Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes

Abstract

Objective: To examine the genome-wide distribution of hydroxymethylated cytosine (5hmC) in osteoarthritic (OA) and normal chondrocytes in order to investigate the effect on OA-specific gene expression.

Methods: Cartilage was obtained from OA patients undergoing total knee arthroplasty or from control patients undergoing anterior cruciate ligament reconstruction. Genome-wide sequencing of 5hmC-enriched DNA was performed in a small cohort of normal and OA chondrocytes to identify differentially hydroxymethylated regions (DhMRs) in OA chondrocytes. Data from the genome-wide sequencing of 5hmC-enriched DNA were intersected with global OA gene expression data to define subsets of genes and pathways potentially affected by increased 5hmC levels in OA chondrocytes.

Results: A total of 70,591 DhMRs were identified in OA chondrocytes as compared to normal chondrocytes, 44,288 (63%) of which were increased in OA chondrocytes. The majority of DhMRs (66%) were gained in gene bodies. Increased DhMRs were observed in ∼50% of genes previously implicated in OA pathology including MMP3, LRP5, GDF5, and COL11A1. Furthermore, analyses of gene expression data revealed gene body gain of 5hmC appears to be preferentially associated with activated, but not repressed, genes in OA chondrocytes.

Conclusion: This study provides the first genome-wide profiling of 5hmC distribution in OA chondrocytes. We had previously reported a global increase in 5hmC levels in OA chondrocytes. Gain of 5hmC in the gene body is found to be characteristic of activated genes in OA chondrocytes, highlighting the influence of 5hmC as an epigenetic mark in OA. In addition, this study identifies multiple OA-associated genes that are potentially regulated either singularly by gain of DNA hydroxymethylation or in combination with loss of DNA methylation.

Figure 1. Genome-wide increases in 5hmC are associated with osteoarthritic (OA) chondrocytes

A. Visualization of the 5hmC distribution across a representative chromosome in normal (donor ages = 24 weeks, 6 months, 27 and 34 years) and OA chondrocytes (donor ages = 61, 65, 73 and 74 years). B. Composite profile of the 5hmC distribution averaged across all genes in representative normal and OA chondrocyte samples. TSS = transcriptional start site, TES = transcriptional end site. C. The number and genomic location of the DhMRs that are increased and decreased in OA chondrocytes when compared to normal chondrocytes. ‘Other Intergenic’ = DhMR identified was not located in any of the other genomic locations. D. E. Pathway analysis of the genes with DhMRs with ≥2-fold increase within 3kb of the transcriptional start site (Promoter 3k) (D) and in the top 10% of DhMRs with a ≥2-fold increase in the gene body (E) in OA when compared to normal chondrocytes. F. G. Pathway analysis of the genes with DhMRs with ≥2-fold decrease within 3kb of the transcriptional start site (Promoter 3k) (F) and in the bottom 10% of DhMRs with ≥2-fold decrease in the gene body in OA when compared to normal chondrocytes. Corresponding p-values are shown.

Figure 2. 5hmC is enriched in the gene bodies of genes activated in OA

A. Heatmap of global gene expression changes between normal (donor ages = 24 weeks, 6 months, 18 months, 27 and 34 years) and OA (donor ages = 64 and 74 years) chondrocytes. Red = higher than mean expression, blue = lower than mean expression. B. C. Pathway analysis of the genes with ≥1.5-fold increases in expression (activated genes) (B) and of the genes with ≥1.5-fold decreases in expression (repressed genes) in OA (C). PEL = Platelet-endothelialleucocyte, dev = development, rearr = rearrangement. Corresponding p-values are shown. D. E. Composite profiles of the average 5hmC distributions in normal (blue) and OA (red) chondrocytes in genes with a ≥1.5-fold increase in expression (activated genes) (D) and in genes with a ≥1.5-fold decrease in expression (repressed genes) in OA when compared to normal chondrocytes. F. G. Composite profile of the average 5hmC distribution in normal (blue) and OA (red) chondrocytes in genes identified by Aigner and colleagues, 2006 (35) as being activated in OA (≥2-fold increase in expression) (F) and repressed in OA (≥2-fold decrease in expression) (G). TSS = transcriptional start site, TES = transcriptional end site.

Figure 3. OA susceptibility genes display increases in 5hmC

A. Composite profile of the average 5hmC distribution in normal (blue) and OA (red) chondrocytes in genes identified as being associated with OA by GWAS studies. TSS = transcriptional start site, TES = transcriptional end site. B. OA susceptibility genes, identified by GWAS studies that have a ≥2-fold increase in 5hmC in OA patients. Out of a total of 103 genes, 52 genes displayed a ≥2-fold increase in 5hmC. C. Compiled list of genes that have been previously identified as being hypomethylated or hypermethyalted in OA. The related study is indicated in parentheses.

Figure 4. Pathways and networks known to be perturbed in OA are regulated by aberrant 5hmC gain in the disease state

A. The genomic locations of DhMRs that show a ≥2-fold increase in OA chondrocytes when compared to normal chondrocytes in genes that have a ≥1.2-fold increase in gene expression in OA. Gene body (All) = All DhMRs, Gene body (Top 10%) = Top 10% of DhMRs in gene bodies. B. C. Pathway analysis of the genes that have increased expression and increased DhMRs within 3kb of the promoter (B) or in the top 10% of DhMRs in the gene body (C) in OA. The top 5 networks, ranked by p-value, are shown. D. The genomic locations of DhMRs that show a ≥2-fold decrease in OA when compared to normal chondrocytes in genes that have a ≥1.2-fold decrease in gene expression in OA. E. F. Pathway analysis of the genes that have decreased expression and decreased DhMRs within 3kb of the promoter (E) or in the bottom10% of DhMRs that are decreased in the gene body (F) in OA. The top 5 networks, ranked by p-value, are shown.

Figure 5. 5hmC containing genes in the Notch signaling pathway in OA

A. The Notch signaling network. Genes with ≥2-fold increased 5hmC and ≥1.2-fold increased expression in OA are marked with a red dot. B. A list of genes in the Notch signaling network that have ≥2-fold increased 5hmC and ≥1.2-fold increased expression in OA chondrocytes when compared to normal chondrocytes.