Hyper-physiologic mechanical cues, as an osteoarthritis disease relevant environmental perturbation, cause a critical shift in set-points of methylation at transcriptionally active CpG sites in neo-cartilage organoids

Abstract

Background: Osteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stiffness, pain, and a significant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (1). It has been shown that post-mitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state.

Results: We showed that hyper-physiological loading evokes consistent changes in ML-tCpGs associated with expression changes in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology, we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity.

Conclusion: Our findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes, concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1, ITGA5, and CD44 are subject to such changes and are central and overlapping with OA-tCPGs of primary chondrocytes, we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting, detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.

Keywords: Chondrocytes; DNA Methylation; Environmental stressors; Mechanical Loading; Osteoarthritis.

Figure 1. Effect of hyper-physiological loading on the genome-wide methylation

(A) A volcano plot of the methylome-wide response to hyper-physiological loading conditions. Blue dots denote CpG sites mapped to a gene body with increased methylation, FDR<0.01, and red dots represent CpG sites mapped to a gene body that are de-methylated in response to hyper-physiological loading conditions as determined by MEAL. (B) Manhattan plot of differentially methylated CpG sites with their genomic mapped genes. The horizontal red line represents the FDR<0.05 threshold. (C) Enrichment of significant DMs within chromatin states; active transcription start site (TSS), proximal promoter states (TssA, TssAFlnk), a transcribed state at the 5′ and 3′ ends of genes showing both promoter and enhancer signatures (TxFlnk), actively transcribed states (Tx, TxWk), enhancer states (Enh, EnhG), and a state associated with zinc finger protein genes (ZNF/Rpts). The inactive states consist of constitutive heterochromatin (Het), bivalent regulatory states (TssBiv, BivFlnk, EnhBiv), repressed Polycomb states (ReprPC, ReprPCWk), and a quiescent state (Quies) (D) Notable examples of differentially methylated mapped CpGs in response to hyper-physiological mechanical loading conditions. The box plots represent the 25th, 50th, and 75th percentiles, and whiskers extend to 1.5 times the interquartile range. Individual samples are depicted by black dots in each graph. *FDR<0.05, **FDR<0.01, ***FDR<0.001.

Figure 2. Circos Plot of Transcriptionally active DM CpG sites.

The inner Circle displays the chromosomes. The middle circle displays change in percentage of the methylation of the 2492 DM-CpG’s that mapped to a gene site. Red bars depict de-methylated CpG-sites, blue bars depict increased Methylated CpG-sites. (FDR<0.01) The outside circle displays the ²log fold change of the 169 unique DE ML-tCpG-Genes. Red dots depict downregulation, blue dots depict upregulation. (FDR<0.05)

Figure 3. Epigenetically regulated transcriptome pathway enrichment analysis.

(A) Protein-protein network of ML-tCpG – genes as determined by STRING-DB. (B) Differential gene expression of genes to which a DM CpG was mapped. (C) Differential methylation of CpG sites mapped to the gene body. The box plots represent 25th, 50th, and 75th percentiles, and whiskers extend to 1.5 times the interquartile range. Individual samples are depicted by black dots in each graph. *FDR<0.05, ***FDR<0.001. (D) Top 10 most significantly enriched pathways of epigenetically regulated DEG. FDR<0.05. (E) Gene-pathway network of notable enriched pathways where lines depict the relationship between the genes and the pathways determined by enrichment analysis. Blue dots depict downregulated DEGs in response to hyper-physiologic mechanical loadings conditions, and red dots depict upregulated DEGs in response to hyper-physiologic mechanical loading conditions.