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• W2338951974 abstract "•Oligodendrocyte-lineage-specific analysis of DNA methylome and transcriptome•No fate switch induced by Dnmt1 ablation in early oligodendrocyte progenitors•DNMT1-dependent regulation of differentiation-specific alternative splicing events•Impaired myelination in Dnmt1 mutants characterized by aberrant ER stress response Oligodendrocytes derive from progenitors (OPCs) through the interplay of epigenomic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing, and multiple transgenic lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPCs, yet genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis but was characterized by a profound defect of differentiation associated with changes in exon-skipping and intron-retention splicing events and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPCs is not sufficient to induce a lineage switch but acts as an important determinant of the coordination between RNA splicing and protein synthesis necessary for myelin formation. Oligodendrocytes derive from progenitors (OPCs) through the interplay of epigenomic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing, and multiple transgenic lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPCs, yet genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis but was characterized by a profound defect of differentiation associated with changes in exon-skipping and intron-retention splicing events and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPCs is not sufficient to induce a lineage switch but acts as an important determinant of the coordination between RNA splicing and protein synthesis necessary for myelin formation. Brain development requires the integration of cell-lineage selection and cell number regulation. This is achieved by coordinating cell proliferation with lineage identity. DNA methylation is a well-recognized epigenetic modification that is carefully regulated during cell division and guarantees faithful transmission of information to the daughter cells. This enzymatic activity is modulated by three proteins: DNMT1 (maintenance DNA methyltransferase, commonly associated with faithful transmission of genomic information from mother to daughter cells during cell division), DNMT3A, and DNMT3B (de novo methyltransferases methylating specific cytosines during development). The activity of these enzymes in the brain is higher than in any other adult tissue (Ono et al., 1993Ono T. Uehara Y. Kurishita A. Tawa R. Sakurai H. Biological significance of DNA methylation in the ageing process.Age Ageing. 1993; 22: S34-S43Crossref PubMed Google Scholar, Tawa et al., 1990Tawa R. Ono T. Kurishita A. Okada S. Hirose S. Changes of DNA methylation level during pre- and postnatal periods in mice.Differentiation. 1990; 45: 44-48Crossref PubMed Scopus (61) Google Scholar), highlighting the importance of DNMTs in neural development (Lister et al., 2013Lister R. Mukamel E.A. Nery J.R. Urich M. Puddifoot C.A. Johnson N.D. Lucero J. Huang Y. Dwork A.J. Schultz M.D. et al.Global epigenomic reconfiguration during mammalian brain development.Science. 2013; 341: 1237905Crossref PubMed Scopus (1280) Google Scholar, Smith and Meissner, 2013Smith Z.D. Meissner A. DNA methylation: roles in mammalian development.Nat. Rev. Genet. 2013; 14: 204-220Crossref PubMed Scopus (1941) Google Scholar). This study addresses the role of DNA methylation in developmental myelination. Genetic loss of Dnmt1 is lethal in mammals (Li et al., 1992Li E. Bestor T.H. Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3216) Google Scholar) and zebrafish (Jacob et al., 2015Jacob V. Chernyavskaya Y. Chen X. Tan P.S. Kent B. Hoshida Y. Sadler K.C. DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.Development. 