Research ArticleFibrosis

Epigenetic activation and memory at a TGFB2 enhancer in systemic sclerosis

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Science Translational Medicine  19 Jun 2019:
Vol. 11, Issue 497, eaaw0790
DOI: 10.1126/scitranslmed.aaw0790
  • Fig. 1 Patient-derived SSc fibroblasts maintain a fibrotic synthetic repertoire ex vivo.

    (A) Heat map of genes differentially expressed (rows) between healthy control and lesional SSc fibroblasts by RNA-seq [n = 3 biological replicates, false discovery rate (FDR) < 0.05]. (B and C) mRNA and protein expression in control and SSc fibroblast lines. (D) mRNA expression in homogenized biopsies taken from diffuse SSc lesional skin (n = 5) versus healthy control skin (n = 4). (E) TGFB2 mRNA expression (green) measured by mRNA in situ hybridization of healthy control skin and lesional SSc skin sections (n = 4). Arrowheads indicate cells with high TGFB2 expression. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F and G) mRNA expression upon stimulation of fibroblasts with exogenous TGFβ2 ligand. (H) TGFB ligand and TGFβ target gene expression upon siRNA knockdown of TGFB2 in primary fibroblasts. *P < 0.05, **P < 0.01, and ***P < 0.001. ncontrol = 5 and nSSc = 6. Two-way Student’s t test for single comparisons or one-factor analysis of variance (ANOVA) with FDR correction for all experiments, except (A) and (D) TGFB2 mRNA (Mann-Whitney). Black points represent individual biological replicates. Y axes are varied across similar plots to aid visualization.

  • Fig. 2 Epigenetic activation of the TGFB2 enhancer promotes profibrotic gene expression in SSc fibroblasts.

    (A) ATAC-seq signals between healthy control and SSc fibroblasts at the TGFB2 locus were compared to ENCODE predictions for proximal promoter (orange) or putative enhancers (green), with the candidate enhancer of interest highlighted on the right. Genome-wide significant peaks at the TGFB2 enhancer are indicated by asterisks. (B) TGFB2 mRNA expression was regressed on estimated chromatin accessibility of the putative enhancer in control and patient fibroblasts. R2, coefficient for determination of linear regression. (C) Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) demonstrated H3K27ac or EP300 occupancy at the putative TGFB2 enhancer in fibroblasts. Values are represented as percent input normalized by immunoglobulin G control. (D) mRNA expression after targeted histone acetylation of the TGFB2 enhancer by coexpression of TGFB2 enhancer-specific guide RNA (sgRNA) and dCas9-EP300 (histone acetyltransferase and dCas9-EP300core) in control and SSc fibroblasts. dCas9-EP300Δ (dCas9-EP300D1399Y) contained a nonfunctional residue substitution at the acetyltransferase domain and was used as a negative control. (E) Targeting of dCas9-KRAB (histone methyltransferase) to the TGFB2 enhancer using TGFB2 enhancer-specific guide RNAs in control and SSc fibroblasts. *P < 0.05, **P < 0.01, and ***P < 0.001. ncontrol = 5 and nSSc = 6. Two-way Student’s t test or one-factor ANOVA with FDR correction were used for all experiments, except for (C) H3K27ac (Mann-Whitney), (D) TGFB1 mRNA (Kruskal-Wallis with FDR correction), and (E) COL1A1 and SERPINH1 mRNAs (Kruskal-Wallis with FDR correction). Black points represent individual biological replicates. Y axes are varied across similar plots to aid visualization.

  • Fig. 3 BRD4 and NF-κB maintain epigenetic activation of the TGFB2 enhancer in SSc.

    (A and B) TGFB2 expression and epigenetic modification of the TGFB2 enhancer were assayed immediately after treatment with EP300 inhibitor (SGC-CBP30) or after 5 days in fresh media sans SGC-CBP30. (C) ChIP-qPCR for BRD4 occupancy at the TGFB2 enhancer in baseline conditions (left) and in response to SGC-CBP30 treatment (right) in fibroblasts ex vivo. (D) ChIP-qPCR for acetylated p65 (activated NF-κB) occupancy at the TGFB2 enhancer in baseline conditions. (E) Chem-seq data revealed specific binding of JQ1, BRD4, and RNA polymerase II (RNApol II) at the TGFB2 enhancer highlighted on the right (21). (F) mRNA expression and epigenetic modifications of the TGFB2 enhancer upon siRNA knockdown of BRD4 mRNA in fibroblasts ex vivo. (G and H) Pulse-chase analysis of TGFB2 enhancer activity in response to JQ1 or BAY 11-7085 treatment up to 5 days after drug removal. (I) TGFβ2 protein expression in vehicle- or JQ1-treated fibroblasts. (J) TGFβ2 target gene expression in cultured control or SSc fibroblasts upon BRD4 inhibition by siRNA knockdown compared to scrambled siRNA control. (K) Collagen and TGFB2 expression upon cotreatment of JQ1 and TGFβ2 ligand in SSc fibroblasts. (L) Heat map of differentially expressed genes between vehicle- or JQ1-treated control and SSc fibroblasts by RNA-seq. n = 3, FDR < 0.05. JQ1-treated SSc fibroblasts clustered together with control fibroblasts in principal components analysis (PC1 and PC2 account for 65 and 14% of total variance, respectively). *P < 0.05, **P < 0.01, and ***P < 0.001. ncontrol = 5 and nSSc = 6, except for ChIP studies, where ncontrol = 4 and nssc = 4. Two-way Student’s t test for single comparisons or one-factor ANOVA with FDR correction were used for all experiments, except for (A) and (F) H3K27ac and BRD4 occupancy (Kruskal-Wallis with corrected FDR) and (D) (Mann-Whitney). Black points represent individual biological replicates.

