Identify novel modulators of regulatory T cell function from the dynamic cis-proteomes Regulatory T (Treg) cells are induced in the thymus or periphery to suppress effector T cells that not only cause autoimmune diseases but also kill tumors. Therefore, Treg cells serve as a therapeutic target to treat a variety of immunological diseases and cancer. Despite intensive investigations, methods to achieve this goal remain to be fully developed. Here, we aim identify novel factors to reprogram Treg immune suppressive function. To this end, we systematically examined the epigenetic and transcriptional mechanisms governing Treg function through Treg master regulator Foxp3. Treg cells are also regulated by environmental cues, including T cell antigen receptor (TCR) agonists and Interleukin-2 (IL-2), that act through transcriptional mechanisms to modulate Treg fitness, homing, and suppressive function. We hypothesize that opposing nuclear programs coordinate to interpret environmental cues, thus balancing Treg suppressive function. Identification and characterization of these programs will produce mechanistic insights into Treg function, leading to novel methods to modulate Treg suppressive activity. To test our hypothesis, we propose to develop a proteomics method to profile signal-induced dynamic protein components at the cis-regulatory elements governing Treg suppressive function. In our preliminary experiments, we adapted a proximity biotinylation–based proteomics method to identify the proteins at Foxp3-associated cis-regulatory elements in the steady state and after cells receiving TCR or IL-2 stimulation. These experiments uncovered unprecedented details of proteins potentially involved in the regulation of Foxp3-target gene expression at the resting state or upon TCR and IL-2 stimulation. As a proof of concept, these results demonstrate the feasibility of proximity biotinylation, when coupled with comparative proteomics, in unbiasedly profiling the nuclear proteins involved in signal-dependent transcriptional regulation. On the basis of these preliminary results, we propose to further define the temporal dynamics of the cis- proteomes in Treg cells after cells receiving TCR and IL-2 stimulation at the early, middle, and later stages of signal transduction. Our in-depth comparative analysis will reveal the proteins recruited to and depleted at Foxp3 targets in response to TCR or IL-2 stimulation. On the other side, we will integrate genetic perturbations and functional assays to identify novel regulators of Treg cell function from signal-dependent dynamic cis-proteomes. We will also determine the mechanisms by which these novel factors control Treg cell function by modulating their response to TCR or IL-2 signaling. Overall, we develop a comparative proteomics method to profile cis-proteomes in situ without engineering new cells or organisms. We will determine the temporal dynamics of the Treg cis-proteomes upon TCR and IL- 2 stimulation and us...