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• W2079701322 abstract "The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species. The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species. The differentiation, maintenance, regeneration, and repair of skeletal muscle requires ongoing interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory program controlled by myogenic transcription factors (1Lassar A. Münsterberg A. Curr. Opin. Cell Biol. 1994; 6: 432-442Crossref PubMed Scopus (143) Google Scholar, 2Naya F.J. Olson E. Curr. Opin Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (258) Google Scholar, 3McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 4Palacios D. Puri P.L. J. Cell. Physiol. 2006; 207: 1-11Crossref PubMed Scopus (105) Google Scholar). Among growth factors with major actions on muscle are the insulin-like growth factors IGF1 and IGF2 (5Glass D.J. Nat. Cell Biol. 2003; 5: 87-90Crossref PubMed Scopus (536) Google Scholar, 6Rotwein P. Growth Horm. IGF Res. 2003; 13: 303-305Crossref PubMed Scopus (21) Google Scholar), 3The abbreviations used are: IGFinsulin-like growth factorICRimprinting control regionAd-MyoDrecombinant adenovirus for MyoDAd-β-Galrecombinant adenovirus for β-galactosidaseDMdifferentiation medium. two closely related single-chain secreted proteins that bind with high affinity to the IGF1 receptor (7Nakae J. Kido Y. Accili D. Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar), leading to activation of several intracellular signal transduction pathways that act downstream of this membrane-spanning protein-tyrosine kinase (7Nakae J. Kido Y. Accili D. Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar). Much experimental evidence supports the importance of IGF actions in muscle. In mice, targeted IGF1 receptor deficiency caused marked muscle hypoplasia and neonatal death secondary to respiratory failure from severe muscle weakness (8Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2585) Google Scholar). In contrast, targeted overexpression of IGF1 stimulated an increase in muscle mass throughout life (9Barton-Davis E.R. Shoturma D.I. Musaro A. Rosenthal N. Sweeney H.L. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 15603-15607Crossref PubMed Scopus (603) Google Scholar), enhanced anabolic responses to exercise (10Paul A.C. Rosenthal N. J. Cell Biol. 2002; 156: 751-760Crossref PubMed Scopus (101) Google Scholar), and slowed the development of experimental muscular dystrophy (11Barton E.R. Morris L. Musaro A. Rosenthal N. Sweeney H.L. J. Cell Biol. 2002; 157: 137-148Crossref PubMed Scopus (399) Google Scholar). Moreover, analysis of quantitative trait loci for muscle in the pig identified a single-nucleotide polymorphism in an IGF2 intron that influenced IGF2 gene expression, with one variant being associated with a 3-fold greater abundance of IGF2 mRNA in muscle and a 3–4% increase in total muscle mass (12Van Laere A.S. Nguyen M. Braunschweig M. Nezer C. Collette C. Moreau L. Archibald A.L. Haley C.S. Buys N. Tally M. Andersson G. Georges M. Andersson L. Nature. 2003; 425: 832-836Crossref PubMed Scopus (710) Google Scholar). insulin-like growth factor imprinting control region recombinant adenovirus for MyoD recombinant adenovirus for β-galactosidase differentiation medium. The IGF2 gene resides on human chromosome 11p15.5 and on a syntenic segment of mouse chromosome 7 and is part of an imprinted cluster with the adjacent upstream insulin gene (Ins2 in mice (13Edwards C.A. Ferguson-Smith A.C. Curr. Opin. Cell Biol. 2007; 19: 281-289Crossref PubMed Scopus (319) Google Scholar)) and downstream H19 (13Edwards C.A. Ferguson-Smith