KMT2C and KMT2D regulate skeletal development through stage-specific epigenetic control of chondrogenesis

Many craniofacial disorders linked to mutations in enhancer-associated chromatin modifiers, including Kabuki syndrome (KS), present with skeletal abnormalities. KS is a craniofacial developmental disorder characterized by mutations in KMT2D, a histone H3 lysine 4 (H3K4) methyltransferase. The cellular origins and molecular pathways leading to KMT2D-dependent skeletal deficits in KS are not well characterized. We previously demonstrated that KMT2D deletion in mouse cranial neural crest cells (cNCCs), multipotent stem cells contributing to craniofacial structures, impairs chondrocyte hypertrophic differentiation. Although we observed defects in cNCC endochondral ossification, KMT2D's broader skeletal role remains unclear and its functional redundancy in skeletal development with its paralog, KMT2C, is understudied. We now find that KMT2D mutation within chondrocyte lineages results in postnatal growth defects. Chondrocytes deficient in both methyltransferases yield severe deficits in bone formation, revealing redundant roles for KMT2C/D in endochondral ossification. These methyltransferases regulate chondrocyte state transitions as hypertrophic differentiation is impaired with loss of KMT2C/D. When both methyltransferases are deleted in stem cell lineages, cNCCs fail to induce factors downstream of SOX9 activation. At the molecular level, H3K4me1 is lost at de novo chondrocyte enhancers essential for skeletal development, accompanied by reduced H3K27ac deposition, indicating KMT2C/D-dependent enhancer priming. We indicate roles for KMT2C/D in facilitating activation of genes downstream of SOX9 and RUNX2 transcription factors. Our findings define a regulatory cascade mediated by chromatin-level modifications to ensure commitment of chondrocyte lineages, expanding the known roles of histone modifiers and offering insights into the epigenetic underpinnings of skeletal dysplasia and related congenital disorders.