Epigenetic regulation of oligodendrocyte myelination in developmental disorders and neurodegenerative diseases

SWI/SNF family members

The SWI/SNF family of ATPase dependent chromatin remodelers have been shown to play critical roles in the development of the oligodendrocyte lineage. Deletion of Brg1/Smarca4, the core helicase component of the SWI/SNF family, in NPCs inhibits oligodendrocyte and astrocyte lineage development while increasing neuronal differentiation in the ventricular zone of the developing brain^80,84. A lineage-specific transcription factor, OLIG2, can recruit the BRG1/SWI/SNF complex to the promoters and enhancers of oligodendrocyte lineage genes such as Sox10 to activate their transcription. BRG1 is also necessary for OPC differentiation. BRG1 expression increases after induction of rat OPC differentiation with T3 thyroid hormone³⁶. These increasing levels are critical for OPC differentiation as conditional knockout in Olig1-expressing oligodendrocyte progenitors and PDGFRa-expressing OPCs in vivo leads to oligodendrocyte differentiation defects and profound dysmyelination defects³⁶ (JW and QL, unpublished). Of note, the loss of Brg1 does not affect OPC survival in culture or in vivo³⁶. However, Brg1 knockout in later OPCs, such as NG2⁺ or CNP⁺, committed or post-mitotic OPCs, respectively, had progressively less severe effects on differentiation³⁷, suggesting that BRG1 effects are stage-dependent. This stage-dependent severity suggests that BRG1 activates early pro-differentiation factors, such as SOX10, that can continue to mediate downstream genetic programs in oligodendrocyte lineage progression despite the upstream loss of BRG1^36,37. In addition, other chromatin remodelers such as CHD8 or CHD7 (discussed below) potentially compensate for the loss of BRG1 at later stages.

CHD family members

CHD7 is highly enriched in the oligodendrocyte lineage, especially in differentiating oligodendrocytes. CHD7 mutations result in a series of birth defects called CHARGE syndrome, which exhibits impaired white matter development and myelination in addition to other congenital developmental abnormalities^85,86. CHD7, like BRG1 above, does not affect OPC formation but instead causes defects in OPC differentiation^6,38,39. In fact, Chd7 is a direct target of the OLIG2/BRG1 complex and its expression is greatly increased by the binding of this complex at its promoter⁶. CHD7 can complex with SOX10 to activate downstream regulators of oligodendrocyte differentiation. CHD7 activates the expression of OPC pro-differentiation regulators, including SOX10 and NKX2-2³⁹, as well as other oligodendrocyte-expressing transcription factors such as Osterix/Sp7 and Creb3l2^6,39. Intriguingly, deletion of Chd7 in PDGFRa⁺ OPCs appears to impair OPC survival via p53 upregulation³⁹. CHD7 binds to the p53 promotor in OPCs and limits p53 expression to maintain the survival of OPCs³⁹.

CHD7 is also required for remyelination after lysolecithin-induced demyelination⁶ or spinal cord laminectomy, wherein it interacts with SOX2 to drive OPC differentiation³⁸. Chd7 deletion impairs OPC proliferation after spinal cord injury³⁸ but not in the developing brain^6,39, suggesting a context-dependent CHD7 regulation of OPC proliferation. However, CHD7 appears to be dispensable for the maturation of oligodendrocytes, possibly due to compensation by other CHD members such as CHD8³⁹, which has been shown to work together with CHD7 to regulate OPC survival and maturation³⁹.