2015; 142: 510-521Crossref PubMed Scopus (43) Google Scholar), because rapidly proliferating cells need to retain a stable epigenetic signature and are eliminated by apoptosis if compromised (Jackson-Grusby et al., 2001Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (566) Google Scholar, Unterberger et al., 2006Unterberger A. Andrews S.D. Weaver I.C. Szyf M. DNA methyltransferase 1 knockdown activates a replication stress checkpoint.Mol. Cell. Biol. 2006; 26: 7575-7586Crossref PubMed Scopus (70) Google Scholar). An example is the apoptotic elimination of neural stem cells and mitotic neuroblasts lacking Dnmt1 (Fan et al., 2001Fan G. Beard C. Chen R.Z. Csankovszki G. Sun Y. Siniaia M. Biniszkiewicz D. Bates B. Lee P.P. Kuhn R. et al.DNA hypomethylation perturbs the function and survival of CNS neurons in postnatal animals.J. Neurosci. 2001; 21: 788-797Crossref PubMed Google Scholar, Hutnick et al., 2009Hutnick L.K. Golshani P. Namihira M. Xue Z. Matynia A. Yang X.W. Silva A.J. Schweizer F.E. Fan G. DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation.Hum. Mol. Genet. 2009; 18: 2875-2888Crossref PubMed Scopus (130) Google Scholar, Milutinovic et al., 2003Milutinovic S. Zhuang Q. Niveleau A. Szyf M. Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes.J. Biol. Chem. 2003; 278: 14985-14995Crossref PubMed Scopus (108) Google Scholar). In contrast, inhibiting DNA methylation in astrocytes or Schwann cells is associated with precocious onset of differentiation, due to the unmasking of critical transcriptional regulatory sites in differentiation genes (Fan et al., 2005Fan G. Martinowich K. Chin M.H. He F. Fouse S.D. Hutnick L. Hattori D. Ge W. Shen Y. Wu H. et al.DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling.Development. 2005; 132: 3345-3356Crossref PubMed Scopus (336) Google Scholar, Takizawa et al., 2001Takizawa T. Nakashima K. Namihira M. Ochiai W. Uemura A. Yanagisawa M. Fujita N. Nakao M. Taga T. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain.Dev. Cell. 2001; 1: 749-758Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Varela-Rey et al., 2014Varela-Rey M. Iruarrizaga-Lejarreta M. Lozano J.J. Aransay A.M. Fernandez A.F. Lavin J.L. Mósen-Ansorena D. Berdasco M. Turmaine M. Luka Z. et al.S-adenosylmethionine levels regulate the schwann cell DNA methylome.Neuron. 2014; 81: 1024-1039Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this study, we show that ablation of Dnmt1 in the oligodendrocyte (OL) lineage does not result in apoptosis or precocious myelination but causes the growth arrest of OL progenitors (OPCs) and a severely disrupted pattern of alternative splicing with the activation of an endoplasmic reticulum (ER) stress response, which precludes differentiation and results in severe and clinically symptomatic hypomyelination. DNA methylation in proliferating progenitors has been shown to prevent untimely differentiation and to guarantee faithful transmission of genomic information from mother to daughter cells during replication (Fan et al., 2005Fan G. Martinowich K. Chin M.H. He F. Fouse S.D. Hutnick L. Hattori D. Ge W. Shen Y. Wu H. et al.DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling.Development. 2005; 132: 3345-3356Crossref PubMed Scopus (336) Google Scholar, Probst et al., 2009Probst A.V. Dunleavy E. Almouzni G. Epigenetic inheritance during the cell cycle.Nat. Rev. Mol. Cell Biol. 2009; 10: 192-206Crossref PubMed Scopus (577) Google Scholar, Sen et al., 2010Sen G.L. Reuter J.A. Webster D.E. Zhu L. Khavari P.A. DNMT1 maintains progenitor function in self-renewing somatic tissue.Nature. 2010; 463: 563-567Crossref PubMed Scopus (336) Google Scholar, Varela-Rey et al., 2014Varela-Rey M. Iruarrizaga-Lejarreta M. Lozano J.J. Aransay A.M. Fernandez A.F. Lavin J.L. Mósen-Ansorena D. Berdasco M. Turmaine M. Luka Z. et al.S-adenosylmethionine levels regulate the schwann cell DNA methylome.Neuron. 