Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.