Increased DNA Methylation and Reduced Expression of Transcription Factors in Human Osteoarthritis Cartilage

Abstract

Objectives To analyze the methylome of normal and osteoarthritis (OA) knee articular cartilage and determine the role of DNA methylation in the regulation of gene expression in vitro. Methods DNA was isolated from human normal (N=11) and OA (N=12) knee articular cartilage and analyzed by an Infinium HumanMethylation450 BeadChip methylation array. To integrate methylation and transcription, RNAseq was performed on normal and OA cartilage validated by qPCR. Functional validation was performed in the human TC28 cell line and primary chondrocytes that were treated with the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5’Aza). Results DNA methylation profiling revealed 929 differentially methylated sites (DMS) between normal and OA cartilage that comprised a total of 500 genes. Among these, 45 transcription factors that harbored DMS were identified. Integrative analysis and subsequent validation showed a subset of 6 transcription factors that were significantly hypermethylated and downregulated in OA cartilage (ATOH8, MAFF, NCOR2, TBX4, ZBTB16 and ZHX2). Upon 5’Aza treatment, TC28 cells showed a significant increase in gene expression for all six transcription factors. In primary chondrocytes, ATOH8 and TBX4 were increased after 5’Aza treatment. Conclusions 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.

Assessment of DNA methylation patterns in the bone and cartilage of a nonhuman primate model of osteoarthritis

“…49 As of yet, none of the remaining DMPs and their annotated genes have been identified as candidate methylation loci in human OA studies. [4][5][6][7][8][9][10] Nevertheless, some DMPs are located in close proximity to genes that have been previously associated with OA, including CRTC1 50 , CRLF1 51 , COMP 52 , SMAD9 53,54 , and ALOX12 55,56 (Table 1). However, the mechanisms by which these DMPs might influence the expression of these distally related genes is unclear.…”

“…In particular, DNA methylation which regulates gene expression, is thought to play an influential role in the development of degenerative skeletal disorders like OA. [4][5][6][7][8][9][10] Animal models, such as mice, rats, rabbits, guinea pigs, dogs, sheep, goats, and horses, have been essential in discerning some of the processes inherent to OA development. 11,12 Nevertheless, all of these animals are limited in their abilities to fully inform our understanding of human OA, so the search to find a gold standard animal model for OA is still ongoing.…”

Phenotypic instability of chondrocytes in osteoarthritis: on a path to hypertrophy

“…152 One study has shown an overall increase in methylation accompanied by reduced expression of transcription factors in OA compared to healthy cartilage. 153 Changes in DNA methylation associated with OA progression are also observed in subchondral bone, with an enrichment of genes involved in morphogenesis and development of the skeletal system in OA samples, 154 highlighting the contributions of developmental pathways in the disease context. Importantly, the transcription of genes encoding regulatory factors, including DIO2, SOX9, RUNX2, ELF3, and GDF5, is in part modulated by DNA methylation.…”

Transcriptional profiling of articular cartilage in a porcine model of early post‐traumatic osteoarthritis

“…However, RNA-seq has the unique ability to determine the relative expression levels of all genes within a cell or tissue. This technique has not yet been widely used on articular cartilage, [8][9][10][11][12][13][14] and to the best of our knowledge, the transcriptome-wide profile of articular cartilage obtained from healthy knee joints has not been reported.…”