TWIST1 homo- and heterodimers orchestrate specificity control in embryonic stem cell lineage differentiation and craniofacial development

Combinations of bHLH factors dimers generate great functional diversity required for cell type specification in development. The bHLH factor TWIST1 plays pleiotropic roles in craniofacial development. However, which combination of TWIST1 dimers are involved in craniofacial development, and what impact each dimer impose on gene regulation network remained elusive. Proteome profiling of human-TWIST1 expressing cell lines and transcriptome analysis of mouse cranial mesenchyme revealed TWIST1 homodimer and heterodimers with TCF3, TCF4 and TCF12 E-proteins as preferred combinations. We found that balance of homo- and heterodimers were impaired by human disease mutations in TWIST1 Helix domains, which may underpin the developmental defects in haploinsufficiency. Further, loss or gain of function analysis of TWIST1-E-protein dimers in differentiating embryonic stem cells revealed their previously unappreciated roles in lineage specification. TWIST1-E-protein heterodimers activate mesoderm and neural crest differentiation through epithelial-to-mesenchymal transition, whilst TWIST1 homodimers maintained a progenitor-like state and blocked entry to endoderm lineages.