev. Biol. 2001; 238: 185-201Google Scholar, 19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar, 20Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Google Scholar, 21Marshak S. Ben-Shushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Diabetes. 2001; 50 Suppl. 1: 37-38Google Scholar, 22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar, 23Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Google Scholar), BETA2 (24Huang H.P. Liu M. El-Hodiri H.M. Chu K. Jamrich M. Tsai M.J. Mol. Cell. Biol. 2000; 20: 3292-3307Google Scholar), pax6 (25Xu Z.P. Saunders G.F. J. Biol. Chem. 1997; 272: 3430-3436Google Scholar, 26Xu P.X. Zhang X. Heaney S. Yoon A. Michelson A.M. Maas R.L. Development. 1999; 126: 383-395Google Scholar),pax4 (27Brink C. Chowdhury K. Gruss P. Mech. Dev. 2001; 100: 37-43Google Scholar), and ngn3 (28Lee J.C. Smith S.B. Watada H. Lin J. Scheel D. Wang J. Mirmira R.G. German M.S. Diabetes. 2001; 50: 928-936Google Scholar). In specific regards topdx-1, expression will likely be mediated by factors involved in both the differentiation and maintenance of functional ॆ cells. Experiments performed in transgenic animals have established that ॆ cell-selective expression of the pdx-1 gene is regulated by sequences 5′ to the transcription start site (18Gannon M. Gamer L.W. Wright C.V. Dev. Biol. 2001; 238: 185-201Google Scholar, 23Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Google Scholar). Control also appears to be largely mediated by those conserved between the vertebrate pdx-1 genes (19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar, 20Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Google Scholar, 22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). Thus, ॆ cell-specific reporter gene expression was driven in transfection assays by areas of sequence identity shared between the chicken, mouse, and human genes (i.e. Area I, −2839/−2520 base pair (bp) (19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar), Area III, −1879/−1799 bp (19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar), Area IV, −6047/−6529 bp), 2R. Stein, unpublished observations. or only the mouse and human genes (Area II, −2141/−1961 bp (19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar, 22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar)). In contrast, the 5′-non-conserved sequences were inactive (18Gannon M. Gamer L.W. Wright C.V. Dev. Biol. 2001; 238: 185-201Google Scholar, 23Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Google Scholar). Apdx-1 gene fragment spanning Areas I and II also directed transgene expression to islet ॆ cells in vivo (termed PstBst, −2917/−1918 bp (18Gannon M. Gamer L.W. Wright C.V. Dev. Biol. 2001; 238: 185-201Google Scholar, 23Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Google Scholar)), although only Area II, and not Areas I (22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar) or III (18Gannon M. Gamer L.W. Wright C.V. Dev. Biol. 2001; 238: 185-201Google Scholar), functioned independently in these in vivo assays. Collectively, these data strongly suggest that Area II represents the core of the mammalian pdx-1 transcription control region. Mutational analysis of 17 conserved sequence blocks within Area II revealed sites for both positive- and negative-acting regulatory factors (22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). Gel shift analysis performed on the activating B8 (−2068/−2060-bp) and B14 (−2006/−1996-bp) elements demonstrated specific binding to Pax6 and Foxa2 (formerly termed HNF3ॆ), respectively. Mutation of the Foxa2 binding site in Area II limited expression of the PstBst transgene to a subset of the islet ॆ cellsin vivo (22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). In addition, conditional deletion of Foxa2 specifically from ॆ cells decreased pdx-1 mRNA and protein expression in mice (29Lee C.S. Sund N.J. Vatamaniuk M.Z. Matschinsky F.M. Stoffers D.A. Kaestner K.H. Diabetes. 