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• W2004708536 abstract "Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. Gsk-3 2The abbreviations used are: Gsk-3glycogen synthase kinase-3Dnmt1DNA methyltransferase 1Dnmt3aDNA methyltransferase 3aDnmt3a2DNA methyltransferase 3a, isoform 2ESCembryonic stem cellDMDdifferentially methylated domainDMR2differentially methylated region 2IAPintracisternal A-particleIgf2insulin-like growth factor IIIgf2rinsulin-like growth factor II receptorNSCneural stem cellsqPCRquantitative polymerase chain reactionDKOdouble knock-outTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is functionally defined as the aggregate activity of both Gsk-3α and Gsk-3β isoforms that are encoded at distinct genetic loci. These highly redundant kinases are constitutively active and generally play an inhibitory role in the signal transduction pathways that they regulate (1.Force T. Woodgett J.R. J. Biol. Chem. 2009; 284: 9643-9647Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), such as insulin signaling and canonical Wnt signaling (2.Kockeritz L. Doble B. Patel S. Woodgett J.R. Curr. Drug. Targets. 2006; 7: 1377-1388Crossref PubMed Scopus (225) Google Scholar). In addition, Gsk-3 has a major role in regulating differentiation of embryonic stem cells (3.Doble B.W. Patel S. Wood G.A. Kockeritz L.K. Woodgett J.R. Dev. Cell. 2007; 12: 957-971Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) and neural progenitors (4.Kim W.Y. Wang X. Wu Y. Doble B.W. Patel S. Woodgett J.R. Snider W.D. Nat. Neurosci. 2009; 12: 1390-1397Crossref PubMed Scopus (296) Google Scholar). Both insulin and Wnt signaling have been implicated in the regulation of stem cell pluripotency (5.Takahashi K. Mitsui K. Yamanaka S. Nature. 2003; 423: 541-545Crossref PubMed Scopus (288) Google Scholar, 6.Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1709) Google Scholar, 7.Paling N.R. Wheadon H. Bone H.K. Welham M.J. J. Biol. 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Nature. 2009; 460: 118-122Crossref PubMed Scopus (654) Google Scholar). Insulin signaling modulates Gsk-3 activity via the activity of upstream effectors; insulin or insulin-like growth factor binds to the insulin receptor, resulting in the activation of PI3K, which in turn phosphorylates and activates Akt (also called protein kinase B; PKB) (14.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4542) Google Scholar). Akt subsequently phosphorylates several substrates, including Gsk-3 isoforms, on N-terminal serine residues (Gsk-3α Ser-21/Gsk-3β Ser-9), resulting in the inhibition of Gsk-3 activity (15.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). glycogen synthase kinase-3 DNA methyltransferase 1 DNA methyltransferase 3a DNA methyltransferase 3a, isoform 2 embryonic stem cell differentially methylated domain differentially methylated region 2 intracisternal A-particle insulin-like growth factor II insulin-like growth factor II receptor neural stem cells quantitative polymerase chain reaction double knock-out N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Gsk-3 activity also represents a key regulatory step in the canonical Wnt signaling pathway. In the absence of ligand, a subset of cytoplasmic Gsk-3 is found in a protein complex that facilitates Gsk-3-mediated phosphorylation of β-catenin, targeting the protein for ubiquitination and degradation via the 26 S proteasome and keeping the Wnt pathway repressed (16.MacDonald B.T. Tamai K. He X. Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (3989) Google Scholar). Upon ligand binding to the Wnt co-receptors, Gsk-3 redistributes to the cell surface (17.Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (638) Google Scholar), rendering the β-catenin destruction complex non-functional, causing the accumulation and subsequent nuclear translocation of β-catenin protein, which leads to the transcription of Wnt target genes. Although activated insulin and Wnt signaling both inhibit Gsk-3 activity, the mechanism of inhibition is distinct for each pathway; Wnt signaling does not affect insulin signaling, and insulin signaling does not activate Wnt target genes (18.Ding V.W. Chen R.H. McCormick F. J. Biol. Chem. 2000; 275: 32475-32481Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 19.Ng S.S. Mahmoudi T. Danenberg E. Bejaoui I. de Lau W. Korswagen H.C. Schutte M. Clevers H. J. Biol. Chem. 2009; 284: 35308-35313Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Gsk-3 function in the insulin and Wnt signaling pathways has been thoroughly investigated, and numerous substrates of Gsk-3 have been described (2.Kockeritz L. Doble B. Patel S. Woodgett J.R. Curr. Drug. Targets. 2006; 7: 1377-1388Crossref PubMed Scopus (225) Google Scholar), yet the downstream effects of such a widely influential enzyme likely extend beyond our current knowledge. Here, we present evidence of a novel role for Gsk-3 isoforms in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs). The de novo DNA methyltransferase, Dnmt3a2, is down-regulated in Gsk-3 double knock-out (DKO) ESCs, resulting in reduced DNA methylation and altered expression of imprinted genes. Inhibition of Gsk-3 activity with lithium mimics the effects of reducing DNA methylation in both wild-type ESCs and wild-type neural stem cells. Furthermore, inactivation of Gsk-3 via components of the insulin signaling pathway results in reduced DNA methylation at imprinted loci. Finally, microarray data reveal that N-myc mRNA is down-regulated in Gsk-3 DKO ESCs. We provide data that demonstrate that a highly conserved N-Myc binding site in the Dnmt3a2 promoter is required for normal expression, and we demonstrate that siRNA knockdown of N-Myc results in a decrease in Dnmt3a2 expression. Therefore, we have identified a novel function for Gsk-3 isoforms as key regulators of the epigenome, and our results add a new perspective on the consequences of altering Gsk-3 activity. Feeder-free wild-type, Gsk-3α−/−, Gsk-3β−/−, and Gsk-3 DKO ESCs (3.Doble B.W. Patel S. Wood G.A. Kockeritz L.K. Woodgett J.R. Dev. Cell. 2007; 12: 957-971Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) were grown on gelatin-coated plates in high glucose DMEM (Invitrogen) supplemented with 15% fetal bovine serum (HyClone), 1× non-essential amino acids, 1× sodium pyruvate, 2 mm l-glutamine, 1× penicillin/streptomycin (Invitrogen), 55 μm 2-mercaptoethanol, and 1000 units/ml ESGRO (Millipore). Media was replenished every other day. Neural stem cells were isolated from 12.5 days postcoitum embryos using NeuroCult neural stem cells (NSC) proliferation media (StemCell Technologies) following the manufacturer's protocol. Integrity of total RNA was evaluated using capillary electrophoresis (Bioanalyzer 2100, Agilent) and quantified using a Nanodrop 1000 (Nanodrop, Wilmington, DE). Following confirmation of RNA quality, Ovation™ biotin RNA amplification and labeling system (NuGen Technologies, Inc., San Carlos, CA) was used to prepare amplified, biotin-labeled cDNA from total RNA following manufacturer's instructions. Briefly, first strand cDNA was synthesized from 25 ng of total RNA using a unique first strand DNA/RNA chimeric primer and reverse transcriptase. Following double strand cDNA generation, amplification of cDNA was achieved by utilizing an isothermal DNA amplification process that involves repeated SPIA™ DNA/RNA primer binding, DNA duplication, strand displacement, and RNA cleavage. The amplified SPIA™ cDNA was purified and subjected to a two-step fragmentation and labeling process. The fragmented/biotinylated cDNA content was measured in a ND-1000 spectrophotometer, and the quality was analyzed on an RNA 6000 Nano LabChip (Agilent) using an Agilent Bioanalyzer 2100. For each array, 2.2 μg of cDNA was hybridized onto the GeneChips® mouse genome 430 2.0 array (Affymetrix Inc.), which contains ∼39,000 transcripts. The sequences from which these probe sets were derived were selected from GenBank™, dbEST, and RefSeq. The sequence clusters were created from the UniGene data base (Build 107, June 2002) and then refined by analysis and comparison with the publicly available draft assembly of the mouse genome from the Whitehead Institute for Genome Research (Mouse Genome Sequencing Consortium (MGSC), April 2002). Hybridization was allowed to continue for 16 h at 45 °C followed by washing and staining of microarrays in a Fluidics Station 450 (Affymetrix Inc.). GeneChip arrays were scanned in a GeneChip Scanner 3000 (Affymetrix Inc.), and CEL files were generated from DAT files using the GeneChip® operating software (GCOS) software (Affymetrix Inc.). The probe set signals were generated using the RMA algorithm in ArrayAssist 3.4 (Stratagene) and were used to determine differential gene expression by pairwise comparisons. The genes that were altered by 2-fold either way and had a false discovery rate of <10% were sorted and used for further interpretation of the microarray data. Microarray data have been deposited in GEO (www.ncbi.nlm.nih.gov/geo) under accession number GSE20015. Mouse Dnmt3a2 cDNA was subcloned into a modified pCAGEN plasmid (from Connie Cepko, Addgene plasmid 11160) with a preceding puromycin resistance gene and internal ribosomal entry site. The plasmid containing a constitutively active myristoylated mouse p110 subunit of PI3K (p110*) ((5.Takahashi K. Mitsui K. Yamanaka S. Nature. 2003; 423: 541-545Crossref PubMed Scopus (288) Google Scholar); from Shinya Yamanaka, Addgene plasmid 15689) expressed a hygromycin resistance cassette. ES cells were transfected with either the Dnmt3a2 or the p110* expression constructs with Lipofectamine 2000 (Invitrogen) in serum-free Opti-MEM (Invitrogen). Cells were allowed to recover in non-selective ES cell media for 24 h, after which puromycin-resistant (Dnmt3a2) or hygromycin-resistant (p110*) cells were grown in ES cell media containing 1 μg/ml puromycin or 200 μg/ml hygromycin, respectively. Puromycin/hygromycin-resistant colonies were isolated after 14 days of selection and expanded in selective media. The Dnmt3a2 promoter was cloned by PCR amplification from bacterial artificial chromosome clone 330c18 RPCI-24 library using primers described in supplemental Table S1. Amplification products were TOPO TA-cloned into pCR8GW (Invitrogen) and then transferred by LR reaction to Gateway-modified pGL3-Basic, pGLF (from Glenn Maston, University of Massachusetts). Mutagenesis of the N-Myc binding site was accomplished by using the QuikChange II site-directed mutagenesis kit (Stratagene) (supplemental Table S1). Plasmids containing the Dnmt3a2 promoter constructs driving firefly luciferase were co-transfected into ESCs with pRL SV40 with Lipofectamine 2000 (Invitrogen) in serum-free Opti-MEM (Invitrogen). Cells were replated in a 24-well plate 24 h after transfection and then lysed 48 h after transfection, at which time firefly and Renilla Luciferase assays were performed according to the manufacturer's protocol (Biotium) in a Veritas microplate luminometer (Turner Biosystems). Three replicates were performed for each transfection. Protein expression was assayed by Western blotting. Cells were resuspended in lysis buffer (137 mm NaCl, 10 mm