Decitabine alters the expression of Mecp2 isoforms via dynamic DNA methylation at the Mecp2 regulatory elements in neural stem cells

Abstract

BackgroundAberrant MeCP2 expression in brain is associated with neurodevelopmental disorders including autism. In the brain of stressed mouse and autistic human patients, reduced MeCP2 expression is correlated with Mecp2/MECP2 promoter hypermethylation. Altered expression of MeCP2 isoforms (MeCP2E1 and MeCP2E2) is associated with neurological disorders, highlighting the importance of proper regulation of both isoforms. While known regulatory elements (REs) within the MECP2/Mecp2 promoter and intron 1 are involved in MECP2/Mecp2 regulation, Mecp2 isoform-specific regulatory mechanisms are unknown. We hypothesized that DNA methylation at these REs may impact the expression of Mecp2 isoforms.MethodsWe used a previously characterized in vitro differentiating neural stem cell (NSC) system to investigate the interplay between Mecp2 isoform-specific expression and DNA methylation at the Mecp2 REs. We studied altered expression of Mecp2 isoforms, affected by global DNA demethylation and remethylation, induced by exposure and withdrawal of decitabine (5-Aza-2′-deoxycytidine). Further, we performed correlation analysis between DNA methylation at the Mecp2 REs and the expression of Mecp2 isoforms after decitabine exposure and withdrawal.ResultsAt different stages of NSC differentiation, Mecp2 isoforms showed reciprocal expression patterns associated with minor, but significant changes in DNA methylation at the Mecp2 REs. Decitabine treatment induced Mecp2e1/MeCP2E1 (but not Mecp2e2) expression at day (D) 2, associated with DNA demethylation at the Mecp2 REs. In contrast, decitabine withdrawal downregulated both Mecp2 isoforms to different extents at D8, without affecting DNA methylation at the Mecp2 REs. NSC cell fate commitment was minimally affected by decitabine under tested conditions. Expression of both isoforms negatively correlated with methylation at specific regions of the Mecp2 promoter, both at D2 and D8. The correlation between intron 1 methylation and Mecp2e1 (but not Mecp2e2) varied depending on the stage of NSC differentiation (D2: negative; D8: positive).ConclusionsOur results show the correlation between the expression of Mecp2 isoforms and DNA methylation in differentiating NSC, providing insights on the potential role of DNA methylation at the Mecp2 REs in Mecp2 isoform-specific expression. The ability of decitabine to induce Mecp2e1/MeCP2E1, but not Mecp2e2 suggests differential sensitivity of Mecp2 isoforms to decitabine and is important for future drug therapies for autism.

Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

“…Primary neurospheres were then dissociated and single cells were differentiated for 8 days, as we have shown to be sufficient for generating neurons, astrocytes and oligodendrocytes ( Fig. 1A) (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). …”

“…To assess the impact of ethanol exposure on Mecp2/MeCP2 transcript and protein expression, we used a previously characterized embryonic NSC differentiation system (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). Brain-derived neural stem cells were isolated from dissected forebrains of E14.5 mouse embryos and were cultured for 7 days in the presence of growth factors to generate neurospheres.…”

“…RNA was converted to cDNA with a reaction set up containing Superscript III Reverse Transcriptase (Invitrogen) using previously described methods (Kobrossy et al, 2006;Liyanage et al, 2013;Nolte et al, 2006;Rastegar et al, 2004). Quantitative RT-PCR was performed using SYBR Green-based RT 2 qPCR Master Mix (Applied Biosystems) in a Fast 7500 Real-Time PCR machine (Applied Biosystems) as previously described (Barber et al, 2013;Liyanage et al, 2013;Olson et al, 2014).…”

“…Neural stem cells isolated from the forebrains of embryonic day (E) 14.5 CD-1 mice were cultured as previously described (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). In brief, dissected forebrain tissues were homogenized in NSC media Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 1:1 (DMEM/F12; Wisent) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glutamine, antibiotic/antimycotic, glucose, recombinant human epidermal growth factor (rhEGF; Sigma, 20 ng/ml), basic fibroblast growth factor (bFGF; Upstate, 20 ng/ml), heparin (Sigma, 2 μg/ml) and hormone mix [DMEM:F12, glucose (0.6%), insulin (0.25 mg/ml), transferrin (1 mg/ml), progesterone (0.2 μM), putrescine (0.097 mg/ml), sodium selenite (0.3 μM)] and plated at a density of 10 5 cells/cm 2 in serum-free full NSC media.…”

“…We have used this neural stem cell model to study the regulation of genes involved in neural development including Mecp2 and Meis1 (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). We have successfully used the same NSC system to rescue the aberrant neuronal morphologies in Mecp2-deficient NSC by MECP2 gene therapy strategies .…”