MeCP2 gene dosage-dependent neurodevelopmentally restricted defects arise by SWI/SNF-mediated aberrant activation of cell fate-determining bivalent genes [SAMMY-seq]
Increased dosage of genes encoding for chromatin factors is thought to inevitably alter gene transcriptional output, leading to cellular dysfunctions. However, a mechanistic understanding of the molecular processes in action and their consistency across different cell types is still lacking. A prototypical gene in this context is MECP2, as its overexpression determines severe neurological deficits in MECP2 duplication syndrome (MDS) and its dosage is considered a crucial safety risk factor in gene therapy for Rett syndrome (RTT). However, in MDS patients, MECP2 dysregulation arises at the embryonic stage while in RTT gene therapy, MECP2 is delivered into the mature brain. Despite these important implications, the molecular consequences of increased MECP2 gene dosage have yet to be fully elucidated. Surprisingly, we found that MeCP2 overexpression induced profound transcriptional alterations in neural progenitor cells (NPCs) while it had minimal or no effects in both mouse and human neurons. Consequently, MeCP2 overexpression in NPCs, but not mature neurons, led to profound functional alterations. Mechanistically, we observed that both endogenous and overexpressed Mecp2 bind to the same CpG island repertoire. In NPCs, lower endogenous Mecp2 levels facilitated stronger deposition of the ectopic protein into CpG islands, driving the transcriptional activation of many developmental bivalent genes, deranging neuronal differentiation. We uncovered that this activation is mediated by the interaction with the SWI/SNF chromatin remodeling complex. Conversely, in neurons, where endogenous Mecp2 is highly expressed, ectopic protein expression leads to minimal CpG island binding and accelerate protein degradation. Our findings reveal that increased gene dosage-dependent effects are highly influenced by cell type, levels of proteins and their mechanisms of action. These results provide a framework to interpret the relationship between Mecp2 chromatin binding and transcriptional output and suggest that Mecp2 overexpression is expected to be well tolerated in gene therapy applications for RTT. Overall design: This study was designed to investigate the cell-type and developmental-stage-specific consequences of Mecp2 overexpression. We used primary mouse Neuronal Progenitor Cells (NPCs) derived from E13.5 embryos and primary mouse Neurons (Ns) from E18.5 embryos, as well as human iPSC-derived neurons, to compare the effects of Mecp2/MECP2 overexpression versus an EGFP control. To assess the functional outcomes, we performed in vivo experiments using in utero electroporation in E13.5 mouse embryos and systemic AAV-PHP.eB vector delivery in adult mice. The molecular consequences of Mecp2 dosage were analyzed using a combination of high-throughput sequencing techniques: RNA-sequencing was used to profile transcriptional changes, CUT&Tag was employed to map the genomic binding of endogenous and exogenous Mecp2 and the chromatin remodeler Smarcb1, and 4f-SAMMY-seq was used to analyze global chromatin accessibility and organization.