A Twist-Box domain of the C. elegans Twist homolog, HLH-8, plays a complex role in transcriptional regulation.

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 (AD) requiring three conserved, equidistant amino acids (LXXXFXXXR). Autosomal dominant mutations in TWIST1, including two reported in these conserved amino acids (F187L and R191M), lead to craniofacial defects in Saethre-Chotzen syndrome (SCS). Caenorhabditis elegans has a single TWIST1 homolog, HLH-8, that functions in the differentiation of the muscles responsible for egg laying and defecation. Null alleles in hlh-8 lead to severely egg-laying defective (Egl) and constipated (Con) 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 three conserved HLH-8 amino acids to investigate their functional role. The TWIST-Box alleles did not phenocopy hlh-8 null 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::gfp and 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.