DNA methylation, nucleic acid structure, and Rett mutations tune MeCP2 binding affinity and cooperativity

ABSTRACT Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-associated factor whose dysfunction causes Rett syndrome. Although MeCP2 preferentially binds methylated DNA, its affinity for methylated substrates is only ∼threefold higher than for unmethylated DNA, raising the question of how MeCP2 selectively recognizes its targets. Here, we quantify binding of full-length wild-type MeCP2 and Rett-associated variants (R106W, T158M, R270X, and R306C) to nucleic acid substrates that vary in length, secondary structure, methylation pattern, CpG symmetry, and strand composition. Wild-type and mutant MeCP2 preferentially bind double-stranded DNA but also interact with single- and double-stranded DNA and RNA with nanomolar affinity. Binding to single-stranded targets is largely driven by the formation of local duplex structures, whereas 5-methylcytosine provides stabilizing contacts and enhances MeCP2 affinity when canonical duplex geometry is absent. Rett-associated mutations segregate into mechanistic classes: mutations within the methyl-binding domain (R106W and T158M) weaken methylation-dependent recognition and reduce binding affinity, whereas C-terminal mutations (R270X and R306C) preserve high-affinity binding but diminish cooperative interactions consistent with impaired higher-order bridging. These findings identify MeCP2 as a methyl-sensitive nucleic acid binder whose interactions are shaped by local nucleic acid topology and modulated by cytosine methylation, providing a mechanistic framework for understanding how Rett-associated mutations disrupt chromatin regulation.