MECP2 mutations disrupt pluripotent stem cell fate through remodeling of the three-dimensional genome

Mutations in the MECP2 gene are the primary cause of Rett syndrome, yet their mechanistic roles during early developmental stages remain poorly understood. In this study, CRISPR-Cas9 technology was applied to generate three loss-of-function mutations in male induced pluripotent stem cells (iPSCs), namely MECP2^del6, MECP2^insA, and MECP2^insT, each targeting distinct functional domains of MECP2. Our results showed that MECP2 mutations led to reduced proliferative capacity and impaired embryoid body formation in iPSCs, and caused premature loss of OCT4 expression during embryoid body development. To explore the molecular mechanisms in depth, we performed integrated multi-omics analyses. MECP2 mutations remodeled three-dimensional genome organization by disrupting chromatin compartmentalization, destabilizing topologically associated domain boundaries, and redistributing frequent interaction hotspots and super-hotspots linked to genes involved in development and chromatin remodeling. These structural alterations were accompanied by genome-wide changes in chromatin accessibility, with differentially open regions enriched for the binding motifs of pluripotency transcription factors OCT4/SOX2 and the 3D genome organizer CTCF. Further analyses confirmed that the MECP2 mutations enhanced CTCF binding at its co-binding sites. Collectively, this study systematically elucidates how MECP2 mutations interfere with iPSC fate determination by reshaping 3D genome organization and chromatin accessibility at multiple levels, providing a new perspective on the early pathogenesis of Rett syndrome.