The long-term goal of this proposal is to understand the molecular activities of methyl CpG-binding protein 2(MeCP2) in order to develop viable treatment options for Rett syndrome (RTT) and other MeCP2-relateddisorders, which range from severe neonatal encephalopathy to autism, juvenile onset schizophrenia and otherneuropsychiatric conditions. Three key discoveries in the past four years have changed the way we think aboutMeCP2. First, we and others have shown that MeCP2 binds to non-CpG methylated dinucleotides (“mCH”) aswell as methylated CpG dinucleotides (“mCG”), and that this binding correlates with transcriptional changes inmouse models of RTT and MECP2 duplication syndrome. Second, we discovered a functional AT-hook domainin MeCP2 and evidence that suggests it remodels chromatin. Third, we have shown that the brain is sensitive tolevels of MeCP2 expression and that antisense oligonucleotides (ASOs) can reduce MeCP2 levels in a mousemodel of the MeCP2 duplication syndrome and reverse the disease. In this proposal we capitalize on these andother recent discoveries to gain deeper insight into RTT pathophysiology and the role of MeCP2 in maintaininghealthy neuronal responsiveness. In Aim 1, we will delineate the contributions of non-CpG methylation to RTTpathogenesis by deleting the mCH “writer”, Dnmt3a, from GABA-expressing neurons (to ablate mCH) andcomparing the resulting phenotype and gene expression changes to those of mice lacking Mecp2 (ourhypothesized mCH “reader”) in precisely the same neurons. In Aim 2, we will find what happens once MeCP2binds its genomic targets, whether mCG or mCH, by using the newly developed in situ Hi-C approach toascertain the 3D chromatin structure in the cerebellum and dentate gyrus in tissue from wild-type, MeCP2 null,and MeCP2 overexpressing mice. Because neuronal activity leads to a multitude of epigenetic changes, as wellas altering the interactions of MeCP2, we will also perform in situ Hi-C in the dentate gyrus both before andafter neuronal stimulation. In Aim 3a, we will expand on our successful ASO studies to prepare for translationby testing them in a MeCP2 duplication syndrome mouse that expresses two human MECP2 alleles (just likethe patients) to titrate the ASO dose that will restore the protein levels from 2X to 1X, and identify theboundaries of safe MeCP2 levels. In Aim 3b, we will apply a novel forward genetic screening strategy that wedeveloped for finding molecules that alter levels of other disease-related proteins to identify druggable targetsthat either decrease or increase MeCP2 levels. (Some Rett-causing MeCP2 mutations reduce the protein'slevel.) Our shRNA screen targets a 7,787 druggable gene collection using a DsRed- IRES-MeCP2-EGFPreporter cell line that allows high-throughput monitoring of MeCP2 levels. Targets will be validated and thosewith most promising safety profiles will be advanced for in vivo studies. In sum, the proposed studies willgreatly advance our understanding of MeCP2 function, the function of chromatin changes duringneurodevelopment, and provide new treatment approaches to MeCP2-related diseases.
Interagency Autism Coordinating Committee (IACC)
Autism Research Database (AFD)