A.C. Curr. Opin. Cell Biol. 2007; 19: 281-289Crossref PubMed Scopus (319) Google Scholar). In mice, the Igf2 gene is composed of six exons (14Rotwein P. Hall L.J. DNA Cell Biol. 1990; 9: 725-735Crossref PubMed Scopus (109) Google Scholar), and gene expression is regulated by three adjacent promoters, termed P1–P3, each with its own unique leader exon, whereas exons 4–6 encode the IGF2 precursor protein (14Rotwein P. Hall L.J. DNA Cell Biol. 1990; 9: 725-735Crossref PubMed Scopus (109) Google Scholar). The human IGF2 gene is more complicated, because it has an additional upstream promoter (15Rotwein P. Rosenfeld R.G. Roberts Jr., C.T. The IGF System. Humana Press, Totowa, NJ1999: 19-35Crossref Google Scholar). In both species, IGF2 is transcribed from the paternally derived chromosome in most tissues, and H19 is expressed from the maternal chromosome by regulation through an imprinting control region (ICR) located between the two genes (13Edwards C.A. Ferguson-Smith A.C. Curr. Opin. Cell Biol. 2007; 19: 281-289Crossref PubMed Scopus (319) Google Scholar, 16Wallace J.A. Felsenfeld G. Curr. Opin. Genet. Dev. 2007; 17: 400-407Crossref PubMed Scopus (330) Google Scholar). The ICR contains binding sites for the nuclear factor, CCTC binding factor (CTCF) (16Wallace J.A. Felsenfeld G. Curr. Opin. Genet. Dev. 2007; 17: 400-407Crossref PubMed Scopus (330) Google Scholar, 17Phillips J.E. Corces V.G. Cell. 2009; 137: 1194-1211Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar), which when bound to DNA in chromatin on the maternally derived chromosome facilitates H19 transcription by directing distal enhancers to the H19 promoter (16Wallace J.A. Felsenfeld G. Curr. Opin. Genet. Dev. 2007; 17: 400-407Crossref PubMed Scopus (330) Google Scholar, 17Phillips J.E. Corces V.G. Cell. 2009; 137: 1194-1211Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar). On the paternal chromosome, DNA in the ICR is methylated, and CTCF cannot bind, and the enhancers have access to the IGF2 promoters (16Wallace J.A. Felsenfeld G. Curr. Opin. Genet. Dev. 2007; 17: 400-407Crossref PubMed Scopus (330) Google Scholar, 17Phillips J.E. Corces V.G. Cell. 2009; 137: 1194-1211Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar). IGF2 gene transcription, mRNA production, and protein biosynthesis are induced as early events during muscle differentiation in culture (18Kou K. Rotwein P. Mol. Endocrinol. 1993; 7: 291-302PubMed Google Scholar, 19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and the secreted IGF2 functions as an autocrine differentiation-promoting factor (20Florini J.R. Magri K.A. Ewton D.Z. James P.L. Grindstaff K. Rotwein P.S. J. Biol. Chem. 1991; 266: 15917-15923Abstract Full Text PDF PubMed Google Scholar, 21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), as evidenced by impaired differentiation when IGF2 synthesis or access to the IGF1 receptor is blocked (19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and by accelerated and enhanced differentiation when IGF2 is overexpressed (20Florini J.R. Magri K.A. Ewton D.Z. James P.L. Grindstaff K. Rotwein P.S. J. Biol. Chem. 1991; 266: 15917-15923Abstract Full Text PDF PubMed Google Scholar, 22Stewart C.E. James P.L. Fant M.E. Rotwein P. J. Cell. Physiol. 1996; 169: 23-32Crossref PubMed Scopus (81) Google Scholar). The molecular mechanisms responsible for IGF2 gene activation during muscle differentiation are unknown, although the single nucleotide porcine IGF2 polymorphism associated with increased muscle mass appears to prevent binding of a putative transcriptional repressor to the IGF2 gene (12Van Laere A.S. Nguyen M. Braunschweig M. Nezer C. Collette C. Moreau L. Archibald A.L. Haley C.S. Buys N. Tally M. Andersson G. Georges M. Andersson L. Nature. 