Ezh2 is essential for activation-induced CD8+ T cell cycle progression via repressing Cdkn1a and Cdkn2c expression (Part I)

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naïve CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ehz2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naïve CD8+ T cells proliferation and apoptosis. When Ezh2 was deleted during thymocyte development (Ezh2fl/flCD4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increased apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increased expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naïve CD8+ T cells as the consequence of reduced levels of H3K27me3 in these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naïve CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn2a and Cdkn1c by Ezh2 plays an essential role in permission of activation-induced CD8+ T cell proliferation. Ezh2 regulates H3K27me3 ofCdkn1candCdkn2alocus.To understand how early the changes in expression of cell cycle related-genes happens in the absence of Ezh2, we included four additional early time points (2, 4, 6, and 8 hours after stimulation) during the course of 72 hours after anti-CD3/CD28 stimulation. Ezh2 mRNA levels were increased at 8 hours, peaked around 48-72 hours after stimulation (Fig. 5A). We then compared expressions of 12 cell-cycle regulators and found thatCdkn1c(p57) was dramatically increased at 2 hours and peaked at 4 hours (>2 folds higher in Ezh2 deficient CD8+T cells than in WT CD8+T cells) after stimulation (Figure 5A). ForCdkn2family, expression ofCdkn2aV1 (Arf)andCdkn2aV2 (p16), key regulators for apoptosis and cell cycle, respectively, were increased in Ezh2 deficient naïve CD8+T cells compared to WT naïve CD8+T cells from 8 to 72 hours after stimulation