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• W2065431340 abstract "The transcription factor Pdx1 is expressed in the pancreatic β-cell, where it is believed to regulate several β-cell-specific genes. Whereas binding by Pdx1 to elements of β-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which β-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from β-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using real-time PCR. We compared these in vivofindings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in β-cells. In contrast, in in vitroimmunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in β-cells and non-β-cells. The transcription factor Pdx1 is expressed in the pancreatic β-cell, where it is believed to regulate several β-cell-specific genes. Whereas binding by Pdx1 to elements of β-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which β-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from β-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using real-time PCR. We compared these in vivofindings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in β-cells. In contrast, in in vitroimmunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in β-cells and non-β-cells. glucose transporter type 2 electrophoretic mobility shift assay chromatin immunoprecipitation islet amyloid polypeptide base pair(s) The expression of genes in a cell type-specific manner is largely dependent upon the restricted expression patterns of the transcription factors that control those genes. Nowhere is cell type-specific gene expression more evident than in the β-cells of the pancreatic islets of Langerhans, where several genes including insulin, glucokinase, glucose transporter type 2 (Glut2),1 and islet amyloid polypeptide (IAPP) demonstrate β-cell-specific expression patterns. Although a network of transcription factors likely directs the overall expression of these β-cell genes (1.Edlund H. Diabetes. 2001; 50 (Suppl. 1): 5-9Crossref Google Scholar, 2.Habener J.F. Stoffers D.A. Proc. Assoc. Am. Physicians. 1998; 110: 12-21PubMed Google Scholar, 3.Madsen O.D. Jensen J. Petersen H.V. Pedersen E.E. Oster A. Andersen F.G. Jorgensen M.C. Jensen P.B. Larsson L.I. Serup P. Horm. Metabol. Res. 1997; 29: 265-270Crossref PubMed Scopus (77) Google Scholar, 4.Sander M. German M.S. J. Mol. Med. 1997; 75: 327-340Crossref PubMed Scopus (282) Google Scholar), one particular factor, Pdx1 (also known as STF1, IDX1, and IPF1 (5.Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 6.Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (760) Google Scholar)), has recently emerged as one of the most important candidates orchestrating β-cell gene regulation. Pdx1 expression has been demonstrated to occur in most cell types that comprise the developing pancreas (5.Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 7.Peshavaria M. Gamer L. Henderson E. Teitelman G. Wright C.V. Stein R. Mol. Endocrinol. 1994; 8: 806-816Crossref PubMed Scopus (138) Google Scholar), and as such, targeted disruption of the Pdx1 gene in mice results in animals that lack pancreas formation (8.Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1546) Google Scholar, 9.Offield 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: 983-995Crossref PubMed Google Scholar). However, Pdx1 expression in the mature pancreas is most prominent in the β-cell (6.Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (760) Google Scholar, 7.Peshavaria M. Gamer L. Henderson E. Teitelman G. Wright C.V. Stein R. Mol. Endocrinol. 1994; 8: 806-816Crossref PubMed Scopus (138) Google Scholar), where it is believed to regulate several β-cell-specific genes. For example, the β-cell-specific disruption of the mouse Pdx1 gene results in animals that develop late-onset diabetes with impairments in insulin, IAPP, and Glut2 expression (10.Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (765) Google Scholar, 11.Lottmann H. Vanselow J. Hessabi B. Walther R. J. Mol. Med. 2001; 79: 321-328Crossref PubMed Scopus (64) Google Scholar). In addition, several functional studies (transient and stable transfections into cell lines, followed by reporter gene analysis) have addressed the role of Pdx1 in β-cell gene regulation; taken together, these studies point to an important role for Pdx1 in the regulation of the insulin (10.Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (765) Google Scholar, 12.Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar, 13.Peers B. Leonard J. Sharma S. Teitelman G. Montminy M.R. Mol. Endocrinol. 1994; 8: 1798-1806PubMed Google Scholar), Glut2 (11.Lottmann H. Vanselow J. Hessabi B. Walther R. J. Mol. Med. 2001; 79: 321-328Crossref PubMed Scopus (64) Google Scholar, 14.Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (321) Google Scholar), IAPP (10.Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (765) Google Scholar, 15.Wang H. Maechler P. Ritz-Laser B. Hagenfeldt K.A. Ishihara H. Philippe J. Wollheim C.B. J. Biol. Chem. 2001; 276: 25279-25286Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 16.Macfarlane W.M. Campbell S.C. Elrick L.J. Oates V. Bermano G. Lindley K.J. Aynsley-Green A. Dunne M.J. James R.F. Docherty K. J. Biol. Chem. 2000; 275: 15330-15335Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17.Watada H. Kajimoto Y. Kaneto H. Matsuoka T. Fujitani Y. Miyazaki J. Yamasaki Y. Biochem. Biophys. Res. Commun. 1996; 229: 746-751Crossref PubMed Scopus (84) Google Scholar), glucokinase (18.Watada H. Kajimoto Y. Miyagawa J. Hanafusa T. Hamaguchi K. Matsuoka T. Yamamoto K. Matsuzawa Y. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1826-1831Crossref PubMed Google Scholar, 19.Watada H. Kajimoto Y. Umayahara Y. Matsuoka T. Kaneto H. Fujitani Y. Kamada T. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1478-1488Crossref PubMed Google Scholar), Pdx1 (20.Gerrish K.E. Cissell M.A. Stein R. J. Biol. Chem. 2001; 276: 47775-47784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), Pax4 (21.Smith S.B. Watada H. Scheel D.W. Mrejen C. German M.S. J. Biol. Chem. 2000; 275: 36910-36919Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and glucagon (10.Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (765) Google Scholar, 11.Lottmann H. Vanselow J. Hessabi B. Walther R. J. Mol. Med. 2001; 79: 321-328Crossref PubMed Scopus (64) Google Scholar, 15.Wang H. Maechler P. Ritz-Laser B. Hagenfeldt K.A. Ishihara H. Philippe J. Wollheim C.B. J. Biol. Chem. 2001; 276: 25279-25286Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) genes. Whereas the functional data described above are important in establishing a link between Pdx1 and the expression of specific genes, these studies do not address the issue of whether Pdx1 is involved in a transcriptional complex that directly controls the expression of these genes. Only circumstantial evidence suggests direct involvement; specific elements within the promoter regions of these β-cell-specific genes have been demonstrated to be bound by Pdx1 in in vitro electrophoretic mobility shift assays (EMSAs) (12.Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar,14.Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (321) Google Scholar, 17.Watada H. Kajimoto Y. Kaneto H. Matsuoka T. Fujitani Y. Miyazaki J. Yamasaki Y. Biochem. Biophys. Res. Commun. 1996; 229: 746-751Crossref PubMed Scopus (84) Google Scholar, 19.Watada H. Kajimoto Y. Umayahara Y. Matsuoka T. Kaneto H. Fujitani Y. Kamada T. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1478-1488Crossref PubMed Google Scholar, 20.Gerrish K.E. Cissell M.A. Stein R. J. Biol. Chem. 2001; 276: 47775-47784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 21.Smith S.B. Watada H. Scheel D.W. Mrejen C. German M.S. J. Biol. Chem. 2000; 275: 36910-36919Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Pdx1 is a homeodomain-containing transcription factor and demonstrates sequence-specific DNA binding (to TAAT-containing motifs), similar to the homeobox genes of Drosophila (22.Draganescu A. Tullius T.D. J. Mol. Biol. 1998; 276: 529-536Crossref PubMed Scopus (18) Google Scholar,23.Ekker S.C. Young K.E. von Kessler D.P. Beachy P.A. EMBO J. 1991; 10: 1179-1186Crossref PubMed Scopus (130) Google Scholar). Because Pdx1 functions as an activator of gene transcription, it is therefore believed to activate gene expression by binding to upstream TAAT sequences (24.Peshavaria M. Henderson E. Sharma A. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 3987-3996Crossref PubMed Scopus (84) Google Scholar). Critical evidence that is lacking to establish Pdx1 as an immediate regulator of β-cell gene expression includes the demonstration that Pdx1 is physically associated with β-cell gene promoters in vivo. An emerging theme in transcriptional regulation is the influence of chromatin structure within the nuclear environment on both DNA binding by transcription factors as well as the resultant regulation of transcription (25.