In the brain, precisely controlled MECP2/MeCP2 transcript and protein expression levels are critical, as even slightly altered expression is associated with severe neurological symptoms [2, 16, 57–60]. However, so far little is known about how MeCP2 expression is regulated in the developing brain. MeCP2 is a major epigenetic regulator in brain, and its reduced expression in the autistic brain is associated with MECP2 promoter hypermethylation . Surprisingly, the role of DNA methylation in MeCP2 expression during brain development is unclear. Currently, most diseases that are associated with aberrant MeCP2 function or expression deficits, including autism and Rett syndrome, have no cure or effective treatment. This underscores an urgent need for investigating how MeCP2 expression is regulated in the brain. Such knowledge for addressing this gap is essential for designing possible future therapeutic strategies. DNA methylation is a reversible epigenetic modification , which can be targeted by existing Food and Drug Administration (FDA)-approved drugs, including decitabine, which is suggested for use in autism [61, 62]. Therefore, investigating the effect of such epigenetic drugs on MeCP2 expression is important. Therapeutic approaches such as gene therapy or restoring MeCP2 expression by genetic engineering have been suggested as possible therapeutic strategies for MeCP2-associated disorders [14, 15, 35]. However, even mild MeCP2 overexpression can lead to severe neurological complications, highlighting the importance of understanding MeCP2 regulatory mechanisms. Since both MeCP2 isoforms have been implicated in severe neurological disorders, investigating MeCP2 regulation is equally important for individual isoforms. This present study is the first report on the potential role of DNA methylation at the Mecp2 REs and the impact on the expression of Mecp2 isoforms.
We observed globally altered DNA methylation upon decitabine exposure and withdrawal. Since DNA methylation is a major epigenetic mechanism that is involved in modulating gene expression and chromatin architecture , these observed changes in 5mC levels may possibly lead to altered chromatin structure and genome-wide changes in gene expression. Furthermore, the presented findings highlight that exposure to drugs that disturb the epigenetic marks during differentiation of brain cells may lead to aberrant DNA methylation profiles. Our observations at D8 indicate that, even after the disturbance factor is withdrawn from the system, an epigenetic memory for this disturbance may be associated throughout cellular differentiation of brain cells. Thus, our findings highlight the biological importance of maintaining proper regulation of epigenetic factors/modifications during brain development with a clear focus on DNA methylation and MeCP2.
Our results show that decitabine alters Mecp2/MeCP2 expression at both the transcript and protein levels. Importantly, even minor changes in Mecp2 transcript expression led to nearly 2- to 3-fold altered protein expression, highlighting the biological significance of proper regulation of Mecp2 expression at the transcript levels. The observed correlation between the Mecp2/MeCP2 (total) and Mecp2e1/MeCP2E1 transcript/protein expression at D2 reinforces the concept that potential changes in Mecp2 transcript levels may reflect possible changes at the protein levels. However, the non-correlated Mecp2/MeCP2 (total) and Mecp2e1/MeCP2E1 transcript/protein expression at D8 indicates that decitabine withdrawal causes not only transcriptional but also, post-transcriptional regulation of MeCP2 expression, leading to reduced expression of MeCP2 (total)/MeCP2E1. One such post-transcriptional regulatory mechanism could be the action of micro-RNAs such as miR132, expression of which has been shown to be increased by 5-aza-2′-deoxycytidine/decitabine , and has the ability to repress MeCP2 expression .
Increased promoter methylation of autistic candidate genes such as RORA, BCL2 and MECP2 are shown to be associated with reduced expression of these genes in autistic patients [2, 19, 62]. Treatment with decitabine was shown to demethylate promoters and restore/induce the expression of the silenced RORA and BCL2 in autistic and patients with fragile X syndrome and hence, the use of DNA demethylating agents in drug therapy for autism and fragile X syndrome has been suggested [61, 62]. A similar strategy to restore/induce MeCP2 expression might be extended to treat such diseases associated with reduced MeCP2 expression, including autism and RTT. Providing insights on such therapeutic strategies, the application of epigenetic drug therapy to induce non-mutated copy of MECP2 expression in Rett syndrome cell lines has been suggested and attempted previously . Therefore, our findings on the ability of decitabine to induce MeCP2 expression in differentiating NSC provide further insights on designing possible drug therapies for autism. Even though the exposure of RTT cell lines (fibroblasts) to lower doses of decitabine for a longer period did not activate MECP2 expression , our results indicate that moderate dose of decitabine can induce Mecp2/MeCP2 expression within a shorter period. However, inhibition of MeCP2 by withdrawal of decitabine as well as other observed changes in DNA methyl marks implies that such drug therapy should be administrated with great caution.
