PROJECT SUMMARY/ABSTRACT The methyl-CpG binding protein 2 (MeCP2) is a highly abundant chromatin-binding protein that recognizes methylated DNA to coordinate the expression of thousands of genes essential for neuronal function. Mutations in MeCP2 cause Rett syndrome, a severe neurological disorder affecting one in every 10,000 females and characterized by psychiatric and motor regression at 6-18 months. Although several treatments are available for improving some isolated features of Rett syndrome, there are currently no approved therapies that directly address the underlying defects of MeCP2 loss of function. One primary reason for the lack of MeCP2-targeted interventions is an inadequate understanding of how MeCP2 reads methylated DNA within hierarchically organized chromosomes. MeCP2 is known to bind to methylated DNA with a higher affinity than unmethylated DNA, but how it navigates the main structural form of packaged DNA in the nucleus, nucleosomes, to reach its canonical methyl-DNA substrate remains unclear. Over 80% of the MeCP2 protein is structurally disordered, resulting in extensive conformational heterogeneity and binding plasticity that could underlie the regulatory capacity of the protein, but simultaneously hamper detailed mechanistic characterization of the protein’s chromatin-binding behavior. To this end, I used a single-molecule platform combining fluorescence microscopy with optical trapping to directly observe the real-time trajectory and dynamics of individual MeCP2 on DNA and nucleosomes. This approach enabled the visualization of how MeCP2 navigates the chromatin landscape harboring methylated CpG sites. I discovered that MeCP2 exhibits long-range, diffusive behavior on bare DNA, whereas methylation drastically suppresses such motions. Unexpectedly, I also found that MeCP2 preferentially and stably binds nucleosomes irrespective of methylation status, suggesting that nucleosomes may serve to regulate the availability of MeCP2 for its canonical methyl-reader activity. Based on my preliminary data, I hypothesize that nucleosomes regulate the availability of MeCP2 for methyl-DNA recognition and biophysically modify MeCP2 dynamics and binding configurations on DNA. To test this hypothesis, I will 1) characterize the dynamics of MeCP2 on DNA with and without methylation, 2) examine the binding and biophysical interaction of MeCP2 with nucleosomes wrapped with methylated or unmethylated DNA and investigate how Rett mutations impact this interaction, and 3) determine the structural basis of the MeCP2-nuclesome interaction. Successful completion of the proposed aims will provide novel insights into how MeCP2 biophysically navigates through a complex chromatin environment to reach its canonical methyl-DNA substrate at unprecedented spatial and temporal resolution. My report will establish an experimental framework for systematic interrogation of Rett syndrome mutations and their impact on fundamental MeCP2-nucleosome and DNA inter...