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

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.

Epigenetic Factors that Control Pericentric Heterochromatin Organization in Mammals

“…Of note, we have recently proposed that, in neurons obtained by in vitro differentiation of mESCs, MeCP2 directly regulates the expression of Hp1β and Hp1γ [ 20 ] ( Figure 3 B), thus strengthening the idea that the two roles of MeCP2 as a chromatin organizer and a transcriptional modulator are tightly interconnected. Moreover, an interaction between MeCP2 and MBD2, another member of the MBD family, has been described ( Figure 2 A, Step 4).…”

“…H3K9me3 and H4K20me3 histone marks represent anchors for the attachment of HP1s to PCH [ 44 , 91 ], with HP1s thus highly enriched in these heterochromatic regions [ 20 ]. HP1s belong to a family of highly conserved proteins [ 92 ] that in mammals includes three isoforms, HP1α, HP1β, and HP1γ [ 93 ], which show different genomic distributions that include both euchromatic loci [ 94 , 95 ] and heterochromatic regions [ 96 ].…”

“…Highly methylated MajSat DNA repeats provide a binding platform for proteins that belong to the methyl-binding domain (MBD) family, a particular group of “readers” of epigenetic marks that establish a functional link between DNA methylation and histone modifications [ 50 ]. MeCP2 is one of these factors, and it is an epigenetic modulator of chromatin architecture that is strongly enriched at PCH [ 20 , 106 ] ( Figure 2 A, Steps 4 and 9). It is mutated in Rett syndrome, a severe neurological disorder [ 107 ].…”

“…MeCP2 co-localizes at chromocenters with ATRX [ 20 ], which is a nuclear epigenetic factor that belongs to the switch/sucrose non-fermentable (SWI-SNF) protein family [ 122 ]. SWI-SNF proteins play roles in several biological processes, such as DNA recombination and repair, transcriptional regulation, and remodeling of nucleosomes [ 123 ].…”

“…The sizes and the numbers of chromocenters is cell-type specific, and is subject to changes during differentiation. These changes generally consist of clustering of the chromocenters, which thus increase in size and decrease in number [ 15 , 17 , 18 , 19 , 20 ]. A number of epigenetic factors contribute to this chromocenter clustering during both myogenic and neural differentiation, such as Methyl-CpG binding protein 2 (MeCP2) [ 18 , 19 ] and Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX) [ 20 , 21 ].…”