PRC1-dependent H2A Monoubiquitination During Human Corticogenesis

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PROJECT SUMMARY Intellectual disability affects 2-3% of the population in industrialized nations, with genetic causes accounting for approximately half of these cases. Whole genome sequencing has led to the discovery of several novel pathogenic variants underlying neurodevelopmental disorders. Among these variants, proteins involved in chromatin post-translational modifications are highly represented. Variants in the polycomb repressive complex 1 (PRC1), the primary catalyst of histone 2A monoubiquitination (H2AUb1), illustrate this trend. RING1 and RNF2 are paralogues that serve as the constituent E3 ubiquitin ligase of PRC1, and missense variants in these genes that blunt H2AUb1 catalysis are associated with a neurodevelopmental disorder characterized by microcephaly, intellectual disability, and early onset schizophrenia. The goal of this proposal is to understand how pathogenic RING1 variants lead to disease genetically and molecularly. To explore the role of PRC1- dependent H2AUb1 in the context of human corticogenesis, we have generated isogenic human embryonic stem cell (hESC) lines harboring pathogenic variants in RING1 and subjected them to neural differentiation to generate 3D cerebral organoids. Previous studies suggest that PRC1-dependent H2AUb1 has roles in transcriptional repression and double strand break repair. Consistent with these previous reports, our preliminary data demonstrates that the RING1G284A/G284A variant diminishes global H2AUb1 levels, leads to increased expression of pro-apoptotic genes and decreased growth in cerebral organoids, and disrupts double strand break repair in neural progenitor cells (NPCs). Aim 1 will explore the genetic mechanism of RING1 pathogenic variants, as experiments will shed light onto whether pathogenesis is due to lost activity of wild type RING1 or due to a dominant negative effect of the mutant allele. Aim 2 will evaluate the molecular mechanism by which pathogenic RING1 variants cause microcephaly. Experiments will evaluate NPCs and organoids differentiated from hESCs harboring RING1 pathogenic variants for defects in DNA damage repair, increased rates of apoptosis, and the ability to silence transcription at sites of DNA damage. Mechanistic insights learned from these experiments will shed light onto the role of PRC1-dependent H2AUb1 in proliferating NPCs and in maintaining genome integrity more broadly. In addition, the results of these studies will inform future efforts to devise therapeutic strategies to restore these functions in individuals with pathogenic RING1 variants.