ABSTRACT Innate lymphocytes represent early sentinels critical in host immunity against pathogens. The best studied innate lymphocyte is the natural killer (NK) cell, which specifically targets virally-infected host cells. Humans deficient in NK cells have severe health complications due to susceptibility to herpesvirus infections. In addition to NK cells, a family of innate lymphoid cells (ILC) has recently been demonstrated to play an important role in protection against pathogen infection at barrier surfaces such as the intestine, lung, and skin. Our long term goals are to understand the the molecular mechanisms underlying the development of these innate lymphocytes, which will increase our understanding of the function of these cells during infection. To this end, we have recently identified a novel role for the transcription factor Nfil3 (also known as E4BP4) in development of NK cells and all members of the ILC family. Using mouse pathogens that accurately model human infectious diseases, along with newly-generated transgenic mouse models that ablate either the Nfil3 gene or evolutionarily-conserved regulatory domains, this R01 grant proposes to investigate how Nfil3 is controling the development and anti-pathogen functions of NK cells and ILCs. In Aim 1, we will investigate how Nfil3 is transcriptionally regulated in innate lymphocyte progenitors by chromatin immunoprecipitation (ChIP) for the transcription factors STAT5, PU.1, TCF-1, and Ets-1 followed by qPCR or deep sequencing (seq). In Aim 2, we will use two novel mouse strains containing a CRISPR/Cas9-targeted deletion of conserved noncoding sequences (termed Nfil3 CNS1 and CNS2) to determine the influence of intronic regulatory regions on innate lymphocyte development and effector function against virus, helminth, and bacterial infection. In Aim 3, we will use ChIP-seq, RNA-seq, and mass spectrometry to determine the genes that Nfil3 is controlling, and binding partners that influence Nfil3-mediated regulation of these target genes. Together, the studies in this proposal will not only increase our understanding of the general molecular mechanisms whereby NK cells and ILCs develop and contribute to host defense during pathogen invasion, but also establish novel clinical paradigms for how the innate lymphocyte compartment may be harnessed for therapeutic strategies against infectious disease.