Tris, pH 7.4, 1% Nonidet P-40) containing protease inhibitor mixture (1:100 Sigma) and sonicated for 10 30-s pulses at 4 °C in a Bioruptor (Diagenode) on the highest setting. Lysates were electrophoresed (8–20 mg/lane) through 7.5% Tris/Tricine gels and transferred onto nitrocellulose membrane (Whatman BA85) at 100 V for 1 h. Blots were blocked for 1 h with 5% milk/TBST (150 mm NaCl, 50 mm Tris, pH 7.4, 0.1% Tween) and incubated in primary antibody diluted in 5% milk or 5% BSA (N-Myc and c-Myc antibodies)/TBST for 16–18 h at 4 °C. Antibodies were used under the following conditions. Anti-tubulin mouse mAb clone B-5-1-2 (Sigma) was diluted 1:10,000; anti-Dnmt3a mouse mAb clone 64B1446 (IMGENEX) was diluted 1:250; and anti-GSK-3α/β mouse mAb clone 1H8 (Calbiochem), anti-phospho-GSK-3α/β (Cell Signaling antibody 9331), anti-N-Myc (Cell Signaling antibody 9405), anti-c-Myc D84C12 XP (Cell Signaling antibody 5605), anti-PI3kinase p110α subunit C73F8 (Cell Signaling antibody 4249), anti-Akt1 2H10 (Cell Signaling antibody 2967), and anti-phospho-Akt Ser-473 193H12 (Cell Signaling antibody 4058) were all used at a 1:1000 dilution. Blots were incubated in anti-mouse or anti-rabbit IgG HRP secondary antibody (GE Healthcare) diluted 1:5000 in 5% milk in TBST for 45 min. Proteins were visualized using ECL detection reagent (GE Healthcare). Blots were stripped in a buffer consisting of 2% SDS, 62.5 mm Tris HCl, pH 6.7, and 100 mm β-mercaptoethanol at 50 °C for 30 min followed by repeated rinsing with TBST prior to reprobing with antibody. High molecular weight genomic DNA was isolated after ESCs were washed in PBS and resuspended in lysis buffer (100 mm NaCl, 10 mm Tris, pH 8.0, 25 mm EDTA, pH 8.0, 0.5% SDS) and lysed 16–18 h at 55 °C. An equal volume of phenol:chloroform:isoamyl alcohol was added and gently rotated at ambient temperature for 2–3 h. DNA was precipitated with 2 volumes of ethanol and 0.1 volumes of sodium acetate, washed in 70% ethanol, and resuspended in sterile water. High molecular weight genomic DNA was digested with methylation-sensitive HpaII or methylation-insensitive MspI isoschizomers (New England Biolabs). Digested DNA was separated on 0.6% agarose in 0.5× Tris-acetate-EDTA and transferred to charged nylon membrane (Osmonics Inc.). The blot was hybridized with a [α-32P]dCTP random primed labeled (Prime-It II, Stratagene) intracisternal A particle (IAP) probe (20.Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (190) Google Scholar) in FBI buffer (21.Budowle B. Baechtel F.S. Appl. Theor. Electrophor. 1990; 1: 181-187PubMed Google Scholar) at 65 °C for 16 h, washed twice with 2× SSC + 0.1% SDS at 65 °C for 30 min, and imaged with a Typhoon 9400 PhosphorImager (GE Healthcare). Cells were resuspended in TRIzol (Invitrogen), and RNA was isolated following the manufacturer's protocol and then further purified with the RNeasy RNA cleanup procedure (Qiagen). Complementary DNA was synthesized with the High Capacity cDNA reverse transcription kit (Applied Biosystems) following the manufacturer's protocol. Quantitative RT-PCR was done on an Applied Biosystems 7500 using TaqMan® master mix and one of the following TaqMan assays (Applied Biosystems): Dnmt3a2 (Mm00463987_m1), Igf2 (Mm00439564_m1), H19 (Mm01156721_g1), Igf2r (Mm01313554_m1), Airn (Mm03943369_s1), Snrpn (Mm02391920_g1), N-Myc (Mm00476449_m1), or c-Myc (Mm00487803_m1). Three biological replicates and three technical replicates were used for each target analyzed. All threshold cycle (Ct) values were normalized to a mouse GAPDH endogenous control (Applied Biosystems), and relative quantitation was calculated from the median Ct value. High molecular weight genomic DNA was fragmented by rapid freezing on dry ice and thawing at 42 °C for five repetitions. 