Histone Code Modifications Repress Glucose Transporter 4 Expression in the Intrauterine Growth-restricted Offspring
2008; Elsevier BV; Volume: 283; Issue: 20 Linguagem: Inglês
10.1074/jbc.m800128200
ISSN1083-351X
AutoresNupur Raychaudhuri, Santanu Raychaudhuri, Manikkavasagar Thamotharan, Sherin U. Devaskar,
Tópico(s)Genetic Syndromes and Imprinting
ResumoWe examined transcriptional and epigenetic mechanism(s) behind diminished skeletal muscle GLUT4 mRNA in intrauterine growth-restricted (IUGR) female rat offspring. An increase in MEF2D (inhibitor) with a decline in MEF2A (activator) and MyoD (co-activator) binding to the glut4 promoter in IUGR versus control was observed. The functional role of MEF2/MyoD-binding sites and neighboring three CpG clusters in glut4 gene transcription was confirmed in C2C12 muscle cells. No differential methylation of these three and other CpG clusters in the glut4 promoter occurred. DNA methyltransferase 1 (DNMT1) in postnatal, DNMT3a, and DNMT3b in adult was differentially recruited with increased MeCP2 (methyl CpG-binding protein) concentrations to bind the IUGR glut4 gene. Covalent modifications of the histone (H) code consisted of H3.K14 de-acetylation by recruitment of histone deacetylase (HDAC) 1 and enhanced association of HDAC4 enzymes. This set the stage for Suv39H1 methylase-mediated di-methylation of H3.K9 and increased recruitment of heterochromatin protein 1α, which partially inactivates postnatal and adult IUGR glut4 gene transcription. Further increased interactions in the adult IUGR between DNMT3a/DNMT3b and HDAC1 and MEF2D and HDAC1/HDAC4 and decreased association between MyoD and MEF2A existed. We conclude that epigenetic mechanisms consisting of histone code modifications repress skeletal muscle glut4 transcription in the postnatal period and persist in the adult female IUGR offspring. We examined transcriptional and epigenetic mechanism(s) behind diminished skeletal muscle GLUT4 mRNA in intrauterine growth-restricted (IUGR) female rat offspring. An increase in MEF2D (inhibitor) with a decline in MEF2A (activator) and MyoD (co-activator) binding to the glut4 promoter in IUGR versus control was observed. The functional role of MEF2/MyoD-binding sites and neighboring three CpG clusters in glut4 gene transcription was confirmed in C2C12 muscle cells. No differential methylation of these three and other CpG clusters in the glut4 promoter occurred. DNA methyltransferase 1 (DNMT1) in postnatal, DNMT3a, and DNMT3b in adult was differentially recruited with increased MeCP2 (methyl CpG-binding protein) concentrations to bind the IUGR glut4 gene. Covalent modifications of the histone (H) code consisted of H3.K14 de-acetylation by recruitment of histone deacetylase (HDAC) 1 and enhanced association of HDAC4 enzymes. This set the stage for Suv39H1 methylase-mediated di-methylation of H3.K9 and increased recruitment of heterochromatin protein 1α, which partially inactivates postnatal and adult IUGR glut4 gene transcription. Further increased interactions in the adult IUGR between DNMT3a/DNMT3b and HDAC1 and MEF2D and HDAC1/HDAC4 and decreased association between MyoD and MEF2A existed. We conclude that epigenetic mechanisms consisting of histone code modifications repress skeletal muscle glut4 transcription in the postnatal period and persist in the adult female IUGR offspring. Pre- and postnatal nutritional deficiency culminating in intrauterine and postnatal growth restriction (IUGR) 2The abbreviations used are: IUGR, intrauterine and postnatal growth restriction; SM, skeletal muscle; CON, control; DNMT1, DNA methyltransferase 1; HDAC, histone deacetylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; MUT, mutated; ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; EMSA, electromobility shift assay; Pipes, 1,4-piperazinediethanesulfonic acid. 2The abbreviations used are: IUGR, intrauterine and postnatal growth restriction; SM, skeletal muscle; CON, control; DNMT1, DNA methyltransferase 1; HDAC, histone deacetylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; MUT, mutated; ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; EMSA, electromobility shift assay; Pipes, 1,4-piperazinediethanesulfonic acid. leads to insulin resistance, a forerunner of gestational and type 2 diabetes mellitus (1Holemans K. 