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2352) Google Scholar, 26.Spencer V.A. Davie J.R. Gene. 1999; 240: 1-12Crossref PubMed Scopus (265) Google Scholar). The highly compacted nature of chromatin could conceivably hinder accessibility to some DNA binding sites but not to others, notwithstanding that these sites may display equal affinity for a given factor in vitro (27.Anderson J.D. Lowary P.T. Widom J. J. Mol. Biol. 2001; 307: 977-985Crossref PubMed Scopus (127) Google Scholar, 28.Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6725) Google Scholar). Thus, we cannot necessarily predict from studies in vitro which promoters Pdx1 binds in vivo. To determine which promoters Pdx1 might directly regulate in vivo, we performed chromatin immunoprecipitation (ChIP) assays using Pdx1 antiserum and chromatin from various cell lines and followed this by quantitative real-time PCR to determine the relative distribution of co-immunoprecipitated promoters. We performed these studies in vivo using a non-β-cell line (NIH3T3) and a β-cell line (β-TC3) as well as in an in vitro setting for comparison. We demonstrate that Pdx1 is associated with several, but not all, β-cell-specific genes in vivo. We show further that there is a significant difference in the binding distribution of Pdx1 to promoters in vitro and in vivo as well as in β-cells and non-β-cells. Our findings suggest a model in which the chromatin structure of a given cell type plays an important role in restricting target promoter binding by Pdx1. The mouse cell lines β-TC3 and α-TC1.6 were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 15% horse serum, 2.5% fetal bovine serum, and 1% penicillin/streptomycin. The mouse fibroblast cell line, NIH3T3, was maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 1% penicillin/streptomycin. For transient mammalian cell transfections, 5 × 105 NIH3T3 cells were plated on 10-cm plates 1 day before transfection. A total of 8 μg of either the cytomegalovirus promoter-driven expression vector pBAT12Pdx1 (12.Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar), or the parent vector without insert (pBAT12) was mixed with 12 μl of Transfast® reagent (Promega), and transfections were performed according to the manufacturer's protocol. Cells were harvested for chromatin immunoprecipitation assays or Western blots ∼48 h later. Single-stranded oligonucleotide probes were 5′ end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. Labeled oligonucleotides were column-purified and annealed to an excess of complementary strand. EMSA buffers and conditions were described previously (29.German M.S. Moss L.G. Wang J. Rutter W.J. Mol. Cell. Biol. 1992; 12: 1777-1788Crossref PubMed Google Scholar). Where supershift assays were performed, 1 μl of Pdx1 antiserum (provided by Dr. M. German) was also added. Oligonucleotide fragments were derived from elements of each promoter known to be important in the transcriptional regulation of that gene and that contained a Pdx1 binding motif (TAAT). The albumin promoter oligonucleotide was selected based entirely upon the presence of a TAAT motif. The following oligonucleotide probes were used (top strands shown): mouse I insulin promoter (A3/A4 element (12.Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar,30.German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. et al.Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar)), 5′-CTTATTAAGACTATAATAACCCTAAGACTA-3′; mutated mouse I insulin promoter (A3/A4 element), 5′-CTTACTAAGACTATAGTAACCCTAAGACTA-3′; mouse Glut2 promoter (Glut2TAAT motif (14.Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (321) Google Scholar)), 5′-ATACACTGACTTAATAATAACAGTAGAAAG-3′; mouse glucokinase promoter (UPE1 element (31.Shelton K.D. Franklin A.J. Khoor A. Beechem J. Magnuson M.A. Mol. Cell. Biol. 1992; 12: 4578-4589Crossref PubMed Scopus (60) Google Scholar)), 5′-GAACAAAACCCCATTATTTACAGATGAGAA-3′; mouse albumin promoter, 5′-TGAAGCTCAGGTTTAATTCCCAGTCACAT-3′; mouse glucagon promoter (G1 element (32.Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar)), 5′-GAACAAAACCCCATTATTTACAGATGAGAA-3′. Chromatin immunoprecipitation assays were performed as described in the literature (33.Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (479) Google Scholar) with some modifications. A total of 1 × 107 NIH3T3 cells (from 3 confluent 10-cm plates) or 2.5 × 107 β-TC3 or α-TC1.6 cells (from 2 confluent 10-cm plates) were treated with 1% formaldehyde by adding 0.27 ml of 37% formaldehyde directly to 10 ml of culture medium. After incubating in formaldehyde for 10 min at room temperature, glycine was added to a final concentration of 0.125 m. The cells were then suspended in 0.6 ml of lysis buffer (50 mm Tris-Cl, pH 8.1, containing 1% Triton X-100, 0.1% deoxycholate, 150 mm NaCl, and 5 mm EDTA) plus protease inhibitors (leupeptin, phenylmethylsulfonyl fluoride, and aprotinin) and subjected to sonication (using a Fisher Scientific model 60 sonic dismembrator with a microtip at a setting of 10). Ten 5-s sonication pulses were required for NIH3T3 cells, and 15 5-s pulses were required for β-TC3 and α-TC1.6 cells to shear chromatin to 1000-bp fragments. The effectiveness of shearing was confirmed by incubating a 10-μl aliquot of the extract at 65 °C for 3 h (to reverse cross-links) and subsequently subjecting it to electrophoresis on a 1% agarose gel. 0.25-ml aliquots of the clarified extracts were diluted to 1 ml in lysis buffer containing protease inhibitors and then incubated with either 5 μl of anti-Pdx1 antiserum or normal rabbit serum (25 μl was also divided into separate aliquots and stored for later PCR analysis as 10% of the input extract). Incubations occurred overnight at 4 °C on a rocking platform, after which 40 μl of protein A-agarose slurry (Santa Cruz Biotechnology) and 2 μl of a 10 mg/ml herring sperm DNA solution (Sigma) were added, and incubation was continued an additional 1 h. The agarose was pelleted by centrifugation, and the pellets were washed consecutively with 1 ml of lysis buffer, lysis buffer plus 500 mm NaCl, lysis buffer plus 0.25 m LiCl, and Tris/EDTA. DNA and protein were eluted from the pellets by incubating the pellets 2 times in 0.25 ml of elution buffer (0.1 mNaHCO3 with 1% SDS and 20 μg/ml herring sperm DNA, and protein-DNA cross-links were reversed by incubating at 65 °C for 3 h. DNA and protein were ethanol-precipitated overnight at −20 °C. The precipitated samples were pelleted and dissolved in proteinase K buffer (10 mm Tris-Cl, pH 7.5 with 1% SDS) and incubated with 1 μg of proteinase K (Roche Molecular Biochemicals) for 1 h at 55 °C. The samples were extracted once with phenol/chloroform and ethanol-precipitated overnight at −20 °C. Samples were pelleted, washed with 70% ethanol, and dissolved in 100 μl of Tris/EDTA. 3-μl aliquots were used for each real time PCR reaction to quantitate co-immunoprecipitated promoter fragments (see below). In vitro reactions were set up similar to EMSA reactions. Briefly, each reaction consisted of 10 μg of poly(dI-dC), 100 μg bovine serum albumin, 1 fmol of each promoter, and 10 μg of either pBAT12- or pBAT12Pdx1-transfected NIH3T3 nuclear extract in a 200 μl solution of 10 mm HEPES, pH 7.9 with 75 mm KCl. The concentration of promoters in the reaction (5 × 10−12m each) was chosen such that the percentage of any given promoter that was immunoprecipitated was in the range 0.1–1.0% of the input quantity (to match the results obtained in the ChIP assays). The promoters used in these reactions (ranging in size from 100 to 250 bp) were excised from their respective pCR2.1 vectors by digestion with EcoRI (see below for details on the construction of these vectors). The reactions were allowed to proceed at room temperature for 15 min, after which formaldehyde was added to a final concentration 1%, and the reactions were incubated an additional 5 min. Glycine was then added to a final concentration of 0.125 m. 90-μl aliquots were removed and diluted to 1 ml using lysis buffer and then incubated with either 5 μl of anti-Pdx1 antiserum or 5 μl of normal rabbit serum (9 μl was also divided in to separate aliquots and stored for later PCR analysis at 10% of input reaction). From this point onward, samples were treated identically to the chromatin immunoprecipitation samples. For standard PCR reactions, 1 μl of sonicated NIH3T3 DNA was used as template in 25-μl reactions containing 3 mm MgCl2, 0.25 mmdNTPs, 10 pmol of primers, and 0.5 units of Platinum®Taq DNA polymerase (Invitrogen). Thermal cycling was performed using an iCycler® (Bio-Rad). Cycling parameters for all amplifications were (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s) × 30 cycles. Amplified promoter fragments were subcloned into the T/A cloning vector pCR2.1 (Invitrogen), and 3–4 clones from each PCR reaction were sequenced to confirm the identity of the amplified fragment. Primers for the PCR reactions are presented in Table I.Table IIdentification of the promoter fragments and their corresponding primer sequences used for PCR amplificationPromoterAmplified regionaNumber of Pdx1 binding sites (TAAT)ReferencePrimer sequencesAlbuminDistal TAAT-containing region2(GenBank™ accession5′-TGGGAAAACTGGGAAAACCATC-3′ no.AF2558025′-CACTCTCACACATACACTCCTGCTG-3′Mouse I−126 to −296 (contains A3/A4 element)2(30.German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. et al.Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar)5′-TCAGCCAAAGATGAAGAAGGTCTC-3′ insulin5′-TCCAAACACTTGCCTGGTGC-3′Glut2−523 to −738 (contains Glut2TAAT motif)3(14.Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (321) Google Scholar)5′-ATCTGGCTCCGCACTCTCATTCTTG-3′5′-CCCTGTGACTTTTCTGTGTTCTTAGG-3′IAPP−97 to −1902(63.Ekawa K. Nishi M. Ohagi S. Sanke T. Nanjo K. J. Mol. Endocrinol. 1997; 19: 79-86Crossref PubMed Scopus (9) Google Scholar)5′-TCACCCACACAAAGGCACTCAG-3′5′-GGTTTCATTGGCAGATGGAGC-3′Glucagon−106 to −231 (contains G1 element)0(64.Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (115) Google Scholar)5′-TCCAAACTGCCCTTTCCATTC-3′ (region 1)5′-ATGATTTCACTCGCCCACTCAC-3′Glucagon+70 to −148 (contains G1 element2(64.Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (115) Google Scholar)5′-CGTAAAAAGCAGATGAGCAAAGTG-3′ (region 2)5′-GAACAGGTGTAGACAGAGGGAGTCC-3′Pax4−1841 to −1966 (contains β-cell-specific enhancer)4(65.Xu W. Murphy L.J. Mol. Cell. Endocrinol. 2000; 170: 79-89Crossref PubMed Scopus (13) Google Scholar)5′-CCAACGATCCAGGCTCTACATC-3′5′-CGGGTTTGGGGCTAATTGTCC-3′Pdx1−2471 to −2598 (contains area 1)2(66.Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar)5′-TGGCTCGGGAAGGCTCTTG-3′5′-CCATCAGGTGGCTAAATCCATTATG-3′Glucokinase−112 to −255 (contains the UPE1 element)2(31.Shelton K.D. Franklin A.J. Khoor A. Beechem J. Magnuson M.A. Mol. Cell. Biol. 1992; 12: 4578-4589Crossref PubMed Scopus (60) Google Scholar)5′-TGATAGGCACCAAGGCACTGAC-3′5′-GCAGAAAACTGGGACTGATTGC-3′a Where indicated, the numbers represent the position of the amplified fragment relative to the known transcriptional start sitedot for the gene. Open table in a new tab a Where indicated, the numbers represent the position of the amplified fragment relative to the known transcriptional start sitedot for the gene. Quantitative real-time PCR reaction conditions were identical to the standard PCR reactions, except that each reaction contained 3 μl of template DNA (from chromatin or in vitroimmunoprecipitations) and a 1:75,000 dilution of SYBR Green I dye (Molecular Probes). Real-time PCR reactions (for a total of 40 cycles) were performed using an iCycler IQ® (Bio-Rad), and continuous SYBR Green I monitoring was done according to the manufacturer's recommendations. The relative proportions of co-immunoprecipitated promoter fragments were determined based on the threshold cycle (TC) value for each PCR reaction. The TC value is determined as the cycle at which fluorescence rises 10 times above the mean S.D. of background levels in all reaction wells. Real time PCR data analysis followed the methodology described in a recent report (34.Christenson L.K. Stouffer R.L. Strauss 3rd., J.F. J. Biol. Chem. 2001; 276: 27392-27399Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). For every promoter studied, a ΔTC value was calculated for each sample by subtracting the TC value for the input (to account for differences in amplification efficiencies and DNA quantities before immunoprecipitation) from the TCvalue obtained for the immunoprecipitated sample. A ΔΔTC value was then calculated by subtracting the ΔTC value for the sample immunoprecipitated with Pdx1 antiserum from the ΔTC value for the corresponding control sample immunoprecipitated with normal rabbit serum. Fold differences (Pdx1 ChIP relative to control ChIP) were then determined by raising 2 to the ΔΔTC power. The equation used in these calculations is summarized as fold difference (Pdx1 ChIP relative to control ChIP) = 2[TC(control) −TC(Pdx1)], where TC =TC(immunoprecipitated sample)TC(input). TC values for immunoprecipitated samples ranged from 25 to 30, whereas the TC values for the 10% input samples ranged from 18 to 24; thus, recovery of promoters in immunoprecipitated samples ranged from 0.1 to 1% of the input values. For every promoter fragment analyzed, each sample was quantitated in duplicate on at least two separate occasions and from at least