known to bind to PREs including the PRC1 subunit Ph, Pho, as well as the PcG and trithorax group (TrxG)-associated epigenetic regulator host cell factor 1 (HCF-1), which antagonizes the action of PcG proteins (Figure 1c). We showed these three proteins are all O-GlcNAz modified and confirmed this using orthogonal methods. These data show that metabolic feeding with Ac4GalNAz followed by biorthogonal labeling and pull down of O-GlcNAz modified proteins can be readily performed using Drosophila tissues.We next tested whether our planned strategy involving PFA fixation, shearing of genomic DNA, followed by enrichment of O-GlcNAcylated protein-bound DNA fragments could be combined with next-generation sequencing of those DNA fragments and used to define the genomic locations to which O-GlcNAcylated proteins bind in Drosophila S2 cells7). Because Pho is largely found at PREs at which silencing occurs, we also carried out ChIP-seq analysis to compare the O-GlcNAz ChIP–seq profile of chromatin we obtained from analysis of S2 cells with the ChIP-seq profile obtained for Pho. This comparison allowed us to compare the presence O-GlcNAcylated proteins and Pho at genomic loci known to contain PREs. In parallel, we also performed ChIP–seq using the lectin WGA and chemoenzymatic GalT labeling, and compared these data to the results obtained when using O-GlcNAz and Pho (Figure 2). We found O-GlcNAz and WGA ChIP–seq data showed similar localization at discrete genomic loci, including those encoding Hox genes, whereas the GalT method revealed poor enrichment (Figure 2). Importantly, these experiments revealed the specificity of the approach and showed we are only detecting DNA sequences at which O-GlcNAz-modified proteins are bound within cells. We next applied this method to intact Drosophila and performed time-resolved experiment to examines the turnover of O-GlcNAc modified proteins on the genome8). We found that flies raised on media containing different concentrations of Ac4GalNAz led to most efficient labeling of proteins, as determined by analysis of whole-organism lysates, of pupae grown on 100 μM Ac4GalNAz. Notably, OGT-null pupae (sxc−/−) had much lower labeling as compared to wild-type pupae (WT). Furthermore, ChIP–seq analysis of O-GlcNAz in WT and OGT-null Ac4GalNAz-fed pupae showed that O-GlcNAc was absent from PRE target genes in OGT-null pupae but was present as expected in WT pupae. This specificity and high enrichment was consistent with our data from S2 cells and supported downstream time-resolved experiments.We next pursued time-resolved analysis of O-GlcNAc on the Drosophila genome using our metabolic labeling strategy8). We experiments (ChIP-seq). Early studies revealed that many chromatin-associated proteins within Drosophila are modified with O-GlcNAc5). However, at the time of this work, the only PcG protein within Drosophila known to be modified was the protein polyhomeotic (Ph)6). Regulation of gene expression by O-GlcNAc, however, is unlikely to only be mediated through modification of Ph. Accordingly, we were interested in improving our understanding of how O-GlcNAc may serve to broadly regulate the expression of genes. We realized, however, that using standard ChIP–seq methods to map PcG proteins having specific post-translational modifications is problematic. There are acknowledged challenges associated with using antibodies for ChIP–seq and top-quality antibodies targeting such modified proteins of interest are not generally known. We therefore decided to implement chemical biology methods that would not depend on antibodies or lectins and which would permit reliable genome-wide mapping of O-GlcNAcylated proteins (Figure 1a). Moreover, we felt that a chemical biology method that exploited metabolic feeding of sugar analogues containing a bioorthogonal functionality that could be incorporated in place of O-GlcNAc could enable performing time-resolved ChIP-sequencing experiments that could not be readily performed using standard ChIP-seq methods. The metabolic labeling strategy that we decided to pursue is based on the known ability of mammalian cells to metabolize azidoacetylgalactosamine (GalNAz) into UDP-azidoacetylglucosamine (UDP-GlcNAz). UDP-GlcNAz is a substrate for OGT, allowing this enzyme to transfer GlcNAz onto proteins after which the azide functionality can be modified using biorthogonal chemistry to append a biotin moiety (Figure 1a). Using this approach, we reasoned we could cross link proteins to DNA using standard paraformaldehyde (PFA) fixation, followed by controlled shearing of genomic DNA by sonication, which would collectively allow streptavidin-based enrichment of genomic DNA fragments. Next generation sequencing of these enriched DNA fragments would permit genome-wide ChIP–seq-like mapping of O-GlcNAz to genomic loci bearing O-GlcNAc (Figure 1b).As a first step7), we tested whether Drosophila S2 cells could metabolize Ac4GlcNAz and Ac4GalNAz in the same way as mammalian cells. We treated cells with these compounds and then labeled O-GlcNAz modified proteins in the resulting cell lysates with biotin using bioorthgonal chemistry. After feeding cells with these sugar analogues and performing bioorthogonal coupling, we purified the resulting biotinylated O-GlcNAz-modified proteins from cell lysates using streptavidin resin (Figure 1b). We then eluted these proteins and probed the released proteins using antibodies against several proteins 67
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