Asymmetric cell division is essential to generate cellular diversity in multicellular organisms. In C. elegans, many asymmetric cell divisions are regulated by the Wnt signaling pathway. To identify genes that function with the Wnt pathway to regulate asymmetric cell division we undertook a genetic screen for
dsh-2 suppressors. Over 60 suppressors were isolated and we have focused our analysis on one of these suppressors, Sup245, which is a strong dominant suppressor of all
dsh-2 phenotypes. In order to determine the molecular identity of Sup245, we performed genetic mapping experiments and whole genome sequencing and identified 3 candidate mutations. Two mutations are intronic (
agef-1,
lagr-1) and one is an exonic mutation in W04A8.6. W04A8.6 is a novel gene and there is no sequence information that lends insight into its function. Since Sup245 is a dominant mutation we could not assume that the exonic mutation is responsible for suppression. For this reason, we undertook a series of RNAi experiments targeting W04A8.6. Knockdown was lethal and therefore these experiments were inconclusive. To unambiguously prove that the mutation in W04A8.6 corresponds to Sup245, we reintroduced the Sup245 specific mutation into the wild-type W04A8.6 gene using CRISPR/Cas9 gene editing technology and have confirmed this mutation suppresses
dsh-2 phenotypes. This mutation is in the 7th and last exon of W04A8.6 and results in a Glutamic Acid to Lysine change. W04A8.6 is located in an operon immediately upstream of
taf-1, a component of TFIID transcriptional complex and predicted to have histone acetyl transferase activity.
taf-1 is notable because our lab has identified another suppressor linked to a histone deacetylase complex. Additionally, the Sup245 mutation is located in a H3K9me1 peak, which is characteristic of transcriptional repression, leading us to hypothesize that the Sup245 mutation might affect
taf-1 expression. We performed RNAi experiments of
taf-1 to test its role in suppression. These experiments were also inconclusive because knockdown of
taf-1 is lethal. However, we have recently molecularly identified a second suppressor, Sup164, which is also a mutation in W04A8.6, but located in the 6th exon. This leads us to suspect that it is alterations of W04A8.6 specifically that are directly responsible for suppression. We are currently undertaking experiments to determine if it is the amino acid changes in W04A8.6 or increase in
taf-1 expression that is responsible for suppression.