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• W2054681277 abstract "Transcription factors binding the insulin enhancer region, RIPE3b, mediate β-cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the β-cell-specific RIPE3b1-binding complex is critical for these actions. The DNA binding activity of the RIPE3b1 activator is induced in response to glucose stimulation and is inhibited under glucotoxic conditions. The C1 element within the RIPE3b region has been implicated as the binding site for RIPE3b1 activator. The RIPE3b region also contains an additional element, A2, which shares homology with the A elements in the insulin enhancer. Transcription factors (PDX-1 and HNF-1α) binding to A elements are critical regulators of insulin gene expression and/or pancreatic development. Hence, to understand the roles of C1 and A2 elements in regulating insulin gene expression, we have systematically mutated the RIPE3b region and analyzed the effect of these mutations on gene expression. Our results demonstrate that both C1 and A2 elements together constitute the binding site for the RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes, three binding complexes that specifically recognize A2 elements are found in nuclear extracts from insulinoma cell lines; the A2.2 complex is detected only in insulin-producing cell lines. Furthermore, two base pairs in the A2 element were critical for binding of both RIPE3b1 and A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin gene expression. In addition, RIPE3b1- and A2-specific activators respond differently to glucose, suggesting that their overlapping binding specificity and functional cooperation may play an important role in regulating insulin gene expression. Transcription factors binding the insulin enhancer region, RIPE3b, mediate β-cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the β-cell-specific RIPE3b1-binding complex is critical for these actions. The DNA binding activity of the RIPE3b1 activator is induced in response to glucose stimulation and is inhibited under glucotoxic conditions. The C1 element within the RIPE3b region has been implicated as the binding site for RIPE3b1 activator. The RIPE3b region also contains an additional element, A2, which shares homology with the A elements in the insulin enhancer. Transcription factors (PDX-1 and HNF-1α) binding to A elements are critical regulators of insulin gene expression and/or pancreatic development. Hence, to understand the roles of C1 and A2 elements in regulating insulin gene expression, we have systematically mutated the RIPE3b region and analyzed the effect of these mutations on gene expression. Our results demonstrate that both C1 and A2 elements together constitute the binding site for the RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes, three binding complexes that specifically recognize A2 elements are found in nuclear extracts from insulinoma cell lines; the A2.2 complex is detected only in insulin-producing cell lines. Furthermore, two base pairs in the A2 element were critical for binding of both RIPE3b1 and A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin gene expression. In addition, RIPE3b1- and A2-specific activators respond differently to glucose, suggesting that their overlapping binding specificity and functional cooperation may play an important role in regulating insulin gene expression. base pair(s) wild type luciferase electrophoretic mobility shift assay(s) glial factor-binding element In adult mammals, the insulin gene is expressed only in the pancreatic β-cells in the islets of Langerhans. In studies using transgenic animals and transient transfection analysis, the proximal 5′-flanking region of the insulin promoter was shown sufficient for directing β-cell-specific expression of the insulin gene (1Dandoy-Dron F. Monthioux E. Jami J. Bucchini D. Nucleic Acids Res. 1991; 19: 4925-4936Crossref PubMed Google Scholar, 2Edlund T. Walker M.D. Barr P.J. Rutter W.J. Science. 1985; 30: 912-916Crossref Scopus (395) Google Scholar, 3Hanahan D. Nature. 1985; 315: 115-122Crossref PubMed Scopus (1006) Google Scholar, 4Crowe D.T. Tsai M.-J. Mol. Cell. Biol. 1989; 9: 1784-1789Crossref PubMed Scopus (103) Google Scholar, 5Stellrecht C.M.M. DeMayo F.J. Finegold M.J. Tsai M.-J. J. Biol. Chem. 1997; 272: 3567-3572Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Stein R. Trends Endocrinol. Metab. 