Effects of hypo O-GlcNAcylation on Drosophila development

Daniel N. Mariappa, Andrew Ferenbach, Daan M. F. van Aalten (Lead / Corresponding author)

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6 Citations (Scopus)
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Abstract

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with N-acetylglucosamine (OGlcNAcylation) 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 OGlcNAc 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.
Original languageEnglish
Pages (from-to)7209-7221
Number of pages13
JournalJournal of Biological Chemistry
Volume293
Issue number19
Early online date27 Mar 2018
DOIs
Publication statusPublished - 11 May 2018

Keywords

  • O-GlcNAc
  • O-GlcNAc transferase
  • host cell factor
  • CRISPR/Cas9
  • Drosophila development
  • polycomb
  • Mediator complex
  • post-translational protein modification
  • hypomorphic sxc mutant

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