2014; 81: 1024-1039Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To begin characterizing the role of DNA methylation in OL-lineage cells, we quantified 5-methylcytosine (5-mC) in developing white matter tracts during embryonic and postnatal development. Quantification of the percentage of OLIG2+ OPCs expressing 5-mC (Figure 1A) revealed a greater proportion of highly methylated cells at late developmental time points (Figure 1B). A similar pattern was detected in cultured OPCs during differentiation into OLs (Figures 1C and 1D). To further understand the role of DNA methylation in OPCs, we also evaluated the transcript (Figures 1E and 1F) and protein (Figure 1G) levels of the maintenance DNA methyltransferase Dnmt1 and the de novo methyltransferases Dnmt3a and Dnmt3b at two stages of development. While Dnmt1 levels decreased with differentiation, Dnmt3a levels did not significantly change, and Dnmt3b levels were undetectable at either stage (data not shown). To generate a genome-wide map of the DNA methylation landscape during the transition from OPC to OL, we used fluorescence-activated cell sorting (FACS) of the brains from transgenic mice expressing GFP driven by OL-lineage-specific promoters at postnatal day (P)2 (Pdgfrα-GFP) and P18 (Plp1-GFP) (Figure 2A). We previously characterized the Pdgfrα-GFP sorted OPC as expressing progenitor markers (e.g., Cspg4 and Pdgfrα) and not expressing myelin genes (e.g., Mog and Mag), while Plp1-GFP sorted OLs were characterized by the expression of differentiation genes and the absence of progenitor markers (Moyon et al., 2015Moyon S. Dubessy A.L. Aigrot M.S. Trotter M. Huang J.K. Dauphinot L. Potier M.C. Kerninon C. Melik Parsadaniantz S. Franklin R.J.M. Lubetzki C. Demyelination causes adult CNS progenitors to revert to an immature state and express immune cues that support their migration.J. Neurosci. 2015; 35: 4-20Crossref PubMed Scopus (168) Google Scholar). DNA methylation mapping was performed by enhanced reduced representation bisulfite sequencing (ERRBS), which combined restriction enzyme digestion (i.e., MspI) of genomic DNA with bisulfite sequencing and provided single-base resolution and highly quantitative data on the methylation state of cytosines throughout the genome. The mean coverage was 1.47 million individual CpGs in the mouse genome (Table S1), with the majority detected at gene promoters (Figure S1C). Among those, we identified 62,807 differentially methylated CpGs as OPCs differentiated (q value <0.01) with 29,707 hypomethylated and 33,100 hypermethylated CpGs. Regions containing at least two CpGs with a minimum difference of 10% between the two stages of differentiation were classified as differentially methylated regions (DMRs). This revealed the clustering of CpGs into 7,386 DMRs characterizing the differentiation of OPC, with 2,385 hypermethylated regions (with an average methylation difference of 22.2% ± 8.5%) (Figure S1B). Biological replicates confirmed the reproducibility of the methylation state (Pearson’s r > 0.9), while comparisons of DMRs in samples obtained at the two developmental time points displayed great disparity (Figure S1A), further supporting the large shift in the DNA methylome during differentiation. To determine the relationship between DNA methylation and transcription, we performed RNA sequencing (RNA-seq) on the same FACS-isolated populations (Figure 2; Table S2). Separate clustering of the samples isolated at distinct time points and differential expression of stage-specific markers (Pdgfrα: fold change, −8.2; q value = 5.5 × 10−220; Cspg4: fold change, −7.0; q value = 4.5 × 10−169; Plp1: fold change, 2.3; q value = 6.4 × 10−3; Mog: fold change, 4.9; q value = 2.09 × 10−34; Mag: fold change, 2.4; q value = 2.6 × 10−26) between the two populations confirmed the selectivity of our cell sorting (Figure S2A). The analysis revealed 3,204 upregulated (average fold change = 3.6 ± 1.8) and 3,547 downregulated transcripts (average fold change = 5.6 ± 2.4) during differentiation. Upregulated genes included gene ontology (GO) categories related to lipid metabolism and axon ensheathment, while downregulated categories included cell cycle, cell migration, and neuronal differentiation (Figures S2B and S2C). The overwhelming overlap (p < 10−244) of our dataset and the published in vitro RNA-seq study (Zhang et al., 2014Zhang Y. Chen K. Sloan S.A. Bennett M.L. Scholze A.R. O’Keeffe S. Phatnani H.P. Guarnieri P. Caneda C. Ruderisch N. et al.An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.J. Neurosci. 