Lysine Acetylation and Foxp3 Function Figure 7. Comparison of gene expression profiles of CD4+ T cells transduced with Foxp3 K17R versus K18R. Heat maps showing distinct gene expression profiles of CD4+ T cells transduced with WT Foxp3, K17R, K18R or EV. doi:10.1371/journal.pone.0029035.g007 ‘signature’ and suppressive genes, we found that CD4+ T cells derived from transduction of Foxp3 K18R were enriched for genes encoding cell division, cell cycle, and phosphoproteins (http:// david.abcc.ncifcrf.gov/). These findings are consistent with our observations that CD4+ T cells transduced with K18R had weaker DNA binding (Fig. 3), and greater CD4+ Thy1.1+ T cell proliferation (Fig. 5), as compared with CD4+ T cells transduced with Foxp3 K17R. These data suggest that K17 and K18 have distinctive differences in the regulation of Foxp3+ Treg gene expression. Discussion We previously demonstrated that dual substitutions of Foxp3 K17-18 with histidine residues led to impaired Treg suppressive function and inhibited Foxp3 DNA binding to the IL-2 promoter [16]. We have now extended our studies to the use of mutations with greater structural similarity and focused on the effects of individual lysines of interest on the Treg phenotype. Our studies suggest that 2 lysine residues (K17 and K18) in the FKH domain of Foxp3 are important for Treg function in vivo and in vitro. Substituting these lysines with arginine impairs Treg suppressive function, alters Foxp3-mediated gene expression, diminishes Foxp3 DNA binding, and alters the chromatin remodeling state of target genes (e.g. IL-2). Although the 2 mutants impaired Treg function, they did not affect Foxp3 mRNA or protein level, indicating detection of Foxp3 mRNA or protein level is insufficient to indicate full functional capacity of Tregs. In addition, we compared the different gene expression profiles of CD4+ T cells transduced with WT, K17R or K18R Foxp3, and found significant differences between CD4+ T cells transduced with WT Foxp3 versus with mutant, as well as between the two mutants themselves, suggesting that the different lysines may play distinct roles in Foxp3 function. Mutation of either K17R or K18R impaired the DNA binding of both unmodified and acetylated Foxp3, with K18R exhibiting a greater effect. There are at least 2 models of the interaction between acetylation and transcription factor DNA binding. First, acetylation can neutralize basic residues and promote transcription factor DNA binding, as was demonstrated in the current studies. In the presence of p300, the enhancement of DNA binding of K18R was lower than that of WT and K17R, suggesting that acetyl group on Foxp3 lysine residues may contribute to conformational changes during DNA recognition and binding. Second, DNA-binding dependent acety- lation can occur, such as was demonstrated for p53 [32], in which transcription factor binding to DNA causes a conformation change which then leads to HAT binding. In our case, p300 acetylation may be an important post-DNA-binding event to stabilize the p300- Foxp3-DNA complex into a very stable state. DNA-bound K17R showed markedly reduced acetyl-bound Foxp3, suggesting that this lysine mutation causes altered conformation, which could affect its affinity for co-factors, including p300. Foxp3 is able to directly bind the promoters of various genes and up- or down-regulate their expression [38,39]. In the current study, the altered expression of multiple Foxp3-dependent genes in CD4+ T cells transduced with Foxp3 K17R or K18R (Fig. 6) could be explained, at least in part, by different DNA binding caused by K17R or K18R. The known association of permissive and repressive chromatin structures at targets that are differentially regulated [33] was also demonstrated in our studies (Fig. 4). Foxp3 may alter target gene promoter chromatin remodeling by recruiting HAT/coactivator or HDAC/corepressor complexes. The identification of a Foxp3 complex containing chromatin-remodeling factors, and data showing a Foxp3-Tip60-HDAC7 complex inhibits IL-2 expres- sion, support this idea [17]. Recent studies showed that the FKH domain mediates Foxp3 association with HDAC1, and that Foxp3 enhances HIV or inhibits IL-2 gene expression in human T cells by inhibiting HDAC1 activity [40]. Studies from our group and from others have shown that various HDAC enzymes contribute to regulation of Foxp3+ Treg production and function [16,17,41,42], but little is known as to how Foxp3 regulates HDAC production and function. The current study shows that Foxp3 can regulate HDAC and HAT levels, and that specific lysines within the FKH play key and distinct roles in this process, pointing to a novel, but as yet unidentified, mechanism by which Foxp3 functions in Treg. While Foxp3 is known to be acetylated by p300 and Tip60 [16,17], no studies have yet mapped the precise lysine residue(s) within Foxp3 that must be acetylated for optimal Treg function, nor tested whether additional HAT enzymes also mediate Foxp3 acetylation. Our findings show that neither K17R nor K18R Foxp3 mutant had impaired p300-dependent acetylation, al- though both mutants led to impaired Treg suppression function. However, conclusions concerning whether K17 and/or K18 mediate Foxp3 acetylation cannot yet be made with confidence for at least 3 reasons. First, the lack of an anti-acetyl-Foxp3 Abs limits our ability to accurately assess Foxp3 acetylation at individual lysines. Second, intrinsic functional redundancy as a result of acetylation on different lysine residues may conceal the impair- ment caused by individual lysines. Third, we cannot exclude the possibility that K17R or K18R is a target of additional HAT enzymes. In addition to acetylation, lysines are the targets of ubiquitina- tion, sumoylation and methylation [43]. It is possible that disruption of one post-translational modification may affect additional post-translational modifications that thereby affect Foxp3 function. For example, acetylation of Foxp3 can prevent its ubiquitination and promote Foxp3 protein stability [18]. Since in our studies, replacement of K residues (K17, 18) with R, Q or E residues each impaired Treg suppressive function, a single molecular mechanism of suppression is unlikely and functional impairment may the result of multiple effects. As demonstrated in the case of Foxo1, acetylation and phosphorylation can cooper- atively regulate the function of a transcription factor. Acetylation of Foxo1 at Lys-242, Lys-245, and Lys-262 alters its affinity for binding to target DNA, and increases its sensitivity for phosphor- ylation [43,44]. Hence, studies are warranted to investigate how the various post-translational modifications, including phosphor- ylation, methylation or sumoylation, may form a dynamic and complex regulatory program to modulate Foxp3. Insights from such studies may ultimately provide new targets for therapeutic application of small molecules (HATi, HDACi or DNMTi) to precisely control and amplify or dampen Treg functions in vivo. Materials and Methods Ethics statement Studies were approved by the Institutional Animal Care and Use Committee of The Children’s Hospital of Philadelphia (approval numbers #2008-7-746 and #2010-6-561). PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29035