2002; 51: 2546-2551Google Scholar). The ability of Pax6 and Foxa2 to bind within Area II of the endogenous pdx-1 gene also strongly supports a direct role in mediating transcription in ॆ cells (22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). In the present study, we show that the B4/5 activator of Area II is RIPE3b1/Maf, a 46-kDa islet ॆ cell-enriched protein(s) essential for both cell type-specific (30Shieh S.Y. Tsai M.J. J. Biol. Chem. 1991; 266: 16708-16714Google Scholar, 31Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476Google Scholar) and glucose-inducible (32Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Google Scholar, 33Kataoka K. Han S.I. Shioda S. Hirai M. Nishizawa M. Handa H. J. Biol. Chem. 2002; 277: 49903-49910Google Scholar) transcription of the insulin gene. Moreover, an antiserum that recognizes the recently isolated large Maf protein(s) of the insulin C1/RIPE3b1 activator revealed binding to Area II of the endogenouspdx-1 gene in ॆ cells. We propose that RIPE3b1/Maf is required for transcription of genes critical to ॆ cell function. The Area II and PstBst reporter constructs were made using human (−2141/−1890-bp) and mouse (Pst/−2917bp:Bst/−1890-bp) pdx-1 sequences (23Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Google Scholar), which were cloned directly upstream of the herpes simplex thymidine kinase (TK) promoter in a chloramphenicol acetyltransferase (CAT) expression vector, pTK(An) (34Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Google Scholar). The block transversion and insulin C1 (InsC1) substitution mutants in B4/5 were constructed in Area II:pTK and PstBst:pTK using the QuikChange mutagenesis kit (Stratagene). Each construct was determined to be correct by DNA sequencing. Monolayer cultures of pancreatic islet ॆ (ॆTC-3, HIT-T15, and Min6) and non-ॆ (NIH3T3) cell lines were maintained as described previously (35Whelan J. Poon D. Weil P.A. Stein R. Mol. Cell. Biol. 1989; 9: 3253-3259Google Scholar). The LipofectAMINE reagent (Invitrogen) was used to introduce 1 ॖg each of Area II:pTK or PstBst:pTK and 0.5 ॖg of pRSVLUC. The activity from the Rous sarcoma virus enhancer-driven luciferase plasmid served as an internal transfection control for the pdx-1:pTK constructs. Luciferase (36de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Google Scholar) and CAT (37Nordeen S.K. Green P.P.D. Fowlkes D.M. DNA. 1987; 6: 173-178Google Scholar) enzymatic assays were performed 40–48 h after transfection. Each experiment was carried out more than three times with at least two independently isolated DNA preparations. Double-stranded Area II block 4 (B4, agcttTCTTTTTGCAAAGCACAGCAt), B5 (agcttAAAGCACAGCAAAAATATTAt), and B4/5 (agcttCTTTTTGCAAAGCACAGCAAAAAt) sequences, in which the lowercase lettering corresponds to linker sequences, were excised from pBluescriptKS2+ and Klenow-labeled with [α32P]dATP. The InsC1 probe spans nucleotides −126 to −101 of the rat insulin II gene and was labeled as described (38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar). Nuclear extracts were prepared as described previously (39Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Google Scholar). Binding reactions (20 ॖl total volume) were conducted with 5–10 ॖg of extract protein and labeled probe (8 × 104 cpm) in binding buffer containing 10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 2 mm dithiothreitol, 1 mm EDTA, 107 glycerol, and 1 ॖg of poly(dGdC) (final concentrations). The conditions for the competition analyses were the same, except that excess (see figures for amounts) of the specific competitor DNA was included in the mixture prior to the addition of probe. Anti-c-Maf antiserum (10 ॖg, M-153, Santa Cruz Biotechnology) was added to binding reactions 10 min prior to addition of the probe for supershift analysis. This antiserum, referred to in the text as αc-Maf M-153, was made to an N-terminal region of c-Maf that is common to the other large Mafs and cross-reacts with each (i.e. MafA, NRL, and MafB). The samples were resolved on a 67 nondenaturing polyacrylamide gel (acrylamide:bisacrylamide ratio 29:1) and run in TGE buffer (50 mm Tris, 380 mm glycine, 2 mm EDTA, pH 8.5). The gel was dried and subjected to autoradiography. ॆTC-3 and Min6 nuclear extracts (30 ॖg) were separated on a 107 SDS-polyacrylamide gel (SDS-PAGE) and then electro-transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore). The extract lanes were cut horizontally into 3-mm slices. The molecular mass range of each lane fraction was determined by