Brain Region-Specific Expression of MeCP2 Isoforms Correlates with DNA Methylation within Mecp2 Regulatory Elements

Abstract

MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs) within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum), whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute towards characterizing the expression profiles of Mecp2/MeCP2 isoforms and thereby provide insights on the potential role of MeCP2 isoforms in the developing and adult brain.

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

“…Recently, we showed that DNA methylation at the REs found within the Mecp2 promoter and intron 1 correlated with the dynamic expression of Mecp2 in differentiating neural stem cells (NSC) and in response to a DNA demethylating drug Decitabine (also called 5-Aza-2′-deoxycytidine) . Recently, our studies in adult murine brain regions demonstrated a correlation between the expression of Mecp2 and DNA methylation at the Mecp2 REs in vivo (Olson et al, 2014). In this current report, we studied whether DNA methylation at these reported Mecp2 REs within the promoter and intron 1 contributes in deregulating Mecp2 expression induced by ethanol.…”

“…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). Transcript levels of Mecp2 and cell type-specific markers were examined using primers listed in Table 1.…”

“…The Ct values (threshold cycle) for all genes were normalized with reference to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh) to obtain ΔCt values for each sample. Relative quantification analysis was carried out using Microsoft Excel 2010 by comparing the 2 −ΔCt of each sample to that of untreated control sample at D2 and D8 as described in our previous studies Olson et al, 2014).…”

“…NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) was used to extract nuclear proteins as previously described Olson et al, 2014). Polyacrylamide gel electrophoresis was performed with 10-30 μg of nuclear extracts and proteins were transferred to nitrocellulose membranes and blocked in 1-2% skim milk in Tris buffered saline with Tween (TBST: TBS + 0.1% Tween) for 1 h at room temperature.…”

“…ACTIN and GAPDH were chosen to be the most reliable and suitable loading controls (for further details see the Results section). Quantification of WB bands was done with Adobe Photoshop CS5 software and normalized to ACTIN or GAPDH signals as described previously Olson et al, 2014). MeCP2 expression in ethanol-treated samples was compared to the corresponding control untreated cells within the same biological replicate.…”