. 2016 Apr 26;15(4):748-760.

doi: 10.1016/j.celrep.2016.03.060. Epub 2016 Apr 14.

Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage

Sarah Moyon¹, Jimmy L Huynh², Dipankar Dutta³, Fan Zhang¹, Dan Ma⁴, Seungyeul Yoo⁵, Rebecca Lawrence¹, Michael Wegner⁶, Gareth R John⁷, Ben Emery⁸, Catherine Lubetzki⁹, Robin J M Franklin⁴, Guoping Fan¹⁰, Jun Zhu⁵, Jeffrey L Dupree¹¹, Patrizia Casaccia¹²

Affiliations

¹ Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
² Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
³ Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁴ Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AH, UK; Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0AH, UK.
⁵ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁶ Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
⁷ Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁸ Jungers Center for Neurosciences Research and Department of Neurology, Oregon Health and Science University, Portland, OR 97239, USA.
⁹ Sorbonne Universités UPMC Université, Paris 06, UMR_S 1127, ICM-GHU Pitié-Salpêtrière, 75013 Paris, France.
¹⁰ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23298, USA.
¹² Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address: patrizia.casaccia@mssm.edu.

PMID: 27149841
PMCID: PMC5063656
DOI: 10.1016/j.celrep.2016.03.060

Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage

Sarah Moyon et al. Cell Rep. 2016.

. 2016 Apr 26;15(4):748-760.

doi: 10.1016/j.celrep.2016.03.060. Epub 2016 Apr 14.

Authors

Affiliations

¹ Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
² Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
³ Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁴ Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AH, UK; Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0AH, UK.
⁵ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁶ Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
⁷ Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁸ Jungers Center for Neurosciences Research and Department of Neurology, Oregon Health and Science University, Portland, OR 97239, USA.
⁹ Sorbonne Universités UPMC Université, Paris 06, UMR_S 1127, ICM-GHU Pitié-Salpêtrière, 75013 Paris, France.
¹⁰ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23298, USA.
¹² Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address: patrizia.casaccia@mssm.edu.

PMID: 27149841
PMCID: PMC5063656
DOI: 10.1016/j.celrep.2016.03.060

Abstract

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.

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Figures

**Figure 1. Increased levels of DNA methylation and dynamic expression of DNA methyltransferases during oligodendrocyte development**
(A) Immunostaining of DNA methylation using 5-mC antibody (green) and OLIG2 (red) in spinal cord sections revealed the presence of cells with low, medium and high 5-mC levels of staining intensity. (B) Quantification of 5-mC immunoreactive OLIG2+ cells in mouse developing spinal cord white matter at E17-18, P1, and P21. (C) Left: protocol for culturing proliferating OPC (PDGF + FGF) and inducing differentiation into OL (T3). Right: dot blot analysis for 5-mC and dsDNA as control. (D) Quantification of 5-mC levels relative to total dsDNA in OPC and OL. (E) Scheme of fluorescence-activated cell sorting of P2 *PDGFRα-GFP* OPC and P18 *PLP-GFP* OL. (F) *Dnmt1* and *Dnmt3a* mRNA levels in sorted OPC and OL relative to OPC. (G) Representative image of nuclear DNMT1 and DNMT3A (green) immunostaining in OLIG2+ OPC and CC1+ OL (red). Scale bar = 10 μm. Data represent mean ± SEM. *p < 0.05 (ANOVA and Student's t-test).

**Figure 2. Negative correlation between DNA methylation and transcript levels in oligodendroglial lineage cells**
(A) Scheme of experimental approach used for RNA-sequencing and high resolution methylomics. (B) Quadrant plot of differentially methylated regions (DMRs) at gene promoters and differentially expressed genes (OL *vs.* OPC). X-axis refers to DNA methylation differences during differentiation. Y-axis indicates log2 fold-change of transcript levels. Horizontal (10% difference) and vertical (2-fold change) dashed lines identify four quadrants: (I) hypomethylated and upregulated genes (red circle), (II) hypomethylated and downregulated (black square), (**III**) hypermethylated and upregulated (black diamond) and (IV) hypermethylated and downregulated (green triangle). Colored genes in quadrants I and IV are characterized by statistically significant differences in methylation and transcript levels (p value indicated in each quadrant).. (C-D) Top gene ontology categories for hypomethylated and upregulated genes (red, C) and for hypermethylated and downregulated (green, D) genes. (E-F) The bar graphs represent statistically significant DNA methylation levels of individual CpGs (blue boxes over gene structures) in OPC (white bars) and OL (gray bars). Note the significantly decreased methylation of CpGs in myelin genes (E) and increased methylation in cell cycle and neuronal genes (F) during differentiation. Data are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.005 (False Discovery Rate). See also Figures S1 and S2.