comparison with colored Rainbow protein markers (Amersham Biosciences). The proteins from each fraction were eluted as previously described (38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar) and analyzed for B4/5 and InsC1 binding activity in electrophoretic mobility shift assays. Min6 or ॆTC-3 nuclear extract (3–5 ॖg) was incubated for 10 min at 4 °C or 30 °C with and without 0.5 units of calf intestinal alkaline phosphatase (CIAP, Promega) in the presence or absence of sodium orthovanadate (Na3VO4, 10 mm) or sodium pyrophosphate (NaPPi, 10 mm) in phosphatase buffer (20 mm Tris-HCl, pH 7.4, 1 mm dithiothreitol, 0.1 mm EGTA, 2 mm MgCl2, 1× protease inhibitor mixture (CØmplete, Roche Diagnostics)) (10 ॖl total volume). The samples were analyzed for InsC1 and B4/5 binding after addition of 10 ॖl of 2× gel shift binding buffer. Immunoprecipitations using anti-Tyr(P) (4G10, Upstate Biotechnology, Lake Placid, NY) were performed as described previously (40Matsuoka T. Zhao L. Stein R. J. Biol. Chem. 2001; 276: 22071-22076Google Scholar). Briefly, SDS was added to a final concentration of 0.57 (w/v) to ॆTC-3 nuclear extract (100 ॖg protein) in a buffer containing 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 107 glycerol, 1 mm Na3VO4, and 2 mm dithiothreitol (final concentrations) and then heated to 65 °C. After diluting the SDS to 0.057, anti-Tyr(P) or control mouse IgG was added along with protein A-Sepharose beads. The washed beads were then resuspended in 1× SDS-PAGE loading buffer and the immunoprecipitated proteins separated on a 107 SDS-polyacrylamide gel. After transfer to an Immobilon PVDF membrane, the 44–47-kDa eluted proteins were assayed for B4/5 and InsC1 binding activity. Pancreata from 6–8-week-old mice were fixed 4–5 h in 47 paraformaldehyde at 4 °C, washed, dehydrated, embedded in paraffin, and then 5-ॖm sections cut and mounted on glass slides. Double immunofluorescence was performed using guinea pig α-human insulin (Linco) and rabbit α-mouse c-Maf (αc-Maf M-153) as primary antibodies at dilutions of 1:2000 and 1:100, respectively. Secondary antibodies were Cy3- or Cy5-labeled donkey anti-guinea pig and anti-rabbit IgG diluted to 1:500 (Jackson ImmunoResearch Laboratories). Fluorescent images were captured on a Zeiss LSM510 confocal microscope at an optical depth of 1 ॖm, false colors were assigned, and the images merged in Photoshop 5 (Adobe). Immunoperoxidase staining was performed with Vectastain Elite kits (Vector Labs) and with 3,3′-diaminobenzidine tetrahydrochloride substrate (Zymed Laboratories Inc.) according to the manufacturer's recommendations. Rabbit anti-c-maf antibody (M-153) was diluted 1:1000. Chromatin immunoprecipitation assays were performed with the following modifications of a described method (19Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Google Scholar, 22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). The αc-Maf M-153 antiserum (10 ॖg) was incubated with sonicated formaldehyde cross-linked ॆTC3 chromatin. Normal rabbit IgG (10 ॖg, sc-2027, Santa Cruz Biotechnology) was used as a control. The protein-DNA complexes were isolated with A/G-agarose beads (Santa Cruz Biotechnology). The PCR oligonucleotides used to detect mouse control sequences were: pdx-1 Area II, −2208 5′-GGTGGGAAATCCTTCCCTCAAG-3′ and −1927 5′-CCTTAGGGATAGACCCCCTGC-3′, and phosphoenolpyruvate carboxykinase (PEPCK), −434 5′-GAGTGACACCTCACAGCTGTGG-3′ and −96 5′-GGCAGGCCTTTGGATCATAGCC-3′. The PCR cycling parameters were 1 cycle of 95 °C/2 min and 28 cycles of 95 °C/30 s, 61 °C/30 s, 72 °C/30 s for PEPCK and 1 cycle of 95 °C/2 min and 28 cycles of 95 °C/30 s, 57.5 °C/30 s, 72 °C/30 s for Area II. Block mutations within conserved B2 (−2131/−2115 bp), B3 (−2110/−2102 bp), B4 (−2100/−2093 bp), and B5 (−2089/−2086 bp) reduce Area II:pTK activity in ॆ cell lines (Fig.1B, HIT-T15,MIN6, and ॆTC-3) (22Samaras S.E. Cissell M.A. Gerrish K. Wright C.V. Gannon M. Stein R. Mol. Cell. Biol. 2002; 22: 4702-4713Google Scholar). To further examine the significance of these elements in Area II activation, each was mutated within the mouse pdx-1 ‘PstBst’ region that spans Area I and Area II (Fig. 1A). In the context of the more active PstBst:pTK expression construct, the B4 and B5 