1993; 4: 96-100Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 7Sander M. German M.S. J. Mol. Med. 1997; 75: 327-340Crossref PubMed Scopus (284) Google Scholar). Further, mutational analysis of the promoter proximal region identified several cis-acting enhancer elements that are important for insulin expression. The insulin enhancer elements, with the exception of E-box elements, have been classified based on the nucleotide sequence of the element (8German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L.G. Olson L.K. Permutt M.A. Philippe J. Robertson R.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.-J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar). Most of these enhancer elements are well conserved in various species, suggesting the presence of a common regulatory mechanism(s) controlling insulin expression.Three conserved insulin enhancer elements, A3 (−201 to −196 bp)1 (9German M.S. Moss L.G. Wang J. Rutter W.J. Mol. Cell. Biol. 1992; 12: 1777-1788Crossref PubMed Google Scholar, 10Peshavaria M. Gamer L. Henderson E. Teitelman G. Wright C.V.E. Stein R. Mol. Endocrinol. 1994; 8: 806-816Crossref PubMed Scopus (139) Google Scholar, 11Petersen H.V. Serup P. Leonard J. Michelsen B.K. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10465-10469Crossref PubMed Scopus (189) Google Scholar, 12Ohlsson H. Thor S. Edlund T. Mol. Endocrinol. 1991; 5: 897-904Crossref PubMed Scopus (95) Google Scholar, 13Boam D.S.W. Docherty K. Biochem. J. 1989; 264: 233-239Crossref PubMed Scopus (66) Google Scholar), RIPE3b/C1-A2 (−126 to −101 bp) (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar), and E1 (−100 to −91 bp) (4Crowe D.T. Tsai M.-J. Mol. Cell. Biol. 1989; 9: 1784-1789Crossref PubMed Scopus (103) Google Scholar, 15Karlsson O. Edlund T. Barnett Moss J. Rutter W.J. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8819Crossref PubMed Scopus (183) Google Scholar, 16Whelan J. Cordle S.R. Henderson E. Weil P.A. Stein R. Mol. Cell. Biol. 1990; 10: 1564-1572Crossref PubMed Google Scholar), play an important role in regulating cell-specific expression of the insulin gene. Factors binding these sites have a very limited cellular distribution. Transcription factors that bind and activate expression from two of the three conserved insulin enhancer elements (A3 and E1) have been cloned. PDX-1, a member of the homeodomain family of transcription factor expressed in cells of the pancreas and duodenum, binds the A3 element (17Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (765) Google Scholar, 18Leonard, J., Peers, B., Johnson, T., Ferreri, K., Lee, S., and Montminy, M. R. (1993) Mol. Endocrinol. 1275–1283Google Scholar, 19Miller C.P. McGehee R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (376) Google Scholar, 20Offield M.F. Jetton T.L. Labosky P. Ray M. Stein R. Magnuson M. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-985Crossref PubMed Google Scholar, 21Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 22Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V.E. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar). Heterodimers of ubiquitously distributed basic helix-loop-helix family members (E2A and HEB (23German M.S. Blanar M.A. Nelson C. Moss L.G. Rutter W.J. Mol. Endocrinol. 1991; 5: 292-299Crossref PubMed Scopus (111) Google Scholar, 24Peyton M. Moss L. Tsai M.J. J. Biol. Chem. 1994; 269: 25936-25941Abstract Full Text PDF PubMed Google Scholar)) and the cell type enriched basic helix-loop-helix member (BETA2 (25Naya F.J. Stellrecht C.M.M. Tsai M.-J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (518) Google Scholar,26Naya F.J. Huang H.P. Qiu Y. Mutoh H. DeMayo F.J. Leiter A.B. Tsai M.-J. Genes Dev. 1997; 11: 2323-2334Crossref PubMed Scopus (844) Google Scholar)) bind the E1 element. BETA2 is expressed in all pancreatic endocrine cell types, in some intestinal endocrine cells, and in the brain (25Naya F.J. Stellrecht C.M.M. Tsai M.-J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (518) Google Scholar, 26Naya F.J. Huang H.P. Qiu Y. Mutoh H. DeMayo F.J. Leiter A.B. Tsai M.-J. Genes Dev. 1997; 11: 2323-2334Crossref PubMed Scopus (844) Google Scholar, 27Robinson G.L.W.G. Peshavaria M. Henderson E. Shieh S.-Y. Tsais M.