Histone Modification Enzyme Regulates Differentiation And Function Of NK Cells

Objectives Part 1: Studies of Natural Killer(NK)cell cytotoxicity have mainly focused on the balance of activated and inhibitory receptors,signaling transduction,calcium influx,formation of immune synapse,and cytolytic degranulation.However,whether the chromatin state of NK cells is changed during target recognition,and what is the impact of this change remain largely unknown.Here,we investigate the contribution of chromatin state dynamics during NK cell activation by comprehensively analyzing a set of microarray data and two sets of ChIP-seq data.And also,a series of small molecule inhibitors,which are specific to H3K4 and H3K27 modification,are utilized to test the degranulation level or the expressions of IFN-? and TNF-?.Thus,our results provide novel insights to the regulation of NK cells cytotoxicity and immunoregulatory function by chromatin state dynamics.Part 2: Although the mechanism of NK cell activation is still partially unclear,the strict calcium-dependence remains the hallmark for lytic granule secretion.There are a large volume of published studies describing that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis with the result of weak target cell lysis.So far,however,there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity.Our previous results indicated that small-molecule inhibitor UNC1999 could induce calcium signal and degranulation level,and the main purpose of this study is to explore the mechanism of UNC1999-dependent calcium signal increasing.What's more,we try to further analyze the relationship among calcium signal,degranulation level and target cell lysis.Part 3: Our previous data indicated that the numbers and frequency of NK cells increase more than twofold in spleen,liver,and BM of mEzh2fl/flVav1 i Cre mice,suggesting that loss of mEzh2 may be associated with improved NK cell early stage differentiation.What's more,we also noticed that the expression of Tox,a transcription factor manipulates NK cell late stage maturation,was induced in mEzh2fl/flVav1 i Cre mice.Based on these data,we hypothesize that mEzh2 may regulate NK cell late stage maturation.We cross mEzh2fl/fl mice with transgenic Ncr1 i Cr mice to delete mEzh2 from NK cells and detect the late stage maturation and cytotoxicity of NK cells in mEzh2fl/flNcr1 i Cre mice.Methods Part 1: 1.Comprehensively analyze a set of microarray data and two sets of ChIP-seq data to investigate the expression level of histone methyltransferase and demethylase and the contribution of chromatin state dynamics during NK cell activation.2.qPCR and Western blot are performed to investigate the expression level of histone methyltransferase and demethylase.3.ChIP-qPCR is performed to analyze the genome-wide modified targets of the H3K4me3 and H3K27me3 before and after NK cell activation.4.We conduct a series of small-molecule compounds,which are specific targeting of H3K4 and H3K27 methyltransferase or demethylase,to impact the chromatin state of NK cell and analyse their immunological function after stimulated with PMA and Ionomycin.Part 2: 1.We use small-molecule inhibitor UNC1999 and GSK343 to inhibit the catalytic activity of EZH2.What' more,NK cells are infected with retrovirus expressing sh RNA to delete EZH2 expression.2.Flow cytometry analysis is performed to investigate calcium signal,degranulation level and target cell lysis.3.We perform imaging studies by confocal microscope to observe conjugates formation,lytic synapse formation and granule polarization of NK cells with or without small-molecule inhibitor UNC1999.4.Gene expression profiling is used to screen out the differentially expressed genes on UNC1999-vs control-samples.5.qPCR,Western blot and ChIP-qPCR are conducted to investigate the mechanism of UNC1999-dependent calcium signal increasing.Part 3: 1.Western blot are conducted to investigate the expression level of mEzh2 in NK cells of mEzh2-/-Ncr1 i Cre?mEzh2fl/-Ncr1 i Cre and mEzh2fl/fl Ncr1 i Cre mice.2.Flow cytometry analysis is performed to analyze NK lineage cells development and cytotoxicity.3.We utilize the B16F10 melanoma model of tumor metastasis to investigate NK cell-mediated tumor surveillance in mEZH2fl/flNcr1 i Cre mice.Result Part 1: 1.Eight histone methytransferase or demethylase are screened out from microarray data.2.The expression of histone methytransferase JARID2,demethylase UTY and KDM6 B are induced during NK cell activation.3.27 genes associated with immune functions are identified and most of these genes(77.8%)are upregulated.4.Bivalent modification of PIK3 CA,NFATC1 and TNFSF9 is altered upon NK cells activation 5.UNC1999 could induce degranulation level of NK cells;OG-L002 and MM102 could increase the expression of IFN-? and TNF-?.Part 2: 1.Surface expression of CD107 a or calcium signal are induced,and cytotoxicity is inhibited after NK cells treated with small molecule inhibitor or NK cells infected with retrovirus expressing sh RNA to delete EZH2 expression.2.The ability of conjugates formation,lytic synapse formation and granule polarization are normal upon NK cells treated with small molecule inhibitor.3.EZH2 deficiency leads to unbalanced granule depletion with the result that only the first or first few target cells could be killed until new lytic granules are being produced.4.EZH2 directly regulated the expression of PKD2 by its enzyme activity.Part 3: 1.The expression of mEzh2 and global modification level of H3K27me3 are decreased in NK cells of mEzh2fl/flNcr1 i Cre mice.2.The numbers and frequency of NK cells increase in spleen,lymph node(LN),and bone marrow(BM)of mEzh2fl/flNcr1 i Cre mice.3.The absence of mEzh2 is associated with the proportion of IFN-?-secreting cells after stimulated with Yac-1 cells.We also observed a marked decrease in the number of pulmonary tumor nodules in mEZH2fl/flNcr1 i Cre mice.Conclusion Part 1: 1.A series of genes that play important roles during NK cell activation are at ‘poised' state prior to activation.2.The expression of some histone methytransferase and demethylase are induced,and the bivalent modification state of some genes' promoter are altered upon NK cells activation.All of these data imply that the histone modification state has profound impact on NK cell activation.3.Some small molecule inhibitors,which are specific to H3K4 and H3K37 modification,enhance the degranulation level or the expressions of IFN-? and TNF-?,which provide novel insights into the regulation of NK cells cytotoxicity and immunoregulatory function by chromatin state dynamics.Part 2: 1.UNC1999-dependent calcium signal increase induces unbalanced granule depletion with the result that only the first or first few target cells could be killed,and finally alters their killing efficiency.2.H3K27me3 and H3K4me3 modification enrich at the promoter of PKD2 in resting NK cells.A significant decrease in H3K27me3 mark at the PKD2 promoter and an obviously induced expression of PKD2 are observed in UNC1999 treated cells compared with DMSO control.Part 3: 1.mEzh2 not only modulates the early-stage differentiation of NK cells but also regulates late-stage maturation of NK cells.2.mEzh2 plays a significant role in NK cell,which controls in vivo tumor progression.