2014; 34: 11929-11947Crossref PubMed Scopus (2922) Google Scholar) further strengthened the validity of our analysis (Figure S2D). To characterize the transcriptional consequences of genome-wide distribution of DNA methylation, we overlapped the differential transcript expression with DNA methylation differences at promoters and represented the analysis as quadrant plot, based on statistical power (Figure 2B). The most statistically significant differences (based on number of events and difference at the two developmental stages) were identified in two quadrants (I and IV). The highest significance was detected in quadrant IV (p = 8.1 × 10−19), which defined hypermethylated genes with decreased expression during differentiation and included genes related to neuronal lineage (e.g., Pax6, Plxnb2, Camk1, and Ephb2) and proliferation (e.g., Cdc6 and Meis2) (Figures 2D and 2F). Quadrant I was also significant (p = 2.5 × 10−7) and included hypomethylated genes with increased expression during differentiation, such as lipid enzymes, myelin components (e.g., Mog and Mag), and enzymes enriched in the myelin compartment such as carbonic anhydrase II (Car2), as well as molecules associated with the differentiated state such as the G-protein-coupled receptor 37 (Gpr37) (Figures 2C and 2E). The other two quadrants (hypomethylated genes with decreased expression or hypermethylated genes with increased expression during differentiation) did not reach statistical significance and, therefore, were not pursued any further. Taken together, these data highlight the relevance of DNA methylation in OPCs for silencing genes related to cell proliferation and neuronal lineage. To define the functional role of DNA methylation in the OL lineage in vivo, we crossed the Dnmt1fl/fl and Dnmt3afl/fl lines with Olig1-cre to target embryonic progenitors and with the Cnp-cre line to target later stages of OL development. Littermates lacking cre expression were used as controls. The cell specificity of gene ablation was confirmed by double immunofluorescence, using antibodies specific for DNMT1 (Figure 3A) or DNMT3A (Figure 3B) and those specific for cell markers. Lack of DNMT1 or DNMT3A immunoreactivity in OLIG2+ cells, but not in glial fibrillary acidic protein (GFAP)-positive (GFAP+) or neuronal-marker (NeuN)-positive (NeuN+) cells, indicated lineage-selective ablation (Figures 3A and 3B). Both Olig1cre/+;Dnmt1fl/fl and Olig1cre/+;Dnmt3afl/fl mice appeared normal at birth; however, by P9, only the Olig1cre/+;Dnmt1fl/fl mice developed tremors and ataxia (Movie S1), eventually leading to decreased survival by the third postnatal week (Figure 3C). Both Olig1cre/+;Dnmt3afl/fl and Cnpcre/+;Dnmt1fl/fl mice showed no obvious phenotype (Figure 3C). Consistent with these observations, myelin basic protein (MBP) staining of spinal cord sections at P16 revealed a dramatic hypomyelination only in the Olig1cre/+;Dnmt1fl/fl mice (Figure 3D). Gross examination of Olig1cre/+;Dnmt1fl/fl mice at P14 revealed only minor differences in body size (Figure 3E) but clear signs of hypomyelination of the spinal cord (see Figure 3F, translucent spinal cord) and brain stem (Figure 3G), which were confirmed by electron microscopy (Figures 3H and S3). Given the early activity of Olig1-cre at the pMN domain (Zhou and Anderson, 2002Zhou Q. Anderson D.J. The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification.Cell. 2002; 109: 61-73Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar) and based on the detection of increased methylation and silencing of neuronal lineage-related genes during OPC differentiation, it was important to determine whether the defective myelination phenotype detected in Olig1cre/+;Dnmt1fl/fl mice could be attributed to impaired specification. To address this question, we used two approaches. First, we processed spinal cord sections from mutants and control siblings at embryonic day (E)12.5 for immunohistochemistry using antibodies specific for motor neurons (i.e., MNX1), as well as ventral (i.e., NKX2.2) and dorsal (i.e., PAX6) markers. Quantification of immunoreactive