Lysine Acetylation and Foxp3 Function Figure 1. Foxp3 lysine mutations affect Treg suppressive function and gene expression. (A) Comparison of amino acid sequences of the C-terminal regions of mouse (m) and human (h) Foxp3, with FKH domains in gray. Lysine at position 332 is K16 and the FKH domain contains K17– K21 (consecutive lysines numbered in green), with residues important for Runx1 (R), DNA (D) or NFAT (N) binding indicated in purple and non- conserved residues shown in red; adapted from [16]. (B) CD4+CD252 T cells were transduced with retroviruses encoding WT Foxp3, Foxp16-19R, Foxp16-19Q or EV; Foxp3 staining showed .85% transduction efficiency (%transduced cells shown in blue in each panel). (C) In vitro Treg suppression assays in which 56105 CFSE-labeled Teff cells were stimulated for 72 h with CD3 mAb in the presence of 56105 irradiated APC and the indicated ratios of Treg to Teff cells. Data are mean 6 SD of duplicate measurements of the percentages of dividing Teff cells, and results are representative of 3 independent experiments; *p value,0.05, **p,0.01 compared to empty vector (EV) in left panel or compared to WT Foxp3 in right panel. (D) RNA derived from CD4+CD252 T cells transduced with WT Foxp3, Foxp3 K16-19R, K16-19Q or EV were analyzed for CTLA4, GITR, IL-2, and IL-10 gene expression by qPCR and data were normalized to 18S; *p value,0.05, **p,0.01 compared to WT Foxp3. Graphs show means 6 SD and results are representative of 3 independent experiments. doi:10.1371/journal.pone.0029035.g001 PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29035