-J. Teitelman G. Stein R. J. Biol. Chem. 1994; 269: 2452-2460Abstract Full Text PDF PubMed Google Scholar). Limited but not identical cellular distribution of transcription factors PDX-1 and BETA2 suggests that cell-specific insulin gene expression may result because of the presence of a unique combination of enhancer element binding factors in the pancreatic β-cells. Interestingly, one of the RIPE3b element-binding complexes is expressed only in insulin-producing cells (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar, 28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and may play a critical role in regulating the cell type specificity of insulin gene expression.The RIPE3b binding activity has been extensively characterized in nuclear extracts from both insulin-producing and non-insulin-producing cell lines (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar, 27Robinson G.L.W.G. Peshavaria M. Henderson E. Shieh S.-Y. Tsais M.-J. Teitelman G. Stein R. J. Biol. Chem. 1994; 269: 2452-2460Abstract Full Text PDF PubMed Google Scholar, 28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Shieh S.Y. Stellrecht C.M.M. Tsai M.J. J. Biol. Chem. 1995; 270: 21503-21508Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar). Two specific RIPE3b-binding complexes have been identified: 1) a cell-specific complex, RIPE3b1, which is detected only in pancreatic β-cell lines, and 2) the RIPE3b2-binding complex that is detected in nuclear extracts from all cell lines examined to date. The importance of the factor(s) binding to the RIPE3b element in mediating cell type-specific insulin gene expression is further emphasized from transgenic mice studies (5Stellrecht C.M.M. DeMayo F.J. Finegold M.J. Tsai M.-J. J. Biol. Chem. 1997; 272: 3567-3572Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). It was demonstrated that an enhancer construct containing both RIPE3b and E1 elements, RIPE3, could correctly regulate temporal and spatial expression of the transgene in vivo (5Stellrecht C.M.M. DeMayo F.J. Finegold M.J. Tsai M.-J. J. Biol. Chem. 1997; 272: 3567-3572Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), whereas a construct containing the E1 element alone was unable to induce expression in transgenic animals (32Dandoy-D F. Monthioux E. Jami J. Bucchini D. Nucleic Acids Res. 1991; 19: 4925-4930Crossref PubMed Scopus (27) Google Scholar). In all RIPE3 transgenic lines, the transgene (growth hormone) expression was detected in β-cells, whereas in other lines, expression of growth hormone was also noted in α-cells (5Stellrecht C.M.M. DeMayo F.J. Finegold M.J. Tsai M.-J. J. Biol. Chem. 1997; 272: 3567-3572Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar).In addition to regulating cell type-specific expression, factors binding to the enhancer elements, A3, E1, and RIPE3b, also regulate glucose-mediated alterations in insulin gene expression (30Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar,33MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (123) Google Scholar, 34Melloul D. Ben-Neriah Y. Cerasi E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3865-3869Crossref PubMed Scopus (160) Google Scholar, 35German M.S. Wang J. Mol. Cell. Biol. 1994; 14: 4067-4075Crossref PubMed Scopus (150) Google Scholar, 36Odagiri H. Wang J. German M.S. J. Biol. Chem. 1996; 271: 1909-1915Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Our earlier studies demonstrated that the binding activity of the cell-specific RIPE3b1 activator play a key role in regulating this glucose responsiveness (30Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar). Although the binding activity of the RIPE3b1 activator is induced in response to acute change in glucose concentration, under glucotoxic conditions, as observed in HIT T-15 cells cultured chronically in the presence of high concentrations of glucose, the RIPE3b1 and PDX-1 binding activities were inhibited (37Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar, 38Olson L.K. Sharma A. Peshavaria M. Wright C.V.E. Towle H.C. Robertson R.P. Stein R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9127-9131Crossref PubMed Scopus (130) Google Scholar, 39Olson L.K. Redmon J.B. Towle H.C. Robertson R.P. J. Clin. Invest. 1993; 92: 514-519Crossref PubMed Scopus (175) Google Scholar). PDX-1 protein and mRNA levels were also inhibited in rat islets, following development of diabetes after partial pancreatectomy (40Zangen D.H. Bonner-Weir S. Lee C.H. Latimer J.B. Miller C.P. Habener J.F. Weir G.C. Diabetes. 1997; 46: 258-264Crossref PubMed Scopus (127) Google Scholar). These observations suggest that in vitro and in vivo glucotoxic conditions regulate the levels and activity of insulin gene transcription factors. Interestingly, the binding activity of the RIPE3b1 factor, but not of PDX-1, is inhibited under glucotoxic conditions in the insulinoma cell line βTC-6 (41Poitout V. Olson L.K. Robertson R.P. J. Clin. Invest. 