MeCP2 neurological diseases: interplay between RNA binding and MeCP2 genomic occupancy

About This Grant

Summary Mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MeCP2) are associated with neurological diseases ranging from impaired intellectual disability, microcephaly, autism, and Rett (RTT) syndrome. MeCP2 is well studied for binding cytosine methylated DNA through its methyl-binding domain (MBD) and interaction with nucleosomes and chromatin remodeling proteins through the NCoR/SMRT interaction domain (NID) and transcriptional repression domain (TRD) to regulate gene transcription. Numerous mutations in these domains are found in patients, highlighting their functional importance. A small highly conserved intervening domain (ID) is directly adjacent to the MBD but the function of the ID is poorly characterized. Basic amino acids in the ID and two other AT-hook domains in the TRD are thought to bind RNA but knowledge is sparse, not quantitative, and the contribution of the ID largely uncharacterized. Our preliminary data provides evidence that the ID is predominant in binding RNA with high nM affinity. As mutations in the ID are mostly classified as variants of unknown significance, we modeled a set of patient mutations and show decreased RNA binding. As the MBD-DNA binding domain and ID-RNA binding domain are directly adjacent we tested their interaction and show that RNA can compete MeCP2 off DNA. To gain in vivo insight into ID function, we deleted the ID in human stem cells and studied the effect of ID deletion on differentiation to neurons and cortical organoids. We observe phenotypic changes in ∆ID cells suggestive of disrupted differentiation. Based on our new findings we propose the following hypothesis: the MeCP2 ID binds RNAs to modulate MeCP2 genome occupancy and thus fine-tunes gene expression to allow robust cell fate transitions during brain development. We will test this hypothesis in three aims. Aim 1 uses biochemical, biophysical and live imaging approaches to examine the affinity and kinetics of the ID in binding RNAs and in displacing MeCP2 from DNA as well as the functionality of patient ID variants in RNA binding and MeCP2-DNA competition. Aim 2 will examine the function of the MeCP2 ID during brain development by creating ID variants in human neurons, organoids and mouse for phenotypic studies and to test the hypothesis that the ID serves to modulate gene expression patterns to allow robust transitions in cell fate. Aim 3 will investigate the ID as a modulator of gene regulation and MeCP2-DNA occupancy in human neurons. Finally, we also examine the functionality of the MeCP2 mini-gene to determine whether it recapitulates normal ID functions of RNA binding and modulation of DNA binding. This is critical and timely as the mini-gene is being used in one-dose clinical trials. Altogether our studies will look beyond the canonical roles of MeCP2 to provide a more in-depth analysis of how the ID and its RNA binding affects MeCP2 molecular and biological functions.