Identification of distinctive DNA methylation patterns correlated with differential gene expression in murine hypertrophic chondrocytes in vitro

Abstract

Purpose: Osteoarthritis (OA) is joint disorder in which the articular cartilage is permanently damaged. Articular chondrocytes undergo profound phenotypic changes in OA, part of which mimic developmental steps observed in hypertrophic growth plate chondrocytes, including increased Runx2, Mmp13 and collagen type X expression. Hypertrophy is a normal step during endochondral ossification processes, which are required for normal chondrogenesis, long bone growth and ossification. Thus, to better understand the molecular mechanisms that trigger a hypertrophy-like reprogramming of articular chondocytes in osteoarthritis, we require a better understanding of gene regulation during hypertrophy. DNA methylation is one of the principal mechanisms by which cells maintain dominant and stable phenotypes, and altered DNA methylation is associated with abnormal gene expression in different pathologies, including OA. This study aims to identify genes that are differentially expressed during hypertrophic differentiation in vitro, and that are correlated with changes in DNA methylation patterns. Methods: Chondrocytes were isolated from articular cartilage obtained from 5 to 6 days old C57BL/6J mice. We used 3D/pellet culture systems to drive chondrocyte hypertrophy in vitro. Pellets were harvested at 7 and 21 days for RNA or DNA isolation. Total RNA was used for RNA sequencing, and DNA was used for Reduced Representation Oxidative Bisulfite Sequencing (RRoxBS) at the Epigenomics Core at Weill Cornell Medicine (EC-WCM). After sequencing, the RNAseq reads were processed using a dedicated RNAseq pipeline, and the expression of selected genes was further validated using SYBR-green-based real-time PCR analyses (RTqPCR). For methylation profiling, libraries were made using the ERRBS protocol. Per sample, 50-60 million ERRBS reads were aligned and processed using the EC-WCM’s in-house bioinformatics pipeline to yield methylation values for each CpG. Oxidative bisulfite (oxBS) technology was applied to distinguish between 5mC and 5hmC. Differential DNA methylation was analyzed using Methylkit and eDMR, and subsequent assessment and annotation of differentially methylated regions was done using R Core Team. Results: Hypertrophic differentiation in vitro was confirmed by RTqPCR analyses, which showed upregulation of Mmp13, collagen X and Runx2 and down-regulation of Sox9 and collagen II. RNAseq analyses uncovered more than 3000 differentially expressed genes between 7 (non hypertrophic) and 21 (hypertrophic) days (p less than 0.01). Among these genes, we identified novel targets with previously unreported roles in chondrocyte hypertrophy. Analysis of RRoxBS sequencing data also revealed changes in DNA methylation patterns accompanying hypertrophic differentiation and differential expression of hypertophic-related genes, highlighting the contribution of DNA methylation to gene expression during chondrocyte hypertrophy. Conclusions: Our results suggest that DNA methylation significantly contributes to changes in phenotype and gene expression during chondrocyte hypertrophy. In addition, we found a number of differentially expressed genes with previously unreported roles in hypertrophic chondrocytes, and with potential relevance to developmental and pathological processes. Ongoing studies are aimed to verify the results obtained in vitro using in vivo systems of chondrocyte hypertrophy during developmental processes (i.e., growth plate chondrocytes) and of hypertrophic-like conversion during OA, using surgical murine models of OA and human samples obtained from OA patients. We believe that our novel integrative approaches have the potential of uncovering new targets for therapeutic intervention.