PROJECT SUMMARY Relapsing remitting multiple sclerosis (RRMS) is an autoimmune disease in which CD4 T cells play a central role. Studying the epigenome of CD4 T cells in RRMS through the integration of genome-wide analysis of DNA methylation, open chromatin, and histone modifications with GWAS variants and RNA sequencing (RNA-seq) data has the potential to elucidate the molecular processes that potentiate disease activity. The studies proposed in this application will examine the epigenomic landscape of CD4 T cells in a well characterized cohort of individuals with RRMS, using longitudinal samples to allow an assessment of how the epigenome is altered in the context of disease flare and remission. The central hypothesis is that differences in the epigenomic landscape of CD4 T cells during flare and remission will identify factors that promote disease flares and the resulting changes in T cell function. The studies in Specific Aim 1 will determine how the epigenomic and transcriptomic landscapes of T cells change during disease flare compared to remission in a longitudinal RRMS cohort. We will profile chromatin accessibility (ATAC-seq) and the transcriptome (RNA-seq) in total CD4, memory CD4, Th17 and regulatory T cells (Tregs). ATAC-seq data will be analyzed using an analytic pipeline integrating capture Hi- C data, transcription factor foot printing and MS genetic risk. The RNA-seq dataset will then be integrated to identify gene expression changes related to epigenomic alterations during disease flare. Findings in the longitudinal cohort will be validated in an independent cross-sectional cohort including individuals flaring and individuals in remission. The studies in Specific Aim 2 will investigate the impact of differential chromatin accessibility at the ZFP36L2 locus on CD4 T cell lineage and function. They will determine whether the changes in chromatin accessibility at the ZFP36L2 locus during disease flare alter ZFP36L2 gene expression in all CD4 T cells or only specific T cell subsets using PrimeFlow. CRISPR editing to either open or close the chromatin at the ZFP36L2 locus will be used to directly confirm the regulation of ZFP36L2 expression by the enhancer. The effect of ZFP36L2 chromatin accessibility on CD4 T cells lineage and function will also be evaluated using samples from RRMS subjects with open or closed chromatin at the ZFP36L2 locus. Collectively, these studies will advance our understanding of how changes in chromatin accessibility in the context of disease activity contribute to the pathogenicity of T cells in RRMS and may also reveal drivers of disease development.