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Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster

Abstract

N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

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Figure 1: A combined metabolic feeding–chemoselective ligation strategy enables labeling of chromatin-associated proteins from Drosophila S2 cells.
Figure 2: Metabolic feeding combined with antibody-free genome-wide chromatin precipitation and sequencing reveals O-GlcNAcylated proteins at discrete loci in Drosophila S2 cells.
Figure 3: Comparative bioinformatics analysis of next-generation sequencing data from S2 cells using Ac4GalNAz feeding, WGA precipitation, and GalT labeling.
Figure 4: Ac4GalNAz feeding enables in vivo labeling of Drosophila at larval, pupal, and fly stages.
Figure 5: O-GlcNAcylated proteins are distributed to genomic loci in Drosophila that contain PREs as well as those that lack PREs and gene expression from these diverse loci is regulated by OGT.

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Acknowledgements

Financial support through a Discovery Grant the Natural Sciences and Engineering Research (D.J.V., NSERC, RGPIN/-2015-05426) and the Canadian Institutes of Health Research (D.J.V., D.S., B.M.H., CIHR, MOP-102756) is gratefully acknowledged. This project was also supported in part by a contract from Genome Canada/Genome British Columbia (173CIC). R.D.M. thanks the CIHR for a new investigator award. D.J.V. acknowledges the kind support of the Canada Research Chairs program for a Tier I Canada Research Chair in Chemical Glycobiology and NSERC for support as an E.W.R. Steacie Memorial Fellow. S.C. thanks the CIHR for a postdoctoral fellowships. M.M. thanks NSERC for a CGS-M graduate fellowship. The authors thank J. Kassis (National Institutes of Health, Bethesda, Maryland, USA), J. Muller (Max Planck Institute of Biochemistry, Frankfurt, Germany, H. Brock (University of British Columbia, Canada), D. Arndt-Jovin (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) and W. Herr (University of Lausanne, Lausanne, Switzerland) for generously providing antibodies, A. Wilson for his assistance in ChIP data analysis, D. Fornika for assistance with performing ChIP sequencing, and T. Cruz-Sanchez, G. Cavalli, and Y. Zhu for early support of this work.

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Contributions

T.-W.L. and D.J.V. designed research; T.-W.L. performed all cell culture experiments and generated DNA samples for sequencing; T.-W.L. and M.M. prepared RNA samples and performed all qPCR experiments, T.-W.L., K.B., and M.M. performed fly labeling experiments; S.C., K.B., B.M.H., and D.A.S. generated specialized materials including unique reagents and wild-type and mutant flies; M.M. performed detailed bioinformatics analyses; T.-W.L., M.M., R.D.M., D.A.S., and D.J.V. analyzed data, T.-W.L., M.M. and D.J.V. wrote the manuscript; all authors provided input into the manuscript.

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Correspondence to David J Vocadlo.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–23 and Supplementary Tables 1–2. (PDF 4593 kb)

Supplementary Dataset 1

O-GlcNAc containing genes in white pre-pupae found by Ac4GalNAz ChIP–seq and colocalization with other PRE marks. (XLSX 95 kb)

Supplementary Dataset 2

O-GlcNAc containing genes in white pre-pupae found by Ac4GalNAz ChIP–seq and colocalization with other PRE marks that were found to be differential expression in wild type and sxc−/− pupae by qPCR. (XLSX 11 kb)

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Liu, TW., Myschyshyn, M., Sinclair, D. et al. Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster. Nat Chem Biol 13, 161–167 (2017). https://doi.org/10.1038/nchembio.2247

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