CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis
Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y.S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin
Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y.S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin
View: Text | PDF
Research Article Cell biology Pulmonology

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis

Abstract

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex, respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator–activated receptor γ coactivator 1 α gene (PPARGC1A, encoding PGC1α). Both TGF-β and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of the CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGF-β– and matrix stiffness–promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Authors

Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y.S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin

×

Figure 8

Inhibition of G9a following bleomycin challenge promotes Ppargc1a gene expression and attenuates lung fibrosis.

Options: View larger image (or click on image) Download as PowerPoint
Inhibition of G9a following bleomycin challenge promotes Ppargc1a gene e...
(A) Schematic showing experimental workflow. (B) qPCR showing Ppargc1a gene repression following bleomycin delivery. One single dose of BIX01294 partially restores Ppargc1a gene expression compared with vehicle-treated animals (n = 5). Data are shown as mean ± SEM (*P < 0.05, ***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (C and D) qPCR shows reduced Fn1 and Ctgf transcripts in BIX01294-treated mice lungs compared with vehicle-treated animals (n = 6). Data are shown as mean ± SEM (*P < 0.05, ***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (E) Representative images of Masson’s trichome–stained lung tissues. Scale bar: 200 μm. (F) H&E-stained lung sections were scored using Ashcroft method. (G) Hydroxyproline assay was used to evaluate collagen deposition in the lungs. Sham control (n = 8), Bleo 20 days control (n = 12), and Bleo 20 days BIX01294 (n = 11). Data are shown as mean ± SEM (***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (H) Strategy for inducible knockout of G9a in COL1α2-expressing cells. Col1a2-CreER:g9afl/fl mice were treated with bleomycin, followed by 5 doses of tamoxifen (0.1 mg/g) starting at day 3. (I) Immunofluorescence images show G9a deletion in PDGFRα+ cells. Arrows indicate cell nuclei that are negative for G9a staining. Scale bar: 10 μm. (J) Representative images of Masson’s trichome–stained lung tissues. Scale bar: 200 μm. (K) Reduced H3K9me2 in fibroblasts from tamoxifen-treated Col1a2-CreER(T):g9afl/fl mice compared with g9afl/fl control mice. (L) H&E-stained lung sections were scored using Ashcroft method. (M) Bleomycin significantly increases hydroxyproline content in the lungs of control mice [Col1a2-CreER(T):g9afl/fl mice not treated with tamoxifen (n = 7) as well as g9afl/fl and Col1a2-CreER(T) control groups treated with tamoxifen (n = 7)] but not in mice with conditional deletion of G9a in fibroblasts [Col1a2-CreER(T):g9afl/fl group treated with tamoxifen, n = 7] compared with uninjured controls (n = 5). Data are shown as mean ± SEM (**P < 0.01, ***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test).