Over 90% of the cases of the devastating neurodevelopmental disorder Rett Syndrome (RTT), which affects 1 in 10,000 girls, is due to loss of function mutations in the X-linked methyl CpG binding protein 2 (MeCP2). MeCP2 is expressed in low levels throughout the body and during neurogenesis, but is expressed at a level equivalent to linker histone H1 in mature neurons, reaching a stoichiometry of one molecule MeCP2 per two nucleosomes, the basic repeating unit of chromatin. Many RTT symptoms have been traced to MeCP2 mutations in neurons within specific regions of the brain and glial cells. Therefore determining the principles of MeCP2 mediated regulation is critical for understanding RTT pathology. MeCP2 and H1 compete to bind nucleosomes at their DNA entry/exit dyad, and lead to distinct chromatin structures with specific transcriptional outcomes.
The first aim of this study will be to determine what nucleosomes throughout the genome are regulated by MeCP2. The genome-wide transcriptional deregulation observed in cells with mutated MeCP2 indicate that MeCP2 binds to specific nucleosomes throughout neurogenesis and during differentiation to drive proper expression patterns. Furthermore, glial cell transcriptional deregulation due to MeCP2 mutations also contribute to RTT phenotypes indicating that MeCP2's regulation is cell type specific. Nucleosome chromatin immunoprecipitation coupled with high throughput sequencing (nuc-ChIP-seq) will be used to generate MeCP2-bound nucleosome maps for neurons during neurogenesis, mature neurons, and for glial cells. In addition to determining the target nucleosomes/genes of MeCP2 regulation these maps will also identify what histone tail post translational modification (PTMs) patterns associate with/signal for MeCP2 binding. By binding to the DNA entry/exit dyad of nucleosome MeCP2 and H1 alter 'nucleosome breathing', the spontaneous dissociation of the DNA from the histone octamer, thereby regulating the accessibility of DNA target sites, usually buried within chromatin, to their binding proteins. Therefore the second aim will be to determine how MeCP2 influences nucleosome stability and dynamics. Frster resonance energy transfer (FRET) will be performed on mononucleosomes and restriction enzyme digest analysis on in vitro multi- nucleosome arrays to determine the intrinsic binding activity of MeCP2 to both the individual nucleosome and to chromatin. These in vitro arrays will be modified to determine how specific epigenetic factors, such as DNA methylation and specific histone tail PTM patterns, regulate this binding. Furthermore, RTT-causing MeCP2 mutations will be utilized in order to determine how these binding properties are altered in disease states. By combining next generation sequencing (aim 1) with detailed binding assays (aim 2) this study will generate a highly detailed and global view of MeCP2 mediated transcriptional regulation in cells important in brain development, thereby providing important insights into RTT pathology and therapeutic targets.
Most cases of the devastating neural developmental disorder Rett Syndrome, characterized by apparently normal development for the first 6-18 months followed by developmental regression and stagnation, complete loss of speech, and hand stereotypies replacing purposeful hand movements, are due to mutations in the neural protein MeCP2 (methyl CpG binding protein 2). This project will determine how MeCP2 interprets its genetic environment to properly regulate gene transcription in neurons, and how this interpretation is changed in Rett Syndrome so that targeted therapies and treatments can be established.
| Riedmann, Caitlyn; Fondufe-Mittendorf, Yvonne N (2016) Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility. Sci Rep 6:33186 |
