DLX2-FLAG IP-MS in embryonic bodies (EBs): Protein complexes identification

The ectomesenchyme generates much of the craniofacial skeleton, sutures, and diverse connective tissues in the mammalian head, yet its derivation from embryonic stem cells (ESCs) and the underlying molecular drivers remain poorly defined. Here, we identified Dlx2 as a key regulator that efficiently directed murine ESCs towards Msx1+ ectomesenchyme, recapitulating the developmental trajectory. These Msx1+ progenitors expressed classical craniofacial markers and retained robust osteochondral bipotency in vitro and in vivo. To unravel the molecular machinery, we performed immunoprecipitation mass spectrometry (IP-MS) of DLX2-Flag in ectomesenchymal progenitors, revealing LAP2α as a critical interactor. Mechanistically, DLX2 utilized a lamina-associated polypeptide 2 (LAP2)-dependent nuclear chaperoning system, wherein its 38-amino-acid homeodomain motif directly bound LAP2α. This interaction facilitated nucleosome engagement, chromatin remodeling, and activation of the craniofacial ectomesenchymal gene network. Functional validation showed that disrupting the DLX2-LAP2α interface (via domain deletion or competitive peptides) or silencing Dlx2 targets abolished ectomesenchymal differentiation. Our findings establish DLX2 as a pioneer factor in ectomesenchyme specification, with the DLX2-LAP2α axis serving as a linchpin for chromatin accessibility and lineage commitment, offering insights into craniofacial development and stem cell engineering.