Modulation of activated T cells by transient EZH2 inhibition [RNA-Seq]. Modulation of activated T cells by transient EZH2 inhibition [RNA-Seq]

The histone methyltransferase enhancer of zeste homolog 2 (EZH2)-mediated epigenetic regulation of T cell differentiation in acute infection has been extensively investigated. However, the role of EZH2 in T cell exhaustion remains under-explored. Here, using in vitro exhaustion models, we demonstrate that transient inhibition of EZH2 in T cells prior to the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetostat, delays their dysfunctional progression and preserves T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetostat induces T-cell epigenetic reprogramming and increases the expression of the self-renewal T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. Transcriptomic profiling revealed a cell division-dependent upregulation of genes associated with thymocytes, memory and effector T cell subsets. Mapping of EZH2-associated genomic regions by ChIP-seq showed an enrichment of promoters among H2K27me3 loci reduced in abundance by tazemetostat, including those genes upregulated in taz-treated T cells. Overall design: OT-I splenocytes were activated with OVA257-264 (SIINFEKL, 500 nM) with 60 IU/mL of IL-2 for three days to obtain CD8+ T cells. The cells were further cultured with or without 1 µM Taz for 24 or 48 h before they are harvested for RNA extraction with the Arcturus™ PicoPure™ RNA Isolation Kit (KIT0204, ThermoFisher Scientific). Traces of genomic DNA were removed using RNase-Free DNase Set (79254, QIAGEN) following the on-column digesting protocol of the RNA isolation kit. Library construction and RNAseq data acquisition was done by the Novogene Corporation (Sacramento, CA) using Illumina HiSeq platform and PE150 strategy.

组蛋白甲基转移酶zeste增强子同源物2（enhancer of zeste homolog 2, EZH2）介导的急性感染中T细胞分化表观遗传调控已被广泛研究。然而，EZH2在T细胞耗竭中的作用仍有待深入探究。
本研究借助体外耗竭模型证实：在T细胞耗竭的表型发作前，采用临床获批的抑制剂他泽司他（Tazemetostat）对EZH2进行瞬时抑制，可延缓T细胞的功能失调进程，维持其干性与多能性，且不会对细胞增殖产生任何负面影响。
他泽司他可诱导T细胞表观遗传重编程，并通过优先在快速增殖的T细胞中降低其启动子区域的H3K27甲基化水平，上调自我更新相关T细胞转录因子T细胞因子1（TCF1）的表达。
转录组测序分析显示，与胸腺细胞、记忆性及效应性T细胞亚群相关的基因呈现细胞分裂依赖性的上调表达。
通过染色质免疫共沉淀测序（ChIP-seq）对EZH2结合的基因组区域进行定位分析发现，经他泽司他处理后丰度降低的H2K27me3位点中，启动子区域显著富集，其中就包括他泽司他处理后上调表达的基因。
实验总体设计如下：将OT-I小鼠脾脏淋巴细胞用OVA257-264（SIINFEKL，500 nM）联合60 IU/mL的IL-2激活3天，以获得CD8+ T细胞。随后将细胞置于添加或不添加1 µM 他泽司他（Taz）的培养基中培养24或48小时，之后收集细胞，使用Arcturus™ PicoPure™ RNA提取试剂盒（KIT0204，赛默飞世尔科技，ThermoFisher Scientific）进行RNA提取。按照该RNA提取试剂盒的柱上消化流程，使用无RNase DNase试剂盒（79254，凯杰，QIAGEN）去除基因组DNA残留。文库构建及RNA测序数据获取由诺禾致源公司（Novogene Corporation，加利福尼亚州萨克拉门托）基于Illumina HiSeq平台及PE150测序策略完成。