Generation and Characterization ofAcetyl‐Mimic and Acetyl‐Deficient Mouse Models
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
ABSTRACT encodes a highly conserved basic helix–loop–helix transcription factor essential for embryonic development from Drosophila to humans. TWIST1 activity is regulated by post‐translational modifications, including phosphorylation during development and cancer metastasis. Recent cancer studies identified acetylation of lysines K73 and K76 as a novel regulatory modification that shifts TWIST1 from a repressive to an activating state during epithelial‐to‐mesenchymal transition (EMT). However, the developmental and in vivo functions of TWIST1 acetylation remain unknown. To investigate the physiological role, we generated the first acetyl‐deficientand acetyl‐mimicmouse models using CRISPR/Cas9‐mediated genome editing. Targeted sequencing confirmed substitutions, and founders were backcrossed onto a C57BL/6J genetic background. Phenotypic analysis revealed thatacetyl‐deficient mice exhibited highly penetrant craniofacial abnormalities, including severe mandibular hypoplasia, mandibular bone fusion, replacement of premaxillary, maxillary, and palatine bones with cartilage, ectopic cartilage‐like structures, ocular malformations, and reduced skull mineralization. In contrast, acetyl‐mimic mutant embryos displayed milder craniofacial defects characterized by reduced mandibular condylar processes and palatine bones, along with decreased skull mineralization. These findings demonstrate that TWIST1 acetylation at K73 and K76 plays a crucial role in normal craniofacial skeletogenesis
Abstract
ABSTRACT encodes a highly conserved basic helix–loop–helix transcription factor essential for embryonic development from Drosophila to humans. TWIST1 activity is regulated by post‐translational modifications, including phosphorylation during development and cancer metastasis. Recent cancer studies identified acetylation of lysines K73 and K76 as a novel regulatory modification that shifts TWIST1 from a repressive to an activating state during epithelial‐to‐mesenchymal transition (EMT). However, the developmental and in vivo functions of TWIST1 acetylation remain unknown. To investigate the physiological role, we generated the first acetyl‐deficientand acetyl‐mimicmouse models using CRISPR/Cas9‐mediated genome editing. Targeted sequencing confirmed substitutions, and founders were backcrossed onto a C57BL/6J genetic background. Phenotypic analysis revealed thatacetyl‐deficient mice exhibited highly penetrant craniofacial abnormalities, including severe mandibular hypoplasia, mandibular bone fusion, replacement of premaxillary, maxillary, and palatine bones with cartilage, ectopic cartilage‐like structures, ocular malformations, and reduced skull mineralization. In contrast, acetyl‐mimic mutant embryos displayed milder craniofacial defects characterized by reduced mandibular condylar processes and palatine bones, along with decreased skull mineralization. These findings demonstrate that TWIST1 acetylation at K73 and K76 plays a crucial role in normal craniofacial skeletogenesis, supporting prior cancer studies showing reduced oncogenic activity following loss of TWIST1 acetylation.