**Figure 3. Conditional ablation of DNA methyltransferases in oligodendrocyte lineage cells results in widespread hypomyelination in the central nervous system of *Olig1^cre/+;Dnmt1^fl/fl* mice**
(A-B) Double immunostaining of P16 spinal cord with antibodies for DNMT1 (red, A) or DNMT3A (red, B) and the cell-specific markers (green): OLIG2 for OL, GFAP for astrocytes, and NeuN for neurons. Note the absence of DNMT1 (A) or DNMT3A (B) in the oligodendrocyte lineage. Scale bar = 10 μm. (C) Kaplan–Meier survival curves for *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, *Olig1^+/+;Dnmt3a^fl/fl* and *Olig1^cre/+;Dnmt3a^fl/fl* mice, and *Cnp^+/+;Dnmt1^fl/fl* and *Cnp^cre/+;Dnmt1^fl/fl* mice. (D) Representative P16 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, *Olig1^+/+;Dnmt3a^fl/fl* and *Olig1^cre/+;Dnmt3a^fl/fl* mice, and *Cnp^+/+;Dnmt1^fl/fl* and *Cnp^cre/+;Dnmt1^fl/fl* mice stained for MBP (green) and OLIG2 (red). Scale bar = 100 μm. (E–G) Gross analysis of P14 *Olig1^cre/+;Dnmt1^fl/fl* mutants revealed minimal differences in body size compared to controls (E) and complete absence of white matter in spinal cord (F) and brain stem (circle in G). Scale bars = 600 μm (E) and 200 μm (F-G). (H) Electron micrograph analysis of P16 spinal cord and corpus callosum sections reveal severe hypomyelination in the *Olig1^cre/+;Dnmt1^fl/fl* mice. Scale bar = 1 μm. See also Figure S3 and Movie S1.

**Figure 4. Normal cell specification in *Olig1^cre/+;Dnmt1^fl/fl* spinal cord**
(A) Representative confocal image of E12.5 spinal cord sections from *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, stained for OLIG2 (red) and MNX1 (green) and quantification of MNX1+ cells in control (white bars) and mutants (gray bars). (B) Quantification of OLIG2+ cells in controls and mutants. (C) Representative E12.5 spinal cord sections stained for OLIG2 (red) and NKX2.2 (green) and relative quantification in controls and mutants. (D) Representative E12.5 spinal cord sections, stained for OLIG2 (red) and PAX6 (green) and relative quantification in controls and mutants. (E-F) Representative P14 spinal cord sections of *Rosa^TdTomato;Olig1^cre/+;Dnmt1^+/+* and *Rosa^TdTomato;Olig1^cre/+;Dnmt1^fl/fl* mice, stained for NeuN (E) or GFAP (F) and percentage of NeuN+/TdTomato+ (E) or GFAP+/TdTomato+ (F) cells among the TdTomato+ cells. Scale bar = 150μm. Data are mean ± SEM. *p < 0.05 (ANOVA and Student's t-test).

**Figure 5. Impaired oligodendrocyte progenitor cell differentiation in *Olig1^cre/+;Dnmt1^fl/fl* mice**
(A) Representative P16 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, stained for OLIG2 (red) and PDGFRα (green) and quantification of PDGFRα+/OLIG2+ cells at indicated time points. Scale bar =40μm. (B) Representative image of P16 spinal cord sections stained for OLIG2 (red) and CC1 (green) and relative quantification. Scale bar =100μm. (C) Representative image of P16 spinal cord sections of mutants and controls, stained for OLIG2 (red) and MBP (green) and relative quantification, showing extensive hypo-myelination. Scale bar =100μm. (D) Schematic of immunopanning from *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* P6 cortex to assess *in vitro* differentiation, and quantification of the number of CC1+/OLIG2+ and MBP+/OLIG2+ cells in T3 medium. Data are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.005 (ANOVA). See also Figure S4.