2003; 425: 832-836Crossref PubMed Scopus (710) Google Scholar, 23Markljung E. Jiang L. Jaffe J.D. Mikkelsen T.S. Wallerman O. Larhammar M. Zhang X. Wang L. Saenz-Vash V. Gnirke A. Lindroth A.M. Barrés R. Yan J. Strömberg S. De S. Pontén F. Lander E.S. Carr S.A. Zierath J.R. Kullander K. Wadelius C. Lindblad-Toh K. Andersson G. Hjälm G. Andersson L. PLoS Biol. 2009; 7: e1000256Crossref PubMed Scopus (125) Google Scholar, 24Butter F. Kappei D. Buchholz F. Vermeulen M. Mann M. EMBO Rep. 2010; 11: 305-311Crossref PubMed Scopus (47) Google Scholar). Here we characterize a conserved distal enhancer that interacts with the mouse Igf2 gene in differentiating myoblasts but not in mesenchymal progenitors and that can promote Igf2 gene transcription in muscle. DMEM, Superscript III first strand synthesis kit, TRIzol reagent, trypsin/EDTA solution, and horse serum were from Invitrogen, and FBS and newborn calf serum were from Hyclone (Logan, UT). Restriction enzymes, buffers, ligases, and polymerases were from New England Biolabs (Beverly, MA), BD Biosciences (Clontech), and Fermentas (Hanover, MD). Protease inhibitor tablets were purchased from Roche Applied Sciences; okadaic acid was from Alexis Biochemicals (San Diego, CA), sodium orthovanadate was from Sigma, and proteinase K was from Roche Applied Sciences. TransIT-LT-1 was from Mirus Corp. (Madison, WI), and Hoechst 33258 nuclear dye was from Polysciences (Warrington, PA). The BCA protein assay kit was from Pierce, Immobilon-FL was from Millipore Corporation (Billerico, MA), and AquaBlock tm/EIA/WIB solution was from East Coast Biologicals (North Berwick, ME). DNA purification kits were from Qiagen, and luciferase assay reagents were from Promega (Madison, WI). The following antibodies were from the Developmental Studies Hybridoma Bank: F5D (anti-myogenin, from W. E. Wright) and CT3 (anti-troponin T, from J. J-C. Lin). The polyclonal antibody to Akt was from Cell Signaling Technology (Beverly, MA). AlexaFluor 680-conjugated goat anti-mouse IgG was from Invitrogen, and IR800-conjugated goat anti-rabbit IgG was from Rockland (Gilbertsville, PA). All other chemicals were reagent grade and were purchased from commercial suppliers. The cells were incubated at 37 °C in humidified air with 5% CO2. C2 myoblasts (passages 4–10) were grown on gelatin-coated tissue culture dishes in DMEM with 10% heat-inactivated FBS and 10% newborn calf serum. At confluent density, the cells were washed, and low serum differentiation medium was added (differentiation medium (DM) was DMEM with 2% horse serum). C3H 10T1/2 mouse embryonic fibroblasts (catalogue number CCL226; ATCC, Manassas, VA) were incubated on gelatin-coated tissue culture dishes in growth medium (DMEM with 10% heat-inactivated FBS) at 37 °C in humidified air with 5% CO2. They were converted to myoblasts by infection at ∼50% of confluent density with a recombinant adenovirus for mouse MyoD (Ad-MyoD), as described (25Wilson E.M. Tureckova J. Rotwein P. Mol. Biol. Cell. 2004; 15: 497-505Crossref PubMed Scopus (58) Google Scholar), followed by incubation in DM after reaching confluent density as above. Male and female C57Bl6 mice were housed at the Oregon Health & Science University Animal Care Facility on a 12-h light/dark schedule with free access to food and water and received care according to institutional and National Institutes of Health guidelines. Pregnant mice were euthanized by exposure to CO2; the pups were isolated by Caesarean section and euthanized after exposure to CO2. Three-month-old male mice were