The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of the global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of “stemness” and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states. Overall design: Spike-in normalized transcriptional profiling of mouse ES cells carrying an auxin-inducible degron system for depletion of Usp9x. Cells were treated with auxin for 8 hours, sorted based on Usp9x status (Usp9x-high or Usp9x-low), and harvested or allowed to recover without auxin for 48 hours. Vehicle-treated and auxin-resistant (Flag-tagged Usp9x) cells serve as controls. Spike-in normalized H3K27me3 ChIP-seq profiles of mouse ES cells carrying an auxin-inducible degron system for depletion of Usp9x. Cells were sorted based on Usp9x status (Usp9x-high or Usp9x-low) following 8 hours of auxin treatment. Transcriptional profiling of single E8.5 male embryos either wild-type or mutant for Usp9x. Usp9x-fl/fl females were crossed to Sox2-Cre males to delete Usp9x in epiblast derivatives in offspring. Litter-matched Cre-negative (Usp9x-fl/Y) embryos serve as controls.