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 7

TGF-β enhances CBX5 recruitment and H3K9me mark enrichment at the PPARGC1A gene promoter.

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TGF-β enhances CBX5 recruitment and H3K9me mark enrichment at the PPARGC...
(A) ChIP analysis showing CBX5 enrichment at the proximal promoter of PPARGC1A gene in human lung fibroblasts cultured on stiff plastic substrates. TGF-β further elevates CBX5 recruitment at the PPARGC1A gene promoter (n = 3). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed, paired t test). (B) ChIP analysis shows increased H3K9me2 mark decoration at the PPARGC1A gene promoter in human lung fibroblasts in response to TGF-β. BIX01294 treatment significantly reduces H3K9 dimethylation at the PPARGC1A gene promoter in response to TGF-β. Three adjacent promoter regions were selected based on their affinity for transcription factors; starting from the transcription starting site (TSS) are CREB1, MEF2, and FoxO1, respectively (n = 3). Data are shown as mean ± SEM (*P < 0.05 by 2-tailed, paired t test) (C) qPCR analysis showing that inhibition of the transcription factor CREB by siRNA in CBX5-silenced fibroblasts reduces PPARGC1A gene expression. Data are shown as mean ± SEM of 3 independent experiments performed in duplicate (*P < 0.05 by 1-way ANOVA with Turkey’s multiple comparisons test). (D) CREB1 knockdown in lung fibroblasts reduces the beneficial effects of inhibiting CBX5 in TGF-β–treated fibroblasts. Data are shown as mean ± SEM of 4 independent experiments performed in duplicate (*P < 0.05, ***P < 0.001 by 1-way ANOVA with Turkey’s multiple comparisons test). (E) Schematic representation of the proposed mechanism by which CBX5 binding to PPARGC1A gene promoter masks the CREB1-binding site and inhibits PPARGC1A gene transcription.