Regulation of Signaling Pathways Involving Nuclear Factor Kappa B

National Institute Of Allergy And Infectious Diseases

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Abstract

NF-kappaB is a major transcription factor that regulates genes responsible for both the innate and adaptive immune response. In this project, we focused on elucidating transcription factors that control the development of immune cytolytic cells. 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. Abnormalities of apoptosis affect many biological processes, including aging, organismal degeneration, and disease. Identification of the molecular checkpoints of Fas-mediated death is still incomplete. We found that 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, closely related to the EBF1 binding site; using a luciferase based reporter, we found a dose- dependent transcriptional response of this motif toEBF4. 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 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.