1996; 97: 1041-1046Crossref PubMed Scopus (127) Google Scholar). This suggests that the inhibition of the RIPE3b1 activator binding may be the primary or initial defect under glucotoxic conditions.The cell type-specific and glucose-responsive transcription factors of the insulin gene also play an important role in pancreatic development and differentiation of β-cells. Mice homozygous for the null mutation in BETA2/NeuroD have a striking reduction in the number of β-cells and fail to develop mature islets, develop severe diabetes, and die perinatally (26Naya F.J. Huang H.P. Qiu Y. Mutoh H. DeMayo F.J. Leiter A.B. Tsai M.-J. Genes Dev. 1997; 11: 2323-2334Crossref PubMed Scopus (844) Google Scholar). The pdx-1 knockout mice have a more profound phenotype, being apancreatic; these animals also develop extreme hyperglycemia and die perinatally (20Offield M.F. Jetton T.L. Labosky P. Ray M. Stein R. Magnuson M. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-985Crossref PubMed Google Scholar, 21Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 42Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1552) Google Scholar). During embryonic development, PDX-1 is also expressed in exocrine and ductal epithelial cells, whereas in the adult pancreas expression is predominantly restricted to β-cells (20Offield M.F. Jetton T.L. Labosky P. Ray M. Stein R. Magnuson M. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-985Crossref PubMed Google Scholar, 21Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 22Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V.E. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 43Slack J.M.W. Development. 1995; 121: 1569-1580Crossref PubMed Google Scholar). However, expression of PDX-1 is induced in the ductal epithelium during pancreatic regeneration in adult animals (44Sharma A. Zangen D.H. Reitz P. Taneja M. Lissauer M.E. Miller C.P. Weir G.C. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 507-513Crossref PubMed Scopus (302) Google Scholar).Similar to the PDX-1 and BETA2 factors, the RIPE3b1 activator, is an important regulator of cell type-specific and glucose-responsive expression of the insulin gene and may play an important role in pancreatic development and/or β-cell differentiation. The C1 element (Fig. 1) in the RIPE3b region has been implicated as the binding site for the RIPE3b1 activator (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar, 28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar). Factors binding the C1 element were shown to functionally co-operate with E1 binding factors (BETA2-E12/E47) in regulating insulin gene expression (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar, 25Naya F.J. Stellrecht C.M.M. Tsai M.-J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (518) Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar, 45Hwung Y.-P. Gu Y.-Z. Tsai M.-J. Mol. Cell. Biol. 1990; 10: 1784-1788Crossref PubMed Scopus (53) Google Scholar). In addition to C1, the RIPE3b region also contains an A element, A2 (Fig. 1 and Refs. 8German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L.G. Olson L.K. Permutt M.A. Philippe J. Robertson R.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.-J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar, 46Boam D.S.W. Clark A.R. Docherty K. J. Biol. Chem. 1990; 265: 8285-8296Abstract Full Text PDF PubMed Google Scholar, and 47Tomonari A. Yoshimoto K. Tanaka M. Iwahana H. Miyazaki J. Itakura M. Diabetologia. 1996; 39: 1462-1468Crossref PubMed Scopus (9) Google Scholar). The homeodomain family of transcription factors (PDX-1, HNF1 α, and Isl-1) that are important for pancreatic development bind insulin A elements (48Karlsson O. Thor S. Norberg T. Ohlsson H. Edlund T. Nature. 1990; 344: 879-882Crossref PubMed Scopus (575) Google Scholar, 49Emens L.A. Landers D.W. Moss L.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7300-7304Crossref PubMed Scopus (122) Google Scholar, 50Ahlgren U. Pfaff S.L. Jessell T.M. Edlund T. Edlund H. Nature. 