mutants reduced activity to a greater extent than in Area II:pTK (Fig. 1B). Combining the B4 with B5 mutations in PstBst:pTK reduced activity further than either individual mutation (Fig. 1B). In contrast, the B2 and B3 mutants had less effect on PstBst:pTK activation (data not shown). The following experiments were designed to characterize the B4 and B5 activators in ॆ cells. To define the factors associated with B4- and B5-mediated regulation, gel shift experiments were performed with probes spanning B4, B5, and B4+B5 (B4/5), and ॆTC-3 or MIN6 cell nuclear extracts (Fig. 2). Identical results were obtained with MIN6 and ॆTC-3 cells, and they were used interchangeably in these analyses. Two common protein-DNA complexes were detected with the B4 and B4/5 probes (labeled as A andB in Fig. 2B), whereas no binding was found with B5 (data not shown). The binding affinity of B4 and B4/5 for these complexes was determined with the wild type and B4/5 double mutant site (B4/5 MT) competitors. As expected, both B4 and B4/5 reduced the levels of these complexes, although B4/5 was roughly 20-fold more effective (Fig. 2B). In contrast, B5 did not compete for binding (data not shown), whereas the B4/5 MT only competed away complex B, consistent with the conclusion that it is unrelated to activation (Fig.2B). These results suggested that B4 and B5 define a single activator-binding site, which is regulated by the factor(s) found within the slower mobility complex A. To determine the distribution of the cellular factor(s) forming complex A, binding reactions were conducted with nuclear extracts from various islet (ॆ: Ins-1, Min6, HIT-T15; α, αTC-6) and non-islet cell types (neuronal, RC2.E10 (41Perez-Villamil B. Schwartz P.T. Vallejo M. Endocrinology. 1999; 140: 3857-3860Google Scholar, 42Schwartz P.T. Perez-Villamil B. Rivera A. Moratalla R. Vallejo M. J. Biol. Chem. 2000; 275: 19106-19114Google Scholar), NCB20 (43West Jr., R.E. Freedman S.B. Dawson G. Miller R.J. Villereal M.L. Life Sci. 1982; 31: 1335-1338Google Scholar, 44Kato K. Higashida H. Umeda Y. Suzuki F. Tanaka T. Biochim. Biophys. Acta. 1981; 660: 30-35Google Scholar); liver, H4IIE (45Darlington G.J. Methods Enzymol. 1987; 151: 19-38Google Scholar), normal rat liver; kidney, MDCK, BHK; fibroblast, NIH 3T3). Complex A was uniquely detected in the ॆ cell extracts (Fig. 2C). These results suggest that the factor(s) in activator complex A is enriched in ॆ cells. To estimate the size of the protein(s) in complex A, Min6 nuclear extracts were separated by SDS-PAGE and transferred to a PVDF membrane that was cut into slices to represent distinct molecular masses. The separated proteins were eluted from the membrane slices, renatured, and tested for binding to the B4/5 probe. The binding specificity of fraction 8 was identical to complex A found in unfractionated Min6 extracts (Fig. 3; data no shown). The molecular mass range of the proteins in fraction 8 was 44–47 kDa. These results indicate that complex A is composed of one or more proteins of ∼46 kDa. Because the RIPE3b1 protein(s) that binds to and activates the InsC1 control element has the same cell-restricted distribution (38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar) and molecular size (see Fig. 3 and Ref. 38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar), we compared the binding properties of B4/5 to InsC1 (Fig.4). Both InsC1 and B4/5 competed effectively for complex A binding when either B4/5 or InsC1 were used as probes (Fig. 4B). In addition, RIPE3b1/complex A activity was affected in the same manner by B4/5 or InsC1 mutations that either modestly (e.g. InsC1mt1, Ref. 38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar) or profoundly (e.g. InsC1mt3, Ref. 38Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Google Scholar) (Fig. 1B,B4/5MT) affected activity. The resulting competition patterns were consistent with each element binding the same factor(s) (Fig. 4B). RIPE3b1 binding activity is inhibited by the actions of a tyrosine phosphatase (40Matsuoka T. Zhao L. Stein R. J. Biol. Chem. 2001; 276: 22071-22076Google Scholar). To test whether complex A formation on B4/5 is also regulated in this manner, Min6 nuclear extracts were incubated in the presence or absence of