500 ng of fragmented DNA was bisulfite-converted with the methyl code bisulfite conversion kit (Invitrogen) per the manufacturer's instructions. Igf2/H19 differentially methylated domain (DMD) and Igf2r differentially methylated region 2 (DMR2) were amplified from bisulfite-converted DNA with Platinum Taq (Invitrogen) using primers shown in supplemental Table S1 (22.Yamasaki Y. Kayashima T. Soejima H. Kinoshita A. Yoshiura K. Matsumoto N. Ohta T. Urano T. Masuzaki H. Ishimaru T. Mukai T. Niikawa N. Kishino T. Hum. Mol. Genet. 2005; 14: 2511-2520Crossref PubMed Scopus (63) Google Scholar) and the following conditions: 94 °C for 10 min, 35 cycles of 94 °C for 45 s, 54 °C for 45 s, and 72 °C for 60 s followed by 72 °C for 7 min. PCR products were then TOPO TA-cloned into pCR8GW (Invitrogen). Plasmid clones were sequenced with M13 reverse primer (supplemental Table S1) using Big Dye version 3.1 chemistry with the following conditions: 96 °C for 10 s, then 25 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min, and reaction products were resolved on an Applied Biosystems 3130XL genetic analyzer. 125 pmol of Nmyc siRNA (Thermo SMARTpool L-058793-01-0005) or GFP siRNA (Dharmacon D-1300-20) was combined with 0.25 ml of Opti-MEM (Invitrogen). In parallel, 7.5 μl of Lipofectamine RNAiMAX (Invitrogen) was combined with 0.25 ml of Opti-MEM per transfection by gentle mixing. siRNA/Opti-MEM and RNAiMAX/Opti-MEM were then combined for a total volume of ∼0.5 ml and incubated for 20 min at room temperature. This mixture was then used to resuspend a pellet of 1 million wild-type ESCs. The cell suspension was then plated into 2 ml of ESC media in an individual well of a gelatin-coated 6-well plate. Media was replaced with fresh ESC media the next day. At 72 h, cells were harvested, and protein and RNA were isolated as described above. In an effort to better understand the downstream targets of Gsk-3, we performed microarray-based global gene expression analysis comparing wild-type (WT) mouse ESCs with those in which Gsk-3α, Gsk-3β, or both isoforms have been genetically deleted (Gsk-3α−/−; Gsk-3β−/− DKO) (3.Doble B.W. Patel S. Wood G.A. Kockeritz L.K. Woodgett J.R. Dev. Cell. 2007; 12: 957-971Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Inspection of our array data revealed a 6.2-fold down-regulation of the de novo DNA methyltransferase Dnmt3a in Gsk-3 DKO ESCs but no effect in Gsk-3α−/− or Gsk-3β−/− ESCs (increased 1.1-fold in each). In addition to full-length Dnmt3a, there is a distinct smaller isoform, Dnmt3a2, which is transcribed from an alternative promoter within the Dnmt3a locus. Dnmt3a2 lacks the amino-terminal 219 amino acids found in Dnmt3a, but the remainder of the protein, including the domain containing methyltransferase activity, is identical between the isoforms (23.Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1250) Google Scholar). Importantly for this study, Dnmt3a2 is the isoform that is predominantly expressed in ESCs (24.Chen T. Ueda Y. Xie S. Li E. J. Biol. Chem. 2002; 277: 38746-38754Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The microarray probes are unable to differentiate between Dnmt3a isoforms, and the microarray expression data represent a composite of the expression of both Dnmt3a and Dnmt3a2. Therefore, we evaluated the protein expression of each individual isoform as the individual isoforms can be resolved by immunoblotting. We found that Dnmt3a2 protein expression is substantially reduced in Gsk-3 DKO cells, whereas Dnmt3a protein levels were essentially unchanged (Fig. 1A). Real-time quantitative PCR (qPCR) revealed that mRNA expression of Dnmt3a2 is reduced 2.7-fold in Gsk-3 DKO ESCs when compared with WT ESCs (Fig. 1B). Dnmt3a/Dnmt3a2 activity is required for the establishment of DNA methylation at imprinted loci in germ cells (25.Kaneda M. Okano M. Hata K. Sado T. Tsujimoto N. Li E. Sasaki H. Nature. 