Functional Comparison to Ezh2 Reveals PRC2-Independent Functions of Jarid2 in Hematopoietic Stem Cell Lineage Commitment

Our previous studies showed the Polycomb Repressive Complex 2 (PRC2) co-factor Jarid2 represses self-renewal transcriptional networks in mouse multipotent progenitor cells (MPPs). But only a fraction of de-repressed HSC-specific genes were associated with loss of H3K27me3, implying Jarid2 may have non-canonical (PRC2-indpendent) in hematopoiesis. Here we sought to delineate any PRC2-independnent functions by comparing stem and progenitor cells genetically deficient for either Jarid2 or Ezh2. Loss of Ezh2 leads to increased myeloid differentiation in transplantation assays. In contrast, loss of Jarid2 enhanced T-cell output. Single cell transcriptomics showed while loss of Jarid2 had minimal impact across progenitor populations, loss of Ezh2 led to accumulation of lymphoid-biased MPP4 cells and B-cell progenitors in the bone marrow. Functional assays confirmed a differentiation block at the pre-pro B-cell stage. The maturational arrest of Ezh2-deficient B-cell progenitors contrasts with increased T-cell output from loss of Jarid2, suggesting Jarid2 has non-canonical functions in hematopoiesis. Overall design: To explore the potentially divergent roles of the PRC2 co-factor Jarid2 and its catalytic subunit Ezh2, we generated Mx1-Cre conditional mouse models with floxed alleles of Jarid2 (Jarid2fl/fl) or Ezh2 (Ezh2fl/fl), enabling the targeted ablation of either gene. We then performed single-cell RNA-seq and single-cell multi-omics sequencing to dissect mechanistic differences between Jarid2 and Ezh2 loss-of-function. For Single Cell RNA-seq studies, primary recipient mice were euthanized 18 weeks post-transplant to collect cells, while two-month-old baseline mice were used for the single-cell multi-omics workflow.To further explore the potentially divergent effects of Jarid2 and Ezh2 loss of function on H3K27me3 repressive mark, we performed Cut and Tag analysis on KSL (c-Kit+, Sca1+, Lineage-) cells sorted from the 3 million WBM secondary transplanted recipients. We then assessed the whole genome and gene specific loci depletion of H3K27me3 repressive marker upon Jarid2 and Ezh2 loss of function.