Search

TWIST1 is a basic helix-loop-helix (bHLH) transcription factor in humans that functions in mesoderm differentiation. TWIST1 primarily regulates genes as a transcriptional repressor often through TWIST-Box domain-mediated protein–protein interactions. The TWIST-Box also can function as an activation domain requiring 3 conserved, equidistant amino acids (LXXXFXXXR). Autosomal dominant mutations in TWIST1, including 2 reported in these conserved amino acids (F187L and R191M), lead to craniofacial defects in Saethre–Chotzen syndrome (SCS). Caenorhabditis eleganshas a single TWIST1 homolog, HLH-8, that functions in the differentiation of the muscles responsible for egg laying and defecation. Null alleles in hlh-8lead to severely egg-laying defective and constipated animals due to defects in the corresponding muscles. TWIST1 and HLH-8 share sequence identity in their bHLH regions; however, the domain responsible for the transcriptional activity of HLH-8 is unknown. Sequence alignment suggests that HLH-8 has a TWIST-Box LXXXFXXXR motif; however, its function also is unknown. CRISPR/Cas9 genome editing was utilized to generate a domain deletion and several missense mutations, including those analogous to SCS patients, in the 3 conserved HLH-8 amino acids to investigate their functional role. The TWIST-Box alleles did not phenocopy hlh-8null mutants. The strongest phenotype detected was a retentive (Ret) phenotype with late-stage embryos in the hermaphrodite uterus. Further, GFP reporters of HLH-8 downstream target genes (arg-1::gfpand egl-15::gfp) revealed tissue-specific, target-specific, and allele-specific defects. Overall, the TWIST-Box in HLH-8 is partially required for the protein's transcriptional activity, and the conserved amino acids contribute unequally to the domain's function.Gruss et al. use CRISPR/Cas9-edited strains to investigate the C. elegansTWIST-Box domain in HLH-8; they use full domain deletion, Saethre-Chotzen patient analogous mutations, and alanine substitutions in protein modeling and phenotypic characterization. Surprisingly, the HLH-8 TWIST-Box isn't required for complete transcriptional activation but drives a subset of tissue and target gene specific transcriptional activity. The amino acid substitution mutants reveal a hierarchy of conserved residue importance in the domain, suggesting a mechanism for the mild patient phenotypes.