MeCP2 epigenetically regulates alpha-smooth muscle actin in human lung fibroblasts.

ABSTRACT: BACKGROUND:A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (?-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating ?-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in ?-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of ?-SMA expression. METHODS:The association of MeCP2 with ?-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor ?1 (TGF-?1). The regulation of ?-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on ?-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of ?-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS:The human lung fibroblasts with increased ?-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced ?-SMA upregulation by TGF-?1. The 5'-Aza-treated cells have decreased ?-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of ?-SMA after being treated by TGF-?1 or 5'-aza. CONCLUSION:Our data indicate that the methyl-binding protein MeCP2 is critical for ?-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of ?-SMA is not a determinative factor for its inducible expression.

SUBMITTER: Xiang Z 

PROVIDER: S-EPMC7263943 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature