Rett syndrome (RTT) is an X-linked postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like syndromes. The disease is caused by loss-of-function mutations of the DNA binding protein MeCP2 (Methyl CpG-binding Protein 2) in the X chromosome and represents the second most common cause of intellectual disability in females. Loss of MeCP2 leads to expression changes in thousands of genes, compromises the majority of brain cells and circuits, and dysregulates all neurotransmitter systems. However, how MeCP2 can act as a global repressor of gene activity as well as an activator for gene expression remains an open question in the field. Microcephaly (the reduction in brain size) has been documented as a hallmark of RTT, and analysis of hESC/iPSC-derived RTT neurons showed a reduced soma size as well. It has been well established that MeCP2 binds to 5-methylcytosine (5-mC) and 5- hydroxymethylcytosine (5-hmC) in the genome. The common RTT mutant of MeCP2 (R133C) represents a unique research tool as this mutation was reported to abolish MeCP2 binding to 5-hmC but not 5-mC. Our preliminary studies on human MeCP2-R133C mutant neurons showed a panel of cellular phenotypes including reduced soma size, impaired electrical properties, and defects in chromosomal structures. Therefore, we hypothesized that MeCP2 is involved in the organization of 3D chromosomal landscape contributing to the regulation of gene expression and subsequent neurobiology. We will use isogenic human neuronal cultures derived from genetically engineered hESCs to characterize the chromosomal landscapes of MeCP2-wild type, -knockout, and -R133C mutant neurons (Aim 1, K99 phase) and to investigate the molecular mechanism of MeCP2-mediated 3D chromosomal organization (Aim 2, K99 phase). Development of RTT-like symptoms in mice can be reversed in RTT adult animals following the restoration of MeCP2 expression. As most female RTT patients still carry a wild type allele of MeCP2 subject to the random X-chromosome inactivation (XCI), it will be of therapeutic benefit if the wild type allele of MeCp2 in the inactive X chromosome (Xi) can be reactivated. Recently, we have developed a DNA methylation editing tool by fusion of a catalytically inactive Cas9 with Tet1/Dnmt3a. As DNA methylation is the major mechanism for XCI, we will use our tool to reverse the RTT phenotypes via reactivation of the wild type MeCP2 allele on Xi by targeted demethylation of the MeCP2 promoter (Aim 3, R00 phase).This project will fill the gaps in our knowledge of MeCP2 function in the organization of 3D chromosomal structure and test the novel therapeutic approach to reverse RTT phenotypes by reactivation of MeCP2 in RTT neurons. During the mentored K99 phase, I will work in collaborations with: 1) Dr. Rick Young to map the 3D chromosomal regulatory landscape of human neurons; 2) Dr. Li-Huei Tsai to characterize the edited RTT neurons.
The proposed research is relevant to public health because it focuses on discovery of the novel role of MeCP2 of which mutations cause Rett Syndrome, the second most common cause of intellectual disability in females whereas male patients are normally more severely affected and die early. These studies will establish new avenues of investigation to develop improved therapeutics to treat this disease. Thus, the proposed research is relevant to NIMH's mission of fostering creative discoveries and innovative research strategies for protecting and improving health and reducing the burdens associated with neurological disorders.