cells revealed no differences in the number of OLIG2+ at the pMN domain or of MNX1+ at the ventrolateral motor neuron domain (Figures 4A and 4B ). The distribution and number of NKX2.2+ (Figure 4C) and PAX6+ cells (Figure 4D) were similar in mice of the two genotypes, and the boundaries with the OLIG2+ domain were preserved. We then conducted a fate-mapping analysis crossing the ROSA26-loxSTOP-lox-TdTomato reporter line with the Olig1cre/+;Dnmt1fl/fl or Olig1cre/+;Dnmt1+/+ mice and stained spinal cord sections at P14 with neuronal (NeuN) or astrocytic (GFAP) markers (Figures 4E and 4F). Only very few cells were identified by the reporter expression and staining for NeuN or GFAP, both in controls and mutants (NeuN controls = 2.5% ± 0.3%, mutants = 3.2% ± 1.0%; GFAP controls = 3.0% ± 0.4%, mutants = 3.7% ± 0.6%), suggesting that only very few OPCs differentiated in neurons or astrocytes in the absence of Dnmt1. To define the potential cause of the hypomyelinating phenotype, we conducted a quantitative immunohistochemical study of the developing spinal cord from E16.5 to P16 using antibodies specific for OLIG2 and platelet-derived growth factor receptor α (PDGFRα) to label OPCs, CC1 to label newly generated OLs, and MBP to label myelinating cells. While a similar number of progenitors was detected in the embryonic spinal cord, a 27% reduction in the number of OPCs was detected in the mutant spinal cords (Figure 5A) and brains (Figure S4A), starting from P2. A greater reduction was observed for newly generated OLs (Figures 5B and S4B), and an even more dramatic impairment was detected when quantifying myelinating MBP+ cells (Figures 5C and S4C), which represent a later stage of differentiation. The cell-autonomous nature of this defect was further validated in vitro, which revealed defective differentiation of mutant OPCs compared to controls (Figure 5D). Together, these data support the critical importance of DNMT1 in the OL lineage to coordinate the late stages of differentiation into myelin-forming cells. To begin understanding the mechanisms underlying the very modest decrease in progenitor numbers and the almost complete absence of myelin detected by electron microscopy, we processed spinal cord sections from controls and mutants at multiple developmental time points for TUNEL (data not shown) or for the presence of active cleaved caspase-3. An accurate assessment of apoptotic nuclei is difficult in vivo, due to rapid clearance, and despite the detection toward modestly increased apoptosis at P9, the measurable differences did not reach statistical significance (Figure 6A). An alternative explanation for the reduced OPC number was impaired proliferation. Co-labeling of OPCs with antibodies specific for OLIG2 or PDGFRα and markers of proliferation (i.e., Ki67) or of mitotic activity (i.e., phosphorylated histone H3) (Figures 6B and 6D) identified a clear proliferation defect in mutants. This result was cell intrinsic, as it was also detected in cultured OPCs (Figures 6C and 6E). We concluded that the lower number of OPCs in the developing spinal cord of mutant mice compared to that of age-matched controls could, at least in part, be attributed to defective expansion of the progenitor pool in the absence of Dnmt1. Since this phenotype was associated with the detection of concurrent hypomethylation and increased transcripts for genes modulating mitosis (e.g., Meis2 and Cdc6), together with those inhibiting the cell cycle (e.g., Cdkn1a), and with the downregulation of positive regulators of proliferation (e.g., Pdgfrα, Rbl2, and Mcm7) (Figures 6F and 6G), we reasoned that OPCs might have activated strategies to counteract the consequences of Dnmt1 ablation after escaping apoptosis. The transcriptional changes leading to opposing effects on proliferation suggested the potential activation of a genotoxic response, possibly resulting in growth arrest. This was validated by the detection of phosphorylated H2AX immunoreactivity, a histone mark demarcating regions of chromatin with double-stranded DNA (dsDNA) breaks (Unterberger et