Multiple Modes of Interaction between the Methylated DNA Binding Protein MeCP2 and Chromatin

Abstract

Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.It is now well established that the severe neurodevelopmental Rett syndrome (RTT) is caused primarily by mutations in the X-linked MeCP2 gene (1). MeCP2 (Fig. 1A) is a member of the family of related proteins that bind specifically to symmetrically methylated CpG dinucleotides via a conserved methyl binding domain (MBD) (17,38,44). The binding of MeCP2 to methylated DNA has been shown to lead to transcriptional repression in a variety of experimental contexts (see, for example, references 13, 35, 44, 45, and 65), a property conferred by a transcriptional repression domain (TRD). Evidence suggests that repression occurs when Sin3A and histone deacetylases (HDACs) are recruited to the TRD, resulting in the deacetylation of nearby nucleosomes (reviewed in reference 48). Additional "AT hook" and "WW" motifs have been identified in MeCP2 (9, 34), as well as a nuclear localization signal (NLS). MeCP2 is widespread and highly conserved in vertebrates, and mice lacking MeCP2 or with a major C-terminal truncation exhibit neurological dysfunctions with remarkable parallels in human RTT patients (12,22,49).Analysis of RTT patients has revealed a small number of single amino acid changes at mutational "hot spots" in MeCP2, many of which are located in the MBD or TRD, as well as a series of C-terminal truncations. In addition to the hot spots, there are a large number of low-frequency mutations that lead to RTT (see the IRSA database at http://mecp2.chw.edu.au /mecp2/). There is growing evidence that...

The Three Methyl-CpG-binding Domains of AtMBD7 Control Its Subnuclear Localization and Mobility

“…multiple chromatin interaction regions in addition to the MBD motif (21,22). In conclusion, the three MBD motifs in AtMBD7 were found to increase the protein binding affinity for chromatin and to determine its subnuclear localization and could potentially induce chromatin compaction of methylated CpG chromosomal regions.…”

Essential Role of MeCP2 in the Regulation of Myofibroblast Differentiation during Pulmonary Fibrosis

“…23,24 These studies implicate a role for the methyl CpG binding protein 2 (MeCP2), a key member of the methyl-DNA binding protein family of proteins. [25][26][27][28][29] While MeCP2 can bind to unmethylated DNA, 29 preferentially it binds methylated DNA at the 5=-CpG residues. 30 -31 MeCP2 is originally considered to be a transcriptional repressor in conjunction with Sin3A and histone deacetylase, but was found later to also have a significant role as a transcriptional activator, as well as in the regulation of chromatin architecture and RNA splicing.…”

Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes

“…More recent evidence suggests that Mecp2-null mice and Rett patients do not ectopically express genes that are regulated by promoter methylation (9,10). More diverse roles for MeCP2 have recently been shown in the direct compaction of chromatin (11,12) and the control of alternative splicing of pre-mRNA (13). The multifunctional nature of MeCP2 is reflected by its intrinsically disordered structure of multiple autonomous domains (14), including a methyl CpG-binding domain (MBD) (15) and a transcriptional repression domain (TRD) (8).…”