TRIM28 mediates Mettl5 ubiquitination to promotes Th2 polarization

Figures

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  Figure 1. KO mice have spontaneous airway Th2 polarization. BALF was collected from KOc mice and KO mice, and analyzed by flow cytometry and ELISA. (A) total cell counts of BALF. (B, C), gating strategy. (D-K) gated plots show indicated cell types on the Y axis. Bars show counts of indicated cells. (L–P) bars show the quantities of indicated cytokines on the Y axis. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: Student’s t-test. *p<0.05; **p<0.01; ****p<0.0001; ns, Not significant. Each group consists of 6 mice. Each experiment was repeated 3 times. KOc: Mettl5 f/f mice. KO: Mettl5 f/f Cd4 CreERT2 mice.

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  Figure 2. Assessment of the correlation between Mettl5 and AA response. An AA mouse model was established. (A–F) bars show the quantities of indicated molecules (denoted on the Y axis) in BALF. (G–L) CD69+CD4+ T cells were isolated from the lung tissues. Bars show the quantities of indicated molecules in cellular extracts. M, a heatmap shows the coefficients between the indicated items. (N–P) CD3+CD4+ T cells were gated from single cells isolated from the lung tissues (N), from which CD69+ cells were gated (O); (P) the histogram shows IL-4+ cells from CD69+ T cells. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: Student’s t-test (A–L) and Pearson correlation coefficient test (M). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Each group consists of 6 mice. Each experiment was repeated 3 times.

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  Figure 3. Assessment of the activities of the Gata3 promoter in airway CD69+CD4+ T cells in AA mice. Airway CD69+CD4+ T cells were isolated from AA mice and NC mice, and analyzed by ChIP assay. (A) quantities of Gata3 promoter DNA in ChIP products. (B, C) quantities of Mettl5 and H3K27me3 at the Gata3 promoter. (D) the Gata3 promoter methylation status. (E) quantity of Pol II at the Gata3 promoter. (F, G) expression of GATA3. (H) correlation coefficients between indicated items. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: Student’s t-test (A–G) and Pearson correlation coefficient test (H). **p<0.01; ***p<0.001. IgG, Isotype IgG (used as a negative control Ab in ChIP assay). Ab, Abs used in ChIP assay. Each group consists of 6 mice. Each experiment was repeated 3 times.

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  Figure 4. Assessment of the ubiquitination status of Mettl5 in CD4+ T cells. (A–C) airway CD69+CD4+ T cells were isolated from NC mice and AA mice. The cells were analyzed by ChIP-cross-ELISA. Bars show the quantities of indicated molecules together with Mettl5 at the Gata3 promoter. (C–G) the treatment for mice is denoted on the X axis. MG132 (DMSO): Mice were treated with nasal instillations (containing MG132, 20 μM, or DMSO) daily in the period of sensitization. Bars show the proteasome complexes at the Gata3 promoter of airway CD69+CD4+ T cells. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: ANOVA + Tukey HSD test. **p<0.01; ***p<0.001. Each group consists of 6 mice. Each experiment was repeated 3 times. IgG, Isotype IgG (used as a negative control Ab in ChIP assay).

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  Figure 5. TRIM28 induces Mettl5 ubiquitination and degradation. (A, B), airway CD69+CD4+ T cells were isolated from AA mice and NC mice. The cells were analyzed using ChIP-cross-ELISA. Bars show a complex of Mettl5 and TRIM28. (C) recombinant Mettl5 protein in HEK293 cells. (D, E) Mettl5-expression HEK293 cells were exposed to TRIM28 (1 μg/ml) in culture overnight. Bars show a complex of TRIM28 and His-Mettl5. (F–I) Mettl5-expression HEK293 cells were exposed to TRIM28 at gradient concentrations overnight. Cellular extracts of HEK293 cells were analyzed using cross-ELISA. The line plots show the quantities of indicated items. (J, K), enforced expression of TRIM28 in EL4 cells. (L) quantity of Mettl5 at the Gata3 promoter in EL4 cells. (M) quantity of GATA3 at the Il4 promoter in EL4 cells. (N) expression of IL-4 in EL4 cells. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: ANOVA + Tukey HSD test (A, B) and Student’s t-test (C–E). **p<0.01; ***p<0.001; ****p<0.0001. The experiments of line plots were repeated 3 times. Ab in (A, B): Anti-HuR Ab (used in ChIP assay to anchor the Gata3 promoter). IgG, Isotype IgG (used as…

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  Figure 6. Inhibition of TRIM28 reconciles epigenetic abnormality at Gata3 gene of airway CD4+ T cells and mitigates AA. (A) quantity of TRIM28 mRNA in KOc mice and KO mice. (B) representative histology images of the lungs (×200). (C) mouse airway resistance changes in response to challenge with methacholine. (D) total cell counts in BALF. (E–J) quantities of indicated molecules in BALF. (K, L) airway CD69+CD4+ T cells were isolated from NC mice and AA mice. The cells were analyzed using ChIP-cross-ELISA. Bars show the quantities of indicated items at the Gata3 promoter. (M) bars show percentage of Gata3 promoter methylation. (N) bars show quantity of GATA3 at the Il4 promoter. The data of bars are presented as mean ± SD. Each bubble in bars presents one sample (tested in triplicate). Statistics: Student’s t-test (A) and ANOVA + Tukey test (C–N). **p<0.01; ***p<0.001. KO mice, Trim28 f/f Cd4 Cre mice; KOc mice, Trim28 f/f mice; IgG, Isotype IgG (used as a negative control Ab in ChIP assay). Each group consists of 6 mice. Each experiment was repeated 3 times.