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These changes include the following: 1) decline in skeletal muscle GLUT4 mRNA and protein concentrations in rats (4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar, 6Ozanne S.E. Jensen C.B. Tingey K.J. Storgaard H. Madsbad S. Vaag A.A. Diabetologia. 2005; 48: 547-552Crossref PubMed Scopus (240) Google Scholar, 14Agote M. Goya L. Ramos S. Alvarez C. Gavette M.L. Pascual-Leone A.M. Escriva F. Am. J. Physiol. 2001; 281: E1101-E1109PubMed Google Scholar) and human (6Ozanne S.E. Jensen C.B. Tingey K.J. Storgaard H. Madsbad S. Vaag A.A. Diabetologia. 2005; 48: 547-552Crossref PubMed Scopus (240) Google Scholar), suggesting aberrations in transcriptional control; and 2) insulin resistance of post-translational translocation of GLUT4 from intracellular vesicles to plasma membrane (4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar). Although we have previously determined the molecular basis for the latter (15Oak S.A. Tran C. Pan G. Thamotharan M. Devaskar S.U. Am. J. Physiol. 2006; 290: E1321-E1330Crossref PubMed Scopus (39) Google Scholar), the molecular mechanisms responsible for the former remain to be investigated. Transgenic investigations established the crucial in vivo role for conserved glut4 promoter regions in skeletal muscle gene expression. Disruption of the myocyte enhancer factor 2 (MEF2)-binding site (–473 to –464 bp) ablated tissue-specific glut4 expression in transgenic mice (16Thai M.V. Guruswamy S. Cao K.T. Pessin J.E. Olson A.L. J. Biol. Chem. 1998; 273: 14285-14292Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 17Liu M.L. Olson A.L. Edgington N.P. Moye-Rowley W.S. Pessin J.E. J. Biol. Chem. 1994; 285: 28514-28521Abstract Full Text PDF Google Scholar). MyoD on the other hand is responsible for glut4 expression in vitro during myoblast to myocyte differentiation (18Moreno H. Serrano A.L. Santalucia T. Guma A. Canto C. Brand N.J. Palacin M. Schiaffino S. Zorzano A. J. Biol. Chem. 2003; 278: 40557-40564Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). MyoD binding with that of MEF2 and TRα1 spans the –502- to –420-bp region of the glut4 gene in skeletal muscle (19Santalucia T. Moreno H. Palacin M. Yacoub M.H. Brand N.J. Zorzano A. J. Mol. Biol. 2001; 314: 195-204Crossref PubMed Scopus (66) Google Scholar). MyoD directly interacts with MEF2 synergistically driving gene expression necessary for myogenesis (19Santalucia T. Moreno H. Palacin M. Yacoub M.H. Brand N.J. Zorzano A. J. Mol. Biol. 2001; 314: 195-204Crossref PubMed Scopus (66) Google Scholar, 20Kaushal S. Schneider J.W. Nadal-Ginard B. Mahdavi V. Science. 1994; 266: 1236-1240Crossref PubMed Scopus (194) Google Scholar, 21Molkentin J.D. Black B.L. Martin J.F. 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Nucleic Acids Res. 2003; 31: 2305-2312Crossref PubMed Scopus (586) Google Scholar). Changes in chromatin structure and locus accessibility predetermine the epigenetic imprint on gene expression (29Li B. Carey M. Workman J.L. Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2643) Google Scholar, 30Kourzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7926) Google Scholar). DNA methylation may permanently silence a gene throughout development (31Dolinoy D.C. Das R. Weidman J.R. Jirtle R.L. Pediatr. Res. 2007; 61: R30-R37Crossref PubMed Scopus (199) Google Scholar). In contrast, the dynamic flexibility of histone post-translational modifications, such as de-acetylation, de-phosphorylation, and methylation of specific amino acid residues in the N-terminal tails, exerts diversified effects on gene transcriptional regulation (30Kourzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7926) Google Scholar). These effects may decrease gene expression rather than complete silencing. An example is the association of MEF2 with class