mutations affected Foxp3 targeting of the NFAT:AP1 complex. We transfected 293T cells with 3x NFAT:AP1-IL-2-Luc reporter, plus expression vectors for NFAT and WT or mutant Foxp3 (K17R, K18R). WT Foxp3 significantly inhibited NFAT tran- scriptional activity, consistent with previous studies [13]. Both K17R and K18R Foxp3 mutants showed similar impairment of NFAT-mediated transactivation, suggesting that Foxp3 K17 and K18 are not involved in Foxp3-NFAT-DNA formation (Figure S1). Foxp3-mediated transcriptional activation and repression are associated with specific histone modifications [33,34]; e.g. Foxp3 binding to the IL-2 promoter leads to histone H3 deacetylation and repressive chromatin remodeling. Accordingly, we assessed acetylated histone 3 (Ac-H3) at the IL-2 promoter using CD4+ T cells transduced with WT or mutant Foxp3 (Fig. 4B). A significant amount of Ac-H3 remained at the IL-2 promoter of CD4+ T cells transduced with empty vector. However, transduction with WT Foxp3 markedly reduced Ac-H3 binding to the IL-2 promoter, whereas cells transduced with mutant Foxp3 (K17R and K18R) exhibited increased Ac-H3 binding to the IL-2 promoter (Fig. 4B). These data indicate that K17 and K18 are involved in Foxp3- mediated remodeling of IL-2 chromatin structure in CD4+ T cells. Lysine Acetylation and Foxp3 Function In addition to recruiting HDAC and HAT enzymes to target genes such as IL-2, Foxp3 could conceivably regulate the expression of these enzymes in Treg cells. Consistent with this concept, we found that transduction with WT versus Foxp3 mutants led to differential expression of HDAC and HAT genes. Compared to EV control, transduction of WT Foxp3 led to significant suppression of HDAC 1, 2, 5, 6, and 7 mRNA. In contrast, mutant Foxp3 transduction resulted in less suppression of HDAC expression (Fig. 4C), and considerably greater HAT expression (Fig. 4D). Collectively, these data indicate that K17 and K18 contribute to Foxp3-dependent chromatin remodeling and modulation of HDAC and HAT expression. K17 and K18 are important for Foxp3 suppressive function in vivo Since Treg cells play an important role in the control of homeostatic proliferation [27,35,36,37], we evaluated the effects of FKH lysine mutations on this process. We adoptively transferred into immunodeficient C57BL/6 Rag12/2 mice one million Thy1.1+ CD4+CD252 cells alone, or along with one million Thy1.2+ CD4+ T cells transduced with EV, WT Foxp3, Foxp3 Figure 4. Foxp3 mutations affect chromatin remodeling and HDAC and HAT expression. CD4+ T cells transduced with WT Foxp3, K17R, K18R or EV were cultured in medium with CD3/CD28 mAb for 20 h. (A) IL-2 levels were detected by ELISA. (B) Chromatin extracts were precipitated with anti-AcH3 Ab or control IgG, and probed for the promoter regions of IL-2 and genes levels were determined by qPCR. Results (mean 6 SD) in panels A and B are representative of 3 independent experiments, and *p,0.05, **p,0.01 compared to WT Foxp3. Heat maps indicating distinct expression profiles of genes encoding (C) HDAC and (D) HAT enzymes. Microarray experiments were performed using whole-mouse-genome oligoarrays (Mouse430a; Affymetrix), and array data were analyzed using MAYDAY 2.12 software [45]. Array data were subjected to robust multiarray average normalization. Normalized data were used for calculating fold changes of up- and downregulated genes using Student’s test, and data with .2x differential expression (p,0.05 with Storey’s FDR,0.1) were included in the analysis. Data underwent z-score transformation for display. doi:10.1371/journal.pone.0029035.g004 PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29035