Project Summary: Human genetic studies have revealed that mutations in transcriptional components such as transcription factors and chromatin modifiers underlie autism spectrum disorders and related neurodevelopmental disorders. Therefore, an important new challenge in molecular neurobiology is to understand normal functions of these proteins in neural development and determine how their disruption alters gene expression to trigger disease. Disruption of methyl-CpG binding protein 2 (MeCP2), a transcriptional regulator with global gene-expression effects, is the primary monogenic cause of Rett syndrome, a severe neurodevelopmental disorder. MeCP2 has been shown to preferentially bind both methylated CG dinucleotides (mCG) as well as neuron-enriched methylation that occurs at cytosines in non-CpG contexts (mCH, where H=C, T, A). Additionally, MeCP2 interacts with transcriptional co-repressor complexes, including NCoR/HDAC3, suggesting it can function as a transcriptional repressor. Depletion of MeCP2 results in an upregulation of long neuronal genes (>100kb) that are enriched for neuron-specific mCH sites within their gene body, suggesting MeCP2 mediates a novel mechanism of neuronal transcriptional control. However, the direct mechanisms of MeCP2-mediated regulation are largely unknown. Our long-term goal is to elucidate the functional importance of MeCP2 and mCA as components of the neuronal-specific epigenome and how their disruption leads to neurological disorders. I will begin to address this by analyzing the mechanism of MeCP2 and mCH in transcriptional regulation of long, highly methylated genes within the cerebral cortex. In this proposal, I will determine how binding of MeCP2 to DNA methylation within gene bodies of very long, highly methylated genes impacts transcription. These analyses will examine how MeCP2 may control transcription through inhibition of transcriptional initiation. In Aim 1, I will test the hypothesis that binding of MeCP2 to mCH within genes leads to repression of transcription. Using ChIP-seq and GRO-seq analysis in MeCP2 knockout mice, I will measure direct changes in transcription at the level of RNA polymerase binding and transcription of pre-mRNA. In Aim 2, I will further dissect how this regulation takes place by determining how MeCP2 engages transcriptional co-regulatory complexes such as NCoR and CBP to exert its effects. This analysis will provide a high resolution read-out of exactly how transcription is affected when MeCP2 is lost, and build a molecular model for how MeCP2 exerts these effects. In Aim 3, I will directly dissect the mechanisms by which mCH and MeCP2 within gene bodies can affect gene regulation. Using CRISPR/dCas9 technology to target mCH to distinct sites in the genome, I will test how the presence of mCH at genic regions affects MeCP2 binding and gene expression. This analysis will provide insight into how MeCP2 and mCH regulate transcription within neurons for proper brain development and how disruption of these components leads to dysregulation that occurs during Rett syndrome and other neurological disorders.