HLH-8 is a transcription factor that is required for a subset of mesoderm development in C. elegans. The human homolog TWIST1 is mutated in craniofacial disease. This work focuses on utilizing CRISPR/Cas9 gene editing techniques to make patient mutations to three equidistant and evolutionarily conserved amino acids in HLH-8 to identify a structure/function relationship of a potential transcriptional activation domain. The three amino acid substitutions in the HLH-8 protein are L95F, F99L, and R103M where the latter two are mutations found in patients. Previously, null mutations of
hlh-8 lead to constipation and egg-laying defects in C. elegans, however these designed mutations seem wild type. Upon confirming the mutant strain establishment by sequencing, fluorescent reporter genes of HLH-8 target genes were crossed in the mutant worms and the expression pattern was assessed as a measure of transcriptional activation. After crossing two different reporter genes,
arg-1::GFP and
egl-15::GFP, into the mutants, only the
arg-1::GFP expression was altered. The severity of
arg-1::GFP expression alteration is allele specific with respect to the HLH-8 mutation. L95F;
arg-1::GFP is the mildest phenotype with only select enteric muscles (ENT) impacted and wild type vulval muscle (VM) and head mesodermal cell (HMC) expression. However, both the F99L;
arg-1::GFP and R103M;
arg-1::GFP strains have a complete loss of expression in the VM, and the F99L;
arg-1::GFP having a more severe depletion in the HMC and ENT in comparison to the R103M;
arg-1::GFP strain. The F99L;
arg-1::GFP and R103M;
arg-1::GFP strains were used in a suppressor screen to restore VM expression. In total, approximately 105,600 haploid genomes for F99L;
arg-1::GFP and 427,200 haploid genomes for R103M;
arg-1::GFP have been screened through and currently work is in progress on following the promising candidates. The aim of this work is to potentially identify new factors to can work with HLH-8 to influence transcription in a spatiotemporal manner throughout development.