ABSTRACT
Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.
Subject(s)
Chromatin/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Animals , Base Sequence , Binding Sites , Cell Nucleus/metabolism , Cerebellum/cytology , Cerebellum/metabolism , DNA/metabolism , DNA Methylation , DNA-Binding Proteins/metabolism , Female , Gene Dosage , Gene Expression Regulation, Developmental , Histones/metabolism , Kinetics , Male , Methyl-CpG-Binding Protein 2/genetics , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Neurons/cytology , Protein Binding , Rett Syndrome/genetics , Transcription Factors/metabolismABSTRACT
Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression and epigenetic events in specific cell types in a range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed gender specific transcriptional differences, cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event evident in three donor samples. Our data establish a comprehensive approach for analysis of molecular events associated with specific circuits and cell types in a wide variety of human conditions.