Our ENCODE proposal aims to functionally characterize changes in gene regulation during the differentiation of mouse CD4+ T cells. We have already accomplished major primary goals of identifying regulatory elements that change activity during cell differentiation, and identifying regulatory elements that are sufficient to drive cell differentiation. We also have ongoing Year 4 studies to achieve our Aim of measuring effects of above-described regulatory elements on changes in gene expression during cell differentiation. Finally, we have completed several cross-ENCODE coordinated studies of human K562 and WTC-11 cells as part of the coordinated functional characterization center effort, and are now contributing to the comparative analysis of those results. We have five objectives of this request for an extension of funding. First, we plan to make highly efficient use of the experimental systems we have established to date to characterize gene targets of regulatory elements involved in CD4 T cell differentiation. Second, we will continue our teams leadership in comparing results from different functional characterization assays across the ENCODE consortium. Third, we will expand coordinated functional characterization efforts to include study of the genomic response to glucocorticoids. Glucocorticoid responses have been studied by ENCODE since 2008, and including functional characterization studies of the system at the conclusion of ENCODE will create new synergies with those previous data production efforts. We also view the theme of studying differential regulatory element activity as particularly important because the elements identified and the methods used are distinct from steady-state studies; and thus comparative analyses of how functional characterization technologies perform when assaying differential regulatory effects will be invaluable for guiding the future environmental response and perturbation studies. Fourth, we commit to submitting all data generated to the ENCODE DCC. Fifth, we also commit to distributing all reagents, protocols and results from comparative analyses for community use. The expected outcome of this extension of funding will be to substantially enhance the utility of ENCODE to inform future genomics research, particularly that involving high-throughput functional characterization assays and/or that involving environmental responses. That outcome aligns with recommendations by the ECP to enhance ENCODE studies of environmental response systems and to enhance coordination between data production and functional characterization centers. Those outcomes will be particularly impactful in the legacy of ENCODE as the NHGRI moves broadly to increase emphasis on studies of environmental responses and genomic perturbations as part of the 2020 Strategic Plan.