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 6

PGC1α inhibition by the G9a/CBX5 pathway is an early and necessary step in TGF-β–mediated fibroblast activation.

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PGC1α inhibition by the G9a/CBX5 pathway is an early and necessary step ...
(A) Western blotting showing reduction of H3K9me2/3 in IPF-derived fibroblasts exposed to BIX01294 for 2, 4, and 6 hours (representative blot of n = 2). (B) qPCR of IPF-derived fibroblasts treated with TGF-β shows an increase of ACTA2 and COL1A1 gene expression at 12 hours following TGF-β exposure. BIX01294 treatment reduces ACTA2 and COL1A1 elevation by TGF-β at 12 hours but has no effect at 4 or 8 hours. Data are shown as mean ± SEM of 4 different IPF cell lines (*P < 0.05 by 2-tailed, paired t test). (C) qPCR analysis showing that TGF-β inhibits PPARA, PPARG, and PPARGC1A gene expression starting at 4 hours following treatment. BIX01294 treatment prevents PPARGC1A gene repression by TGF-β, without significant effect on PPARA and PPARG. Data are shown as mean ± SEM of 4 different IPF cell lines (**P < 0.01 by 2-tailed, paired t test). (D) BIX01294 significantly reduces ACTA2 gene expression and elevated PPARGC1A gene transcripts. Data are shown as mean ± SEM of 4 different IPF cell lines (**P < 0.01, ***P < 0.001 by 2-tailed, paired t test). (E and F) PPARγ or PPARα inhibition by siRNA or antagonists in BIX01294-treated of CBX5-silenced fibroblasts fails to block the beneficial effects of inhibiting G9a or CBX5 in TGF-β–treated fibroblasts. (G) Inhibition of PGC1α by siRNA in BIX01294-treated or CBX5-silenced fibroblasts restores the elevation of ACTA2 by TGF-β. 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). (H) qPCR analysis showing that inhibition of PGC1α in CBX5-silenced fibroblasts promotes COL1A1 gene elevation in response to TGF-β. Data are shown as mean ± SEM of 4 independent experiments performed in duplicate (*P < 0.05 by 1-way ANOVA with Turkey’s multiple comparisons test).