Analysis of 3D Chromatin Structure in Chondrocytes Identifies Putative OA Risk Genes

Genome-wide association studies (GWAS) have identified over 100 loci associated with OA risk, but the majority of OA risk variants are non-coding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multi-omic approach and genome editing to identify and functionally characterize putative OA risk genes. Computational analysis of GWAS and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for OA risk variants. Mapping active enhancers in primary human chondrocytes and intersecting them with OA GWAS variants produced a refined list of putative causal variants. Identifying DNA loops in the chondrocyte cell line C28/I2 using in situ Hi-C allowed us to connect those putative causal variants to target genes and revealed SOCS2 as a putative mediator of OA risk. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from three independent donors led to heightened expression of inflammatory markers in response to a cartilage matrix breakdown product of fibronectin. In total, we identified 56 putative OA risk genes—including 20 that are connected to OA risk SNPs via DNA looping—for further research and potential therapeutic development. Overall design: In situ Hi-C was performed on C-28/I2 cells. C-28/I2 cells were cultured in DMEM/F12 media with 10% fetal bovine serum, 0.1% penicillin streptomycin solution, 0.1% amphotericin B, and 0.04% gentamicin. Cells were treated with DMEM/F12 media with 1% ITS-Plus for 48 hours prior to experiments to promote the chondrocyte phenotype. Cells were then washed with 1X PBS and treated with trypsin-EDTA (0.25%) for 3 minutes. Trypsin was quenched and cells were pelleted at 4°C for 5 minutes at 300g. Cells were resuspended in 1mL DMEM/F12 per million cells and crosslinked in 1% formaldehyde for 10 min with rotation before quenching in a final concentration of 0.2M glycine for 5 min with rotation. Cells were pelleted at 300g for 5 min at 4°C. Pellets were washed with cold PBS and aliquoted into ~3 million cell aliquots. Pellets were flash frozen in liquid nitrogen and stored at -80°C. In situ Hi-C was performed as described by Rao et al. 2014 (PMID: 25497547) for four technical replicates. Cut and run was performed on primary articular chondrocytes. Primary chondrocytes were isolated via enzymatic digestion from human femoral cartilage obtained from two tissue donors without a history of arthritis, as previously described (PMID: 12905472).