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Effects of hypo-O-GlcNAcylation on Drosophila development

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)) d...

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Autores principales: Mariappa, Daniel, Ferenbach, Andrew T., van Aalten, Daan M. F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5950000/
https://www.ncbi.nlm.nih.gov/pubmed/29588363
http://dx.doi.org/10.1074/jbc.RA118.002580
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author Mariappa, Daniel
Ferenbach, Andrew T.
van Aalten, Daan M. F.
author_facet Mariappa, Daniel
Ferenbach, Andrew T.
van Aalten, Daan M. F.
author_sort Mariappa, Daniel
collection PubMed
description 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 sxc(K872M), was recessive lethal, whereas a second mutant, the hypomorphic sxc(H537A), was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxc(H537A) mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc(H537A) 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 sxc(H537A) 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 sxc(H537A) 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.
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spelling pubmed-59500002018-05-14 Effects of hypo-O-GlcNAcylation on Drosophila development Mariappa, Daniel Ferenbach, Andrew T. van Aalten, Daan M. F. J Biol Chem Glycobiology and Extracellular Matrices 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 sxc(K872M), was recessive lethal, whereas a second mutant, the hypomorphic sxc(H537A), was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxc(H537A) mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc(H537A) 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 sxc(H537A) 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 sxc(H537A) 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. American Society for Biochemistry and Molecular Biology 2018-05-11 2018-03-27 /pmc/articles/PMC5950000/ /pubmed/29588363 http://dx.doi.org/10.1074/jbc.RA118.002580 Text en © 2018 Mariappa et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Glycobiology and Extracellular Matrices
Mariappa, Daniel
Ferenbach, Andrew T.
van Aalten, Daan M. F.
Effects of hypo-O-GlcNAcylation on Drosophila development
title Effects of hypo-O-GlcNAcylation on Drosophila development
title_full Effects of hypo-O-GlcNAcylation on Drosophila development
title_fullStr Effects of hypo-O-GlcNAcylation on Drosophila development
title_full_unstemmed Effects of hypo-O-GlcNAcylation on Drosophila development
title_short Effects of hypo-O-GlcNAcylation on Drosophila development
title_sort effects of hypo-o-glcnacylation on drosophila development
topic Glycobiology and Extracellular Matrices
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5950000/
https://www.ncbi.nlm.nih.gov/pubmed/29588363
http://dx.doi.org/10.1074/jbc.RA118.002580
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