II histone deacetylating enzymes suppressing MEF2-mediated downstream gene expression (32Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (413) Google Scholar).Because DNA methylation and concerted changes in the combinatorial histone code regulate tissue-specific gene expression (26Devaskar S.U. Raychaudhuri S. Pediatr. Res. 2007; 61: R1-R4Crossref PubMed Scopus (1) Google Scholar, 27Bernstein B.E. Meissner A. Lander E.S. Cell. 2007; 128: 669-681Abstract Full Text Full Text PDF PubMed Scopus (1662) Google Scholar, 28Fuks F. Hurd P.J. Deplus R. Kouzarides T. Nucleic Acids Res. 2003; 31: 2305-2312Crossref PubMed Scopus (586) Google Scholar, 29Li B. Carey M. Workman J.L. Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2643) Google Scholar, 30Kourzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7926) Google Scholar, 31Dolinoy D.C. Das R. Weidman J.R. Jirtle R.L. Pediatr. Res. 2007; 61: R30-R37Crossref PubMed Scopus (199) Google Scholar), we hypothesized that epigenetic phenomena may underlie aberrations in MEF2- and/or MyoD-mediated transcriptional induction of skeletal muscle glut4 expression in the IUGR offspring. We tested this hypothesis in a well characterized rat model, where the offspring was exposed to prenatal and postnatal nutrient restriction causing intrauterine and postnatal growth restriction (4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar).EXPERIMENTAL PROCEDURESMaterialsOligonucleotides—Synthetic oligonucleotides (Retrogen Inc., Carlsbad, CA; Integrated DNA Technologies, San Diego) were used in these experiments. Double-stranded oligonucleotides were generated by annealing synthetic oligonucleotides with respective complementary sequences under standard conditions.Antibodies—Rabbit polyclonal anti-MyoD, anti-MEF2A, anti-MEF2C, anti-MEF2D, anti-HDAC1, anti-HDAC4, anti-DNMT1, anti-DNMT3a, and anti-DNMT3b were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MEF2D used for Western blot analysis was purchased from BD Biosciences. Anti-acetyl-histone H3, anti-acetyl-histone H4, anti-acetyl-histone H3 (Lys-9), anti-acetyl-histone H3 (Lys-14), anti-acetyl-histone H3 (Lys-27), anti-SUV39H1 clone MG44, anti-HP1α, clone 15.19s2, and anti-MeCP2 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-dimethyl-histone H3 (Lys-9) was purchased from Abcam Inc. (Cambridge, MA), and anti-polymerase II antibody was from Active Motif (Carlsbad, CA). Horseradish peroxidase-linked anti-rabbit and anti-mouse IgGs were from Amersham Biosciences. For gel shift-supershift experiments the more concentrated (2 μg/μl) form (Santa Cruz Biotechnology) of anti-MyoD, anti-MEF2A, anti-MEF2C, and anti-MEF2D antibodies was used.MethodsCells—C2C12 murine cells (American Type Culture Collection, Manassas, VA) were grown at 37 °C with 95% air, 5% CO2 in poly-l-lysine-coated culture flasks and maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, sodium pyruvate (110 mg/ml), penicillin (100 units/ml), streptomycin (100 units/ml), 4.5% glucose, and 10% fetal bovine serum.DNA Cloning and Site-directed Mutagenesis—Standard recombinant molecular biology techniques were used in cloning ∼1-kb rat glut4 upstream DNA sequences obtained by PCR amplification of genomic DNA from 450-day female (control) rat skeletal muscle. Bidirectional cloning of the PCR amplification product obtained by using the Herculase hot start enzyme (Stratagene Inc., La Jolla, CA) was accomplished by ligating the KpnI- and HindIII-restricted glut4 DNA fragment to the pGL3 vector proximal to the contained firefly luciferase reporter gene (Promega Inc., Madison, WI). DNA sequences of the glut4-amplified product and its distal luciferase reporter gene were confirmed by DNA sequencing using specific primers. These sequences were aligned and compared with the rat, mouse, and human glut4 upstream sequences in GenBank™ using the ClustalW alignment search software (33Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55203) Google Scholar). Next, a search for transcription factor-binding sites that demonstrated high fidelity was performed using TRANSFAC 4.0 data base search (IMD = version 1.1, CBIL/GibbsMat = version 1.1, available on line) (34Fu Y. Weng Z. Genome Inform. 