al., 2006Unterberger A. Andrews S.D. Weaver I.C. Szyf M. DNA methyltransferase 1 knockdown activates a replication stress checkpoint.Mol. Cell. Biol. 2006; 26: 7575-7586Crossref PubMed Scopus (70) Google Scholar), both in vivo (Figure 6H) and in vitro (Figure 6I). To better define the dramatic hypomyelinating phenotype of mutant mice, we conducted an unbiased transcriptomic analysis of OPCs sorted from P5 Olig1+/+;Dnmt1fl/fl;Pdgfrα-GFP and Olig1cre/+;Dnmt1fl/fl;Pdgfrα-GFP using RNA-seq (Figure S5A; Table S3). Loss of Dnmt1 resulted in 994 downregulated and 566 upregulated genes in mutant cells, compared to controls (Tables S4 and S5). Downregulated genes included myelin genes, OL-specific factors, and lipid metabolism enzymes (Figure S3B), which were further validated by qPCR (Figure S3D). Upregulated gene categories included cell division and response to DNA stress (Figures 6G and S3C). Consistent with the detection of normal lineage specification, we did not detect any upregulation of neuronally enriched gene categories. Because OL differentiation is characterized by alternatively spliced events (Kevelam et al., 2015Kevelam S.H. Taube J.R. van Spaendonk R.M.L. Bertini E. Sperle K. Tarnopolsky M. Tonduti D. Valente E.M. Travaglini L. Sistermans E.A. et al.Altered PLP1 splicing causes hypomyelination of early myelinating structures.Ann. Clin. Transl. Neurol. 2015; 2: 648-661Crossref PubMed Scopus (24) Google Scholar, Nave et al., 1987Nave K.A. Lai C. Bloom F.E. Milner R.J. Splice site selection in the proteolipid protein (PLP) gene transcript and primary structure of the DM-20 protein of central nervous system myelin.Proc. Natl. Acad. Sci. USA. 1987; 84: 5665-5669Crossref PubMed Scopus (270) Google Scholar), we interrogated our RNA-seq dataset in control and mutant cells (Figure 7A). It has been suggested that DNA methylation in specific genomic regions is critical for exon skipping and intron retention splicing events (Gelfman et al., 2013Gelfman S. Cohen N. Yearim A. Ast G. DNA-methylation effect on cotranscriptional splicing is dependent on GC architecture of the exon-intron structure.Genome Res. 2013; 23: 789-799Crossref PubMed Scopus (158) Google Scholar, Yearim et al., 2015Yearim A. Gelfman S. Shayevitch R. Melcer S. Glaich O. Mallm J.-P. Nissim-Rafinia M. Cohen A.-H.S. Rippe K. Meshorer E. Ast G. HP1 is involved in regulating the global impact of DNA methylation on alternative splicing.Cell Rep. 2015; 10: 1122-1134Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Consistent with these data, we detected severe impairment of exon skipping and intron retention splicing in mutant OPC (Olig1cre/+;Dnmt1fl/fl;Pdgfrα-GFP) compared to wild-type (Figure 7B). These events occurred in GO categories identified as: myelination, lipid metabolism and cell cycle (Figure 7C). An example of splicing defects associated with defective DNA methylation is shown for the gene Mcm7, a ubiquitously expressed cell cycle gene characterized by intron retention (Figure 7D). Hypermethylated CpGs in wild-type OPC within the spliced regions were associated with intron splicing while hypomethylated CpGs in mutant cells were associated with intron-retention (Figure 7E). Together, these data provided molecular validation of the relationship between aberrant DNA methylation and alternatively spliced defect, as suggested by the RNA-seq analysis. It is likely that aberrant splicing might change protein conformation (Kevelam et al., 2015Kevelam S.H. Taube J.R. van Spaendonk R.M.L. Bertini E. Sperle K. Tarnopolsky M. Tonduti D. Valente E.M. Travaglini L. Sistermans E.A. et al.Altered PLP1 splicing causes hypomyelination of early myelinating structures.Ann. Clin. Transl. Neurol. 