PROJECT SUMMARY Identifying causal disease variants and elucidating how they lead to chronic disease remain important challenges in medicine. Genome-wide association studies have shown that polymorphisms associated with different human chronic diseases such as multiple sclerosis, rheumatoid arthritis and type 1 diabetes map predominantly to noncoding cis-regulatory regions of immune cells. Recent work has shown that a substantial fraction of non-coding variants linked to autoimmune disease lie in cis regulatory elements (CREs) of T lymphocytes, a subset of immune cells that play vital roles in preserving self-tolerance and preventing autoimmunity. However, determining which specific genes are regulated by CREs remains a challenge due to the temporal and spatial contexts in which they are modulate gene expression and the lack of suitable models to assess their activity in a manner that preserves their native chromatin structure. Importantly, T cells from autoimmune patients also exhibit marked changes in DNA methylation. However, the link between epigenetic changes and disease-associated variants is unclear. Our work has shown that T cells undergo significant epigenetic programming via DNA demethylation in the thymus and this programming is essential for long-term gene regulation in T cells. Remarkably, we have found that CREs active during T cell development play a critical role in modulating these epigenetic changes. Furthermore, we have shown that a significant number of genes that have critical roles in regulation of signals received the T cell receptor, which is essential for proper T cell function, are programmed in this manner. The goal of this proposal is to elucidate how CREs modulate epigenetic programming in genes with long-term functionality during thymic development and to define their contribution in chronic diseases. We propose a new model to test how human CREs in which disease- associated variants are present, modulate gene expression during thymic development and affect long-term epigenetic programming. We propose a new conceptual framework and mouse models to then investigate the mechanisms by which these CREs alter T cell function and promote chronic disease. Finally, we will investigate the involvement of newly discovered molecular factors that facilitate DNA demethylation during T cell maturation in the thymus. Understanding these mechanisms not only offers crucial insights into the aberrations of normal gene function in T cell driven diseases but also presents opportunities for the development of innovative approaches to enhance treatments for these illnesses. The comprehensive strategies outlined in this proposal will not only establish the foundation for future investigations into non- coding disease variants in autoimmune disorders driven by T cells but also offer a conceptual framework and new models for future investigations into disease-related variants that are prevalent in other cell types such as B cells. .