Another CHD family member, CHD8, is also critical for proper oligodendrocyte development. CHD8 has been linked to a subset of autism disorders, which exhibit a defect in white matter tracts and myelination^{40,41,87–89}. Chd8 knockout in Olig1⁺ progenitors causes defects in CNS myelination, particularly in the spinal cord because of severe reductions in PDGFRα-expressing OPCs in this region⁴¹, suggesting a region-specific role of CHD8 in OPC survival and differentiation. Deletion of Chd8 at the post-natal stages with an inducible PDGFRα-CreER driver also blocks OPC differentiation. The defects in oligodendrocyte differentiation are due to the cell-specific loss of Chd8 in the oligodendrocyte lineage as there are no defects seen after Chd8 knockout in post-mitotic neurons⁴¹. This suggests that the myelination defects seen in CHD8 mutant patients are cell-autonomous defects due to the loss of CHD8. Remyelination after lysophosphatidylcholine (LPC)-induced demyelinating lesions in the spinal cord is also dependent on CHD8 expression⁴¹. CHD8 dysregulation may be an important factor for white matter pathogenesis and remyelination failure given the critical role of CHD8 for OPC replenishment and remyelination in demyelinating lesions.

CHD7 and CHD8 have similar structures and can bind many of the same targets. However, they target different gene regions during oligodendrocyte differentiation. CHD7 predominantly binds to promotor regions in OPCs but switches to enhancer regions in oligodendrocytes³⁹. CHD8, in contrast, binds predominantly to promotor elements near transcription start sites marked by an activating histone mark H3K4me3 in OPCs and oligodendrocytes where it recruits an H3K4 activating histone methyltransferase MLL2 (mixed lineage leukemia 2) complex to drive expression of oligodendrocyte lineage genes⁴¹. MLL2–4 and other family members can form a macromolecular complex called COMPASS (complex of proteins associated with Set1) to methylate H3K4 and regulate gene transcription⁹⁰. Strikingly, blocking lysine demethylase KDM5, an enzyme that erases methylation on H3K4⁹¹, with a pan-KDM5 inhibitor CPI-455 rescues the differentiation defects in Chd8 mutant OPCs⁴¹, suggesting that targeting this eraser to enhance H3K4me3 levels might facilitate the restoration of myelination defects caused by CHD8 defects.

The chromatin remodelers may all work together to regulate oligodendrocyte development but each has its own preferences for regulatory elements and mechanisms to control expression of specific sets of targeted genes. Of these, CHD8 appears to turn on the earliest, eventually promoting BRG1 expression which in turn induces CHD7 expression⁶. This successive signaling cascade enables the progression from OPCs to mature myelinating oligodendrocytes and likely forms convergent points upon which other checkpoints and regulatory mechanisms act to facilitate this development. Nonetheless, these chromatin remodelers could operate simultaneously in a non-linear fashion to promote oligodendrocyte lineage progression.

HATs

The activity of HATs is responsible for the acetylation of histones, leading to relaxed chromatin coiling and increased gene expression. The acetylation of histone H3 on lysine 27 (H3K27ac) is often deposited at active regulatory elements such as enhancers and promoters and is positively correlated with the activation of gene transcription⁹⁵. H3K27 acetylation is catalyzed by multiple HATs, including p300 (also known as EP300), CREB-binding protein (CBP), TIP60, and PCAF^96,97. Histone acetylation status can be further recognized by bromo-, PHD-, Tudor-, or WD40-domain-containing transcription activating regulators, which further modulate target gene expression^95,98–100.

In line with a critical role for histone acetylation in regulating the oligodendrocyte lineage, the loss of multiple HATs can lead to defects in the myelination process. Deletion of EP400, a key subunit of the TIP60 HAT complex, in Cnp-expressing oligodendroglial cells results in a defect in oligodendrocyte terminal differentiation, leading to profound hypomyelination⁴⁵. A genetic disorder, Rubinstein–Taybi syndrome, is associated with mutations in p300 (also known as EP300), which is characterized in part by hypoplasia of the corpus collosum and congenital hypomyelination^42,43. The role of p300 in controlling oligodendrocyte development is still being explored, but p300 has been shown to interact with HDAC3 (discussed in more detail below) partly facilitating the role of HDAC3 in promoting oligodendrocyte as opposed to astrocytic cell lineages during early differentiation from NPCs via its promotion of Olig2 expression¹⁰¹. Also, inhibition of p300 activity itself can lead to pronounced defects in OPC differentiation (JW and QL, unpublished).

