Epigenetic activation during T helper 17 cell differentiation is mediated by Tripartite motif containing 28 - PubMed (original) (raw)
Epigenetic activation during T helper 17 cell differentiation is mediated by Tripartite motif containing 28
Yu Jiang et al. Nat Commun. 2018.
Abstract
Epigenetic regulation is important for T-cell fate decision. Although STAT3 is known to initiate Th17 differentiation program, the downstream mechanism is unclear. Here we show that Tripartite motif containing 28 (Trim28) expression in Th17 cells is required for Th17-mediated cytokine production and experimental autoimmune diseases. Genome-wide occupancy analysis reveals that TRIM28-bound regions overlap with almost all Th17-specific super-enhancers (SE), and that those SEs are impaired by the deficiency of STAT3 or TRIM28, but not of RORγt. Importantly, IL-6-STAT3 signaling facilitates TRIM28 binding to the Il17-Il17f locus, and this process is required for epigenetic activation and high-order chromosomal interaction. TRIM28 also forms a complex with STAT3 and RORγt, and promotes the recruitment of RORγt to its target cytokine genes. Our study thus suggests TRIM28 to be important for the epigenetic activation during Th17 cell differentiation, and prompts the potential use of epigenetic interventions for Th17-related autoimmune diseases.
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1
TRIM28 regulated Th17 cell differentiation in vivo. a Naive T cells (CD4+CD25−CD62LhiCD44lo) isolated from the spleen and peripheral lymph nodes in wild type (WT) and Trim28 −/− mice at the age of 6–7 weeks. b Body weight changes in Rag1_−/−_ mice receiving WT or Trim28_−/−_ naive CD4+CD25−CD62Lhi CD45RBhi T cells in the transfer colitis model (WT: n = 6, Trim28 −/−: n = 6, two mice in WT group died before sacrificed), and two-way ANOVA was used for the statistical test. c H&E staining of the large intestines of Rag1 −/− mice receiving WT or Trim28 −/− naive T cells (scale bars: 100 μm). d Intracellular staining results and statistic data of the IL-17 and IFN-γ expression in the lamina propria (LPL) of large intestine, mesenteric lymph nodes (mLN), and spleen (WT: n = 4, Trim28_−/−_: n = 6) and Student’s t test was used for the statistical test (ns = not significant; *p < 0.05, **p < 0.01, ***p < 0.005). All error bars represent SDs. These experiments were repeated for three times with the consistent results
Fig. 2
TRIM28 KO CD4+ T cells were defective in Th17 differentiation. WT or TRIM28-deficient naive CD4+ T cells were polarized into Th17 cells in the presence of TGF-β and IL-6 for 3 days, and then re-stimulated for intracellular staining and mRNA analysis. a Intracellular staining results and their statistics (Student’s t test; ns not significant; *p < 0.05, **p < 0.01, ***p < 0.005). b The relative mRNA expression levels as determined by real-time PCR assay. c, d Heatmap (left and lower right) and scatter diagram (upper right) of RNA-seq results obtained from WT and TRIM28 KO Th17 cells (anti-CD3 re-stimulated); e Heatmap of pathway analysis from RNA-seq data obtained from WT and TRIM28 KO Th17 cells. All error bars represent SDs. a, b Representative data of three independent experiments
Fig. 3
Th17-specific deletion of TRIM28 resulted in defective Th17 differentiation and resistance to EAE. a Naive T cells isolated from WT and Trim28 _fl/fl_Il17fcre mice were differentiated into Th17 cells under TGF-β plus IL-6 condition for 3 days, then re-stimulated for cytokine staining, and Student’s t test was used for the statistical test. b The disease scores of WT and Trim28 _fl/fl_Il17fcre mice in EAE, and two-way ANOVA was used for the statistical test (WT: n = 7, Trim28 _fl/fl_Il17fcre: n = 7, two mice in WT group died before sacrificed); c Intracellular staining of IL-17, GM-CSF and IFN-γ of infiltrated CD4+ T cells in the central nerve system (CNS) of EAE mice. d Cellularities and percentages of CNS-infiltrating cells, and Student’s t test was used for the statistical test. ns = not significant; *p < 0.05, **p < 0.01, ***p < 0.005. All error bars represent SDs. The data are a representative for three independent experiments