2004; 429: 900-903Crossref PubMed Scopus (999) Google Scholar). Mouse ESCs null for both Dnmt3a and Dnmt3b lose DNA methylation at imprinted loci after extended passaging, demonstrating a role for the de novo methyltransferases in the maintenance of DNA methylation at imprinted loci. Notably, re-expression of Dnmt3a2 alone in Dnmt3a−/−; Dnmt3b−/− ESCs is sufficient to fully restore DNA methylation at paternally imprinted loci, whereas re-expression of Dnmt3a or Dnmt3b1 is only able to minimally rescue DNA methylation at these loci (26.Chen T. Ueda Y. Dodge J.E. Wang Z. Li E. Mol. Cell. Biol. 2003; 23: 5594-5605Crossref PubMed Scopus (565) Google Scholar). Based on these published observations, we hypothesized that reduction of Dnmt3a2 expression in ESCs would result in the loss of DNA methylation and disruption of gene expression at imprinted loci. The imprinted locus containing the genes insulin-like growth factor II (Igf2) and H19 has been well characterized. The reciprocal expression of Igf2 and H19 is regulated by a shared imprinting control region designated the DMD (27.DeChiara T.M. Robertson E.J. Efstratiadis A. Cell. 1991; 64: 849-859Abstract Full Text PDF PubMed Scopus (1424) Google Scholar, 28.Bartolomei M.S. Webber A.L. Brunkow M.E. Tilghman S.M. Genes Dev. 1993; 7: 1663-1673Crossref PubMed Scopus (411) Google Scholar, 29.Tremblay K.D. Duran K.L. Bartolomei M.S. Mol. Cell. Biol. 1997; 17: 4322-4329Crossref PubMed Scopus (291) Google Scholar). In this imprinting control region, the paternal allele is methylated, resulting in expression of Igf2 and silencing of H19, whereas conversely, the maternal allele is unmethylated, promoting H19 expression while silencing Igf2 (30.Ideraabdullah F.Y. Vigneau S. Bartolomei M.S. Mutat. Res. 2008; 647: 77-85Crossref PubMed Scopus (168) Google Scholar). Indeed, the imprinted genes H19 and Igf2 display altered expression in Gsk-3 DKO ESCs when compared with WT ESCs. Igf2 and H19 expression in Gsk-3 DKO cells was assayed by qPCR, and H19 expression is increased 1.4-fold, whereas Igf2 expression is decreased 100-fold (Fig. 1C). This pattern of gene expression is consistent with a scenario for the loss of DNA methylation at the Igf2/H19 DMD (29.Tremblay K.D. Duran K.L. Bartolomei M.S. Mol. Cell. Biol. 1997; 17: 4322-4329Crossref PubMed Scopus (291) Google Scholar, 31.Jinno Y. Sengoku K. Nakao M. Tamate K. Miyamoto T. Matsuzaka T. Sutcliffe J.S. Anan T. Takuma N. Nishiwaki K. Ikeda Y. Ishimaru T. Ishikawa M. Niikawa N. Hum. Mol. Genet. 1996; 5: 1155-1161Crossref PubMed Scopus (88) Google Scholar, 32.Thorvaldsen J.L. Duran K.L. Bartolomei M.S. Genes Dev. 1998; 12: 3693-3702Crossref PubMed Scopus (549) Google Scholar). We directly measured DNA methylation of the Igf2/H19 DMD by bisulfite sequencing and found a 47% reduction of DNA methylation in Gsk-3 DKO ESCs when compared with WT ESCs (Fig. 1D). These results strongly support the hypothesis that the alterations in Igf2 and H19 expression are due to a loss of DNA methylation at the Igf2/H19 DMD. Expression of Air, another imprinted gene that encodes for a long non-coding RNA (34.Lyle R. Watanabe D. te Vruchte D. Lerchner W. Smrzka O.W. Wutz A. Schageman J. Hahner L. Davies C. Barlow D.P. Nat. Genet. 