Functional annotation of proteins determined by Foxp3 PSI MS. (A) Proteins differentially enriched by Foxp3 versus histone H3 PSI MS are independent of their expression levels profiled by whole-cell TMT MS. Data were derived from two to three replicates per condition. (B) Gene Ontology terms of the proteins enriched by Foxp3 and histone H3 PSI MS. (C and D) Identification of the regulators of Foxp3, CTLA-4, and CD25 expression from the proteins identified by Foxp3 PSI MS using CRISPR screening. CD4 Tn cells isolated from Foxp3^gfpRosa^Cas9 mice were induced to iTreg cells by TCR agonists, IL-2, and TGF-β in the presence of ASC. A retroviral sgRNA library targeting 1,493 genes enriched by Foxp3 PSI MS was transduced into iTreg cells on day 3. 5 days later, cells expressing high or low levels of Foxp3, CTLA-4, or CD25 were FACS-sorted to compare sgRNA representation with high-throughput sequencing. (E–G) Cross-comparison of the regulators of Foxp3 versus CD25 expression (E) or CD25 versus CTLA-4 expression (F). Overlaps of regulators are shown (G; FDR < 0.05). Data are derived from three replicates. (H and I) Validation of top candidate regulators of Foxp3 (H) and CD25 (I) expression in Cas9-expressing iTreg cells transduced with individual sgRNAs. CD4 Tn cells isolated from Foxp3^gfpRosa^Cas9 mice were cultured in Treg-induction media. Cells were transduced with retroviral sgRNAs at day 1 and analyzed at day 5. Data show triplicates and means ± SDs and represent two experiments. Two-tailed, unpaired t tests; ns, no significance; **P < 0. 01, ***P < 0.001, ****P < 0.0001.