CIAP and a general (sodium pyrophosphate, NaPPi) or phosphotyrosine-specific (sodium orthovanadate, Na3VO4) phosphatase inhibitor. B4/5 and InsC1 binding activities were monitored in the treated extracts. The binding characteristics of complex A were affected in exactly the same manner with both probes (Fig. 5A). Complex A mobility was shifted upon incubating the ॆ cell nuclear extract at 30 °C with both the B4/5 and InsC1 probes, presumably because of the actions of an endogenous tyrosine phosphatase (40Matsuoka T. Zhao L. Stein R. J. Biol. Chem. 2001; 276: 22071-22076Google Scholar). CIAP treatment reduced binding to each probe, an effect blocked by addition of NaPPi or Na3VO4 (Fig. 5A). In addition, B4/5 bound specifically to the 46-kDa fraction immunoprecipitated from ॆTC-3 nuclear extracts with the anti-phosphotyrosine immunospecific monoclonal antibody, 4G10 (Fig.5B, compare 4G10 immunoprecipitate binding to InsC1 (40Matsuoka T. Zhao L. Stein R. J. Biol. Chem. 2001; 276: 22071-22076Google Scholar) and B4/5). Collectively, these results strongly suggest that RIPE3b1 binds to both the pdx-1 B4/5 and InsC1 elements. Considering the interchangeability of B4/5 and InsC1 in gel shift assays, it was surprising to find only modest sequence identity between human (h) and mouse (m) B4/5 and mouse InsC1 (Fig.6A). However, methylation interference assays over B4/5 suggested some similarity in contact nucleotides for RIPE3b1 with InsC1 (Ref. 30Shieh S.Y. Tsai M.J. J. Biol. Chem. 1991; 266: 16708-16714Google Scholar and Fig. 6A; data not shown). Because of sequence dissimilarity between B4/5 and InsC1, we tested whether InsC1 could substitute for B4/5 in the context of the PstBst:pTK reporter. Replacement of B4/5 with InsC1 maintained the same high level of activation found for wild type PstBst in Min6 ॆ cells (Fig. 6B). Furthermore, mutants in B4/5 (B4mt, B5mt, B4/5mt) and InsC1 (mut3) that compromised complex A/RIPE3b1 binding also reduced PstBst activity only in Min6 cells. These data strongly suggest that the ॆ cell-enriched RIPE3b1 transcription factor activates the B4/5 control element in Area II. The RIPE3b1 transcription factor was recently isolated and shown to be a member of the large Maf transcription factor family, most likely MafA (33Kataoka K. Han S.I. Shioda S. Hirai M. Nishizawa M. Handa H. J. Biol. Chem. 2002; 277: 49903-49910Google Scholar, 46Olbrot M. Rud J. Moss L.G. Sharma A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6737-6742Google Scholar). 3T. Matsuoka, L. Zhao, and R. Stein, unpublished observations.To determine whether the B4/5 binding complex A contained a large Maf protein, Min6 nuclear extract was preincubated with a polyclonal antiserum raised to N-terminal sequences of c-Maf shared with other members of the large Maf family. This c-Maf antiserum, termed αc-Maf M-153, cross-reacts with MafA, MafB, and NRL (46Olbrot M. Rud J. Moss L.G. Sharma A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6737-6742Google Scholar). 4T. Matsuoka, L. Zhao, and R. Stein, unpublished observations. Complex A was completely supershifted by αc-Maf M-153, whereas IgG had no effect (Fig. 4C). These results strongly suggested that complex A contains the RIPE3b1/Maf protein. Immunohistochemical analysis performed with αc-Maf M-153 on adult mouse pancreas also showed that the large Maf protein(s) of the RIPE3b1/Complex A activator was nuclear and expressed almost exclusively in insulin-producing ॆ cells (Fig.7). To directly determine whether RIPE3b1/Maf binds within Area II of the endogenous pdx-1 gene, a chromatin immunoprecipitation assay was performed using formaldehyde cross-linked chromatin from ॆTC-3 cells. The cross-linked DNA was precipitated with the Maf antiserum and PCR-amplified with Area II and PEPCK promoter-specific primers. The Maf antibody was capable of immunoprecipitating Area II sequences, whereas the control IgG could not (Fig. 8). However" @default.
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• W2078561941 date "2003-04-01" @default.
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• W2078561941 title "The Islet ॆ Cell-enriched RIPE3b1/Maf Transcription Factor Regulates pdx-1 Expression" @default.
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