Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation

Emily A. Rowell,Andrew D. Wells,Chunxia Chen,Rajan M. Thomas,Wayne W. Hancock

doi:10.1074/jbc.m608848200

Emily A. Rowell, Andrew D. Wells + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m608848200

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Abstract

Regulatory T cells (T(reg)) express Foxp3, a forkhead family member that is necessary and sufficient for T(reg) lineage choice and function. Ectopic expression of Foxp3 in non-T(reg) leads to repression of the interleukin 2 (IL-2) and interferon gamma (IFNgamma) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNgamma loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNgamma genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.

Highlights

Recent studies have shown that Foxp3 overexpressed in transformed cell lines can repress transcription from artificial forkhead, nuclear factor of activated T cell (NFAT),3 or NF␬B consensus binding elements in transiently transfected plasmids [7,8,9]
Treg constitutively express GITR, CTLA-4, and TGF␤ (Ref. 11 and Fig. 2, A–C, left panels), and consistent with previous studies [1, 7, 8, 16, 17] we found that ectopic expression of Foxp3 in CD4ϩ T cells recapitulates this genetic program (Fig. 2, A–C, right panels)
This activation-induced Foxp3 binding could be simulated by the combination of diacylglycerol analog and Ca2ϩ ionophore (Fig. 3K), implicating protein kinase C, RasGRP, and/or Ca2ϩ signaling in promoting Foxp3 chromatin binding. Consistent with this interpretation, inhibition of Ca2ϩ-dependent calcineurin phosphatase activity during T cell activation using cyclosporine A (CsA) abrogated Foxp3 binding to the interleukin 2 (IL-2) promoter (Fig. 3L) and the IFN␥ enhancer (Fig. 3M). These data demonstrate that T cell receptor (TCR)-coupled Ca2ϩ signaling through calcineurin is required for Foxp3 binding to chromatin at the IL-2 and IFN␥ genes and provides a molecular framework for why Treg suppressor function requires activation and is CsA sensitive [11]

Summary

Introduction

Recent studies have shown that Foxp3 overexpressed in transformed cell lines can repress transcription from artificial forkhead, nuclear factor of activated T cell (NFAT),3 or NF␬B consensus binding elements in transiently transfected plasmids [7,8,9]. Like natural CD25ϩ Treg (Fig. 2E), Foxp3-transduced T cells were defective in activation-induced IL-2 gene expression at the level of both mRNA and protein (Fig. 2, F and G).

Results

Conclusion