1997; 385: 257-260Crossref PubMed Scopus (581) Google Scholar, 51Rudnick A. Ling T.Y. Odagiri H. Rutter W.J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12203-12207Crossref PubMed Scopus (143) Google Scholar). Furthermore, factors binding to A elements are implicated in synergistically interacting with other insulin gene transcription factors and regulating gene expression (52German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (359) Google Scholar, 53Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar, 54Peshavaria M. Henderson E. Sharma A. Wright C.V.E. Stein R. Mol. Cell. Biol. 1997; 17: 3987-3996Crossref PubMed Scopus (84) Google Scholar).Hence, in this study, we have further characterized the RIPE3b region to better understand the role of C1 and A2 elements in regulating insulin gene expression. We demonstrate that the presence of both C1 and A2 elements are essential for binding of the RIPE3b1 activator. We also identified factors that require the A2 element but not the C1 element for binding. Interestingly, one of the A2 element-binding complexes, A2.2, is selectively expressed in insulin-producing cell lines. We further demonstrate that two base pairs in the A2 element are critical for the formation of both β-cell-specific DNA-binding complexes, RIPE3b1 and A2.2. Results from transient transfection analysis indicate that C1-A2 (RIPE3b1) and A2-specific factors positively regulate insulin gene expression. A mutation that prevents binding of both activators shows greater inhibition of insulin gene expression than a mutation that prevents binding of either of these factors alone. Based on these results we suggest that the presence of two β-cell-specific factors with overlapping DNA-binding sites may provide an important means of regulating insulin gene expression in response to various metabolic/environmental signals.DISCUSSIONThe present study demonstrates that β-cell-specific RIPE3b1 and A2.2 factors recognize overlapping DNA-binding sites within the insulin enhancer. These factors can each positively regulate insulin gene expression as well as cooperatively activate insulin gene expression. Because the binding activity of RIPE3b1 activator is regulated by glucose (Fig. 7 and Refs. 28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar, and 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar) and phosphorylation (28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), overlapping binding specificity and functional cooperation between A2.2 and RIPE3b1 activator may play major roles in regulating insulin expression in response to various metabolic stimulus. These observations suggest that like RIPE3b1, the A2.2 activator is an important regulator of insulin gene expression. Initial characterization indicates that A2-specific binding factors are distinct from PDX-1 (Fig. 6 A). Excess unlabeled A3 oligonucleotide did not prevent formation of A2-specific complexes (Fig. 6 A), suggesting that A2 binding factors might belong to a different family of transcription factors than the PDX-1 and other A element binding homeodomain factors. Similarly, anti-Nkx2.2 antibody did not recognize any of the A2-specific complexes, and mutating the Nk2 consensus sequence did not affect the formation of A2 complexes. These results indicate that A2-specific activators are novel and distinct from the Nk2 and other homeodomain family of transcription factors.The importance of the RIPE3b region in mediating β-cell-specific and glucose-responsive insulin gene expression is well established (5Stellrecht C.M.M. DeMayo F.J. Finegold M.J. Tsai M.-J. J. Biol. Chem. 1997; 272: 3567-3572Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar,27Robinson G.L.W.G. Peshavaria M. Henderson E. Shieh S.-Y. Tsais M.-J. Teitelman G. Stein R. J. Biol. Chem. 1994; 269: 2452-2460Abstract Full Text PDF PubMed Google Scholar, 28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Shieh S.Y. Stellrecht C.M.M. Tsai M.J. J. Biol. Chem. 1995; 270: 21503-21508Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar, 31Sharma A. Stein R. Mol. Cell. Biol. 1994; 14: 871-879Crossref PubMed Google Scholar, 37Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar, 41Poitout V. Olson L.K. Robertson R.P. J. Clin. Invest. 1996; 97: 1041-1046Crossref PubMed Scopus (127) Google Scholar). Although two insulin enhancer elements, C1 and A2, were described in the RIPE3b region, we were unable to detect any C1 selective binding activity in insulin-producing cells. Our results demonstrate that C1 binding factors (RIPE3b1 and RIPE3b2) require sequences present in the A2 element. Similarly, A2-specific factors require additional nucleotides upstream of the RIPE3b region for their binding (Figs. 2 and 3). These observations suggest a need to redefine the enhancer elements in the RIPE3b region. Because we were unable to identify any C1-specific binding factor, we suggest that designation of the C1 region as a DNA-binding element may not be accurate. We suggest that the binding element for RIPE3b1 and RIPE3b2 activator may accurately be described as C1-A2 or the CA element. Similarly, additional nucleotides must be included within the A2 element to describe it as a binding site for A2-specific factors. These criteria would help interpret earlier studies that describe mutations and deletions in C1 and A2 elements and their effect on insulin gene expression (47Tomonari A. Yoshimoto K. Tanaka M. Iwahana H. Miyazaki J. Itakura M. Diabetologia. 