Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape

Abstract

DNA methylation (5-mC) is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation (5-hmC) is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and Western blotting. Reduced representation bisulfite sequencing (RRBS) method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. Our data suggest that DNA 5-mC/5-hmC is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.