Effects of Hypo--GlcNAcylation on Development

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc (-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In , null mutants of the Polycomb gene -GlcNAc transferase (; also known as super sex combs ()) display homeotic phenotypes. To dissect the requirement for -GlcNAc signaling in development, we used CRISPR/Cas9 gene editing to generate rationally designed catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive , was recessive lethal, whereas a second mutant, the hypomorphic , was homozygous viable. We observed that reduced total protein -GlcNAcylation in the mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between and a null allele of host cell factor (), encoding an extensively -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in flies lacking a copy of skuld (), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant enabled us to identify pleiotropic effects of globally reduced protein -GlcNAc during development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for development.