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
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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

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Figure 3

Nuclear G9a and H3K9 methylation are elevated in fibrotic lung tissues.

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Nuclear G9a and H3K9 methylation are elevated in fibrotic lung tissues. ...
(A) Lungs from Col1α1-GFP mice show GFP-labeled fibroblast expansion following bleomycin challenge (day 10) compared with control lungs. Scale bar: 100 μm. (B) Immunofluorescence images show increased G9a expression in GFP+ fibroblasts from bleomycin-treated lungs compared with fibroblasts from healthy lungs. Fluorescence intensity was measured in GFP+ fibroblasts in normal and bleomycin-treated lung tissues using ImageJ software (n = 10). Data are expressed as mean ± SEM of 3 normal and 4 bleomycin-treated lungs (*P < 0.05 by 2-tailed, paired t test). Scale bar: 50 μm. (C) Immunofluorescence images showing elevated H3K9me2 in GFP+ fibroblasts from bleomycin-treated lungs compared with GFP+ fibroblasts from healthy lungs (n = 10). Data are expressed as mean ± SEM of 4 normal and 4 bleomycin-treated lungs (*P < 0.05 by 2-tailed, paired t test). Scale bar: 50 μm. (D) Immunofluorescence images showing increased H3K9me2 in lung tissues from IPF patients compared with healthy lungs (n = 10). Data are expressed as mean ± SEM of 3 normal and 3 IPF lungs (**P < 0.01 by 2-tailed, paired t test). Scale bar: 50 μM. (E) Western blotting showing CBX5 and G9a expression in IPF-derived and normal lung fibroblasts. Densitometry analysis demonstrated significant upregulation of CBX5 in IPF-derived fibroblasts compared with normal fibroblasts. No significant difference was observed in G9a expression (n = 5). Data are expressed as mean ± SEM of 5 normal and 5 IPF-derived fibroblasts (*P < 0.05 by 2-tailed, paired t test).