Abstract
Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49−79%), H3K27me3 (44−81%), and DNA methylation (61−97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.—Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.
Highlights
Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF)
We found that under unstimulated conditions, histone H3 lysine trimethylation (H3K4me3) at the COX-2 promoter was slightly lower in fibroblast from IPF lung (F-IPF) than in fibroblast from nonfibrotic lung (F-NL), whereas it was the opposite for histone H3 lysine trimethylation (H3K9me3) and H3K27me3
Treatment with IL-1 resulted in a marked increase in H3K4me3 in F-NLs (PϽ0.01 at 1 h after stimulation compared with unstimulated) but not in F-IPFs, and the level of H3K4me3 at the COX-2 promoter was significantly lower in F-IPFs than in F-NLs (Fig. 1A)
Summary
Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt inhibitor markedly reduced H3K9me (49؊79%), H3K27me (44؊81%), and DNA methylation (61؊97%) at the COX-2 promoter. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt inhibitor markedly reduced H3K9me (49؊79%), H3K27me (44؊81%), and DNA methylation (61؊97%) at the COX-2 promoter DNA methylation by Dnmts at 5-cytosine at CpG islands of gene promoters is the most common epigenetic modification associated with transcriptional silencing. Recent studies suggested that intimate communication and mutual dependence exist between DNA methylation and histone modifications in the process of gene silencing. It is likely that neither of the repressive epigenetic mechanisms can be universally applied to the silencing of specific genes, because this may be dependent on cell type and physiological or pathophysiological context
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