Fadial, E.; Hansen, V.; Kulzhanova, G.; Tashbib, E. T.; Chinta, D.; Klee, A.; Shammas, H.; Pradhan, G.; Wu, C.-L.

Cartilage development and homeostasis require precise regulation by transcriptional and epigenetic networks. PRDM16 is a transcription factor containing zinc finger domains that enable protein-DNA and protein-protein interactions, as well as domains with the capacity for histone methyltransferase activity. However, the detailed molecular mechanisms by which PRDM16 regulates chondrogenesis and chondrocyte identities remain largely unknown. Using our osteochondral lineage-specific, conditional knockout mouse model (Col2a1Cre;Prdm16flox/flox, Prdm16 cKO), we found that loss of Prdm16 in osteochondral lineage cells delays, but does not fully inhibit, endochondral ossification and bone formation in the knee joint. Furthermore, Prdm16 cKO male mice exhibit comparable OA severity between injured and non-injured joints, suggesting that PRDM16 may exert a chondroprotective function. In our hiPSC-derived chondrocyte model, we observed significantly reduced pellet size and DNA content in cells with modulated PRDM16 expression compared to Control, implying a link between PRDM16 and chondrocyte viability. Integrated analysis of single cell RNA-sequencing and CUT&RUN-sequencing revealed that PRDM16 regulates chondrocyte cell fate decisions by altering chromatin accessibility and DNA binding at promoter/enhancer regions of genes essential for chondrogenesis and chondrocyte hypertrophy. Indeed, PRDM16 governs the expression of key chondrogenic regulators including SOX9, ARID5A, SMOC2, HAND2, and hypertrophic driver MEF2C. Overall, our results provide evidence that PRDM16 serves as an essential genetic and epigenetic regulator of chondrogenesis and chondrocyte phenotype specification in the knee joint through DNA binding and by modulating H3K4me3 histone mark deposition.