two (and in many cases, three) independent immunoprecipitations to give a minimum of four independent quantitations of each sample for each promoter. Mean ± S.D. values were determined for each fold difference, and these values were subsequently used in two-tailed paired t tests to determine statistical significance, as reported in the figures. A melt curve analysis was performed for each sample after PCR amplification to ensure that a single product of expected melt curve characteristics was obtained. Nuclear extracts from NIH3T3, β-TC3, and α-TC1.6 cells were prepared from single, confluent 10-cm plates of cells according to methods described previously (35.Sadowski H.B. Gilman M.Z. Nature. 1993; 362: 79-83Crossref PubMed Scopus (234) Google Scholar). 5 μg of nuclear extract were subject to Western blot analysis after electrophoresis on a 4–20% gradient SDS-polyacrylamide gel using Pdx1 antiserum. Western blots were visualized using the ECL-Plus® system (Amersham Biosciences). Western blots from immunoprecipitated samples proceeded similarly, except that 20 μl of protein after elution (in 100 μl of Laemmli buffer) from the protein A-agarose was used in the analysis. Several studies document the sequence-specific DNA binding properties of Pdx1 (12.Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar, 13.Peers B. Leonard J. Sharma S. Teitelman G. Montminy M.R. Mol. Endocrinol. 1994; 8: 1798-1806PubMed Google Scholar, 36.Ohlsson H. Thor S. Edlund T. Mol. Endocrinol. 1991; 5: 897-904Crossref PubMed Scopus (95) Google Scholar). To demonstrate that Pdx1 can bind to key promoter elements from a variety of β-cell-specific genes, we performed EMSAs (Fig. 1) using Pdx1 protein from transfected NIH3T3 nuclear extract and duplexed oligonucleotide fragments corresponding to known regulatory elements of the insulin, Glut2, and glucokinase genes. Fig. 1 shows that Pdx1 binds to upstream promoter elements from these genes and also demonstrates that when the TAAT sequences in the insulin promoter are mutated by a single base pair (TAAT to TAGT), DNA binding by Pdx1 is completely abolished (Fig. 1, lanes 4 and 5). Fig. 1 also shows that Pdx1 is capable of binding to promoter elements from genes not expressed in the β-cell (Albumin and Glucagon, lanes 8–11), suggesting that Pdx1 binding to DNA elements in vitro is dependent primarily upon the presence of binding sites. Importantly, it should be noted that variations do exist in the extent of binding to each of these promoter elements, due presumably to variations in the DNA sequences that flank the TAAT motif (37.Catron K.M. Iler N. Abate C. Mol. Cell. Biol. 1993; 13: 2354-2365Crossref PubMed Scopus (177) Google Scholar). The data in Fig. 1 are unchanged when Pdx1 protein is obtained from β-cell nuclear extract (β-TC3 cells) or from an in vitro translation system using rabbit reticulocyte lysate (data not shown). Because the data in Fig. 1 demonstrate poor promoter selectivity by Pdx1 in vitro, we sought to determine the distribution of Pdx1 promoter binding in vivo by use of the ChIP assay. The ChIP assay is a recently developed method that has typically been used to assess the association of abundant histone complexes with specific genes (33.Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (479) Google Scholar). When the ChIP assay is coupled to PCR-based detection methods, however, the association of far less abundant transcription factor complexes with specific genes can be reliably assessed (38.Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell. Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (207) Google Scholar). Fig. 2a shows the general scheme of our ChIP assays. Assays were performed using chromatin from three cell types: (a) NIH3T3 cells (a mouse fibroblast-derived cell line, which serves as a non-β-cell line control for our studies) transfected with either a vector containing the Pdx1 cDNA (referred to as “NIH3T3/Pdx1”) or the empty vector (referred to as “NIH3T3/EV”), (b) β-TC3 cells, an insulin-secreting mouse β-cell-derived cell line (39.Efrat S. Linde S. Kofod H. Spector D. Delannoy M. Grant S. Hanahan D. Baekkeskov S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 90" @default.
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• W2065431340 title "Quantitative Assessment of Gene Targeting in Vitroand in Vivo by the Pancreatic Transcription Factor, Pdx1" @default.
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