Early B Cell Factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells [RNA-seq]

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Bio-ID - mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3, and a strong pathway relationship to IL-2 regulated genes which are known to govern cytotoxicity pathways. ChIP-seq analysis defined a canonical EBF4 binding motif 5’-CCCNNGG/AG-3’ closely related to the EBF1 binding site and using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted ATAC-seq in EBF4 overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function. Total RNA-seq libraries were sequenced in triplicate of the following conditions: EBF4 knock out vs non-target guide RNA treated and Lentiviral transduction of EBF4 vs empty vector.

细胞凋亡（apoptosis）是一种受遗传调控的细胞死亡程序，在免疫疾病进程中发挥关键作用。我们在针对Fas/APO-1/CD95介导的T细胞死亡调控因子的全基因组CRISPR筛选中，鉴定出了早期B细胞因子（EBF）家族中研究较少的转录因子成员EBF4。EBF4缺失会延长c-FLIP蛋白的半衰期，其在Fas信号复合物中的存在会抑制半胱天冬酶-8的切割与细胞凋亡。转录组分析显示，EBF4可调控对人类自然杀伤（NK）细胞及CD8+ T细胞功能至关重要的TBX21、EOMES、颗粒酶、穿孔素等分子。Bio-ID质谱分析结果表明，EBF4可与STAT3、STAT5及丝裂原活化蛋白激酶3（MAP kinase 3）结合，且与已知调控细胞毒性通路的白细胞介素2（IL-2）调控基因存在显著的通路关联。染色质免疫共沉淀测序（ChIP-seq）鉴定出典型的EBF4结合基序5’-CCCNNGG/AG-3’，该基序与EBF1结合位点高度相似；通过荧光素酶报告基因实验，我们发现该基序对EBF4存在剂量依赖性的转录响应。我们还在EBF4过表达细胞中开展了转座酶可及性测序（ATAC-seq），发现颗粒酶与穿孔素上游区域以及人类淋巴细胞的拓扑关联结构域内的染色质可及性显著升高。最终我们发现，EBF4在人类而非小鼠的NK细胞及CD8+ T细胞中存在基础表达，且在活化刺激后表达消失。综上，本研究数据揭示了此前未知的人类细胞毒性免疫功能转录调控因子的关键特征。本研究对以下两种条件下的样本进行了三次重复的总RNA测序：EBF4基因敲除组与非靶向向导RNA处理组，以及EBF4慢病毒转导组与空载体转导组。