Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 33; p. e2208522119
• Main Authors: Kubo, Satoshi, Kataria, Rhea, Yao, Yikun, Gabrielski, Justin Q, Zheng, Lixin, Markowitz, Tovah E, Chan, Waipan, Song, Jian, Boddapati, Arun K, Saeki, Keita, Häupl, Björn, Park, Ann Y, Cheng, Yan H, Cui, Jing, Oellerich, Thomas, Lenardo, Michael J
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 16.08.2022
• Subjects: Accessibility; Animals; APO-1 protein; Apoptosis; Apoptosis - physiology; Binding sites; Biological Sciences; Biotin; c-FLIP protein; Caspase-8; CD8 antigen; CD8-Positive T-Lymphocytes; CD95 antigen; Cell death; Chromatin; Chromatin - metabolism; Cytotoxicity; Cytotoxicity, Immunologic - genetics; DNA sequencing; Fas antigen; Fas Ligand Protein - metabolism; FLIP protein; Genomes; Granzymes - genetics; Humans; Immune response; Immune system; Immunological diseases; Immunoprecipitation; Interleukin 2; Kinases; Lymphocytes; Lymphocytes T; MAP kinase; Mass spectrometry; Mass spectroscopy; Mice; Natural killer cells; Perforin; Perforin - metabolism; Stat3 protein; Stat5 protein; T-Lymphocytes, Cytotoxic; Toxicity; Transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism; EBF; apoptosis; cytotoxic lymphocyte
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2208522119

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.