Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity

Abstract

Regulatory T (Treg) cells are important for the maintenance of immune homoeostasis and prevention of autoimmune diseases. Epigenetic modifications have been reported to modulate autoimmunity by altering Treg cell fate. Here we show that the H3K4 methyltransferase Ash1l facilitates TGF-β-induced Treg cell polarization in vitro and protects mice from T cell-mediated colitis in vivo. Ash1l upregulates Smad3 expression by directly targeting Smad3 promoter to increase local H3K4 trimethylation. Furthermore, we identify an lncRNA, namely lnc-Smad3, which interacts with the histone deacetylase HDAC1 and silences Smad3 transcription. After TGF-β stimulation, activated Smad3 suppresses lnc-Smad3 transcription, thereby recovering the Smad3 promoter accessibility to Ash1l. By revealing the opposite regulatory functions of Ash1l and lnc-Smad3 in Smad3 expression, our data provide insights for the epigenetic control of Treg cell fate to potentially aid in the development of therapeutic intervention for autoimmune diseases.

Figure 1. Ash1l facilitates TGF-β-mediated Treg cell induction via promoting Foxp3 expression.

(a,b) Representative flow cytometry (a) and quantification (b) of the percentage of Foxp3 in the CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. (c) mRNA expression of Foxp3 in the WT and Ash1l-silenced CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. (d,e) Representative flow cytometry (d) and quantification (e) of the percentage of Foxp3 in WT and Ash1l-silenced CD4⁺ T cells transduced with Ash1l-fragment-expressing lentivirus (Lenti-Ash1l-F1, Lenti-Ash1l-F2, Lenti-Ash1l-F3 or Lenti-Ash1l-ΔN) and cultured under iTreg cell-skewing conditions (with TGF-β) for 3 days. (f) mRNA expression of Foxp3 in WT and Ash1l-silenced CD4⁺ T cells transduced and cultured as in d. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. Data are representative of three independent experiments (a,d) or are from three independent experiments (b,c,e,f; mean±s.d.).

Figure 2. In vivo silencing of Ash1l renders mice more susceptible to experimental colitis.

(a–c) TNBS-colitis was induced in 8-week-old WT and Ash1l-silenced mice (n=5 mice per group) and monitored for disease development for 3 days after TNBS induction. (a) Weight change over time of WT and Ash1l-silenced mice after TNBS induction, presented relative to weight at day 0, set as 100%. (b,c) Appearances (b) of colons, reduction of colon length (c) were examined at day3 after TNBS induction. (d–f) T cell adoptive transfer-mediated colitis was induced in 6-week-old Rag1^−/− mice via transferring with WT CD4⁺CD25⁻ T cells with or without WT or Ash1l-silenced iTreg cells (n=5 mice per group). Disease development was monitored for 6 weeks. (d) Weight change over time of Rag1^−/− mice transferred with WT CD4⁺CD25⁻ T cells with or without WT or Ash1l-silenced iTreg cells, presented relative to weight at week 1, set as 100%. (e,f) Appearances (e) and length (f) of colons were examined at week 6 after T cell transfer. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. Data are from three independent experiments (a,c,d,f; mean±s.d. of five mice) or are representative of three independent experiments (b,e).

Figure 3. Ash1l enhances TGF-β-Smad2/3 signalling by promoting Smad2/3 expression.

(a) mRNA expression of Smad2 (left) and Smad3 (right) in the WT and Ash1l-silenced CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. Results are relative to those in unstimulated WT CD4⁺ T cells, set as 1. (b) ELISA assay of Smad2/3 in 5 μg whole-cell extract (left) and 5 μg nuclear extract (right) of WT and Ash1l-silenced CD4⁺ T cells under iTreg cell-skewing conditions (with TGF-β) for indicated times. OD₄₅₀ represents absorbance at 450 nm. (c) mRNA expression of Smad2 and Smad3 in the WT and Ash1l-silenced CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or Ash1l-fragment-expressing lentivirus (Lenti-Ash1l-F1, Lenti-Ash1l-F2, Lenti-Ash1l-F3 or Lenti-Ash1l-ΔN) and cultured under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to those in WT CD4⁺ T cells transduced with Lenti-CTR, set as 1. (d) ELISA assay of Smad2/3 in 5 μg whole-cell extract (left) and 5 μg nuclear extract (right) of WT and Ash1l-silenced CD4⁺ T cells transduced and cultured as in c. OD₄₅₀ represents absorbance at 450 nm. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. All data are from three independent experiments (mean±s.d. of technical triplicates).