2005; 16: 68-72PubMed Google Scholar). In particular, the highly conserved MEF2- and MyoD-binding sites were delineated within the rat glut4 upstream sequences. In addition, using the CpG islander searcher software (35Takai D. Jones P.A. In Silico Biol. 2003; 2: 35-40Google Scholar), we identified multiple highly stringent CpG dinucleotide-enriched sequences in and around the proximal region of the glut4 promoter. These CpG dinucleotides in close proximity to the MEF2/MyoD-binding site were artificially grouped into three clusters, CpG-I, CpG-II, and CpG-III to facilitate analysis without disrupting the transcriptional binding sites. An additional eight CpG islands (numbered 21–28) 3′ to the MEF2/MyoD-binding site but 5′ to the transcription start site were also identified for analysis. Both primer-specific PCR-mediated (QuikChange mutagenesis kits, Stratagene) deletions and/or substitutions of target sequences were performed and confirmed by DNA sequence analyses. The primer sequences employed for DNA cloning and mutational analysis are shown in supplemental Table 1.Transient Transfection and Reporter Activity Assays—Transient transfection of cultured C2C12 cells was achieved by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, 2–4 μg of glut4-luciferase DNA constructs along with 0.5 μg of the β-galactosidase expressing plasmid were incubated with Lipofectamine 2000 at room temperature, in reduced serum containing Opti-MEM medium to facilitate DNA-liposome complex formation. After a 6–8-h incubation of the washed adherent cells with the DNA-liposome complex in either 6-well or 60-mm plates, fresh medium was added and incubation continued for varying durations. Following cell lysis, luciferase reporter activity was assessed in 20 μl of the cell extract that was mixed with 100 μl of the luciferase assay reagent, and firefly luciferase activity was measured as light output (10 s) by a standard luminometer (Monolight 2010, Analytical Luminescence Laboratory, San Diego). To determine the transfection efficiency, β-galactosidase activity was also assayed by the luminometer according to the manufacturer's protocol (Promega, Madison, WI). The glut4 promoter-driven luciferase enzyme activity was expressed as a ratio to the corresponding β-galactosidase activity per cellular protein concentrations (36Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Effect of in Vitro Manipulations on Reporter Gene Activity—To methylate DNA in vitro, 10–20 μg of pglut4-Luc containing pGL3 plasmid DNA was treated with 10–20 units of SssI DNA methylase enzyme (New England Biolab, Ipswich, MA) for 6 h at 37 °C in a 20–50-μl volume and then purified in a quick column (Qiagen Inc., Valencia, CA) after deactivation. The CpG-methylated reporter DNA was digested by HpaII and BstU1 and compared with the corresponding wild type DNA on agarose gel electrophoresis. To assess the effect of DNA methylation on glut4 promoter activity, the WT and modified glut4-luciferase DNA were transiently transfected into C2C12 cells, and luciferase reporter activity was assessed after 48 h (36Rajakumar R.A. Thamotharan S. Menon R.K. Devaskar S.U. J. Biol. Chem. 1998; 273: 27474-27483Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).To inhibit histone deacetylase (HDAC) enzymes in general, 0, 0.3, and 0.6 μm trichostatin A (Upstate Inc., Lake Placid, NY) was added to cultured cells for 12 h prior to assessing reporter luciferase enzyme activity. In addition, the effect of glucose and amino acid deprivation was examined in vitro by culturing C2C12 cells in starvation medium (Hanks' balanced salt solution with calcium chloride, magnesium chloride, sodium pyruvate and dialyzed 10% fetal bovine serum) (Sigma). The cells were initially maintained in complete Dulbecco's modified Eagle's medium with high glucose (4.5 g/liter), l-glutamine, and sodium pyruvate along with 10% fetal bovine serum. Following overnight transfection in Opti-MEM media of