Foxp3 Acetylation Research Articles

Lysosome-dependent p300/FOXP3 degradation and limits Treg cell functions and enhances targeted therapy against cancers

p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (Treg) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185her2/neu (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit Treg functions in targeted therapies for cancers.

Histone deacetylase inhibition by ITF2357 reduces SLE pathogenesis in vivo (P5161)

Abstract Histone deacetylase (HDAC) inhibitors are important modifiers of gene and protein expression. In our studies, we sought to determine if a specific HDAC I and II inhibitor (ITF2357) was able to decrease disease in lupus-prone New Zealand Black/White (NZB/W) F1 female mice through regulation of T cell profiles. From 22 - 38 weeks-of-age, NZB/W and non-lupus New Zealand White (NZW) mice were injected intraperitoneally with 10mg/kg of ITF2357 (high dose), 5mg/kg of ITF2357 (low-dose), or the vehicle control. Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA and cytokine levels were measured every 4 weeks. Kidney disease was determined by sera BUN and creatinine, immune complex deposition, and renal pathology. T lymphocyte profiles were assessed using flow cytometric analyses and T regulatory cell function was determined by CFSE assay. Our results showed NZB/W mice treated with the high-dose of ITF2357 had increased histone acetylation resulting in a decrease in overall renal disease and inflammatory cytokines in the sera. Treatment with ITF2357 at 10 mg/kg decreased the Th17 phenotype while increasing the percentage and function of Treg cells as well as Foxp3 acetylation. These results suggest that specific HDAC inhibition may decrease disease by altering T cell differentiation and function.

Loss of Epigenetic Modification Driven by the Foxp3 Transcription Factor Leads to Regulatory T Cell Insufficiency

Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and invitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-β-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments.

Inhibition of p300 impairs Foxp3+ T-regulatory (Treg) function and promotes anti-tumor immunity (127.40)

Abstract Foxp3+ Treg cells limit autoimmunity and maintain immune homeostasis but also restrict immune responses to cancer. Thus, Tregs are a promising target for modulation of host anti-tumor immune responses. We have shown that targeting of Foxp3+ Tregs using histone/protein deacetylase inhibitors (HDACi) promotes Foxp3 acetylation and increases Treg production and suppression function. By contrast, there are no studies concerning the role of histone/protein acetyltransferases (HAT) targeting in Tregs. HATs are classified into 3 main families; GNAT, MYST and p300/CBP. Here, we found that conditional deletion of p300 in Tregs caused lymphoproliferation, weight loss, arthritis and dermatitis in mice. Likewise, deletion of p300 in Tregs, or its pharmacologic inhibition, decreased Foxp3+ Treg production and impaired Treg-mediated suppression of homeostatic proliferation, and blocked Treg-dependent donor-specific allograft tolerance. In contrast with these data, p300 targeting decreased TC1 lung tumor volume in immunocompetent C57BL/6 mice, but had no direct effect on TC1 tumor cell proliferation in vitro, or on the growth of TC1 implanted tumors in RAG1-/- immunodeficient mice. We conclude that in real world tumor models using immunocompetent mice, p300 targeting impaired Treg function without overt impairing effector T cell responses or inducing autoimmunity. We thereby identify p300 as a critical target for modulation of Foxp3+ Treg function in the context of anticancer therapy.

Abstract 4842: p300 targeting impairs treg function and promotes host anti-tumor immunity

