NK cells are innate lymphoid cells that play important roles in tumor eradication and viral clearance. We previously found that deletion or inhibition of the histone methyltransferase Ezh2 (enhancer of zeste homolog 2) in hematopoietic stem and progenitor cells (HSPCs) from both mice and humans enhanced the commitment and cytotoxicity of NK cells to tumor cells. This study tested the hypothesis that inhibiting Ezh2, especially in NK lineage cells, could also affect NK cell development and function. We crossed Ezh2fl/fl mice with Ncr1iCre mice to delete the Ezh2 gene in immature NK cells and downstream progeny. Ezh2 deficiency increased the total number of NK cells and promoted NK cell terminal differentiation, as the percentages of the most mature CD27–CD11b+ subsets were increased. The NK cell cytotoxicity against tumor cells in vitro was enhanced, with increased degranulation and IFN‐γ production. In addition, during the process of human NK cells differentiating from HSPCs , inhibiting EZH2 catalytic activity at day 14 (when NK lineage commitment began) also resulted in increased proportions of mature NK cells and cytotoxicity. Furthermore, RNA‐seq and CUT&RUN‐qPCR assays showed that the effects of Ezh2 may be based on its direct modulation of the expression of the transcription factor Pbx1 (pre‐B‐cell leukemia transcription factor 1), which has been reported to promote NK cell development. In summary, we demonstrate that Ezh2 is a negative regulator of NK cell terminal maturation and function.

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and exhibit important functional properties in antimicrobial and antitumoral responses. Our previous work indicated that the enhancer of zeste homolog 2 (Ezh2) is a negative regulator of early NK cell differentiation and function through trimethylation of histone H3 lysine 27 (H3K27me3). Here, we deleted Ezh2 from immature NK cells and downstream progeny to explore its role in NK cell maturation by single-cell RNA sequencing (scRNA-seq). We identified six distinct NK stages based on the transcriptional signature during NK cell maturation. Conditional deletion of Ezh2 in NK cells resulted in a maturation trajectory toward NK cell arrest in CD11b SP stage 5, which was clustered with genes related to the activating function of NK cells. Mechanistically, we speculated that Ezh2 plays a critical role in NK development by activating AP-1 family gene expression independent of PRC2 function. Our results implied a novel role for the Ezh2-AP-1-Klrg1 axis in altering the NK cell maturation trajectory and NK cell-mediated cytotoxicity.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lostage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.