the glut4 promoter-luciferase DNA construct, 50% glucose and amino acid content in culturing media was achieved for 24 h. The reporter luciferase gene activity was measured after cell lysis according to the manufacturer's protocol.Animals—Sprague-Dawley rats (Charles River Breeding Laboratories, Hollister, CA) were housed in individual cages, exposed to 12:12-h light-dark cycles at 21–23 °C, and allowed ad libitum access to standard rat chow. Animal care and use were approved by the Animal Research Committee at UCLA in accordance with the guidelines from the National Institutes of Health.Prenatal Semi-nutrient Restriction Model—Pregnant rats received 50% of their daily food intake (11 g/day) beginning from day 11 through day 21 of gestation, which constitutes mid- to late gestation, as compared with their control counterparts that received ad libitum access to rat chow (∼22g/day). Both groups had ad libitum access to drinking water (3Garg M. Thamotharan M. Rogers L. Bassilian S. Lee W.N.P. Devaskar S.U. Am. J. Physiol. 2006; 290: 1218-1226Crossref PubMed Scopus (23) Google Scholar, 4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar). At birth, the litter size was culled to six. The newborn rats born to semi-nutrient-restricted mothers were reared by the same mother that continued to be semi-nutrient restricted by receiving 20g/day food intake through lactation (IUGR) (3Garg M. Thamotharan M. Rogers L. Bassilian S. Lee W.N.P. Devaskar S.U. Am. J. Physiol. 2006; 290: 1218-1226Crossref PubMed Scopus (23) Google Scholar). Similarly, newborn pups born to control mothers were reared by the control mother with ad libitum access to rat chow (∼40g/day) (CON). This food restriction scheme ensured that the semi-nutrient restricted maternal rats received about ∼50% of the ad libitum food intake through mid- to late pregnancy and lactation (3Garg M. Thamotharan M. Rogers L. Bassilian S. Lee W.N.P. Devaskar S.U. Am. J. Physiol. 2006; 290: 1218-1226Crossref PubMed Scopus (23) Google Scholar). At day 21, in both experimental groups, the pups were weaned from the mother and maintained in individual cages with ad libitum access to a similar diet of standard rat chow (3Garg M. Thamotharan M. Rogers L. Bassilian S. Lee W.N.P. Devaskar S.U. Am. J. Physiol. 2006; 290: 1218-1226Crossref PubMed Scopus (23) Google Scholar, 4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar).Skeletal Muscle Preparation—Hind limb skeletal muscle was rapidly separated from surrounding tissues, quickly snap-frozen in liquid nitrogen, and stored at –70 °C as described previously (4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar). Skeletal muscle was powdered under liquid nitrogen prior to use for varying extractions.Northern Blot Analysis—Total RNA was isolated using the TRIzol reagent (Invitrogen). The extracted RNA (15 μg/lane) was subjected to Northern blot analysis as described previously (4Thamotharan M. Shin B.C. Suddirikku D.T. Thamotharan S. Garg M. Devaskar S.U. Am. J. Physiol. 2005; 288: E935-E947Crossref PubMed Scopus (72) Google Scholar). A 32P-labeled 439-bp fragment of the rat glut4 cDNA served as the probe. The probe was prepared by amplifying rat glut4 cDNA containing the coding region spanning exons 4–7, with the forward primer 5′-ccggaattcctatgctggccaacaatgtc-3′ that primed rat glut4 cDNA at the beginning of exon 4 and the reverse primer 5′-cacacaagcttagtgcatcagacacatcagc-3′ that primed rat glut4 cDNA at the beginning of exon 7. PCR parameters for the glut4 cDNA amplification over 30 cycles consisted of 95 °C for 2 min initially, followed by denaturation at 95 °C for 30 s, annealing at 60 °C for 60 s, extension at 72 °C for 30 s, and the last step consisting of 72 °C for 5 min. Inter-lane loading variability of Northern blots was standardized by re-hybridization of stripped filters with a 32P-labeled rat 589-bp fragment of the β-actin cDNA probe (37Nudel U. Zaket R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acid Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1017) Google Scholar). The rat β-actin probe was amplified