Abstract The long-held view that tumors were not sufficiently immunogenic to promote host immune responses has now been revised to the assessment that at least for some tumors, Foxp3+ T-regulatory (Treg) cells actively suppress anti-tumor immunity. Seeking to build on our ongoing assessment of the regulation of Foxp3+ Treg biology by HDAC and HAT enzymes, we have begun to use combined genetic and pharmacologic approaches to identify and modulate key mechanism within Tregs that limit host immunity. Microarray studies of gene expression in WT, p300-/- and CBP-/- Tregs showed that p300 or CBP deletion led to down-regulation of Foxp3 expression, as well as that of a number of signature Treg genes. Cells co-transfected with Foxp3 and increasing amounts of p300 expression vectors showed that Foxp3 acetylation was enhanced by increasing levels of p300 levels, whereas a p300 small molecule inhibitor, C646 (p300i), impaired Foxp3 acetylation and inhibited Treg function using in vitro suppression assays. In vivo use of C646 showed negligible effects on CD4, CD8 or Treg cell numbers, but qPCR studies showed C646 decreased Treg expression of CTLA-4, GITR, IL-10 and TGF-b, in vitro assays again showed impaired Treg suppression. Additional studies showed, critically, that use of the p300i impaired Treg but not T effector cells in allograft recipients. Thus, parent-to-F1 assays involving alloactivation and proliferation of CD4 an dCD8 T cells were unaffected by C646, and this p300i restored allograft rejection in Treg-dependent models of allograft survival. Turning to tumor models, TC1 lung cancer cells were injected in the flanks of WT B6 mice, B6 mice in which p300 was deleted in CD4+ T cells (Treg plus Teff using CD4-Cre) or just in Tregs (Foxp3-Cre) (n=10/grp), p300 was inhibited using C646 (Alzet pumps, 14 d, 0.9 mg/kg/d) from day 6 post-tumor injection; in both genetic and pharmacologic approaches, p300 targeting suppressed tumor cell growth. qPCR analysis of tumors in C646-treated mice showed increased CD4 and granzyme B mRNA but decreased Foxp3; these data were confirmed by immunohistology, along with increased IFN-g production by purified CD8 cells (ELISPOT), along with reductions in tumor volumes and weights in these C646-treated mice. C646 use did not impair tumor growth in immunodeficient (RAG-/-) mice, in which tumors were exposed to drug in RAG-/- mice as seen by decreased acetylation of histone 3 in tumor extracts (Western blot); no effects on acetylation of certain p300-independent targets within tumor extracts was seen (e.g. acetylation of alpha-tubulin was unimpaired). The p300i also had no direct effect on tumor cell proliferation in vitro. We conclude that our comprehensive genetic and pharmacologic studies show that p300 is an important target for modulation of host Foxp3+ Treg functions, such that Treg suppression can be incrementally reduced and host anti-tumor responses promoted without inducing concomitant autoimmunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4842. doi:1538-7445.AM2012-4842

Rapid Temporal Control of Foxp3 Protein Degradation by Sirtuin-1

Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2–7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2–7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.

FoxP3: A Key Player in T Regulatory Biology

The immune system has adapted mechanisms to recognize and eliminate threats, as well as to defend against self-destruction. Tolerance to self-antigens and defence against non self pathogens are maintained by the different immune cells. If there is an imbalance in T reg cells population, may leads to auto immunity. The generation of natural Treg (nTreg)/induced Treg (iTreg) and its maintenance of suppressive function are crucial for immune homeostasis. There are various surface markers on T cells like CD25, CTLA-4, OX-40 etc. and intracellular marker like FoxP3 involved in the generation and maintenance of suppressive function of Treg cells. Recent work has shown that forkhead DNA-binding proteins FoxP3, is X-chromosome-encoded transcription factor uniquely involved in the development and function of the T-regulatory cells. Lack of FoxP3 leads to development of fatal autoimmune lymphoproliferative disease in humans known as IPEX; (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) furthermore, ectopic FoxP3 expression induces conversion of effector T cells to regulatory T cells. The regulation of FoxP3 expression, and its role in maintenance of CD4+CD25+/CD25− Treg fitness still a matter of debate. Being a transcription factor FoxP3 activates/represses various effectors of the T cell activation/inhibition signal pathways. FoxP3 is an acetylated, oligomeric component of large molecular complex, actively represses transcription by recruiting enzymatic corepressors, including histone modifiers during its function. Chromatin remodeling through FoxP3 associated complexes may dynamically affect the multiple immunological signaling networks, eventually in summation lead to immune suppression. FoxP3 interacting proteins (CTLA-4, IL-2, IL-2RA, NFATC2 etc.) complex plays a central role in inducing gene transcription during the immune response. Transcription factors function as a carrier to take histone modifiers at particular locus at which they impose their effect. FoxP3 interacting with histone acetyl transferases, helps to maintain the acetylated state of FoxP3. Dynamic regulation of HAT and HDAC in FoxP3 acetylation confers the suppressive index of nTreg and iTreg cells.

