DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Matt J Barter,David A Young,Catherine Bui,Kathleen Cheung,Julia Falk,Andrew J Skelton,Hannah R Elliott,Rodolfo Gómez,Louise N Reynard

doi:10.1038/s41598-020-58093-5

Abstract

Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450 K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis.

Highlights

Our skeleton acts as an essential framework for the overall structure of our body
A number of DMLs are found at key cartilage gene loci such as ACAN and SOX9 (Fig. 1C,D). 12 of the DMLs associated with genes exhibiting significant expression changes during chondrogenesis were selected for pyrosequencing analysis in an independent chondrogenic differentiation of mesenchymal stem cells (MSCs) (Supplementary Fig. 1)[23]
We defined differentially methylated regions (DMRs) as regions containing >1 differentially methylated CpG with a maximum separation of 1000 bp. 1276 DMRs are found during MSC chondrogenesis (Supplementary Table 2), including a DMR found at the promoter of SNORC (C2orf82), the transcript of which we found previously as the most upregulated during MSC chondrogenesis, and a DMR in WWP2 a key cartilage development protein upregulated during chondrogenesis (Fig. 1E,F)[23,26]

Summary

Introduction

Our skeleton acts as an essential framework for the overall structure of our body. Accurate generation of this frame is dependent upon carefully controlled chondrocyte differentiation and cartilage formation, processes that underpin the development of the long and short bones of the skeleton[1]. During development and cell differentiation DNA methylation is dynamic, correlating with changes in gene expression[8,9]. In order to identify CpG methylation changes during MSC chondrogenic differentiation a DNA methylation profile was generated with the Infinium HumanMethylation[450] BeadChip.

Results

Conclusion