HDACs

Histone acetylation status can be reversed by HDACs. Mammals possess four classes of HDACs. Class I contains HDACs 1–3 and 8, class II contains HDACs 4–7 and 9 and 10, class III are NAD-dependent HDACs (also known as sirtuins, encompassing SIRT1–7), and finally class IV contains one HDAC, HDAC11^102,103. Pharmacological studies using HDAC inhibitors have indicated the importance of HDACs in oligodendrocyte fate specification and differentiation. Treating rat NPCs with valproic acid inhibited oligodendrogenesis and astrogenesis while promoting neurogenesis likely through NeuroD1 upregulation following the inhibition of HDAC activity¹⁰⁴. Blocking HDAC activity with pan inhibitors also disrupts the differentiation of OPCs into mature oligodendrocytes¹⁰⁵. The timing of this treatment appears to be critical. Treating cells with pan-HDAC inhibitors after the differentiation process has been shown to have minimal effect on oligodendrocyte differentiation¹⁰⁶. These studies indicate that HDACs play various roles at different stages during OPC differentiation and subsequent myelination. However, classic pan-HDAC inhibitors are non-specific and target HDACs across multiple classes. Genetic manipulation can specifically target individual HDACs to define their specific functions during oligodendrocyte lineage progression.

Class I HDACs

The expression levels and functions of class I HDACs are important for oligodendrocyte fate specification and differentiation (Figure 2). HDAC1 and HDAC2, when knocked out individually in the oligodendrocyte lineage, have no obvious effects on OPC formation, proliferation, or differentiation⁴⁶. However, the double-knockout animals die shortly after birth, and analysis revealed a severe defect in OPC proliferation or differentiation in these animals, suggesting that HDAC1 and HDAC2 can functionally compensate for the loss of the other in oligodendrocyte lineage determination⁴⁶. Another HDAC class I family member, HDAC3, has been implicated in the control of oligodendrocyte lineage specification⁴⁴ but differs from those effects observed in HDAC1. HDAC3 deletion at the same stages as HDAC1 and 2 above results in a switch from oligodendrocyte to astrocytic fates, suggesting that HDAC3 regulates the fate choice of primitive OPCs between astrocytic and oligodendrocytic cell lineages⁴⁴.

HDACs have been shown to exhibit non–histone dependent functions during oligodendrocyte development. HDAC1/2 co-repressor complexes can compete with β-catenin for binding to TCF7L2 (TCF4), a member of the TCF transcription factor family, resulting in the disinhibition of TCF7L2, which then is free to promote OPC differentiation^46,47,48. This is an example of how non-enzymatic activity of HDACs through protein–protein interaction in addition to the deacetylase activity can function to regulate genetic expression. HDAC1 can also be recruited by YY1 transcription factor to the promotor of oligodendrocyte differentiation inhibitory genes such as Id4 to reduce their expression^107,108. In addition, HDAC1 and HDAC3 can deacetylate the OLIG1 transcription factor, increasing its likelihood of nuclear translocation and ultimate promotion of OPC differentiation⁴⁹.

HDAC activity can be modulated through co-regulators or covalent modifications¹⁰⁹. For example, casein kinase 2 (CK2) phosphorylates HDAC3 to activate its activity while phosphatase 4 dephosphorylates it¹¹⁰. Of interest, in vitro experiments revealed that the CK2 kinase, which activates HDAC3, also elevates expression of OLIG2, a critical transcription factor for initiating oligodendroglial cell fate¹¹¹. HDAC3 also forms protein complexes with co-repressor complexes such as NCOR and SMRT to regulate its activity¹¹². NCOR has been shown to negatively regulate astrogenesis through inhibiting JAK-STAT signaling, activation of which leads to astrocyte differentiation¹¹³. In addition, HDAC3/NCOR can deacetylate and inactivate astrocyte-promoting factor STAT3 and therefore promote oligodendrogenesis while inhibiting the astroglial fate⁴⁴. HDAC3 also forms complexes with the HAT p300, to regulate OLIG2 expression levels during OPC specification⁴⁴. This interaction likely indicates that the coordinated activity of two opposing factors is required for oligodendrocyte and astrocytic lineage fate decisions.

HDAC3 functions not only as a transcriptional co-repressor, as one may assume from its histone deacetylation activity, but also as a transcriptional co-activator as in its role in the activation of retinoic acid response elements^114,115. It is worth noting that HDAC3 deacetylase activity may not be vital for oligodendrocyte development. HDAC3 requires NCOR and SMRT to promote its deacetylation activity. Deleting the deacetylase-activating domains (DADs) in NCOR and SMRT abrogates the deacetylase activity of HDAC3. However, the DAD deletion mice survive to adulthood and exhibit normal myelination whereas the ablation of HDAC3 is embryonic lethal¹¹⁶. Although the function of HDAC3 catalytic site mutants remains to be determined, the current data suggest that HDAC3 may serve as a scaffold for multi-component transcriptional regulatory complexes vital for oligodendrocyte myelination.

Other HDAC classes

Among class II HDACs, HDAC6 has been shown in rat oligodendrocyte cultures to acetylate the microtubule-associated protein tau and α-tubulin, both of which are required for normal oligodendrocyte development⁵². HDAC10, along with HDAC1 and HDAC3, has also been shown to regulate the nuclear localization of OLIG1 for oligodendrocyte maturation⁴⁹. It is worth noting that the enzymatic activity of class II HDACs is dependent on the HDAC3/SMRT/N-CoR complex¹¹⁷.

The class III HDACs SIRT1 and SIRT2 have been shown to regulate early oligodendrocyte lineage determination^50,51,118. SIRT2, in particular, is highly expressed in mature oligodendrocytes¹¹⁹ and regulates the differentiation of oligodendrocytes as blocking its activity or overexpressing it prevents or promotes differentiation of CG4 oligodendroglial cells, respectively^120–122. This class of HDACs also relies on NAD as a co-factor for deacetylase activity¹²³. The loss of NAMPT, the rate-limiting enzyme for NAD biosynthesis in mammals, leads to defective oligodendrocyte development^118,124. Like those of many other epigenetic factors, the effects that SIRT1 and SIRT2 have on development are stage-dependent. For example, Sirt1 knockout in NPCs increases OPC proliferation⁵⁰ while Sirt1 knockout in PDGFRa⁺ OPCs promotes cell cycle exit and OPC differentiation¹²⁵. Notably, SIRT2 is depleted in myelin sheathes of PLP-deficient oligodendrocytes, a model for spastic paraplegia, suggesting that SIRT2 might have a role in myelin sheath maintenance and provide trophic support of axons¹²⁶.

Finally, the class IV HDAC, HDAC11 has been shown to regulate H3K9 and H3K14 acetylation and expression levels of Mbp and Plp genes^53,54. HDAC11 overexpression enhances the maturation of an oligodendrocyte cell line (OL-1) in vitro^53,54, suggesting a potential role of HDAC11 in regulating myelin gene expression. At present, how the function of each HAT and HDAC is controlled, individually and coordinately, on a system-wide level to regulate the complex processes of oligodendrocyte development and myelination remains to be defined. This is of particular importance given the reiterative involvement of many HAT and HDAC enzymes in the gene regulatory network during CNS development and regeneration.