**Figure 6. Proliferation defect and genotoxic damage in *Olig1^cre/+;Dnmt1^fl/fl* oligodendrocyte progenitors**
(A) Representative P9 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^{c re/+};Dnmt1^fl/fl* mice, stained for OLIG2 (red) and cleaved-CASPASE3 (green) and quantification of cCASPASE3+ cells in the spinal cord at the indicated time points. (B) Representative P9 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, stained for OLIG2 (red) and Ki67 (green) and quantification of Ki67+/OLIG2+ cells at P2 and P9. (C) Representative *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* immunopanned OPC, stained for PDGFRα (red) and Ki67 (green) and quantification of the number of Ki67+/PDGFRα+ cells in PDGF + FGF medium. (D) Representative P9 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, stained for OLIG2 (red) and phospho-H3 (green) and quantification of phospho-H3+/OLIG2+ cells at P9. (E) Representative *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* immunopanned OPC, stained for OLIG2 (red) and phospho-H3 (green) and quantification of the number of phophos-H3+/OLIG2+ cells in PDGF + FGF medium. White arrowheads indicate double-positive cells. (F) Methylation of individual CpGs assessed by MassARRAY EpiTYPER of *Meis2* and *Cdc6* reveal hypomethylation. In blue is the complete gene structure, including hypermethylated DMRs observed in differentiating OL (blue rectangles). Dotted lines identify the zoomed region containing the DMRs. Methylation levels of indicated CpGs is shown for control (white) and mutant (gray) sorted OPC. (G) Quantitative real-time PCR analysis of transcript levels of the indicated genes in P2 and P16 spinal cord of *Olig1^cre/+;Dnmt1^fl/fl* mice (gray) relative to the levels in controls (white). (H) Representative P9 spinal cord sections of *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* mice, stained for OLIG2 (red) and the genotoxic stress marker phospho-H2AX (green) with relative quantification. (I) Representative *Olig1^+/+;Dnmt1^fl/fl* and *Olig1^cre/+;Dnmt1^fl/fl* immunopanned OPC, stained for OLIG2 (red) and phospho-H2AX (green) and relative quantification in proliferating OPC. Scale bar = 25 μm. Data are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.005 (ANOVA and Student's t-test).

**Figure 7. Ablation of *Dnmt1* in oligodendrocyte progenitors results in aberrant alternative splicing events and endoplasmic reticulum stress**
(A) Histogram and pie chart showing respectively number and proportion of alternative splicing events during normal oligodendrocyte development (classified in 5 categories). (B) Histogram and pie chart respectively showing number and proportion of alternative splicing events in mutant OPC compared to controls. (C) Gene ontology of genes alternatively spliced in mutant *Olig1^cre*^/⁺;*Dnmt1^fl/fl* OPC compared to *Olig1⁺*^/⁺;*Dnmt1^fl/fl* OPC. (D) Validation of retained-intron splicing of *Mcm7* in *Olig1^cre*^/⁺;*Dnmt1^fl/fl* OPC by PCR. (E) Methylation differences in individual CpGs between control (white) and mutant (gray) DNA isolated from OPC and assessed by MassARRAY EpiTYPER. (F) Ultrastructural analysis identifies a dilated ER with inclusions (red arrows) in *Olig1^cre/+;Dnmt1^fl/fl* mice, compared to *Olig1^+/+;Dnmt1^fl/fl* controls (blue arrows). (G) Quantitative real-time PCR analysis of genes involved in ER stress response, including the ATF6, PERK, and IRE1 pathways, analyzed in P2, P9 and P16 spinal cord of controls (*Olig1^+/+;Dnmt1^fl/fl*) and mutants (*Olig1^cre/+;Dnmt1^fl/fl*). (H) Quantitative real-time PCR analysis of ER stress response target genes assessed in P9 spinal cord of *Cnp^+/+;Dnmt1^fl/fl* and *Cnp^cre/+;Dnmt1^fl/fl* mice. Data are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.005. (ANOVA). See also Figure S5.

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Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage

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Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage

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