Lysine Acetylation and Foxp3 Function Figure 3. Foxp3 mutants impair Foxp3 DNA binding ability. 293T cells were transfected with EV, WT Foxp3, K17R or K18R without or with p300 expression vectors, and 48 h later, cell lysates were harvested. (A) Equal amounts of cell lysates were incubated with biotin-labeled Foxp3 binding site nucleotide, and Foxp3 DNA binding was detected with anti-Foxp3 or anti-acetyl-lysine Abs. The protein expression levels of Foxp3 and loading control b-actin were detected by western blotting; arrow indicates acetylated Foxp3 bound to DNA, and star indicates non-specific binding. (B–D) The densities of Foxp3 DNA-binding bands were measured using Image-J software and normalized with Foxp3 input levels. (B) The relative Foxp3 DNA binding ability in the absence of p300 is shown. (C) Foxp3 and mutant DNA binding ability was increased in the presence of p300. (D) Comparison of relative Foxp3 DNA binding between WT and mutants in the presence of p300 is shown. (E) Comparison of relative acetylated Foxp3 binding level between WT and mutants is shown. Results are representative of 2 independent experiments. doi:10.1371/journal.pone.0029035.g003 FKH K17 and K18 residues regulate IL-2 promoter acetylation and HDAC and HAT expression Foxp3 can bind to the IL-2 promoter, in both natural Tregs and CD4+ T cells transduced with Foxp3 [13], at a site adjacent to the ARRE-2 NFAT/AP-1 response element [13,32,33]. Given the reduced DNA binding of our Foxp3 mutants, we measured IL-2 protein levels in supernatants of T cells transduced with WT Foxp3, K17R or K18R Foxp3, or EV. Both K17R and K18R mutations resulted in greater production of IL-2 than cells transduced with WT Foxp3 (Fig. 4A). In addition, since Foxp3 controls Treg function by competing with AP1 and cooperatively binding with NFAT [13], we assessed if K17R and K18R PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29035