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.
Highlights
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, O-GlcNAc transferase (OGT) ( known as sxc, referred to only as sxc) mutants die as pharate adults [21]. sxc is a Polycomb group (PcG) gene that contributes to control of HOX gene expression and specification of segmental identity [16]
To enable reliable and physiological phenotypic characterization of the requirement of the O-GlcNAc modification for Drosophila development, we embarked on producing a precise hypomorphic OGT point mutant, sxcH537A and a catalytically dead mutant, sxcK872M utilizing the CRISPR/Cas9 gene-editing technology in combination with homologous recombination (Fig. 1A, Table S1)
Summary
Given that sxc is a maternal effect gene and resides at a locus that is not amenable to producing germ line clones lacking the maternal copy using the FRT-flipase system, current approaches to eliminate the maternal copy have relied on using the UAS-GAL4 system [8, 46]. In addition to the necessary mutations changing the codon to Ala in place of His at position 537 or Met in place of Lys at position 872, silent mutations were introduced in wobble positions of adjacent codons (Fig. 1C) This strategy was employed to decrease the chances of the repaired DNA being subjected to further Cas nuclease cleavage and to enable a robust screening assay exploiting the elimination of TaqI (H537A) or XhoI (K872M) restriction enzyme sites (Fig. 1C). Both the sgRNA and the repair plasmids were injected into the vasa::Cas fly line [47]. At least five individual F1 males from each of the 23 (H537A) and 8 (K872M) fertile parental lines were assessed in this manner
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