The PRC2 Co-Factor JARID2 Regulates Hematopoiesis through Non-Canonical Mechanisms

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell fate through the modification of chromatin architecture. Jarid2 is a co-factor of the polycomb repressive complex 2 (PRC2) that recruits this complex to different sites in the genome to catalyze the repressive epigenetic mark of Histone 3 Lysine 27 tri-methylation (H3K27me3). Previous studies from our lab have demonstrated that Jarid2 functions as a tumor suppressor in chronic myeloid neoplasms by recruiting PCR2 to silence self-renewal gene expression programs in committed hematopoietic progenitor cells. In the absence of Jarid2, mouse multipotent progenitor (MPP) cells gain ectopic self-renewal in normal hematopoiesis, and neoplastic transformation is accelerated from JAK2V617F-mutant MPN. However, our previous studies showed that in Jarid2-null MPPs, only about half of the upregulated genes were associated with a loss of H3K27me3, suggesting that Jarid2 might have important non-canonical (PCR2-independent) functions in hematopoiesis, which forms the scientific premise of the current study. When bound to PRC2, the lysine 116 (K116) residue of Jarid2 becomes methylated by the catalytic subunit Ezh2. Jarid2^K116 methylation was shown to be indispensable for the regulation of the PRC2 catalytic activity through competing with the H3K27me3 binding to PRC2.

Here, we created a separation of function mouse model to study the non-canonical functions of Jarid2 in hematopoiesis. Through CRISPR/Cas9 gene targeting, we created a genetic knock in mouse model by mutating lysine 116 to alanine (K116A), a point mutation that cannot be methylated by PRC2. We generated a Vav-cre:Jarid2^fl/K116A(K116A) mouse model, that express one K116A mutated copy of Jarid2, while the other floxed copy will be conditionally knocked out by Vav-cre recombinase system in hematopoietic stem and progenitor cells (HSPCs). We hypothesized if Jarid2K116A HSPCs functionally phenocopy Vav-Cre:Jarid2^fl/fl (Jarid2-KO) HSPCs then the function of Jarid2 in these cells is largely PCR2-dependent. However, if the output of Jarid2-K116A HSPCs resembles that of Vav-Cre:Jarid2^fl/+ (Jarid2-HET) HSPCs, then this suggests Jarid2 has previously undescribed non-canonical functions in hematopoiesis.

To characterize the functional effects of this mutation, we performed competitive transplantation of stem and progenitor subsets. Transplantation of MPP1 (Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150-) cells revealed that Jarid2-K116A MPPs behaved similarly to Jarid2-KO MPPs. Recipients of both Jarid2-K116A and Jarid2-KO MPPs displayed higher peripheral blood (53% and 60%, respectively) and bone marrow (33% and 43%, respectively) engraftment compared to the control (12% and 3%, respectively) and Jarid2-HET (37.5% and 11%, respectively) MPPs. Additionally, Jarid2-K116A mutation enhanced donor MPP1 chimerism over recipient cells and promoted MPP1 self-renewal, similarly to what we previously described for Jarid2-KO MPP1 cells. These results suggest that Jarid2 function in MPP1 cells is largely dependent on PRC2. However, competitive transplantation of Jarid2-K116A HSCs (HSCs; Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150+) revealed a striking phenotype. Jarid2-K116A HSCs showed a neomorphic phenotype compared to control, Jarid2-HET and Jarid2-KO HSCs in both primary and secondary transplantations. Jarid2-K116A mutation significantly enhanced the peripheral blood and bone marrow myeloid engraftment (both 40% increase). Furthermore, Jarid2-K116A enhanced HSC fitness and self-renewal with these mutant HSCs able to gain increased chimerism in the HSC pool and increased donor-derived HSC number in the bone marrow of recipient mice (Figure 1). These results suggest that the unmethylated version of Jarid2-K116 exerts a neomorphic effect on HSC function confirming the importance of the post-translational modification at the K116 residue of Jarid2 in hematopoiesis. Thus far, our studies suggest that Jarid2 function is dependent on PRC2 in MPPs, but may act independently of PRC2 in HSCs. Ongoing studies are focused on examining the importance of PCR2-related Jarid2 function in malignant hematopoiesis, and determining if the ratio of methylated to unmethylated JARID2 influences leukemic transformation of human chronic myeloid disorders.

Celik:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Challen:Incyte: Consultancy, Other: Sponsored Research agreements.