MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Abstract

Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.DNA methylation constitutes an important epigenetic component in transcriptional regulation, with methylation generally leading to repression of nearby genes (6). However, the mechanism by which the epigenetic signal is passed to the regulatory machinery is not well understood. Research in this area has been focused on a small family of methyl-CpG binding proteins, best characterized by MeCP2 (19), mutations in which result in Rett syndrome (RTT), a debilitating neurodevelopmental disease in humans (2).A mechanism of MeCP2-mediated gene silencing may involve recruitment of histone deacetylases upon methyl-specific binding (57). However, other mechanisms, which are not necessarily mutually exclusive, such as stabilization of large chromatin loops (29) and promotion of chromatin compaction (51), have also been suggested (14). Studies on in vivo distribution of MeCP2 in nuclei have revealed that, in addition to the expected occupancy of sites of CpG methylation, MeCP2 shows significant binding to unmethylated DNA (71). However, a recent analysis of MeCP2 occupancy has revealed that the genomic distribution of MeCP2 in mammalian neurons closely tracks methyl-CpG density (60). These results highlight our current lack of understanding of key questions pertinent to the binding of MeCP2 to DNA and chromatin. It is especially important, for example, to quantitate the modulation of binding by factors such as methylation density (8,37,48,60) and the presence of adjacent A/T-rich sequences (31) that are reported to influence binding. In the present work, we have used a variety of quantitative approaches to show that, when bound to DNA, MeCP2 exhibits a cooperative monomerdimer equilibrium, which is influenced by DNA length, methylation density, and the presence of nearby A/T repeats.Th...

Synaptic Dysfunction in Neurodevelopmental Disorders Associated with Autism and Intellectual Disabilities

“…In addition to the gene-specific binding, chromatin-immunoprecipitation (ChIP) studies revealed that MeCP2 binds widely throughout the genome (Yasui et al 2007;Skene et al 2010) and is as abundant as one molecule for every two nucleosomes (Skene et al 2010). The findings that MeCP2 and H1 compete for DNA binding sites support the hypothesis that MeCP2 might act as an alternative linker histone (Ghosh et al 2010;Skene et al 2010). However, how such competition causes the specific Rett and MeCP2 duplication phenotypes and the root of the very specific opposing gene expression changes are not clear at this time.…”

Sumoylated Human Histone H4 Prevents Chromatin Compaction by Inhibiting Long-range Internucleosomal Interactions

“…Samples were diluted as needed, applied to glow-discharged carbon-coated grids, stained with 0.1% aqueous uranyl acetate, and washed extensively with water. Grids were examined in a Tecnai 12 TEM LaB6 filament operated at 100 kV in the tilted darkfield mode, and digital images were recorded using a TVIPS 2024 ϫ 2024 CCD camera (31).…”

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

“…MeCP2 minimally binds 11 bp of DNA (9). Under these conditions, unmethylated and methylated DNA binding sites were present in molar excess over MeCP2, and DNA concentrations were always above the K d (5) so that H/DX was being measured under saturated binding conditions and dual population (i.e. bound and unbound to DNA) affects were avoided.…”