Methyl-CpG-binding protein 2 reads histone methylation via an aromatic cage to regulate gene expression and chromatin association.

Methyl-CpG binding protein 2 (MeCP2) is a DNA methylation reader, which is highly expressed in the central nervous system. Loss-of-function mutation of MeCP2, which impairs DNA binding, causes Rett syndrome, while gain of MeCP2 function causes MeCP2 duplication syndrome. MeCP2 functions as both a transcription activator and a repressor, but its full range of activity remains largely unknown. A recent study suggests that beyond DNA binding, MBD domain of MeCP2 also interacts with methylated lysine residues on histone H3 tail. In this study, we aimed to characterize the methyllysine-binding pocket of MeCP2-MBD. Structural analysis reveals the presence of an aromatic cage formed by five residues, namely, W104, F132, Y141, F142, and F155, where the trimethyllysine of H3K27me3 binds, as confirmed in docking studies. Alanine substitution of these residues abolished the binding of MeCP2-MBD with its reported ligand H3K27me3 in ITC and pull-down experiments. Genomic analysis of publicly available ChIP-seq data reveals that MeCP2 localizes with canonical histone methylation marks like H3K4me3, H3K9me3, H3K27me3, and H4K20me3 and regulates critical biological and cancer-related pathways. Promoters of MeCP2-regulated genes show a signature of H3K4me3 and H3K27me3. At the genomic level, mutation of aromatic cage residue W104A significantly reduced chromatin occupancy of MeCP2 as demonstrated by ChIP-qPCR experiments. Furthermore, our qRT-PCR experiment shows that this aromatic cage-dependent binding is necessary for regulating the expression of MeCP2 target genes. Overall, our study establishes MeCP2 as a histone methylation reader and highlights the functional significance of its histone binding capacity in chromatin association and gene regulation.