Molecular Development of the Immune System Section, Laboratory of Immune System Biology and Clinical Genomics Program, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
National Institute of Allergy and Infectious Diseases Collaborative Bioinformatics Resource, Frederick National Laboratory for Cancer Research Leidos Biomedical Research, Inc., Frederick, MD 21702.
Proc Natl Acad Sci U S A. 2022 Aug 16;119(33):e2208522119. doi: 10.1073/pnas.2208522119. Epub 2022 Aug 8.
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.
细胞凋亡是一种受基因调控的细胞死亡程序，在免疫疾病过程中发挥着关键作用。我们在全基因组 CRISPR 筛选 Fas/APO-1/CD95 介导的 T 细胞死亡的调节因子时，鉴定出 EBF4，这是早期 B 细胞因子 (EBF) 家族转录因子中一个研究较少的成员。EBF4 的缺失会增加 c-FLIP 蛋白的半衰期，并且其存在于 Fas 信号复合物中会损害半胱天冬酶-8 的切割和细胞凋亡。转录组分析显示，EBF4 调节 TBX21、EOMES、颗粒酶和穿孔素等分子，这些分子对人类自然杀伤 (NK) 和 CD8 T 细胞功能很重要。邻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，与白细胞介素-2 调节基因有很强的途径关系，这些基因已知可调控细胞毒性途径。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，我们发现该基序对 EBF4 有剂量依赖性的转录反应。我们还在 EBF4 过表达细胞中进行了转座酶可及染色质测序分析，发现颗粒酶和穿孔素上游以及人类淋巴细胞中拓扑相关结构域的染色质可及性增加。最后，我们发现 EBF4 在人类而不是小鼠 NK 细胞和 CD8 T 细胞中有基础表达，并且在激活刺激后消失。总之，我们的数据揭示了一个以前未知的人类细胞毒性免疫功能的关键转录调节因子的关键特征。
