Reversing SKI–SMAD4-mediated suppression is essential for TH17 cell differentiation

Abstract

T helper 17 (TH17) cells are critically involved in host defence, inflammation, and autoimmunity1,2,3,4,5. Transforming growth factor β (TGFβ) is instrumental in TH17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables TH17 cell differentiation remains elusive. Here we reveal that TGFβ enables TH17 cell differentiation by reversing SKI–SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into TH17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and TH17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and TH17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and TH17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI–SMAD4-mediated suppression of RORγt to enable TH17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls TH17 cell differentiation and uncovers the SKI–SMAD4 axis as a potential therapeutic target for treating TH17-related diseases.

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Figure 1: SMAD4 deletion leads to a TH17 cell differentiation in the absence of TGFβ signalling.
Figure 2: SMAD4 controls TH17 cell program by directly suppressing Rorc expression.
Figure 3: TGFβ signalling disrupts SKI–SMAD4 complex to enable TH17 cell differentiation.
Figure 4: SKI suppresses TH17 cell differentiation in a SMAD4-dependent manner.

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Acknowledgements

We thank E. Robertson and E. Bikoff for Smad4fl/fl mice, H. Moses for Tgfbr2fl/fl mice, D. Littman for Rorc−/− mice, F. Zhang for Cre-dependent Cas9 knock-in mice, N. Fisher for cell sorting, W. Chen and D. Zhang for discussion, and J. Massagué for the suggestion on SMAD4 ChIP–seq analysis. This study was supported by the National Natural Science Foundation of China (81402549, LJQ2015033) (G.Z.), the National Institutes of Health (NIH) (AI029564) and the National Multiple Sclerosis Society (CA10068) (J.P.Y.T.), the Intramural Research Program of the National Institute of Environmental Health Science (ES101965 to P.A.W. and ES102025 to D.N.C.), and by the NIH (AI097392; AI123193), the National Multiple Sclerosis Society (RG4654), and a Yang Family Biomedical Scholars Award (Y.Y.W.).

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Contributions

S.Z. contributed to the design and implementation of the cellular, molecular, biochemical, and animal experiments, and the writing of the manuscript. M.T., X.X., S.Y.T., P.A.W., and D.N.C. contributed to ChIP–seq and RNA-seq experiments and bio-informatic analysis. L.Z., Q.K., and X.C. contributed to proteomic and biochemical experiments and data analysis. A.D.G. contributed to the in vitro assays. W.C. and J.P.T. contributed to the EAE experiments. G.Z. contributed to ChIP analysis. B.W. contributed to qRT–PCR analysis. J.S.S. contributed critical reagents. Y.Y.W. conceived the project, designed experiments, and wrote the manuscript.

Corresponding author

Correspondence to Yisong Y. Wan.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks T. Egawa and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 TH17 cell differentiation in the absence of SMAD4.

a, Naive CD4+ T cells isolated from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were activated in the presence of TGFβR inhibitor (i), IL-6 + TGFβR inhibitor (IL-6 + inhibitor), or IL-6 + TGFβ (IL-6 + TGFβ). IL-17A+ cells were assessed by flow cytometry 1 and 2 days later. Representative results (left) and statistical analysis (right) of five experiments are shown. b, The percentages of IL-17A+CD4+ and IFN-γ+CD4+ cells in the peripheral lymph nodes (pLN) and spleens from wild-type, Cd4-cre;Smad4fl/fl (S4 KO), Cd4-cre;Tgfbr2fl/fl (RII KO), and Cd4-cre;Smad4fl/fl;Tgfbr2fl/fl (S4–RII DKO) mice under steady state were assessed by flow cytometry. Representative results (left) and statistics from eight mice (right) are shown. (***P < 0.001; two-sided t-test; NS, not significant; centres indicate mean values.) Source data

Extended Data Figure 2 SMAD4 suppresses RORγt expression.

a, CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. The mRNA expression of TH17-related genes was analysed at the indicated time points after activation by qRT–PCR. Means ± s.d. of three experiments are shown. b, Naive CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor for 3 and 12 h. Total RNA was then collected for RNA-seq analysis. All genes were analysed and presented as volcano plots based on the log2(fold change) of SMAD4 knockout versus wild type and −log10(P value). Differentially expressed genes (P < 0.05) are highlighted in red. c, Naive CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor. The mRNA expression of TH17-related genes was analysed at the indicated time points after activation by qRT–PCR. Means ± s.d. of three experiments are shown. d, Naive CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl;Tgfbr2fl/fl (S4–RII DKO) mice were sorted and activated in the presence of IL-6. The mRNA expression of TH17-related genes was analysed at the indicated time points after activation by qRT–PCR. Means ± s.d. of three experiments are shown. e, Naive CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor. The RORγt protein expression was assessed by immunoblotting 1 and 4 days after activation. Results are representative of three experiments with similar results. f, CD4+ T cells from wild-type and Cd4-cre;Tgfbr2fl/fl;Smad4fl/fl (S4–RII DKO) mice were activated in the presence of IL-6. The RORγt protein expression was assessed by immunoblotting 1 day after activation. Results are representative of three experiments with similar results. g, CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells were harvested after 12 h. ChIP assay was performed with control IgG antibody and SMAD4 antibody. The enrichment of SMAD4 binding to the Rorc promoter was determined. Means ± s.d. of three samples in one experiment of three are shown. h, CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells were harvested after 12 h. ChIP–seq assay was performed with SMAD4 antibody. The enrichment of SMAD4 binding to the Il17a and Il17f loci was determined by the mapped read coverage of SMAD4 ChIP–seq data. The results of two independent experiments were show as ‘#1’ and ‘#2’. (*P < 0.05, **P < 0.01, ***P < 0.001; two-sided t-test; NS, not significant). Source data

