function in vitro and in vivo, and altered expression of Treg- associated markers, cytokines, and levels of HAT and HDAC enzymes. Our data suggest that point mutations in Foxp3 can have different effects than the widely recognized mutations that lead to the development of autoimmunity in scurfy mice and patients with IPEX syndrome. The complex effects of these mutations point to the importance of FKH lysine residues in control of Foxp3 function. Results C-terminal Foxp3 lysine mutations affect Treg suppressive function and gene expression Five FKH domain lysines (K17, 18, 19, 20, and K21) are conserved between humans and mice (Fig. 1A). K17 and 18, but not K20, are important for Treg suppressive function in vitro [16], whereas K20 and K21 are important for Foxp3 localization within the nucleus [7]. An additional lysine, K16, adjacent to the FKH domain, is important for the interaction of Foxp3 with AML/ RunX1 [14]. We therefore focused on K16, 17, 18 and 19 and investigated whether these residues were important for Foxp3 function. We used site-directed mutagenesis to substitute the 4 residues (K16-19) with arginine (R), so as to prevent acetylation but conserve the positive charge, or with glutamine (Q), so as to change the positive charge of lysine to neutral and possibly mimic features of acetylation [21]. Wild-type mouse Foxp3 (WT-Foxp3) or mouse Foxp3 constructs containing mutated K16-19R or K16- 19Q were cloned into a retroviral vector (MinR1) encoding nonfunctional nerve growth factor receptor (NGFR), MinR1 constructs were transfected into packaging cell lines, and supernatants from packaging cell lines were used to infect activated mouse primary CD4+ T cells. Flow cytometric analysis (Fig. 1B) indicated transduction efficiencies of .85% for mutant and WT Foxp3, and comparable levels of Foxp3 expression, indicating the mutations did not affect Foxp3 expression in Treg cells. We next examined whether these Foxp3 mutations affected Treg suppressive function. Mouse primary CD4+ T cells transduced with vectors encoding the WT-Foxp3 exhibited comparable or better suppressive function than native CD4+CD25+ Treg cells isolated from C57BL/6 mouse (B6-Treg), whereas empty-vector (EV) transduction essentially lack any suppressive function (Fig. 1C, left panel). However, compared with transduction of CD4+ T cells with vectors encoding WT- Foxp3, transduction of vectors containing lysine mutations (K16- 19R or K16-19Q) led to markedly impaired Treg suppressive ability, with glutamine mutations (K16-19Q) displaying more impaired function (Fig. 1C, right panel). These data indicated these four lysines (K16-19) are important for Treg suppression function, at least in vitro. We used qPCR to examine the mRNA levels of several genes of functional significance for Tregs [22,23,24,25,26,27]. Com- pared with WT-Foxp3 transduced CD4+ T cells, CD4+ T cell transduction with vectors containing Foxp3 K16-19R or K16- 19Q showed lower expression of cytotoxic T lymphocyte- associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor-receptor-related protein (GITR), and IL-10, with K16-19Q mutations showing even lower mRNA expression than K16-K19R mutations (Fig. 1D). Moreover, compared with WT- Foxp3-transduced CD4+ T cells, CD4+ T cells transduced with Foxp3-K16-19R or K16-19Q exhibited a higher level of IL-2 (Fig. 1D), indicating that these four lysines are likely involved in regulating expression of key Treg genes. Since the mutation of lysine to glutamine, which mimics acetylation in some systems but Lysine Acetylation and Foxp3 Function not others [28], unexpectedly impaired Treg function to a greater extent than arginine mutations, we focused our studies on the arginine mutants that maintain the same polarity as lysine. Single Foxp3 residues, K17 or K18, are important for Treg suppressive function and gene expression We tested the roles of individual lysines in Treg function by transducing mouse primary CD4+ T cells with MinR-Foxp3 constructs containing K16R, K17R, K18R or K19R. Transduc- tion efficiency in each case was .80% as indicated by Foxp3 expression (Fig. 2A), and there were no significant differences between mutants. However, Treg suppression assays showed that compared with CD4+ T cells transduced with WT-Foxp3, cells transduced with Foxp3 K17R or K18R mutant exhibited significantly impaired Treg suppression (Fig. 2B), whereas Foxp3 K16R increased Treg suppressive function at certain Treg to Teff cell ratios, and K19R mutation did not impair Treg suppression. Additionally, CD4+ T cells transduced with Foxp3 K17Q, K18Q, K17E, or K18E mutant also resulted in impaired Treg suppressive function to varying extents (data not shown). These data thereby suggested that K17 and K18 of the Foxp3 FKH domain were especially important for Treg suppressive function in vitro. In addition, compared with CD4+ T cells transduced with WT-Foxp3, CD4+ T cells transduced with Foxp3 K17R or K18R mutant exhibited markedly decreased mRNA level of CTLA-4 and slightly decreased levels of GITR, CD62L, and IL- 10 (Fig. 2C and data not shown). Foxp3 K17R or K18R mutants showed significantly increased levels of IL-2, IL-4, IL-17, and IL- 21 upon stimulation of CD3/CD28 mAbs (Fig. 2C). These observations demonstrated that the K17 and K18 residues contribute to Foxp3-mediated control of cytokine expression within Treg cells and to maintenance of Treg suppressive function. K17 and K18 mutants exhibit impaired Foxp3 DNA binding ability While p300 is known to acetylate Foxp3 [18], the single point mutations (K17R, K18R) did not reduce overall Foxp3 protein acetylation (data not shown). However, as the C-terminal FKH domain mediates Foxp3 DNA binding [29,30], we next compared the DNA binding of WT Foxp3 and mutants. Biotin-labeled Foxp3 binding site nucleotide [30] was incubated with cell lysates from 293T cells expressing WT or mutant Foxp3, with or without p300. DNA-bound biotinylated Foxp3 nucleotide was precipitated using streptavidin beads and detected by immunoblotting with anti-Foxp3 mAb. Compared to WT Foxp3, K18R had markedly decreased DNA binding ability, whereas K17R binding was only modestly decreased (Fig. 3A and B). Since transcription factor DNA binding ability is frequently increased by acetylation [31], we further investigated Foxp3 DNA binding ability after acetylation by p300. After adding p300, the DNA binding ability of both WT Foxp3 and mutants was increased up to two-fold, with a greater effect on K17R than K18R (Fig. 3A and C). Remarkably, the presence of p300 enhanced the differences between Foxp3 DNA binding of WT and mutant Foxp3, revealing a more than two-fold decrease in K18R mutant Foxp3 binding (Fig. 3B and D). We also assessed the level of acetylated-Foxp3 bound to DNA level by immuno- blotting with anti-acetyl-lysine antibody after nucleotide pull- down of Foxp3, and found that K17R mutant had markedly decreased acetylated-Foxp3 bound to DNA (Fig. 3A and E). These data suggest that K17 and K18 contribute to regulation of Foxp3 DNA binding. PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29035