DNA methylation in oligodendroglial cells during developmental myelination and in disease

ABSTRACT

Oligodendrocyte progenitor cells (OPC) are the myelinating cells of the central nervous system (CNS). During development, they differentiate into mature oligodendrocytes (OL) and ensheath axons, providing trophic and functional support to the neurons. This process is regulated by the dynamic expression of specific transcription factors, which, in turn, is controlled by epigenetic marks such as DNA methylation. Here we discuss recent findings showing that DNA methylation levels are differentially regulated in the oligodendrocyte lineage during developmental myelination, affecting both genes expression and alternative splicing events. Based on the phenotypic characterization of mice with genetic ablation of DNA methyltransferase 1 (Dnmt1) we conclude that DNA methylation is critical for efficient OPC expansion and for developmental myelination. Previous work suggests that in the context of diseases such as multiple sclerosis (MS) or gliomas, DNA methylation is differentially regulated in the CNS of affected individuals compared with healthy controls. In this commentary, based on the results of previous work, we propose the potential role of DNA methylation in adult oligodendroglial lineage cells in physiologic and pathological conditions, and delineate potential research approaches to be undertaken to test this hypothesis. A better understanding of this epigenetic modification in adult oligodendrocyte progenitor cells is essential, as it can potentially result in the design of new therapeutic strategies to enhance remyelination in MS patients or reduce proliferation in glioma patients.

Introduction

Oligodendrocyte progenitor cells (OPC) are widely recognized by the expression of platelet-derived growth factor α receptor (PDGFαR) and chondroitin sulfate proteoglycan (Cspg4 or NG2).^1,2 Differentiation into mature oligodendrocytes is regulated by the successive expression of transcription factors (recently reviewed by Küspert et al.³). Briefly, OPC are specified by the combined expression of Ascl1, Dlx1/Dlx2 and Olig1/Olig2, then Sox10 and Nkx2.2.^4-8 Upon differentiation, cells express a different set of transcription factors-Sox17, Myt1, Yy1, Myrf and Zfp191,^9-13 as well as new membrane markers such as Gal-C, CNPase, MBP, PLP, MAG and MOG.^4-17 The mature OLs take on a more complex morphology, forming condensed myelin sheaths around adjacent axons for metabolic support and functional conduction.

Epigenetic modifications represent another layer of regulation of OL differentiation. Genomic DNA is compacted into nucleosomes that are further assembled into a higher degree structure inside the nucleus.¹⁸ This structure can be modified by epigenetic marks that enable or prevent accessibility of transcription factors to the genome.¹⁹ We and others have shown that various epigenetic phenomena, including histone modifications, microRNAs and chromatin remodelers are required for OPC differentiation, during myelination and remyelination^20-24 (recently reviewed by Moyon et al.²⁵). Another epigenetic mark, DNA methylation, has been shown to be particularly important for brain development and function,^26-30 but its role has been studied almost exclusively in neurons and astrocytes.^31,32 In particular, DNA demethylation of lineage specific genes and subsequent transcriptional activation has been proposed for neuronal, astrocytic, and Schwann cell differentiation.^26,33,34 Therefore, we aimed at further characterizing the role of DNA methylation in the oligodendroglial lineage and in associated pathologies (i.e MS), by combining histological and bioinformatic analysis on animal models and human tissues.^35,36

Here, we review several studies on the dysregulation of DNA methylation in disease-affected tissues and more recent findings describing the role of DNA methylation in neonatal oligodendrocyte progenitor cells during developmental myelination. We then propose a potential beneficial role for DNA methylation in adult oligodendroglial lineage cells, which - if better characterized - might allow the development of new therapeutic strategies for myelin regeneration in the adult brain.

