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 5

Inhibition of CBX5 in activated lung fibroblasts promotes upregulation of peroxisomal genes in vitro.

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Inhibition of CBX5 in activated lung fibroblasts promotes upregulation o...
(A) Western blotting analysis shows that TGF-β potently inhibits peroxisomal protein expression in normal human lung fibroblasts (representative blot of n = 3). Cells were incubated with TGF-β for 24 hours. (B) qPCR analysis reveals that inhibition of CBX5 in normal human lung fibroblasts prevents TGF-β–inhibited peroxisomal gene expression (n = 3). Data are shown as mean ± SEM of 3 independent experiments performed in duplicate. (*P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (C) Wester blotting analysis shows strong repression of peroxisomal proteins by TGF-β in IPF-derived fibroblasts (representative blot of n = 3). (D) Similarly to normal lung fibroblasts, inhibition of CBX5 in IPF-derived fibroblasts blocks TGF-β–inhibited peroxisomal gene expression (n = 3). Data are shown as mean ± SEM of 3 independent experiments performed in duplicate (*P < 0.05,***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (E) Gene expression analysis shows PPARA and PPARGC1A gene repression in normal lung fibroblasts seeded on stiff substrates (64 kPa) compared with soft (0.2 kPa) (n = 3). Data are shown as mean ± SEM of 3 independent experiments performed in duplicate (*P < 0.05 by 2-tailed, paired t test). (F) Western blotting analysis shows downregulation of PPARα and PGC1α in lung fibroblasts cultured on stiff compared with soft substrates. CBX5 silencing blocks matrix stiffness–mediated PPARα and PGC1α downregulation in lung fibroblasts representative blot of (n = 2).