euthanized by cervical dislocation. Freshly isolated tissues were flash-frozen in liquid nitrogen and pulverized prior to RNA extraction. The Oregon Health & Science University Animal Care and Use Committee approved all animal studies. Recombinant adenoviruses for MyoD (Ad-MyoD) and β-galactosidase (Ad-β-Gal) were purified on discontinuous cesium chloride gradients and titered by optical density, as described (25Wilson E.M. Tureckova J. Rotwein P. Mol. Biol. Cell. 2004; 15: 497-505Crossref PubMed Scopus (58) Google Scholar). Prior to infection, the viruses were diluted in DMEM plus 2% fetal calf serum, filtered through a Gelman syringe filter (0.45 μm), and then were added to cells at 37 °C for 120 min. After the addition of an equal volume of DMEM with 20% fetal bovine serum, the cells were incubated for a further 24 h. The cells then were washed twice with phosphate-buffered saline and incubated in DM. Whole cell and nuclear RNA were isolated as described (21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). RNA concentrations were determined spectrophotometrically at 260 nm and quality assessed by agarose gel electrophoresis. RNA (2.5 μg) was reverse transcribed in a final volume of 20 μl, with either oligo(dT) primers (for total RNA) or random hexamers (for nuclear RNA), and PCR was performed with 0.1 μl of cDNA and the primer pairs in Table 1. The linear range of product amplification was established in pilot studies for each primer pair, and the cycle number that reflected the approximate midpoint was used in final experiments. This varied from 20 to 27 cycles for total RNA and from 25 to 30 cycles for nuclear RNA. The results were visualized after electrophoresis through 1.0% agarose gels.TABLE 1Primers used for RT-PCRGeneLocationPrimer sequenceProductbpNuclear RNA Igf2Exon 35′-GCAAACTGGACATTAGCTTCT-3′597Intron 3–45′-CCCTTGGGTAACTAAAATCATCTT-3′ MyogeninIntron 2–35′-GGGATCACTCAGTCAGTGTTGTAA-3′537Exon 35′-TCTCTGCTTTAAGGAGTCAGCTAAA-3′ S17Exon 25′-ATCCCCAGCAAGAAGCTTCGGAACA-3′439Intron 2–35′-GAACCGACTTTGTCTCTACATCAAG-3′Total RNA Igf2Exon 15′-CAGCAGCTCCCACTTCATCCG-3′400Exon55′-TGGCACGGCTTGAAGGCCTGC-3 Igf2Exon 25′-CGGCCTCTGCGACTCGGGCAG-3′485Exon 55′-TGGCACGGCTTGAAGGCCTGC-3 Igf2Exon 35′-CCTGTGAGAACCTTCCAGCCT-3′396Exon 55′-TGGCACGGCTTGAAGGCCTGC-3 MyogeninExon 15′-GGGGACCCCTGAGCATTGTCC-3′512Exon 35′-CAGCCTGACAGACAATCTCAGTT-3′ Open table in a new tab Mouse myogenin and mouse Igf2 promoter-luciferase plasmids have been described (18Kou K. Rotwein P. Mol. Endocrinol. 1993; 7: 291-302PubMed Google Scholar). For these experiments, Igf2 P3 was inserted in plasmid pGL3 (Promega). DNA fragments pictured in Fig. 2 were isolated from mouse genomic DNA after PCR by standard methods, except for 4.3-kb element D, which was obtained from Dr. Jie Chen (University of Illinois, Urbana, IL). Region 1, CS6, and CS9 were cloned via 5′ SalI and 3′ XbaI linkers into the corresponding sites in the Igf2 P3 pGL3 plasmid (see supplemental Table S1 for details). All of the subfragments were generated by restriction enzyme digestion or PCR and were purified after preparative agarose gel electrophoresis by ion exchange chromatography (Qiaex II gel extraction kit; Qiagen) and subcloned into Igf2 promoter-luciferase plasmids. All of the DNA manipulations were confirmed by sequencing at the Oregon Health & Science University DNA Services Core. C2 and C3H 10T1/2 cells were plated onto gelatin-coated 12-well plates and were transfected at 50 or 25% of confluent density, respectively, with individual Igf2 promoter-luciferase reporter plasmids or with mouse myogenin promoter-luciferase (0.4 μg of plasmid DNA/well for C2 cells and 0.2 