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 4

Fibroblasts isolated from fibrotic lungs display sustained peroxisomal gene repression in vitro.

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Fibroblasts isolated from fibrotic lungs display sustained peroxisomal g...
(A) Lungs from bleomycin- or vehicle-treated Col1α1-GFP mice were harvested 2 weeks following bleomycin administration. FACS sorting was employed to isolate GFP+ fibroblasts from diseased and healthy lungs. Antibodies against markers for epithelial (CD326), endothelial cells (CD31), and leukocytes (CD45) were included in the analysis. Gating strategy includes exclusion of CD45+ cells, doublets and dead cells (data not shown). FACS analysis reveals an increased number of GFP+ fibroblasts and higher GFP intensity in fibroblasts from diseased lungs compared with healthy lungs. (B) qPCR on FACS-sorted lung fibroblasts shows elevation of Fn1 gene expression and repression of peroxisomal genes, including Ppara and Ppargc1a, but not Pparg, in diseased compared with normal fibroblasts (n = 5). Results are expressed as mean ± SEM (**P < 0.05, ***P < 0.001 by 2-tailed, paired t test). (C) Representative images showing cultured GFP+ fibroblasts from bleomycin-treated or healthy lungs. Scale bar: 10 μm. Wester blotting showing elevated αSMA expression and sustained PGC1α and PPARα repression in cultured GFP+ lung fibroblasts from diseased lungs compared with healthy GFP+ fibroblasts (Sham, n = 2; Bleo, n = 2). (D) Immuno-ECM assay analysis shows that diseased mouse lung fibroblasts display increased collagen I deposition compared with normal ones. Inhibition of G9a by BIX01294 in diseased fibroblasts significantly reduces collagen I deposition to a level comparable to normal fibroblasts. Results are expressed as mean ± SEM (*P < 0.05, **P < 0.01 by 1-way ANOVA with Turkey’s multiple comparisons test). (E) Experimental strategy to evaluate peroxisomal gene expression in diseased lung fibroblasts in vivo following G9a inhibition by BIX01294. (F) Western blotting image showing reduced H3K92/3me in whole lung at 24 and 48 hours following BIX01294 administration (representative blot of n = 2). (G) qPCR shows Ppargc1a and Ppara elevation in fibroblasts from BIX01294-treated mice compared with control mice (n = 6). Results are expressed as mean ± SEM (*P < 0.05, ***P < 0.001 by 2-tailed, paired t test).