Background/Purpose:

DNA methylation is the most characterized epigenetic mechanism and has been recently linked to knee osteoarthritis (OA) pathogenesis. The present study was design to comprehensively compare the methylome and transcriptome of normal and OA knee articular cartilage using the Illumina Infinium HumanMethylation450 beadchip array and next generation RNA sequencing, respectively. Furthermore, we used different in vitroapproaches to experimentally validate the link between DNA methylation and gene expression in human chondrocytes.

Methods:

Genomic DNA was isolated from human macroscopically preserved (N=11) and OA (N=12) knee articular cartilage. After bisulphite treatment, DNA was profiled using the Illumina Infinium HumanMethylation450 beadchip. For transcriptomic analysis, RNA isolated from normal (N=8) and OA (N=10) knee articular cartilage was sequenced using the Illumina HiSeq 2000 platform. In both cases, normalized data was analyzed using a custom-made R bioconductor package. For in vitroexperiments, primary chondrocytes isolated from donors with normal (N=5) cartilage, and TC28 immortalized chondrocytes were used. Cells were cultured in monolayer until confluence, treated with different doses of the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5’Aza) or vehicle, and gene expression was assessed by qPCR.

Results: DNA methylation profiling revealed 2833 differentially methylated sites (DMS) between normal and OA cartilage (p<0.05, FDR<1%, delta β-value>0.15). DMS were significantly enriched in gene bodies and enhancer regions of the genome, and comprised a total of 1279 genes. Among these, 102 transcription factors that harbored DMS were identified. Integrative analysis and subsequent validation showed a subset of 8 transcription factors that were significantly hypermethylated and downregulated in OA cartilage (ATOH8, FOXO3, KLF15, MAFF, NCOR2, RARA, TBX4, and ZBTB16). Upon 5’Aza treatment, TC28 cells showed a significant increase in gene expression for all eight transcription factors. In primary chondrocytes, ATOH8, FOXO3 and TBX4 were increased after 5’Aza treatment

Conclusion:

Our findings reveal that normal and OA knee articular cartilage have significantly different methylomes. The identification of a subset of epigenetically regulated transcription factors with reduced expression in OA may represent an important mechanism to explain changes in the chondrocyte transcriptome and function during OA pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-dna-methylation-and-reduced-expression-of-transcription-factors-in-human-oa-cartilage/