Lysine Acetylation and Foxp3 Function Figure 6. Comparison of gene expression profiles of CD4+ T cells transduced with WT Foxp3, K17R, K18R or EV. (A) Venn diagrams summarizing overlapping upregulated (left) or downregulated (right) gene expression profiles of CD4+ T cells transduced with WT Foxp3 (blue), K17R (purple) or K18R (yellow) compared with CD4+ T cells transduced with EV (cutoff was set as Log2 fold .1). (B–E) Heat maps showing gene expression profiles of CD4+ T cells transduced with EV, WT Foxp3, K17R or K18R. (B) Heat maps showing distinct Treg ‘signature’ gene expression profiles. (C) Heat maps indicating the change in gene expression profiles of Treg cell identified and putative suppressive genes. (D) Heat maps indicating the distinct gene expression profiles of Foxp3 directly bound genes. (E) Heat maps indicating distinct gene expression profiles of Treg-related cytokine genes. (F) qPCR assays of Treg-specific genes selected from microarray analyses; results are representative of 3 independent experiments, and *p,0.05, **p,0.01 compared to WT Foxp3. doi:10.1371/journal.pone.0029035.g006 Further examination of the dataset resulted in several findings. First, compared with CD4+ T cells transduced with WT Foxp3, CD4+ T cells transduced with the 2 mutants had altered expression of multiple Treg ‘signature’ genes. WT Foxp3 transduction led to marked down-regulation of Igfbp4, Pole2, Enc1, Tgfbr3 and Arhgap29, whereas expression of these genes was only modestly downregulated or increased in CD4+ T cells transduced with Foxp3 K17R and K18R (Fig. 6B). Conversely, Nrp1, Gpr83, Rgs16, Tnfrsf4, Tnfrsf9, Klrg1, Mdfic, CTLA-4, Tiam1, Tnfrf18, and Pdcd1Ig2 were up-regulated in CD4+ T cells transduced with WT Foxp3 but were decreased in CD4+ T cells transduced with Foxp3 K17R and K18R (Fig. 6B). Second, we compared expression across the groups of known or putative suppressor genes found in Treg cells, including Nrp1, Lag3, CTLA- 4, IL-10, Epstein-Barr virus-induced gene 3 (Ebi3), Tgfb1, ICOS, Crem, Fgl2, Mmp9, Gzmk, and Gpr83 (Fig. 6C). Except for Grp83, and in contrast to data from WT Foxp3, the expression of each of these genes was decreased in CD4+ T cells transduced with Foxp3 K17R or 18R. These data are consistent with the impaired suppressive function of CD4+ T cells transduced with either mutant as compared with that of CD4+ T cells transduced with WT Foxp3. Third, recent studies demonstrated that Foxp3 is able to directly bind to the promoters of a number of genes and up- or down-regulate their expression in Treg cells [34,38]. Foxp3- dependent genes downregulated in CD4+ T cells transduced with Foxp3 K17R or K18R compared with WT Foxp3 included cell surface molecules: Nrp-1, Abcb1a, CTLA-4 and ICOS; transcrip- tional factors: Prdm1, Irf6 and Crem; and a vesicular trafficking gene: Snx9 (Fig. 6D). Fourth, with respect to effects on cytokine gene expression, CD4+ T cells transduced with Foxp3 K17R or K18R exhibited higher levels of IL-4 and IL-13 and a lower level of IL-18 compared with CD4+ T cells transduced with WT Foxp3 (Fig. 6E). These data suggest that each lysine residue may directly mediate Foxp3 binding to the binding sites on a range of target promoters. To validate the microarray results, we used quantita- tive PCR analysis to determine selected genes, including Igfbp4, Nrp1, Lag3 and Mmp9 (Fig. 6F); the results were consistent with the microarray results. Lastly, we compared the gene expression profile of CD4+ T cells transduced with Foxp3 K17R versus K18R (Fig. 7). In addition to the differences in expression of Treg PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29035