2015; 2: 648-661Crossref PubMed Scopus (24) Google Scholar, Yura et al., 2006Yura K. Shionyu M. Hagino K. Hijikata A. Hirashima Y. Nakahara T. Eguchi T. Shinoda K. Yamaguchi A. Takahashi K. et al.Alternative splicing in human transcriptome: functional and structural influence on proteins.Gene. 2006; 380: 63-71Crossref PubMed Scopus (53) Google Scholar) and lead to the potential accumulation of incorrectly folded proteins inducing ER stress. Consistent with this possibility, we observed dilated ERs in mutant OLs, with characteristic and unique electron-dense inclusions (Figure 7F), suggestive of protein accumulations and activation of ER stress response. This interpretation was further supported by the detection of specific downstream targets of ER response pathways in mutant mice, including Bip (downstream of ATF6), Chop (downstream of PERK), and spliced Xbp1 (downstream of the IRE1 pathway) (Figure 7G). The ER stress response was only detected in Olig1cre/+;Dnmt1fl/fl mice, as ablation of Dnmt1 at later stages in Cnpcre/+;Dnmt1fl/fl mice did not induce any change (Figure 7H). These results suggested that Dnmt1 ablation in OPCs resulted in inappropriate protein folding, likely as a result of the dramatic changes in alternative splicing events detected in mutants. Overall, this study supports a role for DNA methylation in OL differentiation that goes beyond the repression of progenitor stage genes and includes the regulation of alternative splicing events at later stages of differentiation, which are critical for the attainment of the myelinating phenotype. OPCs are the last cells to differentiate in the developing CNS, and their maturation is characterized by the silencing of alternative lineages and genome-wide deposition of repressive histone K9 and K27 methylation marks (Liu et al., 2015Liu J. Magri L. Zhang F. Marsh N.O. Albrecht S. Huynh J.L. Kaur J. Kuhlmann T. Zhang W. Slesinger P.A. Casaccia P. Chromatin landscape defined by repressive histone methylation during oligodendrocyte differentiation.J. Neurosci. 2015; 35: 352-365Crossref PubMed Scopus (80) Google Scholar, Sher et al., 2008Sher F. Rössler R. Brouwer N. Balasubramaniyan V. Boddeke E. Copray S. Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2.Stem Cells. 2008; 26: 2875-2883Crossref PubMed Scopus (143) Google Scholar, Sher et al., 2012Sher F. Boddeke E. Olah M. Copray S. Dynamic changes in Ezh2 gene occupancy underlie its involvement in neural stem cell self-renewal and differentiation towards oligodendrocytes.PLoS ONE. 2012; 7: e40399Crossref PubMed Scopus (52) Google Scholar). Our genome-wide analysis of differentially methylated genes during the differentiation of OPCs into OLs directly sorted from developing brains revealed hypermethylation at the promoter of OPC-specific genes in response to mitogens (such as Pdgfra), as well as regulators of DNA replication (such as Cdc6), and neuronal-lineage genes (such as Pax6). This suggested that DNA methylation contributes to the transition of OPCs to OLs by regulating cell-cycle exit and, possibly, lineage choice decisions. The concept of fine-tuning of the DNA methylome during mammalian brain development was previously suggested (Kessler et al., 2016Kessler N.J. Van Baak T.E. Baker M.S. Laritsky E. Coarfa C. Waterland R.A. CpG methylation differences between neurons and glia are highly conserved from mouse to human.Hum. Mol. Genet. 2016; 25: 223-232Crossref PubMed Scopus (13) Google Scholar, Lister et al., 2013Lister R. Mukamel E.A. Nery J.R. Urich M. Puddifoot C.A. Johnson N.D. Lucero J. Huang Y. Dwork A.J. Schultz M.D. et al.Global epigenomic reconfiguration during mammalian brain development.Science. 2013; 341: 1237905Crossref PubMed Scopus (1280) Google Scholar), and we had predicted two potential outcomes to the ablation of Dnmts in OPCs: increased OPC proliferation and potential lineage-choice switch. In neural stem cells, Dnmt1 ablation led to decreased survival and astroglial differentiation (Fan et al., 2001Fan G. Beard C. Chen R.Z. Csankovszki G. Sun Y. Siniaia M. Biniszkiewicz D. Bates B. Lee P.P. Kuhn R. et al.DNA hypomethylation perturbs the function and survival of CNS neurons in postn" @default.
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• W2338951974 date "2016-04-01" @default.
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• W2338951974 title "Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage" @default.
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