Two Lysines in the Forkhead Domain of Foxp3 Are Key to T Regulatory Cell Function Yujie Liu1, Liqing Wang 1 , Rongxiang Han1, Ulf H. Beier2, Wayne W. Hancock1* 1 Laboratory of Medicine, Division of Transplant Immunology, Department of Pathology, School of Medicine, University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America, 2 Division of Nephrology, Department of Pediatrics, School of Medicine, University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America Abstract Background: The forkhead box transcription factor, Foxp3, is master regulator of the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. The carboxyl-terminal forkhead (FKH) domain is required for the nuclear localization and DNA binding of Foxp3. We assessed how individual FKH lysines contribute to the functions of Foxp3 in Treg cells. Methodology/Principal Findings: We found that mutation of FKH lysines at position 382 (K17) and at position 393 (K18) impaired Foxp3 DNA binding and inhibited Treg suppressive function in vivo and in vitro. These lysine mutations did not affect the level of expression of Foxp3 but inhibited IL-2 promoter remodeling and had important and differing effects on Treg-associated gene expression. Conclusions/Significance: These data point to complex effects of post-translational modifications at individual lysines within the Foxp3 FKH domain that affect Treg function. Modulation of these events using small molecule inhibitors may allow regulation of Foxp3+ Treg function clinically. Citation: Liu Y, Wang L, Han R, Beier UH, Hancock WW (2012) Two Lysines in the Forkhead Domain of Foxp3 Are Key to T Regulatory Cell Function. PLoS ONE 7(1): e29035. doi:10.1371/journal.pone.0029035 Editor: Pierre Bobe´, Institut Jacques Monod, France Received February 7, 2011; Accepted November 19, 2011; Published January 11, 2012 Copyright: 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to WWH (AI073489, CA158941) and UHB (K08AI095353) from the National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: whancock@mail.med.upenn.edu Introduction CD4+CD25+ T-regulatory (Treg) cells are important to the maintenance of immunological homeostasis and self-tolerance [1,2]. The forkhead box transcription factor, Foxp3, is now recognized as the master regulator of the development and function of CD4+CD25+ Treg cells [3,4,5]. Deletion or mutations of Foxp3 cause lethal autoimmunity in scurfy mice and profound morbidity in patients suffering from IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) [5,6]. In contrast, retroviral transduction of CD4+ T cells with Foxp3 induces a Treg phenotype and the capacity to suppress lymphocyte proliferation [3]. Murine and human Foxp3 protein share a high degree of homology, with 429 and 431 amino acids, respectively. Both proteins contain a repressor domain, a zinc finger domain, a leucine zipper domain, and a conserved DNA binding C-terminal forkhead domain (FKH) important for the nuclear translocation and DNA binding of Foxp3 [7,8]. Mutations in the FKH and leucine zipper domains are associated with clinical autoimmunity [9,10,11,12], indicating the importance of these regions. For example, mutations within the FKH domain that disrupt the interaction of Foxp3 and NFAT result in loss of the ability of Foxp3 to downregulate IL-2 expression and upregulate CTLA4 and CD25 expression [13]. Foxp3 also inhibits IL-2 expression by interacting with AML1/Runx1, which is normally an activator of IL-2 expression. Three amino acids located immediately N- terminal to the FKH domain are important for this inhibition, and their mutation (D329V, Y330H, and K332L) impaired Treg suppressive function [14]. Similarly, a single glutamic acid mutation (E251) or deletion of E250 in the leucine-zipper domain inhibited Foxp3 dimerization and abrogated its repressor functions [7,15]. Studies from mice and human demonstrated that the histone acetyltransferases (HAT), Tat-interactive protein (Tip60) and p300, increase lysine (K) e-acetylation of Foxp3, as does use of histone/protein deacetylase inhibitors (HDACi) [16,17,18]. Inter- estingly, Foxp3 acetylation promotes Treg function by increasing DNA binding to several promoters, including that of IL-2 [16], as well as by increasing the resistance of Foxp3 to polyubiquitination and proteasomal degradation [18,19]. Cytokine-dependent signals play central roles in promoting the DNA binding of Foxp3. Thus, TGFb increases binding of acetylated human Foxp3 to the IL-2 promoter, whereas TGFb and IL-6 together decrease this binding, and binding is restored by treatment with an HDACi [20]. While the contributions of individual lysines have not been determined, it is clear that acetylation of Foxp3 plays an important role in the regulation of Foxp3 production and function in Treg cells. In the current study, we found that mutations of single lysines in the FKH domain of Foxp3, Lys17Arg (K17R) or Lys18Arg (K18R), affected Foxp3 DNA binding, impaired Treg suppressive PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29035