The study of the prevalence of cartilage age-CpGs in articular joint tissues and the consequence on target-gene expression, homeostasis and pathogenesis

Abstract

Osteoarthritis is a debilitating musculoskeletal disease having age as one of the biggest risk factors with DNA methylation being reported as a risk factor. DNA methylation is an epigenetic mechanism of gene regulation occurring at cytosine-guanine dinucleotides (CpG). Our group performed a genome-wide DNA methylation analysis of 179 human knee cartilage samples with 716-CpGs showing age-related DNA methylation changes (age-CpGs). The impact on gene expression is unknown, although DNA methylation is usually associated with repression of gene expression. The aims were to assess age-related DNA methylation changes in musculoskeletal tissues and the impact on gene expression, determine DNA methylation impact on transcription factor binding and the cellular consequences of these changes. In total 23 age-CpGs were identified in cartilage, synovium and fat pad tissues using bisulphite pyrosequencing with 17 being across multiple tissues. These were predominantly located in promoter regions with 19 in total, with four in enhancer regions, where methylation has been observed to have a repressive activity on gene transcription. DNA methylation was correlated with repressed target gene expression at 27 CpG sites across musculoskeletal tissues. The promoter region of FHL2 contains highly correlated age-CpGs in musculoskeletal tissues and publicly available datasets and the analysis further focused on the FHL2 region using the electrophoretic mobility shift assay. DNA methylation increased the binding of multiple transcription factors such as Sp1 and Sp3 by up to 2.3-fold. The impact of CRISPR-Cas9 mediated knockdown and a plasmid overexpression vector of FHL2 was studied by RNAsequencing in SW1353 chondrosarcoma cells, and it was observed that this impacted several cellular pathways such as cellular proliferation, cellular metabolism and extracellular matrix synthesis. In conclusion, the current study indicates the importance of age-CpG sites in gene expression regulation during ageing and the study of how these changes could impact cellular functionality