1996; 39: 1462-1468Crossref PubMed Scopus (9) Google Scholar, 64Lu M. Seufert J. Habner J. J. Biol. Chem. 1997; 272: 28349-28359Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For example, Tomonari and co-workers (47Tomonari A. Yoshimoto K. Tanaka M. Iwahana H. Miyazaki J. Itakura M. Diabetologia. 1996; 39: 1462-1468Crossref PubMed Scopus (9) Google Scholar) reported the role of the A2 (GG1) element in regulating human insulin gene expression. Their results obtained from an A2 deletion construct would not only inhibit activation from the A2-specific factor but also from the RIPE3b1 activator. Similarly, other mutations in the A2 region described in the their study would also inhibit activation mediated by both RIPE3b1 and A2.2 factors. In addition, functional cooperation between the C1-A2 and A2-specific factors should be considered prior to interpreting results from earlier studies. We therefore suggest that a clear designation of the C1-A2 and A2 elements would be helpful in analyzing the role of this region in insulin gene expression.Mutational analysis of the RIPE3b region has provided important information regarding the RIPE3b1 and RIPE3b2 activators. Binding of these factors requires at least a 16-bp-long region (−123 to −107 bp; Figs. 2 and 8) of the rat insulin II enhancer. Nucleotides critical for binding of these factors are separated into two distinct regions, −112 to −107 bp and −123 to −117 bp. Although the nucleotides between these regions can be mutated without any effect on binding activity and insulin gene expression (Fig. 2 A, data not shown, and Ref.28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), attempts to alter spacing between these regions by either insertion or deletion mutations prevented DNA binding (Fig.2 C). These observations suggest that RIPE3b1 and RIPE3b2 factors not only recognize specific nucleotide sequence but also the recognize specific structure/organization of these nucleotides. The two critical nucleotide regions are spaced apart by about one helical turn of DNA, suggesting that RIPE3b1 and RIPE3b2 activators may recognize only one surface of the DNA helix. This is consistent with the results from methylation interference analysis of RIPE3b1 and RIPE3b2 complexes by Shieh and colleagues (14Shieh S-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar, 29Shieh S.Y. Stellrecht C.M.M. Tsai M.J. J. Biol. Chem. 1995; 270: 21503-21508Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), showing a strong interference at Gs −107, −108, and −111 and a weak interference at −114 bp. Furthermore, results from the methylation interference experiment demonstrated interference at Gs only on the bottom strand and not on the top strand. Our mutational analysis (Fig. 2) and transient transfection data (Fig. 9) are consistent with the importance of nucleotides −107, −108, and −111 bp in insulin gene expression. The role of weak interference at −114 bp is unclear, because −113 and −114 bp are not critical for binding nor gene expression (data not shown). Also, Zhao and co-workers (28Zhao L. Cissell M.A. Henderson E. Colbran R. Stein R. J. Biol. Chem. 2000; 275: 10532-10537Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) demonstrated that mutating −114 bp had no effect on insulin gene expression, suggesting that weak interference at −114 bp is not critical for binding of the RIPE3b1 factor and insulin gene expression. Our results demonstrate the requirement of a large binding region for the RIPE3b1 and the RIPE3b2 factors (−123 to −107 bp; Figs. 2 and 8); however, it is unclear why no interference was observed at Gs on the bottom strand at positions −117 and −120 bp.RIPE3b1 and RIPE3b2 factors have iden" @default.
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• W2054681277 title "Transcription Factors Recognizing Overlapping C1-A2 Binding Sites Positively Regulate Insulin Gene Expression" @default.
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