μg for 10T1/2 cells). C2 cell extracts were harvested 1 day later (undifferentiated), or DM was added, and cellular proteins were isolated after an additional 48 h (differentiated). For 10T1/2 cells, 1 day after transfection the cells were infected with Ad-MyoD or adenoviruses for β-galactosidase, and after an additional day in growth medium, the extracts were harvested (undifferentiated), or DM was added, and the cells were incubated for an additional 24 h before protein isolation (differentiated). Cell extracts from an individual experiment were stored at −80 °C until luciferase assay, and the results were normalized to cellular protein concentrations (21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). At least three experiments were performed for each promoter-reporter plasmid using duplicate transfections per experiment. Whole cell protein lysates were prepared as described (19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and stored in aliquots at −80 °C until use. Protein concentrations were determined with the BCA protein assay kit, and aliquots (25 μg/lane) were separated by SDS-PAGE, transferred to Immobilon-FL, blocked in AquaBlock, and incubated with primary and secondary antibodies, as described (21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The membranes were washed according to a protocol from LiCoR and scanned on an Odyssey Infrared Imaging System using v3.0 analysis software (LiCoR Biosciences, Lincoln, NE). Primary antibodies were used at the following dilutions: anti-myogenin (1:100), anti-troponin T (1:1000), and anti-Akt (1:2000). The cells were fixed, permeabilized, blocked, and incubated with antibodies as described (21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The primary antibodies were added in blocking buffer for 16 h at 4 °C (anti-troponin T, 1:1000 dilution; anti-myogenin, 1:50; and secondary antibodies at 1:1000). The images were captured with a Roper Scientific Cool Snap FX CCD camera attached to a Nikon Eclipse T300 fluorescent microscope using IP Labs 3.5 software. Hoechst staining was performed as described (21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Isolated nuclei from 1 × 107 cells were incubated overnight at 37 °C in 100 μl of AluI buffer (New England Biolabs; 50 mm NaCl, 10 mm Tris-Cl, 10 mm MgCl2, 1 mm DTT, pH 7.9) with 1 unit/μl of AluI. The nuclei incubated in the same buffer without AluI served as a negative control. Following the addition of 100 μl of 2× proteinase K digestion buffer (100 mm TrisCl, 200 mm NaCl, 2 mm Na2EDTA, 1% SDS, pH 7.5) for 2 h at 55 °C, 100 μl of AluI buffer plus 100 μl of 2× proteinase K buffer containing 100 μg of proteinase K were added and incubated overnight at 37 °C. DNA was isolated by extraction with a phenol-chloroform-isoamyl alcohol solution and ethanol precipitation and dissolved in 500 μl of 10 mm TrisCl, 1 mm Na2EDTA, pH 7.9. PCR was performed using 25–50 ng of this DNA per reaction (see Table 2 for primers). The linear range of product amplification was established for each primer pair in pilot studies, and the cycle number that reflected the approximate midpoint was used in the final experiments. This varied from 30 to 35 cycles. The results were visualized after electrophoresis through 1.0% agarose gels.TABLE 2Primers for restriction accessibility assayDNA fragmentDNA strandPrimer sequenceProductbpFragment 1Top5′-CTTCCAGACTCATCAAGAATA-3′299Bottom5′-GAACAACTGTGGGGACCAAAG-3′Fragment 2Top5′-ATTGCAGGCAGTGGGTGGA-3′300Bottom5′-ATAGAAATGCCTCTTAAGAGT-3′Fragment 3Top5′-GGCTTCCCGCCATCTCGA-3′285Bottom5′-TGGGGTTAGGAGCAGCTGT-3′Fragment 4Top5′-AAGGAGGATTTAGCTCGGGAG-3′399Bottom5′-CTGGGGTCCGGCTCACAT-3′Fragment 5Top5′-ATGTGACCCGGACCCCAGGCC-3′291Bottom5′-GACAGGCCTTGTGTTCTTGCA-3′ Open table in a new tab These studies followed published protocols (26Ling J.Q. Li T. Hu J.F. Vu T.H. Chen H.L. Qiu X.W. Cherry A.M. Hoffman A.R. Science. 