Integration of transcriptomic and epigenomic analyses uncovered Lrrc15 as a potential contributing factor to cartilage damage in osteoarthritis

Objective: We used the destabilization of the medial meniscus (DMM) model to identify temporal changes in DNA methylation patterns associated with structural and transcriptomic changes in cartilage during osteoarthritis (OA) progression. Methods: RNA sequencing (RNAseq) and Reduced Representation Oxidative Bisulfite Sequencing (RRoxBS) analyses were done in total RNA and DNA obtained from micro-dissected cartilage at 4 and 12 weeks after DMM surgery. Murine and human primary chondrocytes were used to evaluate the cytokine- and methylation-dependent changes in the expression of Lrrc15, and its contribution to IL-1beta-induced changes in chondrocytes. Results: We identified time-dependent alterations in epigenomic patterns in cartilage after DMM, with significant changes in 5mC and 5hmC methylation comparing samples retrieved at 4 and 12 weeks after surgery. Integration of RNAseq and RRoxBS datasets identified Lrrc15 as a hypomethylated gene with increased expression at 4 weeks after surgery. We confirmed LRRC15 immunostaining in OA cartilage, and experiments in human and murine primary chondrocytes showed that the expression of Lrrc15 is DNA methylation-dependent and induced by IL1beta and TNFalfa in vitro. Knockdown experiments showed that Lrrc15 contributes to the IL1beta-driven expression of catabolic genes relevant to OA, including Mmp13. Significance: Our integrative analyses showed that the structural progression of OA is accompanied by transcriptomic and dynamic epigenomic changes in articular cartilage. We found that Lrrc15 is differentially methylated and expressed in OA cartilage, and that it may contribute to the cytokine-driven responses of OA chondrocytes. A better understanding of the role of Lrrc15 in cartilage homeostasis and osteoarthritis may help us uncover targets with therapeutic potential. Knee articular cartilage was harvested from DMM-operated and contralateral/non-operated legs at 4 and 12 weeks post-surgery. Cartilage samples were used for RNA or DNA isolation. Total RNA was used for RNA-seq, and DNA was used for Reduced Representation Oxidative Bisulfite Sequencing (RRoxBS) at the Epigenomics Core at Weill Cornell Medical College. CpG sites in the resulting RRoxBS data were interrogated for methylation patterns and differential methylation and hydroxymethylation using methylKit. The differential methylation data was queried for differentially methylated regions using eDMR. RNA-Seq reads passing Illumina’s purity filter were adapter trimmed using FLEXBAR barcode and adapter processing tool. The trimmed reads were aligned to the Gencode mm10/ GRC38.p3 build of the mouse genome using STAR aligner with default parameters. Read counts for RefSeq (NCBI) transcripts were then quantified from the alignments using the feature Counts software package. Significant differential expression (adjusted p-value < 0.05) was assessed between the read counts for the control and DMM sample groups using the DESeq2 R Bioconductor package. Gene Ontology (GO) enrichment analysis for Biological Processes and Molecular Functions was performed for differentially expressed genes and genes associated with differentially methylated regions using the clusterProfiler Biocondutor R package.