Extended Data Figure 3 SKI identification and its degradation upon low doses of TGFβ.

a, Schematic of quantitative immunoprecipitation and mass spectrometry proteomic strategy to identify SMAD4-binding proteins under different conditions. In one scheme, to identify SMAD4-binding protein in the absence of TGFβ signalling, CD4+ T cells from Cd4-cre;Smad4fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 + inhibitor in the SILAC/AACT medium provided either with amino acids containing light (L) isotopes or with amino acids containing heavy (H) isotopes. Cells were then transduced with retroviruses expressing either Flag tag (RV Flag) or Flag tag and SMAD4 fusion protein (RV Flag-S4). Cells were harvested and mixed 4 days after activation. Immunoprecipitation was performed using anti-Flag. Immunoprecipitated proteins were processed and subjected to quantitative mass spectromtery analysis. Proteins with a heavy/light ratio of more than 2 were identified. In the other scheme, to identify the proteins whose SMAD4 interaction was perturbed upon TGFβ stimulation, CD4+ T cells from wild-type mice were sorted and activated either in the presence of IL-6 + inhibitor in the SILAC/AACT medium provided with amino acids containing light isotopes or in the presence of IL-6 + TGFβ in the SILAC/AACT medium provided with amino acids containing heavy isotopes. Cells were harvested and mixed 4 days after activation. Immunoprecipitation was performed using anti-SMAD4 antibody. Immunoprecipitated proteins were processed and subjected to quantitative mass spectrometry analysis. Proteins with a heavy/light ratio of less than 1 were identified. The commonly identified protein SKI in the two experiments was subjected to further investigation. b, CD4+ T cells from wild-type mice were activated in the presence of IL-6 and the indicated doses of TGFβ. Cells were harvested after 24 h. SKI protein expression was detected by immunoblotting. Results are representative of three experiments with similar results. c, CD4+ T cells from wild-type mice were activated in the presence of IL-6 and the indicated doses of TGFβ. IL-17A+ and Foxp3+ cells were assessed by flow cytometry on day 4. Representative results (top) and statistical analysis (bottom) of five experiments are shown (centres indicate mean values). d, CD4+ T cells from wild-type or Cd4-cre;Smad2fl/fl (S2 KO) mice were activated in the presence of IL-6 for 24 h. Cells were then stimulated with the indicated conditions for an additional 1 h with or without 10 μM SIS3 (specific inhibitor of Smad3 phosphorylation). SKI protein expression and Smad3 phosphorylation were assessed by immunoblotting. Results are representative of three experiments with similar results. Source data

Extended Data Figure 4 SKI and SMAD4 cooperatively suppress TH17 cell differentiation.

a, Bone marrow cells were isolated from the femur bones of sex- and age-matched Cd4-cre;CdC (Cas9, CD45.2+) mice and wild-type (wild type, CD45.1+) mice. Cells were mixed, and transduced with two different gRNA-expressing viruses (as indicated) and transferred into sublethally irradiated (400 cGy) Rag1−/− recipient mice. CD4+ T cells isolated from lymph nodes and spleen of generated bone marrow chimaeric mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells transduced with gRNA in wild-type donors are indicated as wild type. Cells transduced with gRNA in CD4-cre;CdC donor are indicated as Ski knockout. IL-17A+ cells were assessed by flow cytometry on day 4. Representative results (left) and statistical analysis (right) of five experiments are shown. b, CD4+ T cells from wild-type mice were activated in the presence of IL-6 and TGFβ, and then transduced with MSCV-IRES–GFP (RV), MSCV-SKI-IRES–GFP (RV SKI), or co-transduced with MSCV-SKI-IRES–GFP and MSCV-RORγt-IRES-Thy1.1 (RV SKI+RORγt) retroviruses. IL-17A expression of transduced (GFP+) or co-transduced (GFP+Thy1.1+) T cells was assessed by flow cytometry. Representative results (left) and statistical analysis (right) of five experiments are shown. c, CD4+ T cells from wild-type and Cd4-cre;Smad4fl/fl (S4 KO) mice were activated in the presence of IL-6 + TGFβR inhibitor (i) or IL-6 + TGFβ. Cells were harvested 3 days later. ChIP assay was performed with control IgG antibody or SKI antibody. The relative enrichment of SKI binding to the Rorc locus was determined. Means ± s.d. of three samples in one experiment of three are shown. (**P < 0.01, ***P < 0.001; two-sided t-test; NS, not significant; centres indicate mean values.) Source data

Extended Data Figure 5 TGFβ superfamily signalling overcomes SKI–SMAD4 complex-mediated suppression of RORγt expression in activated T cells to enable TH17 cell differentiation.

a, RORγt expression is potentiated by IL-6–STAT3 signalling but restrained by the histone deacetylase activity-containing SKI–SMAD4 complex that associates with and deacetylates the Rorc locus. b, Additional TGFβ or activin signalling triggers SKI degradation. The disruption of SKI–SMAD4 complex dissociates histone deacetylase activity from the Rorc locus and enables RORγt expression and TH17 cell differentiation.

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This file contains Supplementary Figure 1, the uncropped gels with size marker indications and Supplementary Figure 2, gating strategies for flow cytometry analysis.

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Zhang, S., Takaku, M., Zou, L. et al. Reversing SKI–SMAD4-mediated suppression is essential for TH17 cell differentiation. Nature 551, 105–109 (2017). https://doi.org/10.1038/nature24283

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