Stage-Specific Epigenetic Landscapes in Skeletal Development: Coordination of Histone Methyltransferases and Transcription Factor Function

Skeletal development requires the precise coordination of epigenetic modifiers and lineage-specific transcription factors, such as SOX9 and RUNX2, to drive chondrocyte and osteoblast differentiation. While SOX9 canonically precedes RUNX2 activation and osteochondral differentiation, we observe an alternative regulatory logic by characterization of the temporal activation of these factors in mouse cranial neural crest (cNCC)-derived tissues. We find that RUNX2 is broadly expressed in the first branchial arch prior to lineage determination, validated by a green fluorescent protein (GFP) reporter inserted in the 3′ untranslated region (3′UTR) of Runx2. Although this novel Runx2 reporter preserves 3’UTR sequences, homozygous Runx2GFP/GFP exhibited enhanced RUNX2 protein levels, leading to accelerated chondrocyte hypertrophic differentiation and impaired ossification. These phenotypes recapitulate human metaphyseal dysplasia with maxillary hypoplasia and brachydactyly, demonstrating that skeletal maturation is acutely sensitive to RUNX2 dosage. To define the epigenetic landscape required for these factors to function, we investigated the histone modifiers KMT2C and KMT2D. Kabuki Syndrome (KS) is a craniofacial development disorder characterized by mutations in KMT2D, a histone H3 lysine 4 (H3K4) methyltransferase. KMT2D cellular origins and molecular pathways leading to skeletal deficits in KS are not well characterized. Although we previously 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 KMT2C and KMT2D mutation within chondrocyte lineages result in severe bone formation deficits, revealing partially redundant roles in chondrocyte specification, though KMT2D remains the predominant methyltransferase required for the transition to hypertrophic differentiation. At the molecular level, loss of KMT2C/D leads to a global loss of H3K4me1 and reduced H3K27ac at de novo chondrogenic enhancers, suggesting that KMT2C/D are essential for enhancer priming. We also indicate roles for KMT2C/D in facilitating the activation of gene programs downstream of SOX9 and RUNX2. Together, our findings delineate essential interactions between the epigenetic landscape and lineage-specific transcription factors during chondrogenesis. This work expands the current paradigm of cell identity regulation in skeletal development and provides a clear molecular basis for some of the systemic pathologies observed in human skeletal dysplasia and chromatinopathies.