Identification and characterization of mCpH binding proteins in neurons

PROJECT SUMMARY DNA methylation is a major epigenetic modification that plays an important role in key biological processes, including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, and carcinogenesis. Although it has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes, emerging evidence over the past decade has shown that CpH (H=A/C/T) methylation is present in mammalian genomes, including cultured pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, the mouse germ line, and especially at a relatively high level in human and mouse brains. Given that CpGs only represent 4% of the metazoan genomes, CpH methylation greatly expands the proportion of the genome that is subject to regulation by cytosine methylation and represents a new mechanism of transcriptional regulation. In our previous studies, we generated neuronal DNA methylation profiles at a single base-resolution of the adult mouse dentate gyrus in which 80-90% of the cells are NeuN positive granule neurons, and our team was one of the first to show that ~25% of cytosine methylations are located in the CpH context. Notably, we identified the first mCpH reader, MeCP2, both in vitro and in postmitotic neurons in vivo. In addition, we found that CpH methylation was established postnatally and required DNMT3A for its active maintenance in neurons in vivo. Mutations on both the reader and writer lead to neurodevelopmental disorders, such as fragile X syndrome (FXS), amyotrophic lateral sclerosis (ALS), and Rett syndrome. Loss of either Dnmt3A or MeCP2 in the mouse models causes overlapping and distinct phenotypes in behavioral and molecular tests, suggesting the existence of additional mCpH-binding proteins. We believe that a critical step towards understanding the biological functions of mCpH is to identify its binding proteins. In this proposal, our goal is to identify additional mCpH binding proteins. We hypothesize that mCpH regulates transcription directly or indirectly via recruiting sequence-independent and/or -dependent mCpH-binding proteins in neurons. We will use protein array (Aim 1) and Digital Affinity Profiling via Proximity Ligation (DAPPL; Aim 2) to identify sequence-independent and -dependent mCpH-binding proteins and validate candidates using gel-shift (EMSA), OCTET and luciferase assays in vitro. We will employ a viral in vivo delivery system and high- throughput sequencing technologies to characterize their roles in transcriptional and chromatin regulation in the adult mouse brain (Aim 3). The effectiveness of our strategy will be rigorously evaluated via a series of in vitro and in vivo assays. If funded, the success of this project is expected to provide a rich resource of sequence-dependent and independent mCpH-binding proteins that will lay the foundation to elucidate the roles of CpH methylation in neurons, stem cells and other tissues. The insights into the mechanism of CpH methylation is expected to provide novel drug targets for treating neurodevelopmental disorders.