PROJECT SUMMARY T helper (Th) cells are CD4-expressing T lymphocytes that diversify into several subclasses to support distinct immune responses. Among the diversity of Th subset differentiation options, IL-17A-producing inflammatory Th17 cells stand out as unique by virtue of their relatively high level of inherent plasticity. Indeed, this subset that normally functions in mucosal immunity against bacteria and fungi can easily adopt features of other T helper subsets when environmental conditions change. While this feature can be advantageous during the clearance of an infection, dysregulated Th17 cell function has been implicated in numerous autoimmune conditions, including Inflammatory Bowel Disease, Multiple Sclerosis, and Rheumatoid Arthritis. Moreover, Th17 cell plasticity exhibited in the context of inflammatory disease tends to take on Th1-like traits, such as expression of IFNg or T-bet, that are also associated with increased pathology. In our current funded studies, we identified a JunB-T-bet axis as a central node in the regulatory network governing Th17 cell lineage stability versus plasticity. While several regulators influencing the transcriptional program of Th17 cell identity and flexibility have been identified, less is known about the regulators of the three-dimensional genome architecture that differs between cell types and states and is a critical determinant of gene regulation. Thus, we hypothesize that regulation of chromatin confirmation by lineage-specific factors is key to the control of CD4 T cell effector conversions. Here, we propose to comprehensively evaluate the dynamic changes in chromatin configuration and the cis genomic elements that govern Th17 cell effector conversions in vivo. To this end, in Aim 1, we will apply genetic fate-mapping mouse models, global chromatin conformation assays, and epigenomic profiling tools to characterize the chromatin looping dynamics during Th17 cell plasticity in a mouse model of experimental autoimmune encephalomyelitis (EAE). We will determine the contribution of looping to Th17 cell plastic conversion by assessing the role of both chromatin organizing proteins and lineage-regulating transcription factors. In Aim 2, we plan to determine the cis regulatory network that governs Th17 cell plasticity. For this, we will apply scATACseq profiling of Th cells in EAE to identify plasticity-associated cis elements. We will validate candidates for activity and function using novel in vivo high throughput reporter assays and CRISPR/Cas9 epigenomic screens. This will identify new regulatory networks, regulators, and gene targets critical to Th17 cell effector plasticity. Taken together, the proposed work will fill an important gap in knowledge concerning the molecular mechanism governing stability versus plasticity of Th17 cells.