2006; 312: 269-272Crossref PubMed Scopus (382) Google Scholar, 27Kurukuti S. Tiwari V.K. Tavoosidana G. Pugacheva E. Murrell A. Zhao Z. Lobanenkov V. Reik W. Ohlsson R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10684-10689Crossref PubMed Scopus (397) Google Scholar, 28Yoon Y.S. Jeong S. Rong Q. Park K.Y. Chung J.H. Pfeifer K. Mol. Cell. Biol. 2007; 27: 3499-3510Crossref PubMed Scopus (93) Google Scholar). The nuclei from 1 × 107 cells were fixed by the addition of 0.5 ml of lysis buffer (10 mm Hepes, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 1% Triton X-100, pH 7.9) plus protease inhibitors and 2% formaldehyde for 2–3 min at 15 °C and quenched by the addition of glycine to 0.125 m for 5 min on ice. Following two washes with ice-cold lysis buffer, the fixed nuclei were resuspended in 0.5 ml of 1.2× BglII restriction enzyme buffer (100 mm NaCl, 50 mm Tris-HCl, 10 mm MgCl2, 1 mm DTT, pH 7.9) containing 0.3% SDS and incubated at 37 °C for 1 h with shaking at 1000 rpm. Triton X-100 was added to a 1.8% final concentration, and samples were incubated for 1 h at 37 °C with shaking followed by the addition of 1500 units of BglII and incubation for 16 h at 37 °C and 1000 rpm. After the addition of SDS to 1.3% for 20 min at 65 °C, each sample was mixed with 7 ml of 1× DNA ligation buffer, Triton X-100 was added to 1%, and the nuclei were incubated for 1 h at 37 °C and 400 rpm. After equilibration at 16 °C, T4 DNA ligase was added (100 units), followed by incubation for 5 h at 16 °C with slow agitation. After sequential incubation with proteinase K (300 μg for 16 h at 65 °C) and RNase A (3 μl of a 100 mg/ml solution for 30 min at 37 °C), DNA was isolated by phenol-chloroform extraction and ethanol precipitation and dissolved in 1 ml of 10 mm TrisCl, 1 mm Na2EDTA, pH 7.9. PCRs were performed with 1 μl of DNA, and the primers are listed in Table 3. The linear range of product amplification was established for each primer pair in pilot studies using an artificial template that was generated by overlap extension PCR (29Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar), and the cycle number that reflected the approximate midpoint was used in the final experiments. This varied from 30 to 35 cycles. The results were visualized after electrophoresis through 1.2% agarose gels.TABLE 3Primers for chromatin conformation capture assayDNA fragmentPrimer sequenceEnhancer 15′-AAACAGCATCCTTAGCCTATGATG-3′Enhancer 25′-ACCAGCCTGGCTACCACCTG-3′Promoter 15′-TAGAGGGTTACAAGGTAGG-3′Promoter 25′-AGAGAGGCCAAACGTCATCGT-3′Enhancer 35′-ATGTGACCCGGACCCCAGGCC-3′Enhancer 45′-GACAGGCCTTGTGTTCTTGCA-3′ Open table in a new tab The data are presented as the means ± S.D. The statistical significance was determined using a paired Student's t test. The results were considered statistically significant when p < 0.05. Previous studies have found that IGF2 gene expression and protein secretion were induced within early stages of differentiation of skeletal myoblasts in culture (18Kou K. Rotwein P. Mol. Endocrinol. 