PRC2 GATEKEEPS THE BALANCE BETWEEN DIRECT AND INDIRECT NEUROGENESIS AND CONTROLS NEURONAL MIGRATION DURING HUMAN CORTICOGENESIS

Weaver syndrome (WVS) is a rare, autosomal dominant multisystem disorder, characterized by pre- and post-natal overgrowth, macrocephaly, facial dysmorphisms and varying degrees of intellectual disability. WVS’ genetic cause was identified in heterozygous mutations in Polycomb repressive complex 2 (PRC2) components. This complex catalyzes the trimethylation of Lysine 27 on histone 3 (H3K27me3), thereby repressing transcription during embryonic development. However, our understanding of how PRC2 dysfunction affects cortical development, and how WVS heterozygous mutations impact H3K27 trimethylation landscape and subsequent transcriptional changes, remains limited and primarily based on mouse studies. To better understand the molecular mechanisms underlying WVS, we conducted comprehensive transcriptomic and epigenomic analyses of patient-derived cortical brain organoids (CBOs) cultured for up to 250 days in vitro. Our multi-omic approach integrated bulk and single-cell RNA sequencing, EZH2 and H3K27me3 genomic distribution mapping, and DNA methylation profiling. These analyses revealed disruptions in neuronal maturation and migration processes from day 25 to 250 of differentiation. We observed temporally regulated gene expression defects and asynchronous cell type emergence in WVS, particularly affecting indirect neurogenesis. By identifying specific transcription factors and mediators responsible for cell-type dysregulation, we highlighted defects in cell fate commitment and migration - fundamental processes in cortical development. This finding is consistent with clinical data from WVS patients, whose MRI profiles show altered cortical development associated with defects in later stages of neuronal migration. Moreover, we identified AJAP1 as one of the most significantly upregulated genes in WVS- CBOs at day 25 of differentiation. AJAP1 cooperates with multiple proteins to organize the adherens junctions (AJs) belt, which determines radial glial cell (RGC) fate. Disruption of AJs can lead to ventricular zone disruption, loss of normal apicobasal polarity, and RGC detachment from the apical surface. Since proper cortical layering depends on RGC detachment from the AJ belt, differentiation and subsequent neuronal migration, we developed a CRISPRa-based system to upregulate AJAP1 in control CBOs. This approach allowed us to isolate the molecular and cellular effects of AJAP1 dysregulation in WVS and demonstrate its regulatory relationship with EZH2 in neuronal migration. This study provides the first comprehensive molecular characterization of PRC2's role in human cortical development and its involvement in neuronal migration, addressing a significant knowledge gap. Our findings advance the understanding of WVS pathogenesis and establish new resources for investigating neurodevelopmental disorders (NDDs).