Histone Deacetylase 9 (HDAC9) Regulates the Functions of the ATDC (TRIM29) Protein

Histone deacetylase 9 (HDAC9), like most Class II HDACs, catalyzes the removal of acetyl moieties from the ε-amino groups of conserved lysine residues in the N-terminal tail of histones. Biologically, HDAC9 regulates a wide variety of normal and abnormal physiological functions, including cardiac growth, T-regulatory cell function, neuronal disorders, muscle differentiation, development, and cancer. In a biochemical approach to identify non-histone substrates of HDAC9, we found that HDAC9 co-purifies specifically with the ataxia telangiectasia group D-complementing (ATDC; also called TRIM29) protein. HDAC9 deacetylates ATDC, alters the ability of ATDC to associate with p53, and consequently inhibits the cell proliferation-promoting activity of ATDC. These results implicate the importance of non-histone deacetylation by HDAC9 and confirm and further extend the multifunctions of this Class II deacetylase.

Epigenetic Regulation of Regulatory T-Cells: Impact on Autoimmunity and Graft Rejection

AbstractAbstract SCI-23Mutations of Foxp3, a transcription factor characteristic of T regulatory (Treg) cells, often cause lethal autoimmunity, leading to much research into how Foxp3+ Tregs control inflammatory and immune responses. This presentation will emphasize therapeutic aspects of our ongoing studies showing that the functions of Foxp3 are regulated by histone/protein deacetylases (HDAC), histone acetyltransferases (HAT), and DNA methyltransferases (DNMT), and for the sake of time will focus on the effects of HDAC inhibitors (HDACi). We have found that Foxp3 acetylation promotes DNA binding and can induce or suppress expression of multiple Foxp3-dependent genes in Tregs. Acetylation is catalyzed by specific HATs whose neutralization can diminish Treg function, with relevance to cancer and HIV, whereas use of selected HDAC inhibitors (HDACi) can increase acetylation and Treg suppression, with relevance to control of autoimmunity and transplant rejection. Many HDAC inhibitors (HDACi) were evaluated for their effects on Treg function. As class I-selective HDACi agents (e.g. benzamides) had little or no effect on Treg function, but pan-HDACi (e.g. hydroxymates) enhanced Treg function, we focused on class II HDACs. There are 2 class II subfamilies; class IIa members are thought to largely function in a tissue-specific manner through recruitment of other proteins since they display only weak catalytic activity, whereas class IIb family members display bona fide catalytic activity. We therefore analyzed class IIb members, of which HDAC6 is the best established and for which selective HDACi are available. HDAC6 exists in the cytoplasm and regulates acetylation of alpha-tubulin and other proteins, including HSP90. Blocking HDAC6 through the use of an HDAC6-specific inhibitor promotes HSP90 acetylation and release of HSP90 client proteins. Use of HDAC6 or HSP90 inhibitors increased Foxp3 expression and enhanced Treg function in vitro and in vivo, and could prevent, or treat pre-existing, autoimmunity in a Treg-dependent manner. We have also investigated the various class IIa family members, of which HDAC9 is of particular interest since its expression is increased 30-fold in Treg vs. regular T cells. HDAC9 decreases Foxp3 expression and function, and its neutralization promotes Treg survival by regulating expression of HSP70 and related HSPs. Our ongoing studies show that HDAC6 neutralization leads to acetylation of HSP90, release of HSF-1 and induction of HSP70, and also suggest that HDAC9 may regulate the acetylation and stabilization of HSF-1. Once produced, HSP70 can chaperone and promote Foxp3 nuclear translocation and function, such that the HDAC6 and HDAC9 pathways are closely intertwined with regard to control of Treg biology. In summary, acetylation, methylation and other epigenetic mechanisms in Tregs are being probed using genetic and pharmacologic approaches. Various currently approved drugs influence Foxp3-dependent Treg functions by affecting epigenetic mechanisms, and while additional HDAC-specific regulators are needed, a rationale is now in place for use of HDAC inhibitors as powerful tools to promote the development and functions of Foxp3+ Tregs in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization

AbstractRegulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

Using histone deacetylase inhibitors to enhance Foxp3+ regulatory T‐cell function and induce allograft tolerance

The histone/protein deacetylase inhibitor (HDACi), trichostatin A (TsA), increases the production and suppressive function of Foxp3(+) regulatory T cells (T(regs)), at least in part, by promoting the acetylation of Foxp3 protein itself. Acetylation of Foxp3 is required for effective binding of Foxp3 to the promoter of the interleukin-2 (IL-2) gene and the suppression of IL-2 expression. We have sought to identify agents that had similar effects on T(regs), but without the associated toxicity of TsA. This review summarizes the contrasting effects of various HDACis on T(reg) functions in vitro and in vivo. Agents that block primarily class I HDAC had minimal or no effect on T(reg) suppression, whereas multiple inhibitors of both class I and class II HDAC enhanced T(reg) suppression in vitro and in vivo. These data indicate tools for further analysis of T(reg) functions, and point to a critical role of class II HDAC in the regulation of T(regs). Such knowledge has direct implications for the development of in vivo approaches to treat autoimmune and other inflammatory diseases.

Deacetylase inhibition promotes the generation and function of regulatory T cells

Histone/protein deacetylases (HDACs) regulate chromatin remodeling and gene expression as well as the functions of more than 50 transcription factors and nonhistone proteins. We found that administration of an HDAC inhibitor (HDACi) in vivo increased Foxp3 gene expression, as well as the production and suppressive function of regulatory T cells (T(reg) cells). Although T(reg) cells express multiple HDACs, HDAC9 proved particularly important in regulating Foxp3-dependent suppression. Optimal T(reg) function required acetylation of several lysines in the forkhead domain of Foxp3, and Foxp3 acetylation enhanced binding of Foxp3 to the Il2 promoter and suppressed endogenous IL-2 production. HDACi therapy in vivo enhanced T(reg)-mediated suppression of homeostatic proliferation, decreased inflammatory bowel disease through T(reg)-dependent effects, and, in conjunction with a short course of low-dose rapamycin, induced permanent, T(reg)-dependent cardiac and islet allograft survival and donor-specific allograft tolerance. Our data show that use of HDACi allows the beneficial pharmacologic enhancement of both the numbers and suppressive function of Foxp3(+) T(reg) cells.

FOXP3 is a homo-oligomer, and it interactions with histone acetyltransferase and class II histone deacetylases are required for repression (88.30)

Abstract We have found that FOXP3 is an oligomeric component of a large supramolecular complex. The Zinc-Leuzip region of FOXP3 is sufficient to mediate its homotetramerization. Wild type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient’s T cells disrupts its homo-oligomerization, heteromerization with FOXP1, its association with the IL-2 promoter, and limits repression of IL-2 transcription after T cell activation. Additionally we report that transcriptional repression by FOXP3 involves a histone acetylase-deacetylase complex that includes TIP60 and HDAC7 and HDAC9. FOXP3 can be acetylated, and TIP60 promotes FOXP3 acetylation in vivo. Knockdown of endogenous TIP60 relieved FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation, and can be restored by the Trichostatin A indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a novel complex based mechanism by which FOXP3 actively mediates transcriptional repression.

FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression.

The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase-deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106-190 aa of FOXP3 are required for TIP60-FOXP3, HDAC7-FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression.