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Different molecular complexes that mediate transcriptional induction and repression by FoxP3

Nature Immunology volume 18, pages 1238–1248 (2017)Cite this article

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Abstract

FoxP3 conditions the transcriptional signature and functional facets of regulatory T cells (Treg cells). Its mechanism of action, whether as an activator or a repressor, has remained unclear. Here, chromatin analysis showed that FoxP3 bound active enhancer elements, not repressed chromatin, around loci over- or under-expressed in Treg cells. We evaluated the impact of a panel of FoxP3 mutants on its transcriptional activity and interactions with DNA, transcriptional cofactors and chromatin. Computational integration, confirmed by biochemical interaction and size analyses, showed that FoxP3 existed in distinct multimolecular complexes. It was active and primarily an activator when complexed with the transcriptional factors RELA, IKZF2 and KAT5. In contrast, FoxP3 was inactive when complexed with the histone methyltransferase EZH2 and transcription factors YY1 and IKZF3. The latter complex partitioned to a peripheral region of the nucleus, as shown by super-resolution microscopy. Thus, FoxP3 acts in multimodal fashion to directly activate or repress transcription, in a context- and partner-dependent manner, to govern Treg cell phenotypes.

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Figure 1: FoxP3 binds to active enhancers around both Treg-up loci and Treg-down loci.
Figure 2: Transcriptional activity of the FoxP3 mutants.
Figure 3: Effects mutations in FoxP3 on its binding to chromatin.
Figure 4: Cofactor-binding activity of the FoxP3 mutants.
Figure 5: Connecting cofactor interaction and transcriptional activity.
Figure 6: FoxP3′s active and inactive cofactors form different complexes.
Figure 7: Different FoxP3 molecular complexes distribute to different regions of the nucleus.

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Acknowledgements

We thank A. Arvey, L. Chen, T. Chatila, K. Struhl, J. Waters and A. Rudensky for discussions: K. Hattori, C. Araneo and A. Rhoads for help with mice, cell sorting, profiling and software; and the HMS Cell Biology Microscopy Facility and L. Shao for super-resolution microscopy. Supported by the US National Institutes of Health (AI116834), GlaxoSmithKline (Sponsored Research Agreement+ and NRF (357-2011-1C00084 to H.-K.K.).

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Authors and Affiliations

  1. Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, and Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA

    Ho-Keun Kwon, Hui-Min Chen, Diane Mathis & Christophe Benoist

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  1. Ho-Keun Kwon
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Contributions

H.-K.K. and H.-M.C. performed the experiments; all authors designed the study and analyzed and interpreted the data, and H.-K.K., C.B. and D.M. wrote the manuscript.

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Correspondence to Diane Mathis or Christophe Benoist.

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Integrated supplementary information

Supplementary Figure 1 Determinants of FoxP3’s transcriptional functions.

(a) Representative flow cytometry plots of FoxP3 expression after transduction of EV, WT and mutant FoxP3 vectors into activated CD4+ T cells. Surface staining detected the vector-encoded Thy1.1 reporter (which serves as a standard and to guide cell sorting in later experiments) and intracellular staining detected FoxP3 levels. Data are representative of biological triplicates (quantitative results tabulated in Table S4). (b) After retroviral transduction as in a, the size and expression of the FoxP3 proteins were detected by immunoblotting of nuclear extracts with anti-FLAG antibody. Data representative of biological triplicates. (c) WT or mutant FoxP3 were transduced into HEK293 cells, and after 72 hr of transfection, cells were stained with anti-FLAG (red) and counterstained for nuclear DNA with DAPI (blue). Data representative of biological triplicates. (d, e): Evaluation of DNA-binding capacity of WT or mutant FoxP3. Specificity controls: nuclear extracts prepared from CD4+ T cells 72 hrs after transduced with EV or WT FoxP3 vectors as in a were incubated with FKRE2 or control (scrambled sequence) oligos, and binding detected by ELISA with anti-FLAG (results shown as mean of biological duplicates, ± SD) (e) Binding activity of WT and mutant FoxP3 in the same assay (values standardized to binding by WT FoxP3 in each experiment; mean +/- SD, Data representative for 2-3 biological experiments). (f) Correlation between the suppressive activity of CD4+ T cells transduced with the mutant FoxP3 panel and the induction (or repression) of individual FoxP3 target genes by the same mutant panel (as Fig. 2f, except for individual transcripts). t.test, P* < 0.05, P** < 0.005 and P*** < 0.001

Supplementary Figure 2 Determinants of FoxP3’s transcriptional functions.

(a) Specificity controls for the co-IP experiments (Fig. 4). To validate the interaction between WT FoxP3 and 17 selected co-factors, FLAG-tagged FoxP3 and each co-factor (tagged with HA, 6xHIS or GFP) were co-transduced into HEK293 cells and immuno-precipitated with anti-FLAG or isotype-matched IgG. Specific co-IP of each co-factor was detected by specific anti-tag antibody. Data representative of biological triplicates. (b, c): Co-immunoprecipitation of FoxP3 and cofactors does not depend on DNA as an intermediate. Co-IPs were performed as in Fig. 4 in the presence of 10μg/ml Ethidium bromide (b) or 1 mg/ml DNAse I (c). Representative immunoblots from biological duplicates. (d) The co-IP experiments primarily detect pre-formed multimolecular complexes, not in vitro-association. To test for artefactual complex formation, lysates of cells singly or doubly transduced with WT FoxP3 and/or STAT3 or RELA were incubated alone or after mixing for 30 minutes under co-IP conditions, then co-IPed with anti-FLAG (FoxP3) and STAT3 or RELA detected by immunoblotting (as for Fig. 4 and Supplementary Fig. 2B). Interaction is only detected robustly with lysates from doubly-transduced cells, although a low amount of interaction can be detected by co-incubating lysates from singly-transduced cells. Results representative of biological duplicates.

Supplementary Figure 3 FoxP3 forms two distinct complexes.

3D-SIM detection of FoxP3, RELA and IKZF3 in ex vivo Treg cells. Image representative of >20 cells in biological triplicates as for Fig. 7. Scale bars (left and middle: 1 μm, right: 0.1 μm).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 515 kb)

Life Sciences Reporting Summary (PDF 211 kb)

Supplementary Table 1

Probe sequences for each transcript in Treg specific code-set (XLSX 65 kb)

Supplementary Table 2

Mutant information (XLSX 11 kb)

Supplementary Table 3

FoxP3 expression (MFI) for WT and mutant constructs (XLSX 9 kb)

Supplementary Table 4

Nanostring data (XLSX 57 kb)

Supplementary Table 5

References for co-factor and FoxP3 interaction (XLSX 12 kb)

Supplementary Table 6

Co-factor interaction (XLSX 23 kb)

Supplementary Video 1

3D-SIM for FoxP3, RelA and EZH2 in FoxP3 transduced CD4+ T cells (MP4 15365 kb)

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Kwon, HK., Chen, HM., Mathis, D. et al. Different molecular complexes that mediate transcriptional induction and repression by FoxP3. Nat Immunol 18, 1238–1248 (2017). https://doi.org/10.1038/ni.3835

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