2000; 25: 19-21Crossref PubMed Scopus (221) Google Scholar), is increased in Gsk-3 DKO ESCs. Air mRNA expression in Gsk-3 DKO ESCs was assayed by qPCR and is increased by 3.8-fold (Fig. 2A). We then determined whether the change in expression of Air is due to a loss of DNA methylation. We performed bisulfite sequencing on DMR2 of Igf2r, from which Air is transcribed and known to be methylated on the maternal allele (22.Yamasaki Y. Kayashima T. Soejima H. Kinoshita A. Yoshiura K. Matsumoto N. Ohta T. Urano T. Masuzaki H. Ishimaru T. Mukai T. Niikawa N. Kishino T. Hum. Mol. Genet. 2005; 14: 2511-2520Crossref PubMed Scopus (63) Google Scholar, 35.Stöger R. Kubicka P. Liu C.G. Kafri T. Razin A. Cedar H. Barlow D.P. Cell. 1993; 73: 61-71Abstract Full Text PDF PubMed Scopus (499) Google Scholar). DNA methylation at DMR2 is reduced by 67% in Gsk-3 DKO ESCs (Fig. 2B). These data suggest that the increase in Air expression is likely due to DNA hypomethylation within the Igf2r DMR2 and demonstrate that the loss of Gsk-3 activity on DNA methylation extends beyond the Igf2/H19 DMD, supporting a possible broader role for Gsk-3 in the regulation of imprinted genes in the mouse. We next determined whether the DNA hypomethylation we observed at imprinted loci is due to a defect in global DNA methylation. Because Dnmt1 is the primary enzyme responsible for maintaining genome-wide DNA methylation (36.Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3159) Google Scholar), we examined whether the loss of Gsk-3 activity affects Dnmt1 function. Repetitive DNA sequences, such as IAP repeats, are highly methylated in WT mouse ESCs (37.Howlett S.K. Reik W. Development. 1991; 113: 119-127Crossref PubMed Google Scholar) and largely unmethylated in Dnmt1−/− and Dnmt1 hypomorph ESCs (38.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4348) Google Scholar). IAP repeat methylation, although reduced, is largely retained in ESCs deficient in de novo DNA methyltransferase activity (Dnmt3a−/−; Dnmt3b−/−) (38.Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4348) Google Scholar, 39.Lei H. Oh S.P. Okano M. Jüttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar). Therefore, analyzing DNA methylation of IAP repeats provides an assay for Dnmt1 activity. Southern blotting using methylation-sensitive restriction enzymes revealed that Gsk-3 DKO ESCs do not exhibit a significant difference in DNA methylation of IAP repeats when compared with wild-type ESCs (Fig. 2C), suggesting that there is not a general defect in DNA maintenance methylation in Gsk-3 DKO cells. These data strengthen our hypothesis that the loss of DNA methylation observed at imprinted loci in Gsk-3 DKO ESCs is likely the result of specific down-regulation of Dnmt3a2 expression. To evaluate whether exogenous expression of Dnmt3a2 could rescue the loss of DNA methylation at imprinted loci in Gsk-3 DKO ESCs, we isolated puromycin-resistant Gsk-3 DKO ESCs stably expressing Dnmt3a2 under the control of the chicken β-actin (CAG) promoter and evaluated DNA methylation at the Igf2/H19 DMD by bisulfite sequencing. A puromycin-resistant clone in which Dnmt3a2 is stably overexpressed in DKO ESCs (Dnmt3a2) was selected for bisulfite sequencing analysis, and a puromycin-resistant clone that does not overexpress Dnmt3a2 (Vector) served as a negative control (Fig. 3A). Bisulfite sequencing of the Igf2/H19 DMD revealed that DNA methylation is restored to levels previously observed in WT ESCs in the overexpressing clone (92.7%; Fig. 3B). Rescue of the Igf2/H19 DMD methylation defect by stably expressing Dnmt3a2 in Gsk-3 DKO ESCs strongly supports our hypothesis that reduced Dnmt3a2 expression in Gsk-3 DKO ESCs is the cause of decreased DNA methylation at imprinted loci. Based on the data obtained from Gsk-3 DKO ESCs, we hypothesized that the Gsk-3 inhibitor lithium (40.Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 8455-8459Crossref PubMed Scopus (2056) Google Scholar" @default.
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