Fig. 4
TRIM28 bound to the Il17-Il17f gene locus in Th17 cells. a−d Naive CD4+ T cells were cultured under Th17 condition (TGF-β plus IL-6) for 1 day and then prepared for ChIP-seq or ChIP-qPCR assay using anti-TRIM28 antibody. a Distribution of TRIM28-binding peaks in Th17 cells. b Alignment of TRIM28 binding peaks with histone modification and DNA methylation/demethylation markers in Th17 cells. c IGV browser view of TRIM28 binding peaks with H3K4me3, H3K27me3, 5hmc and 5mc markers at the Il17-Il17f gene locus. d ChIP-qPCR analysis of TRIM28 binding at representative gene loci; the data shown are a representative result for more than three independent experiments. e WT or Trim28 −/− naive CD4+ T cells were cultured at Th17 condition (TGF-β plus IL-6) for 3 days, and then prepared for ChIP-qPCR assay performed by anti-H3K4Me3 antibody or MeDIP assay performed by 5hmc antibody. All error bars represent SDs. The experiments were repeated twice with consistent results
Fig. 5
TRIM28-deficiency impaired epigenetic activation in Th17 cells. TRIM28 ChIP-seq was performed as in Fig. 4. a Overlap of TRIM28 binding peaks with SE and TE regions in Th17 cells. b IGV browser view of TRIM28, p300 binding peaks and SE regions at the Il17-Il17f gene locus (right). c Volcano plot showing p300 binding peak change in WT and Trim28_−/_− Th17 cells cultured for 3 days (TGF-β+IL-6). d Overlay of p300 binding peaks in WT and TRIM28KO Th17 cells over Th1-, Th2 and Th17-specific SEs with decreased p300 intensity. e Heatmap of pathway analysis in genes associated with Th17-SEs that had decreased (left) or increased (right) p300 recruitment in KO cells. f Transcriptional comparison of T helper-specific SE-associated genes analyzed from WT Th17 RNA-seq data (p values, Mann−Whitney test). g Fold change of Th17-SE-related genes with increased (up) or decreased (down) p300 binding at the SE regions (p values, Mann-Whitney test). h 3C-PCR experiments were performed in WT and TRIM28KO Th17 cells cultured as in c and the PCR products were acquired by nest PCR. M is short for marker, and the whole DNA gels are shown in Supplementary Fig. 8. *p < 0.05, **p < 0.01, ***p < 0.005. All error bars represent SDs. The experiments were repeat twice with consistent results
Fig. 6
TRIM28 functions as a cofactor for RORγt and STAT3. a Overlay of TRIM28 binding peaks with that of RORγt and STAT3 in Th17 cells. b Genome-wide distributions of binding peaks of RORγt, STAT3, and p300 at TRIM28 binding sites. c IGV browser view of RORγt, STAT3, and TRIM28 binding peaks at the Il17-Il17f gene locus. d Binding of RORγt and STAT3 to the Il17-Il17f gene locus in WT and Trim28 _−/_− Th17 cells cultured at TGF-β plus IL-6 condition. e Overexpression of RORγt in WT or TRIM28 KO CD4+ T cells polarized under neutral (Th0) or Th17 (TGF-β plus IL-6) conditions. d, e The experiments were repeated 2–3 times with consistent results, and Student’s t test was used for the statistical test (ns = not significant; *p < 0.05, **p < 0.01, ***p < 0.005)
Fig. 7
Cytokine signaling regulated TRIM28 recruitment. a Expression of TRIM28 in different T-cell subsets and naive CD4+ T cells. b TRIM28 ChIP-qPCR results in WT Th0, WT Th17, STAT3 KO Th17 or RORγt KO Th17 cells. c RORγt overexpression in WT or STAT3 KO CD4+ T cells polarized under neutral (Th0) condition. d Overlay of p300 binding peaks in WT/STAT3KO(Upper) or WT/RORγKO (lower) Th17 cells over Th17-specific SEs with decreased p300 intensity. e 3C-PCR experiments were performed in WT and TRIM28KO Th17 cells cultured for 3 days and the PCR products were acquired by nest PCR. M is short for marker, and the whole DNA gels are shown in Supplementary Fig. 8. f Immunoblot results of TRIM28 immunoprecipitated protein complexes as detected by RORγt and STAT3 antibodies in Th17 cells. The whole WB gels are shown in Supplementary Fig. 9. Those experiments were repeated twice with consistent results, and Student’s t test was used for the statistical test (ns = not significant; *p < 0.05, **p < 0.01, ***p < 0.005). All error bars represent SDs
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