Our findings on the changes in DNA methylation at the Mecp2 REs are in agreement with the previous reports on MECP2 promoter methylation, which demonstrate that an approximate difference of 2.0 to 2.5% overall methylation over a region -233 to -531 upstream of the MECP2 promoter is correlated with reduced MECP2 expression in autistic male brains. The authors report that within the 15 CpG sites found in this MECP2 promoter region, two CpG sites are specifically altered in the autistic males . Furthermore, our results are in agreement with a previous report on significantly reduced MeCP2 expression in the postnatal mouse brain (under stress), which is associated with 2 to 5% increased methylation at the individual CpG sites within a 164-bp region of the Mecp2 promoter . Supporting these observations, studies have also shown minor differences, such as 2 to 5% DNA methylation causing significant changes in the expression of other genes, such as RASSF1, in the human brain , AMOTL2 in the human heart , and PGC1α in human muscles . Therefore, although the detected DNA methylation changes in this current study are not considerably high (they varied between 2 to 15%), they were statistically significant for average DNA methylation (within R1, R3 and R5) during NSC differentiation, and for several specific CpG dinucleotides subsequent to decitabine treatment (within R2, R4, R5, and R6), and are likely to be biologically important.
The Mecp2 promoter CpG island studied by Franklin et al.,  overlaps with the R1 and R2 of the Mecp2 promoter that we studied here. The significantly methylated CpGs reported in their study coincides with the R2 CpGs, where we observed changes at individual CpG sites as well as average methylation upon decitabine treatment. However, in our study we did not see any significant change in the R1 CpG sites (both D2 and D8), where Franklin et al., reported DNA methylation changes. Importantly, the results we obtained for one of the promoter regions studied (R2) are in agreement with this previous report, which showed a biological and functional importance of the methylation changes in regulating MeCP2 expression in response to stress in vivo. Therefore, it is likely that the detected changes we observed in the Mecp2 REs in our study also have biological importance. The hypermethylation of this R2 region in mouse brain was associated with MeCP2 downregulation , and hence it is possible that the hypomethylation/demethylation of the same R2 region causes Mecp2/ MeCP2 upregulation.
Our results on the ability of 2.5 μM decitabine to upregulate Mecp2e1 (but not Mecp2e2) suggest that the two isoforms may have different sensitivities to drugs/chemicals. This observation is in agreement with the previous report on the higher sensitivity of Mecp2e1 than Mecp2e2 to Bisphenol A . These observations further suggest that the differential sensitivity to drugs might be used to specifically induce only one Mecp2 isoform. This is also important because overexpression of Mecp2e2, but not Mecp2e1 causes neuronal cell death . Hence, our study provides a functional relevance of DNA demethylation at the Mecp2 REs by decitabine causing upregulation of Mecp2e1, but not Mecp2e2.
The observed negative correlation between the expression of both Mecp2 isoforms and Mecp2 promoter elements are novel and are in accordance with previous correlation studies on the human MECP2 expression and promoter DNA methylation [2, 19]. Furthermore, our study is novel in demonstrating a dynamic (positive/negative) correlation between the intronic DNA methylation and expression of Mecp2 isoforms in differentiating brain cells. It is possible that the promoter regions analyzed in our study (which also overlap with the core Mecp2 promoter ) might be shared by both Mecp2 isoforms, whereas depending on the stage of neural differentiation, intron 1 regions may add another layer of regulation for Mecp2 isoform-specific expression. Supporting our findings, the role of intronic DNA methylation in regulating gene expression of other genes has been previously reported [70, 71]. Several other reports also show evidence that gene expression negatively correlates with promoter methylation and positively correlates with gene-body methylation [67, 72].
Intronic DNA methylation is reported to be involved in regulating alternative splicing [27, 28]. Although, it is known that Mecp2 isoforms are generated by alternative splicing [4, 5], the underlying molecular mechanisms are still unclear. We observed that the expression ratio of Mecp2e1/Mecp2e2 changed during NSC differentiation. The observed correlation between the splice ratio and intron 1 R4 DNA methylation in differentiating NSC at D2 would provide insights on the potential importance of this region in Mecp2 alternative splicing.
The intron 1 regions analyzed in this study were designated as part of a silencer element, which has been previously proposed to regulate MECP2 alternative splicing and tissue-specific expression . Our findings are in agreement with possible involvement of these regions in Mecp2 isoform-specific expression. Although the link between DNA methylation and Mecp2 expression is supported by our results in the NSC system, the contribution of other epigenetic modifications such as histone acetylation and histone methylation should not be excluded [73, 74].