Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. METHODS AND RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Transcriptomic and Epigenomic Landscape in Rett Syndrome.

Rett syndrome (RTT) is an extremely invalidating, cureless, developmental disorder, and it is considered one of the leading causes of intellectual disability in female individuals. The vast majority of RTT cases are caused by de novo mutations in the X-linked Methyl-CpG binding protein 2 (MECP2) gene, which encodes a multifunctional reader of methylated DNA. MeCP2 is a master epigenetic modulator of gene expression, with a role in the organization of global chromatin architecture. Based on its interaction with multiple molecular partners and the diverse epigenetic scenario, MeCP2 triggers several downstream mechanisms, also influencing the epigenetic context, and thus leading to transcriptional activation or repression. In this frame, it is conceivable that defects in such a multifaceted factor as MeCP2 lead to large-scale alterations of the epigenome, ranging from an unbalanced deposition of epigenetic modifications to a transcriptional alteration of both protein-coding and non-coding genes, with critical consequences on multiple downstream biological processes. In this review, we provide an overview of the current knowledge concerning the transcriptomic and epigenomic alterations found in RTT patients and animal models.

MeCP2 and Major Satellite Forward RNA Cooperate for Pericentric Heterochromatin Organization.

Methyl-CpG binding protein 2 (MeCP2) has historically been linked to heterochromatin organization, and in mouse cells it accumulates at pericentric heterochromatin (PCH), closely following major satellite (MajSat) DNA distribution. However, little is known about the specific function of MeCP2 in these regions. We describe the first evidence of a role in neurons for MeCP2 and MajSat forward (MajSat-fw) RNA in reciprocal targeting to PCH through their physical interaction. Moreover, MeCP2 contributes to maintenance of PCH by promoting deposition of H3K9me3 and H4K20me3. We highlight that the MeCP2B isoform is required for correct higher-order PCH organization, and underline involvement of the methyl-binding and transcriptional repression domains. The T158 residue, which is commonly mutated in Rett patients, is directly involved in this process. Our findings support the hypothesis that MeCP2 and the MajSat-fw transcript are mutually dependent for PCH organization, and contribute to clarify MeCP2 function in the regulation of chromatin architecture.

Epigenetic Factors That Control Pericentric Heterochromatin Organization in Mammals.

Pericentric heterochromatin (PCH) is a particular form of constitutive heterochromatin that is localized to both sides of centromeres and that forms silent compartments enriched in repressive marks. These genomic regions contain species-specific repetitive satellite DNA that differs in terms of nucleotide sequences and repeat lengths. In spite of this sequence diversity, PCH is involved in many biological phenomena that are conserved among species, including centromere function, the preservation of genome integrity, the suppression of spurious recombination during meiosis, and the organization of genomic silent compartments in the nucleus. PCH organization and maintenance of its repressive state is tightly regulated by a plethora of factors, including enzymes (e.g., DNA methyltransferases, histone deacetylases, and histone methyltransferases), DNA and histone methylation binding factors (e.g., MECP2 and HP1), chromatin remodeling proteins (e.g., ATRX and DAXX), and non-coding RNAs. This evidence helps us to understand how PCH organization is crucial for genome integrity. It then follows that alterations to the molecular signature of PCH might contribute to the onset of many genetic pathologies and to cancer progression. Here, we describe the most recent updates on the molecular mechanisms known to underlie PCH organization and function.

ATRX Contributes to MeCP2-Mediated Pericentric Heterochromatin Organization during Neural Differentiation.

Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2, mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH.