Ezh2Regulates Transcriptional And Post-translational Expression Of T-bet And Promotes Th1Cell Responses Mediating Aplastic Anemia In Mice

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome.IFN-γ-producing T helper (Th)1CD4+T cells mediate the immune destruction ofhematopoietic cells, and are central to the pathogenesis.However, the molecular events that control the development of BM-destructive Th1cellsremain largely unknown. Ezh2is a chromatin-modifying enzyme that regulates multiplecellular processes primarily by silencing gene expression. We recently reported that Ezh2is crucial for inflammatory T cell responses after allogeneic BM transplantation.To elucidate whether Ezh2mediates pathogenic Th1responses in AA and the mechanismof Ezh2action in regulating Th1cells, we studied the effects of Ezh2inhibition in CD4+Tcells using a mouse model of human AA. Conditionally deleting Ezh2in mature T cellsdramatically reduced the production of BM-destructive Th1cells in vivo, decreasedBM-infiltrating Th1cells, and rescued mice from BM failure.Ezh2inhibition resulted in marked decrease in the expression of Tbx21and Stat4(whichencode transcription factors T-bet and STAT4, respectively). Introduction of T-bet but notSTAT4into Ezh2-deficient T cells fully rescued their differentiation into Th1cells. Ezh2bound to the Tbx21promoter in Th1cells, and directly activated Tbx21transcription.Unexpectedly, Ezh2was also required to prevent proteasome-mediated degradation ofT-bet protein in Th1cells.Our results identify T-bet as the transcriptional and post-translational Ezh2target that actstogether to generate BM-destructive Th1cells, and highlight the therapeutic potential ofEzh2inhibition in reducing AA and other autoimmune diseases.Part One: In the absence of Ezh2, LN cells are defective in mediating AA in mice.1. Gerneration of T cell-specific Ezh2conditional knockout (T-KO) B6mice.2. The establishment of mouse model for human AA.3. The impact of Ezh2to mediate AA.Conclusion: Ezh2is required to mediate AA; Ezh2inhibition resulted in survivalwithout clinical signs and histological evidence of AA in mouse model.Part Two: Ezh2is required for the development of Th1cells inducing AA in mice. 1. The phenotype of sorted CD4+donor cells in vivo.2. The related Th gene expression of sorted CD4+donor cells in vivo.3. Tracking the longitudinal proliferation and differentiation of transferred WT andT-KO LN cells in vivoConclusion: The inflammatory stimuli produced during the AA processpredominantly induces Th1cell differentiation in vivo; Ezh2is critically involved inregulating the development of Th1cells, but has little impact on Th2or Th17cells.Part Three: Ezh2promotes in vitro Th1cell differentiation in cultures underTh1-skewing conditions.1. The division, expansion and activation of CD4+T cells in the absence of Ezh2.2. The establishment of in vitro CD4+T cell cultures under Th1-skewing condition.3. The phenotype of WT and T-KO Th1cells in vitro.4. Ezh2promotes Th1cell differentiation through a mechanism independent of IL-4and GATA3.Conclusion: In the absence of Ezh2, CD4+T cells can be normally activated toundergo cell division and expansion upon stimulation with Th1-skewing conditions; Ezh2promotes in vitro Th1cell differentiation in cultures under Th1-skewing conditionsthrough a mechanism independent of IL-4and GATA3.Part Four：Ezh2associates with Th1gene loci.1. The presence of histone methylation markers on Th1gene loci innaive T cells andTh1cells.2. The binding of Ezh2on Th1gene loci innaive T cells and Th1cells.3. The expression of Ezh2and Th1related genes innaive T cells and Th1cells.4. The co-expression of Ezh2and IFN-γin Th1cells in vitro.5. The presence of Ezh2and histone methylation markers on Th1gene loci in WTand T-KO Th1cells.Conclusion: The amount of H3K27me3in differentiated Th1cells was markedlyreduced at the promoter region of Th1gene loci. In contrast, the permissive H3K4me3was increased. Interestingly, there was no significant reduction in the amount of Ezh2atthese loci during differentiation; Ezh2was positively correlated with expression of Th1related genes and all CD4+T cells producing IFN-γalso expressed high levels of Ezh2; Loss of Ezh2leads to the switch to a permissive histone methylation signature for Th1genes, which is favorable for their activation. However, our results showed that loss ofEzh2impaired Th1cell development.Part Five：Ezh2regulates T-bet at both the transcriptional level and post-translationallevel.1. The mRNA expression of T-bet and STAT4related genes in WT and T-KO Th1cells.2. The construction of Tbx21-specific dual reporter system.3. The protein express of T-bet and STAT4related genes in WT and T-KO Th1cells.4. The impact of proteasome inhibitor on the expression of T-betin WT and T-KOTh1cells.Conclusion: Ezh2promotes the expression o T-bet at the transcriptional level and candirectly activate Tbx21transcription; The dramatic reduction of T-bet protein in T-KO Th1cells can not be completely supported by moderate reduction of T-bet mRNA in these cells;Treatment with the proteasome inhibitor prevented the down-regulation of T-bet proteinlevels in T-KO Th1cells.Part Six： Introduction of T-bet into T-KO CD4+T cells fully rescues theirdifferentiation into Th1cells.1. The construction of MigR1virus bicistronically encoding T-bet or STAT4andGFP.2. The phenotype of infected Th1cells in vitro.3. The related gene expression of sorted GFP+cells.Conclusion: Viral expression of T-bet fully rescues the ability of Ezh2-deficient CD4+T cells to differentiate into Th1cells, whereas overexpression of STAT4only partiallyimproves Th1cell differentiation of activated T-KO T cells.Part Seven：Introduction of T-bet to T-KO T cells rescues their ability to mediate AAin mice1. The construction of MigR1virus bicistronically encoding T-bet or STAT4andGFP. 2. The establishment of mouse model for human AA with infected T-KO LN cells.3. The phenotype of infected Th1cells with ectopic expression of T-bet or STAT4inAA mouse model.Conclusion: Ectopic expression of T-bet but not STAT4in T-KO T cells rescues theirability to mediate AA in mice. Ezh2regulation of T-bet is important for production ofTh1cells mediating AA.