Figure 4. Ash1l accumulates at the Smad2/3 promoter regions upon TGF-β stimulation.

(a) CHIP analysis of the recruitment of Ash1l to the Smad2 and Smad3 promoter regions with anti-Ash1l antibody in CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. (b) H3K4me3 modifications of the Smad2 and Smad3 promoter regions. CHIP analysis of the trimethylation of histone H3 lysine 4 (H3K4me3) at the Smad2 and Smad3 promoter regions in WT and Ash1l-silenced CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for 1 day. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. All data are from three independent experiments (mean±s.d. of technical triplicates).

Figure 5. Lnc-Smad3 inhibits iTreg cell polarization via suppressing Smad3 transcription.

(a) A schematic outlining the genomic loci of lnc-Smad3 and Smad3. Semi-quantitative PCR (b) and gel electrophoresis (c) detection of lnc-Smad3 in CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. Results are relative to the baseline lnc-Smad3 expression in unstimulated CD4⁺ T cells, set as 1. (d) Relative expression of lnc-Smad3 in WT and Smad3-KO CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to the lnc-Smad3 expression in unstimulated WT CD4⁺ T cells, set as 1. (e) CHIP analysis of the accumulation of Smad3 at the lnc-Smad3 promoter regions with anti-Smad3 antibody in CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for 2 days. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. (f) Relative expression of lnc-Smad3, Smad3 and Foxp3 in CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to those in CD4⁺ T cells transduced with Lenti-CTR, set as 1. (g,h) The percentages of Foxp3⁺ Tregs in CD4⁺ T cells transduced and cultured as in f were analysed by flow cytometry (g) and quantified (h). Numbers in quadrants indicate per cent cells in each. Error bars represent s.d. Student's t test. NS, not significant. *P<0.05, **P<0.01. Data are from three independent experiments (b,d-f,h; mean±s.d. of technical triplicates) or are representative of three independent experiments (c,g).

Figure 6. Lnc-Smad3 reduces the accessibility of the Smad3 promoter to Ash1l.

(a) CHIP analysis of the H3K4me3 and H3K27ac modifications, and Pol II occupancy around lnc-Smad3 and Smad3 loci in CD4⁺ T cells left unstimulated (day 0) or cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days (day 2). Four regions (capital letters A–D) across lnc-Smad3 gene locus and three regions (capital letters E–G) across Smad3 gene locus were analysed by CHIP assay. Normalized data are shown as percentage of input control. (b) HepG2 cells were transfected with a Smad3 promoter reporter construct (1 kb upstream of the transcription start site) and lnc-Smad3 expression vector (lnc-Smad3) or empty control vector (CTR), and stimulated with TGF-β. Luciferase activity was measured 48 h later. Data were normalized to renilla luciferase and presented with respect to CTR, set as 1. (c) Chromatin accessibility of the Smad3 promoter region by quantitative PCR with DNase I pretreated nucleus of CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days. Changed fold are concluded using 2^ΔCt with respect to CD4⁺ T cells transduced with Lenti-CTR, set as 1. (d) CHIP analysis of the accumulation of Ash1l and H3K4me3 modification at Smad3 promoter regions in CD4⁺ T cells transduced and cultured as in c. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. All data are from three independent experiments (mean±s.d. of technical triplicates).

Figure 7. Lnc-Smad3 interacts with HDAC1 at the Smad3 promoter.

(a) A schematic outlining the conserved sequences of the Smad3 promoter regions in Mus musculus and Homo sapiens. (b) CHIP analysis of the accumulation of HDAC1 at Smad3 promoter region in naive CD4⁺ T cells and iTreg cells. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. (c) Quantitative PCR detection of the lnc-Smad3 retrieved by HDAC1 specific antibody compared with immunoglobulin G (IgG) in the RIP assay within CD4⁺ T cells. Normalized data are shown as percentage of input control (% Inp). IgG serves as a RIP control. (d) CHIP analysis of the accumulation of HDAC1 at Smad3 promoter region in CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. (e) Relative expression of HDAC1 in CD4⁺ T cells transduced and cultured as in d. Results are relative to the baseline expression of HDAC1 in unstimulated CD4⁺ T cells transduced with Lenti-CTR, set as 1. Error bars represent s.d. Student's t test. **P<0.01. NS, not significant. All data are from three independent experiments (mean±s.d. of technical triplicates).