Cell type-specific regulation of the TGF-beta-responsive alpha2(I) collagen gene by CpG methylation

The alpha2(I) collagen gene shows cell type-specific expression, however, the mechanism behind this specificity remains to be determined. We demonstrate here that transforming growth factor-beta (TGF-beta)-mediated induction of alpha2(I) collagen gene is regulated by DNA methylation in a cell type-specific manner. Human alpha2(I) collagen mRNA and type I collagen protein were expressed in normal human fibroblasts (NHF), and also strongly enhanced by TGF-beta; they were not detected in HaCaT, HeLa, or HepG2 cells (termed "collagen-induction resistant (CIR) cells") even following stimulation with TGF-beta. On the other hand, the transcriptional activity of exogenously transfected alpha2(I) collagen promoter was clearly up-regulated by TGF-beta in the CIR cells as well as in NHF. In the CIR cells, CpG clusters around the transcription start site of the alpha2(I) collagen gene were heavily methylated, whereas no methylation was detected in NHF. Moreover, alpha2(I) collagen gene was reactivated in the CIR cells by 5-Aza-2-deoxycytidine (5-AdC) treatment to some extent. However, demethylation by 5-AdC was limited and it was unable to recover the TGF-beta responsiveness. In NHF, the alpha2(I) collagen gene has a Smad3-accessible chromatin structure and acetylated histones in the promoter regions. By contrast, in the CIR cells, Smad3 failed to bind to the chromatin and histones were not acetylated in this area. Furthermore, in vitro methylation of the reporter gene containing the alpha2(I) collagen promoter significantly reduced both basal and TGF-beta-induced enhancement of the transcriptional activity in NHF. Thus, we propose that alpha2(I) collagen gene provides the first example of the TGF-beta responsive gene whose cell type-specificity is regulated by CpG methylation.