  • Fig. 4 Organ culture with JQ1 mitigates profibrotic gene expression in SSc skin.

    mRNA in situ hybridization for TGFB2 (green foci) and COL1A1 (red foci) in the dermis of healthy control skin and SSc lesional skin biopsies maintained in organ culture with dimethyl sulfoxide (DMSO) or JQ1 for 10 days. Scale bars, 20 μm. Total number of fluorescent foci normalized to the number of nuclei was quantified and averaged from three different sections per biological replicate. ***P < 0.001. ncontrol = 4 and nssc = 4. One-factor ANOVA with FDR correction was used for all experiments. Black points represent individual biological replicates.

  • Fig. 5 Organ culture with JQ1 promotes collagen clearance in SSc skin.

    (A) Picrosirius red staining in the dermis of healthy control skin and SSc lesional skin biopsies that were maintained in organ culture with DMSO or JQ1 for 10 days. Type 1 collagen (yellow/orange fibers) was visualized and quantified on the basis of light birefringence under polarized light. Dashed lines indicate the epidermal-dermal junction. Scale bars, 50 μm. (B) mRNA in situ hybridization for MMP1 (yellow foci) in the dermis of control and SSc skin biopsies maintained in organ culture with DMSO or JQ1 for 10 days. Scale bars, 20 μm. Total number of fluorescent foci was normalized by the total number of nuclei and averaged from two different sections per biological replicate. *P < 0.05, **P < 0.01, and ***P < 0.001. ncontrol = 4 and nssc = 4. One-factor ANOVA with FDR correction was used for all experiments except for (B) (Kruskall-Wallis with FDR correction). Black points represent individual biological replicates.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/497/eaaw0790/DC1

    Fig. S1. SSc fibroblasts maintain profibrotic gene expression pathways.

    Fig. S2. SSc fibroblasts actively transcribe TGFB2 mRNA.

    Fig. S3. Nonlesional SSc fibroblasts exhibit profibrotic gene expression.

    Fig. S4. Quantification of TGFB2 mRNA by in situ hybridization.

    Fig. S5. mRNA in situ hybridization for TGFB isoform expression in SSc lesional skin.

    Fig. S6. SSc fibroblasts exhibit increased chromatin accessibility in genes related to profibrotic pathways.

    Fig. S7. TGFβ2-expressing cell lines contain unique predicted enhancers at the TGFB2 locus.

    Fig. S8. TGFβ2 enhancer accessibility correlates with TGFB2 mRNA expression.

    Fig. S9. TGFB2 enhancer is inactive in nonlesional SSc fibroblasts.

    Fig. S10. The TGFB2 enhancer exhibits minimal H3K27me3 modification in lesional SSc fibroblasts.

    Fig. S11. SSc fibroblasts exhibit high NF-κB signaling activity.

    Fig. S12. TNFα induces TGFB2 enhancer activity in a NF-κB– and BRD4-dependent manner.

    Fig. S13. siRNA knockdown of BRD4 abrogates BRD4 mRNA expression.

    Fig. S14. JQ1 treatment reduces collagen content in SSc skin explants.

    Fig. S15. TGFβ2 inhibition by JQ1 or siRNA induces MMP1 expression in SSc fibroblasts.

    Table S1. Patient data on SSc skin biopsy donors and healthy volunteers.

    Data file S1. Sanger sequencing results.

    Data file S2. Shapiro-Wilk test results for normally distributed datasets.

  • The PDF file includes:

    • Fig. S1. SSc fibroblasts maintain profibrotic gene expression pathways.
    • Fig. S2. SSc fibroblasts actively transcribe TGFB2 mRNA.
    • Fig. S3. Nonlesional SSc fibroblasts exhibit profibrotic gene expression.
    • Fig. S4. Quantification of TGFB2 mRNA by in situ hybridization.
    • Fig. S5. mRNA in situ hybridization for TGFB isoform expression in SSc lesional skin.
    • Fig. S6. SSc fibroblasts exhibit increased chromatin accessibility in genes related to profibrotic pathways.
    • Fig. S7. TGFβ2-expressing cell lines contain unique predicted enhancers at the TGFB2 locus.
    • Fig. S8. TGFβ2 enhancer accessibility correlates with TGFB2 mRNA expression.
    • Fig. S9. TGFB2 enhancer is inactive in nonlesional SSc fibroblasts.
    • Fig. S10. The TGFB2 enhancer exhibits minimal H3K27me3 modification in lesional SSc fibroblasts.
    • Fig. S11. SSc fibroblasts exhibit high NF-κB signaling activity.
    • Fig. S12. TNFα induces TGFB2 enhancer activity in a NF-κB– and BRD4-dependent manner.
    • Fig. S13. siRNA knockdown of BRD4 abrogates BRD4 mRNA expression.
    • Fig. S14. JQ1 treatment reduces collagen content in SSc skin explants.
    • Fig. S15. TGFβ2 inhibition by JQ1 or siRNA induces MMP1 expression in SSc fibroblasts.
    • Table S1. Patient data on SSc skin biopsy donors and healthy volunteers.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.txt format). Sanger sequencing results.
    • Data file S2 (Microsoft Excel format). Shapiro-Wilk test results for normally distributed datasets.

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