Loss of function mutations in Methyl-CpG-binding Protein 2 (MeCP2) cause the severe neurological disorder Rett Syndrome. MeCP2 is a highly abundant nuclear protein particularly enriched in neurons. Although biochemical and genomic analyses of MeCP2-DNA interaction and genomic distribution demonstrate that MeCP2 binding on chromatin is dependent on DNA modification state, the dynamic behavior of individual MeCP2 proteins in live neurons has not been explored. Here we use live-cell single-molecule imaging to assess the detailed kinetic features of MeCP2 in distinct sub-nuclear regions at high spatial and temporal resolution. Surprisingly, we found that, in granule cell nuclei, MeCP2 has unique diffusion and chromatin binding kinetics that are distinct from highly mobile sequence-specific transcription factors (TF) and immobile histone proteins. Approximately, half of MeCP2 is bound to DNA in a transiently stable mode that is similar to TF binding to their cognate sites. The binding of meCP2 to DNA requires its methyl-binding domain (MBD) and is sensitive to the levels of both DNA methylation and hydroxymethylation. However, when not stably bound, MeCP2 moves slowly in the nucleus most closely resembling histone H1.0. The rate of MeCP2 diffusion in compact, granule cell nuclei is determined by weak, transient DNA interactions mediated primarily by the MBD and three AT-hook domains located in the C-terminal portion of the protein. Both the fraction of stably bound MeCP2 and its rate of diffusion depend on the level of chromatin compaction and neuronal cell type. Our data reveal new features of MeCP2 that dictate its dynamic behavior in neuronal nuclei and suggest that the limited nuclear diffusion of MeCP2 in live neurons may contribute to its local impact on chromatin structure and gene expression.

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions