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Project description: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.
2022-07-27 | GSE206219 | GEO
Project description: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.
2022-07-27 | GSE206221 | GEO
Project description: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.
2022-07-27 | GSE206220 | GEO
Project description:Early B Cell Factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells.
| PRJNA849644 | ENA
Project description:Early B Cell Factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells [ChIP-seq]
| PRJNA849648 | ENA
Project description:Early B Cell Factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells [ATAC-seq]
| PRJNA849647 | ENA
Project description:Early B Cell Factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells [RNA-seq]
| PRJNA849649 | ENA
Project description:Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.
| S-EPMC40509 | biostudies-other
Project description:Lipid rafts are subdomains of the cell membrane with distinct protein composition and high concentrations of cholesterol and glycosphingolipids. Raft proteins are thought to mediate diverse cellular processes including signal transduction. However, its cellular mechanisms remain unclear. Caveolin-1 (cav-1, marker protein of caveolae) has been thought as a switchboard between extracellular matrix (ECM) stimuli and intracellular signals. Flotillin-2/reggie-1(Flot-2) is another ubiquitously expressed raft protein which defines non-caveolar raft microdomains (planar raft). Its cellular function is largely uncharacterized. Our novel studies demonstrated that Flot-2, in conjunction with cav-1, played important functions on controlling cell death via regulating Fas pathways. Using Beas2B epithelial cells, we found that in contrast to cav-1, Flot-2 conferred cytoprotection via preventing Fas mediated death-inducing signaling complex (DISC) formation, subsequently suppressed caspase-8 mediated extrinsic apoptosis. Moreover, Flot-2 reduced the mitochondria mediated intrinsic apoptosis by regulating the Bcl-2 family and suppressing cytochrome C release from mitochondria to cytosol. Flot-2 further modulated the common apoptosis pathway and inhibited caspase-3 activation via up-regulating the members in the inhibitor of apoptosis (IAP) family. Last, Flot-2 interacted with cav-1 and limited its expression. Taken together, we found that Flot-2 protected cells from Fas induced apoptosis and counterbalanced the pro-apoptotic effects of cav-1. Thus, Flot-2 played crucial functions in cellular homeostasis and cell survival, suggesting a differential role of individual raft proteins.
| S-EPMC3799625 | biostudies-literature
Project description:We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.
| S-EPMC2858774 | biostudies-literature