with a forward primer 5′-acctgacagactacctcatg-3′ and a reverse primer 5′-taacagtccgcctagaagca-3′ with similar PCR conditions except for annealing at 55 °C for 30 s and extension at 72 °C for 90 s. In the case of insulin, pancreatic RNA was extracted and Northern blot analysis performed using similar conditions with a PCR-amplified rat I insulin probe (38Devaskar S.U. Giddings S.J. Rajakumar P.A. Carnaghi L.R. Menon R.K. Zahm D.S. J. Biol. Chem. 1994; 269: 8445-8454Abstract Full Text PDF PubMed Google Scholar) using an internal control of rat β-actin (37Nudel U. Zaket R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acid Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1017) Google Scholar). The rat insulin I probe was amplified using the following primers: forward primer 5′-atagaccatcagcagcaagcagg-3′ and reverse primer 5′-tccagttgtggcacttgcg-3′ (GenBank™ accession number V01242) in PCR beginning at 94 °C for 5 min followed by denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min over 35 cycles followed by 72 °C for 5 min. The insulin I probe was digoxigenin-labeled (Roche Diagnostics) and used with 15 μg of RNA/lane loading. The signals on blots were quantified with a Variable Mode Imager (Typhoon 9410, Amersham Biosciences). The results were expressed as a ratio between glut4 and β-actin mRNA or insulin and β-actin PhosphorImager values.Electromobility Shift Assay (EMSA)—The 1-kb upstream glut4 wild type and mutant clones carrying deletions of MyoD-I, MyoD-II, and MEF2 binding domains were amplified by PCR. Primers encompassing MyoD-I-, MyoD-II-, and MEF2-binding sites individually were employed to produce ∼100-bp size DNA fragments carrying either only one binding site each or a deleted binding site that was then end-labeled with [γ-32P]ATP (Perkin Elmer Life Sciences) and T4 polynucleotide kinase. The primers used for amplifying the wild type and mutated probes are listed in supplemental Table 2. Approximately 6 fmol (specific activity = 3000 Ci/mmol) of the labeled DNA probe was added to 5 μg of skeletal muscle nuclear extract in a final volume of 20 μl containing 1 μg of poly(dI-dC), 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.5 mm EDTA, 1 mm MgCl2, 4% glycerol, 1 mm dithiothreitol and incubated for 15 min at room temperature. Subsequently, the DNA-protein complexes were separated from unbound DNA by electrophoresis through a 5% nondenaturing polyacrylamide gel in a 90 mm Tris borate, 2 mm EDTA buffer. The gels were dried and subjected to autoradiography (39Rajakumar R.A. Thamotharan S. Raychaudhuri N. Menon R.K. Devaskar S.U. J. Biol. Chem. 2004; 279: 26768-26779Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Competition occurred in the presence of 10–100-fold excess of unlabeled DNA oligonucleotides. In electrophoretic mobility super-shift assays, 2 μg of the respective antibody (IgG) was included in the reaction mix and incubated for 30 min. DNA target sequences (oligonucleotides) used in gel shift reactions are listed in supplemental Table 3.DNA Bisulfite Modification Assay—Genomic DNA was extracted from 450-day female CON and IUGR and 2-day male and female CON and IUGR skeletal muscle using the DNeasy tissue kit (Qiagen Inc., Valencia, CA). The extracted DNA was subjected to bisulfite modification using the CpGenome fast DNA modification kit (Chemicon International Inc., Temecula, CA). The bisulfite-modified naked DNA served as the template in a PCR where specific regions containing the CpG-I, CpG-II, and CpG-III islands and other 3′-CpG-rich regions of the upstream glut4 promoter 5′ to the transcription start site were amplified with primers created by the MethPrimer software (40Pattyn F. Hoebeeck J. Robbrecht P. Michels E. De Paepe A. Bottu G. Coornaert D. Herzog R. Speleman F. Vandesompele J. BMC Bioinformatics. 2006; 7: 1-9PubMed Google Scholar). The PCR-amplified DNA was cloned in a TOPO-TA vector (Invitrogen) after the addition of a 3′-A. These clones were then sequenced to detect either unmodified CpG islands prot
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