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. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5′-CCCNNGG/AG-3′, closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing 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.

中文翻译：

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早期 B 细胞因子 4 调节 FAS 介导的细胞凋亡并促进人体免疫细胞的细胞毒功能

细胞凋亡是一种基因调控的细胞死亡程序，在免疫疾病过程中起着关键作用。我们在 Fas/APO-1/CD95 介导的 T 细胞死亡的全基因组 CRISPR 筛选中鉴定了 EBF4，这是早期 B 细胞因子 (EBF) 转录因子家族中研究较少的成员。EBF4 的缺失会增加 c-FLIP 蛋白的半衰期，并且它在 Fas 信号复合物中的存在会损害 caspase-8 的切割和细胞凋亡。转录组分析显示 EBF4 调节 TBX21、EOMES、颗粒酶和穿孔素等对人类自然杀伤 (NK) 和 CD8 很重要的分子+T 细胞功能。接近依赖性生物素鉴定 (Bio-ID) 质谱分析显示 EBF4 与 STAT3、STAT5 和 MAP 激酶 3 结合，并且与白细胞介素 2 调节基因有很强的通路关系，已知这些基因控制细胞毒性通路。染色质免疫沉淀和 DNA 测序分析定义了一个典型的 EBF4 结合基序，5'-CCCNNGG/AG-3'，与 EBF1 结合位点密切相关；使用基于荧光素酶的报告基因，我们发现了该基序对 EBF4 的剂量依赖性转录反应。我们还在 EBF4 过表达细胞中进行了转座酶可及染色质测序测定，发现颗粒酶和穿孔素上游以及人类淋巴细胞拓扑相关区域的染色质可及性增加。最后，+T 细胞在激活刺激后消失。总之，我们的数据揭示了以前未知的人类细胞毒性免疫功能转录调节因子的关键特征。