A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

FASEB J. 2014 Jul;28(7):3183-96. doi: 10.1096/fj.13-241760. Epub 2014 Mar 20.


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.

Keywords: DNA methylation; antifibrotic gene; histone deacetylation; histone hypermethylation; lung fibroblasts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Adult
  • Aged
  • Cells, Cultured
  • Chromatin Immunoprecipitation / methods
  • Cyclooxygenase 2 / genetics*
  • Cyclooxygenase 2 / metabolism
  • DNA Methylation / genetics
  • Enhancer of Zeste Homolog 2 Protein
  • Epigenesis, Genetic / genetics*
  • Fibroblasts / metabolism
  • Gene Silencing / physiology*
  • Histocompatibility Antigens / genetics
  • Histocompatibility Antigens / metabolism*
  • Histone-Lysine N-Methyltransferase / genetics
  • Histone-Lysine N-Methyltransferase / metabolism*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Idiopathic Pulmonary Fibrosis / genetics*
  • Idiopathic Pulmonary Fibrosis / metabolism*
  • Male
  • Middle Aged
  • Polycomb Repressive Complex 2 / genetics
  • Polycomb Repressive Complex 2 / metabolism*
  • Promoter Regions, Genetic / genetics
  • RNA, Messenger / genetics
  • Young Adult


  • Histocompatibility Antigens
  • Histones
  • RNA, Messenger
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • EHMT2 protein, human
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein
  • Histone-Lysine N-Methyltransferase
  • Polycomb Repressive Complex 2