miRNAs

Comparisons between OPCs and immature and mature oligodendrocytes revealed a set of miRNAs enriched during oligodendrocyte differentiation, including miR-219, miR-138, and miR-338^64–66,150. miR-219 is necessary and sufficient to induce differentiation and can even partially rescue the Dicer knockout phenotype^65,66. Knockout of miR-219–encoding genes (miR-219-1 and miR-219-2) led to reduced myelination throughout the CNS⁶⁷. In contrast to miR-219, miR-338 is dispensable for OPC differentiation or myelination in vivo. However, there was a synergistic effect on the myelination defects in miR-219 and miR-338 double-conditional knockout mice. miR-219 is also required for remyelination after LPC-induced demyelination. Overexpression of miR-219 in OPCs increased oligodendrocyte differentiation and could even promote repair when overexpressed genetically or administered with intrathecal injections of miR-219 mimics⁶⁷. miR-219 likely functions by repressing inhibitors of OPC differentiation, including Lingo1 and Etv5⁶⁷. miR-219 has been suggested in zebrafish to regulate oligodendrocyte lineage specification from NPCs¹⁵¹. However, there were no defects of OPC specification in the miR-219-1/2 double-null animals⁶⁷, suggesting a species-specific effect. Other miRNAs have been associated with oligodendrocyte development (reviewed in more detail in 152), but of these miR-219 exhibits the strongest effects.

A set of miRNAs have been identified to negatively regulate oligodendrocyte differentiation. One of these, miR-212 was found to be downregulated in oligodendrocytes after spinal cord injuries in rats, where it appears to repress expression of differentiation-associated genes⁶⁸. Another such miRNA, miR-125a-3p, was enriched in cerebrospinal fluid from MS patients with active demyelinating lesions. miR-125a-3p overexpression impaired oligodendrocyte differentiation whereas knocking it down promoted differentiation⁶⁹. Similarly, overexpression of miR-27a inhibits oligodendrocyte differentiation and myelination by activating the Wnt/beta-catenin signaling pathway¹⁵³. Such negative regulatory miRNAs are potential targets for enhancing remyelination.

LncRNAs

LncRNAs are long RNA sequences (more than 200 nucleotides) that are highly conserved across species but have no coding potential¹⁵⁴. LncRNAs have been implicated in the regulation of both normal development^155,156 and diseases^157,158. LncRNAs can be very specifically expressed in the oligodendrocyte lineage; for instance, lncOL1 and Pcdh17it were recently identified as specific markers for oligodendrocytes and immature premyelinating oligodendrocytes, respectively^56,71. Gene-chip microarrays were initially used to identify lncRNAs such as SOX8OT (SOX8 opposite transcript) in cultured OPCs. SOX8OT might have a role in regulating oligodendrocyte differentiation through its regulation of SOX8^72,73.

Combining RNA sequencing and chromatin mapping across oligodendrocyte lineage stages revealed several lncRNAs that are actively transcribed and restricted to this lineage⁵⁶. Of these, lncOL1 was identified as a top candidate on the basis of its abundance, regulation during oligodendrocyte differentiation, and preliminary screening for effects on myelin-associated gene expression. lncOL1 overexpression led to precocious oligodendrocyte differentiation in mouse embryos, and lncOL1 knockout led to defects in OPC differentiation while having no effect on OPC formation. Interestingly, these myelination defects were seen only during development but not in adulthood, suggesting a role of lncOL1 in regulating the timing of myelinogenesis and not the maintenance of myelin. lncOL1 mediates this effect in part by its interaction with SUZ12, a member of the PRC2 complex which mediates histone methylation through EZH2. lncOL1 directs the PRC2 complex to silence the expression of OPC-associated genes via H3K27me3 deposition⁵⁶. In contrast to lncOL1, lnc-OPC, another lncRNA found in the oligodendrocyte lineage, is enriched in OPCs and regulated by OLIG2⁷⁰. Knockdown of lnc-OPC in cultured NPCs limited their differentiation into OPCs without affecting NPC proliferation⁷⁰. In similar fashion, lnc158 expression directly correlated with oligodendrocyte-associated protein expression and differentiation along the oligodendrocyte lineage⁷⁵. In addition, another lncRNA, Neat1, was downregulated in schizophrenia. Neat1 knockout mice exhibited a reduction in the number of oligodendrocytes in the frontal cortex because of a failure in the retention of oligodendrocyte transcription factors in the nucleus⁷⁴. These studies indicate that lncRNAs regulate oligodendrocyte development and myelination via various processes such as controlling mRNA transcripts, nuclear localization of transcription factors, or interactions with chromatin remodelers.