DNA methylation is differentially regulated in diseases

Alterations in DNA methylation have been implicated in oligodendroglial pathologies, suggesting an important role for DNA methylation in oligodendrocyte function. In addition to neuropathy, dementia and hearing loss, patients with DNA methyltransferases (DNMT1) mutations presented slight CNS hypomyelination.³⁷ Additionally, several studies in gliomas have described an extensive global DNA hypomethylation associated with aberrant activation of genes and non-coding regions^38-40 but also site-specific DNA hypermethylation that could contribute to tumorigenesis by silencing tumor suppressor genes.^41,42

Recently, our laboratory has provided the first evidence of DNA methylation changes in post-mortem tissues from patients affected by multiple sclerosis (MS), a common immune-mediated demyelinating disease.³⁵ We performed in parallel RNA-Sequencing and whole-genome bisulfite sequencing to directly address and correlate the transcriptomic and methylomic changes occurring in MS-affected brain tissues compared with controls. Genes known to regulate oligodendrocyte survival (i.e. BCL2L2, NDRG1) were hypermethylated and downregulated, whereas genes implicated in proteolytic processing (i.e., LGMN, CTSZ) were hypomethylated and upregulated in MS tissues. Our findings suggested that DNA methylation changes in OPC and OL contributed to MS pathology, possibly by affecting demyelination. To address the role of DNA methylation on OPC and OL gene expression and function we turned to cell-specific genetic approaches.

DNA methylation is a key regulator of oligodendrocyte differentiation during developmental myelination

Previous studies have shown that specific mature myelin genes (e.g. Mag) were demethylated upon oligodendrocyte differentiation but that blockade of DNA methylation enzymes during rat CNS development delays myelination, which suggested a more complex role of DNA methylation in the oligodendroglial lineage.^43,44 Our recent work addressed in detail the role of DNA methylation of OPC differentiation into OL during developmental myelination by combining whole-genome transcriptomic and methylomic analysis with loss-of-function experiments using conditional knockout mouse models.³⁶

We first observed that DNA methylation levels (5-mC levels) and expression of DNA methyltransferases (e.g., DNMT1 and DNMT3A) were dynamically regulated during the transition from OPC to OL in development. We then identified genome-wide changes in DNA methylation between OPC and OL and overlapped them with transcriptomic changes, revealing a negative correlation between DNA methylation at the promoter region of genes and transcription of those genes. The most significant methylomic and transcriptomic changes between OPC and OL were detected on genes with hypermethylated promoters and decreased expression during OPC differentiation (including genes related to neuronal lineage, cell cycle regulation and proliferation) and on genes with hypomethylated promoters and increased expression during OPC differentiation (including genes related to lipid enzymes enriched in the myelin compartment and myelin components, e.g., Mag). Indeed, DNA methylation at promoter regions is mainly associated with transcriptional repression, either by directly preventing the access of transcription factors to their binding sequence or by recruiting cofactors that modulate the chromatin environment.^45,46 For example, the E2F consensus motif contains two CpGs, whose methylation levels affect the affinity for E2F members including activator E2F1 that cannot bind to methylated motif and repressor E2F4 that can bind to one methylated CpG motif.⁴⁷ Magri et al. have shown that E2F1 binding sites in proliferative OPC are targeted and silenced by E2F4 during their differentiation, suggesting that DNA hypermethylation at these sites could directly modulate TF binding and induce gene repression in the oligodendroglial lineage.⁴⁸

To define the functional role of DNA methylation in the OL lineage in vivo, we crossed the Dnmt1^fl/fl line with the Olig1-cre line, to target the ablation of Dnmt1 specifically in OPC. Interestingly, this resulted in severe CNS hypomyelination in mutants, associated with tremors and decreased survival.^31,34,36 OPC were able to undergo lineage specification correctly, but despite the hypomethylation of myelin genes, they did not precociously differentiate or proliferate, either in vitro or in vivo. In highly proliferative embryonic cells, ablation of Dnmt1 has been associated with activation of genotoxic stress and apoptosis, eventually leading to embryonic lethality.^28,49 Here, in OPC lacking Dnmt1 we detected phosphorylated histone H2AX, a measure of genotoxic stress, but no massive apoptosis, suggesting that altered methylation in OPC was not threatening their survival. In addition, the lack of phenotype observed when crossing the Dnmt1^fl/fl line with the Cnp-cre line to target later stages of OL development, suggest that DNA methylation is essential to activate the first steps of OPC differentiation once they exit cell cycle.

