Effects of hypo-O-GlcNAcylation on Drosophila development

Abstract

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the Polycomb gene O-GlcNAc transferase (OGT; also known as super sex combs (sxc)) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc 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 sxcK872M, was recessive lethal, whereas a second mutant, the hypomorphic sxcH537A, was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxcH537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxcH537A and a null allele of Drosophila host cell factor (dHcf), encoding an extensively O-GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxcH537A flies lacking a copy of skuld (skd), 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 sxcH537A enabled us to identify pleiotropic effects of globally reduced protein O-GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development.

Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability

“…Lack of functional OGT causes lethality and developmental arrest in most metazoan model organisms, including mice, frogs, zebrafish, and fruit flies (19, 22, 24, 61). To probe if the N567K mutation directly affects OGT catalytic activity in vivo, we exploited the genetically tractable system D. melanogaster , where phenotypes arisen due to null and hypomorphic alleles of sxc (the fly ogt ortholog) have been described (62, 63). Drosophila sxc null mutants ( sxc 1 and sxc 6 ) die at the pharate adult stage with homeotic transformation defects (62).…”

Intellectual disability and autism spectrum disorders ‘on the fly’: insights from Drosophila

“…For such attempts, either transgenes expressing the human mutant proteins, or transgenes expressing the Drosophila genes with engineered, analogous mutations, can be used. Alternatively, gene replacement by homologous recombination and CRISPR/Cas9 genome-editing approaches now allow manipulation of the fly gene at its endogenous locus (de Brouwer et al, 2018; Mariappa et al, 2018) (Fig. 2B).…”

Regulation of BMP Signaling by O-GlcNAcylation

“…In this regard, sxc 1 and sxc 6 mutant cuticles display no denticle belts ( Figure 1E) while sxc 3 , sxc 5 , and sxc 7 cuticles display hypotrophied denticle belts ( Figure 1F). We also showed that the cuticle phenotype of animals harboring the sxc 1 allele in trans to a non-complementing deficiency is indistinguishable from that of sxc 1 homozygotes ( Figure 1G), providing genetic evidence that the sxc 1 nonsense allele is, as previously suspected, null (Mariappa et al, 2018;Sinclair et al, 2009). Given that sxc 1 exhibits the greatest level of embryonic lethality, the strongest cuticle defect, and is genetically defined as null, we chose this allele for all further experiments.…”