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

Domenico Marano,Salvatore Fioriniello,Maurizio D'Esposito,Francesca Fiorillo,Floriana Della Ragione,Richard J Gibbons

doi:10.3390/ijms20215371

Abstract

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.

Highlights

Methyl-CpG binding protein 2 (MeCP2), encoded by the X-linked MECP2 gene, was identified in 1992 as a protein able to bind methylated DNA [1]
We analyzed FL-Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX) enrichment at pericentric heterochromatin (PCH) in terminally differentiated, untreated TK23_WT and Mecp2-/y neurons, and in terminally differentiated TK23_WT neurons after RNase A treatment, by chromatin immunoprecipitation (ChIP), a more sensitive and quantitative method, by using H300 antibody. These experiments highlighted a decreased accumulation of ATRX at PCH in both Mecp2-/y and in RNase A-treated TK23_WT neurons, in comparison with untreated TK23_WT cells; the RNase A treatment elicited a greater effect than the lack of MeCP2 (Figure 2B). These results suggest that both MeCP2 and some RNA components play a role in the targeting of full length ATRX (FL-ATRX) to PCH
We reported that FL-ATRX targeting to PCH is dependent on an RNA component in terminally differentiated neurons (Figure 2A,B) and that this protein partially co-localizes with major satellite (MajSat)-fw transcript to chromocenters (Figure 2C)

Summary

Introduction

Methyl-CpG binding protein 2 (MeCP2), encoded by the X-linked MECP2 gene, was identified in 1992 as a protein able to bind methylated DNA [1]. MeCP2 is a master epigenetic modulator of transcription [2,3,4] that mediates gene silencing via methylation-dependent chromatin remodeling, through the recruitment of histone deacetylases and co-repressors, such as histone deacetylase 1 (HDAC1) and switch-independent 3A (Sin3A), respectively [5]. More recent findings indicated that MeCP2 acts as a transcriptional activator in specific brain subregions [2,3,6]. Several RTT phenotypes are successfully recapitulated by constitutive and brain-specific knockout (ko) of Mecp2 [16,17]. Despite the large number of studies achieved in the last couple of decades designed to unravel the molecular function of MeCP2, to date, it is still not clear how dysfunction of this protein contributes to RTT pathogenesis

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