DNA methylation is modulating alternative splicing events in oligodendrocyte lineage

Recently, DNA methylation marks have been shown to be correlated with alternative splicing regulation.^50,51 According to a kinetic model, the elongation rate of RNA polymerase could be directly modulated by chromatin structure, including 5-mC marks.^52,53 A recruitment model, mediated by binding partners (i.e., MeCP2, HP1 and PRMT5) with DNA methylation-dependent affinities, could also explain the link between methylation and alternative splicing.^4-56 Yearim et al. have shown the first direct evidence that DNA methylation regulates alternative splicing, acting as a splicing enhancer or silencer by recruiting splicing factors (i.e., HP1 in their model).⁵⁶ Alternative splicing events are occurring during oligodendrocyte differentiation and this mechanism is necessary for normal myelination.^7-61 Indeed, further analysis of our RNA-Sequencing have shown that 831 transcripts are alternatively spliced (70% concerning skipped exons) during the transition from OPC to OL in development.³⁶ By comparing the transcriptomic profile of control OPC and mutant OPC lacking Dnmt1, we identified 994 downregulated and 566 upregulated genes, as well as 341 alternatively spliced transcripts (51% concerning skipped exons and 26% concerning retained introns) in mutant cells compared with control cells. The regions targeted by differential alternative splicing events were associated with massive hypomethylation at the exon-intron boundaries in mutant OPC, suggesting a link between DNA methylation and alternative splicing in oligodendroglial cells. The gene ontology of the alternative spliced transcripts was enriched for genes involved in cell cycle process and myelination which indicated that lack of DNA methylation, in addition to modulate gene expression, might alter alternative splicing events, thus directly affecting oligodendrocyte differentiation.

Perspectives: DNA methylation a potential target of therapeutic strategies for oligodendroglial pathologies?

We concluded that the role of DNMT1 in oligodendroglial lineage cells is more complex than originally anticipated and encompasses the regulation of the proliferative state of OPC, as well as a tight coordination between alternative splicing and protein synthesis in the generation of myelinating OL (Fig. 1). The dysregulation of DNA methylation observed in many neurologic pathologies,^35,37-40,42 and the recapitulative hypothesis, suggesting that adult OPC differentiation during remyelination mimics neonatal OPC differentiation during developmental myelination,^62-66 let us speculate that DNA methylation might also be essential to adult OPC proliferation and differentiation regulation in the adult CNS. However, the transcriptomic profiles of neonatal OPC and adult OPC differ,⁶⁵ implying that epigenetic marks might accumulated in adult OPC and that DNA methylation might regulate slightly differently their differentiation.

Figure 1.

During development, DNA methylation, mediated by DNMT1, regulates oligodendrocyte progenitor cell (OPC) growth arrest and differentiation into mature oligodendrocyte (OL), by modulating gene expression and alternative splicing events. After ablation of Dnmt1, OPC present DNA damage, aberrant alternative splicing and endoplasmic reticulum stress, and fail to differentiate into mature OL, resulting in severe CNS hypomyelination and early death of the mutant mice.

Further epigenome-wide studies should be performed on adult OPC to specifically identify genomic loci that might be hypo- or hyper-methylated during their proliferation and their differentiation, in control conditions and after demyelination or in gliomas. Use of DNA methylation modulatory compounds could address the possibility to directly target and modulate specific gene expression and alternative splicing events in the oligodendroglial cells.^67,68 From a therapeutic standpoint, it would open new strategies to regulate OPC proliferation or differentiation that would have beneficial implications for oligodendroglial-related pathologies such as MS or glioma.

Abbreviations

CNS

Central nervous system

DNMT

DNA methyltransferase

MS

Multiple Sclerosis

OPC

Oligodendrocyte progenitor cell

OL

Oligodendrocyte

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Candice Chapouly and Kamilah Castro for their comments on the manuscript.

Funding

This work was supported by NIH-NINDS R37NS042925 and NS-R0152738 to P.C. and by postdoctoral fellowships from the Paralyzed Veterans of America (3061) and National Multiple Sclerosis Society (FG-1507–04996) to S.M.