1993; 7: 291-302PubMed Google Scholar, 19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Wilson E.M. Rotwein P. J. Biol. Chem. 2006; 281: 29962-29971Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and have shown that IGF2 actions, mediated by autocrine activation of the IGF1 receptor and the phosphatidylinositol 3-kinase-Akt pathway, are necessary to sustain muscle differentiation (19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 30Wilson E.M. Rotwein P. J. Biol. Chem. 2007; 282: 5106-5110Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In C3H110T1/2 mouse mesenchymal stem cells, adenoviral-mediated expression of the myogenic transcription factor, MyoD, can potently convert these uncommitted progenitors to a myoblast fate (31Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar), with rapid and robust up-regulation of muscle genes and proteins and myotube formation occurring once DM is added (19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) (Fig. 1, A–C). Under these conditions we find that transcription of the endogenous Igf2 gene is also rapidly stimulated after the addition of DM, with kinetics of activation very similar to those of the early differentiation gene myogenin (Fig. 1D). The Igf2 gene on mouse chromosome 7 is composed of six exons and five introns, and its transcription is governed by three tandem promoters, P1–P3, each of which regulate a unique leader exon (14Rotwein P. Hall L.J. DNA Cell Biol. 1990; 9: 725-735Crossref PubMed Scopus (109) Google Scholar). In mouse fetal tissues all three promoters are active (14Rotwein P. Hall L.J. DNA Cell Biol. 1990; 9: 725-735Crossref PubMed Scopus (109) Google Scholar). To examine promoter usage in Ad-MyoD-converted muscle cells, we developed a selective RT-PCR assay in which transcripts containing individual leader exons are assessed. In mouse fetal liver all three promoters are active, as evidenced by positive RT-PCR products with primers derived from each leader exon coupled to a shared coding exon primer, whereas in Ad-MyoD-converted 10T1/2 cells, only transcripts directed by P3 accumulate during muscle differentiation (Fig. 2). In addition, in mouse gastrocnemius muscle, mRNAs directed by P3 were more abundant than transcripts controlled by P1 or P2 (Fig. 2). Although it has been established that IGF2 is highly expressed in skeletal muscle in vivo (32DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar) and that its gene expression is induced during myoblast differentiation in culture (FIGURE 1, FIGURE 2 and Refs. 18Kou K. Rotwein P. Mol. Endocrinol. 1993; 7: 291-302PubMed Google Scholar, 19Wilson E.M. Hsieh M.M. Rotwein P. J. Biol. Chem. 2003; 278: 41109-41113Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, and 33Erbay E. Park I.H. Nuzzi P.D. Schoenherr C.J. Chen J. J. Cell Biol. 2003; 163: 931-936Crossref PubMed Scopus (127) Google Scholar), very little is known about the mechanisms of Igf2 gene regulation in muscle. No transcriptional response elements have been identified, and no transcription factors have been characterized. Studies using transgenic mice have been employed to investigate aspects of regulation of the Igf2-H19 locus and have defined several chromosomal segments that could direct gene activity to mesenchymal tissues, including muscle (34Ainscough J.F. John R.M. Barton S.C. Surani M.A. Development. 2000; 127: 3923-3930Crossref PubMed Google Scholar, 35Ishihara K. Hatano N. Furuumi H. Kato R. Iwaki T. Miura K. Jinno Y. Sasaki H. Genome Res. 2000; 10: 664-671Crossref PubMed Scopus (73) Google Scholar, 36Kaffer C.R. Srivastava M. Park K.Y. Ives E." @default.
• W2079701322 created "2016-06-24" @default.
• W2079701322 creator A5017687289 @default.
• W2079701322 creator A5042933624 @default.
• W2079701322 creator A5090842769 @default.
• W2079701322 date "2010-12-01" @default.
• W2079701322 modified "2023-10-06" @default.
• W2079701322 title "Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation" @default.
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