B Normalized heatmap of sequencing coverage at each of the 1883 loci that overlap between WT and OGA-C Sigmoidal curve fit of WT (circles) and OGA-null (triangles) Drosophila using TDCA. Figure reproduced from modified proteins from chromatin followed by their degradation. We therefore performed the same experiment using both WT and OGA-null larvae. By doing this parallel experiment we could thereby distinguish between the rates of OGA-mediated removal of O-GlcNAz from proteins and the rates at which chromatin associated proteins bearing O-GlcNAz modified proteins are degraded.We performed two independent replicate experiments, obtaining data for each time point in the time course O-GlcNAc-seq experiment using both OGA-null and WT larvae (Figure 4). We identified O-GlcNAc-containing genomic regions by quantifying overlapping sequencing reads at genomic loci and defined 1883 loci bearing O-GlcNAz modified proteins in both OGA-null and WT samples. As expected, a time dependent decrease in sequencing reads was observed (Figure 4). We quantified these changes using the time-dependent ChIP-seq analyzer (TDCA) software9) and found the average O-GlcNAz modification half-life was 14.4 ± 1.6 h for OGA-null and 4.6 ± 0.9 h for WT Drosophila (Figure 4). Notably, only 3% (56/1883) of the Figure 4. OGA-null Drosophila retains O-GlcNAz genome-wide longer compared to WT Drosophila A Tracks for WT and OGA-null Drosophila Ac4GalNAz TC experiment. The gene bithoraxoid (bxd) O-GlcNAc enriched loci showed an observable OGA resistant behavior and these OGA resistant loci were significantly absent from exons and gene bodies. These collective data show OGA acts on most chromatin-associated proteins, which is reflected by the small (~3%) fraction of loci resistant to OGA. These data show this metabolic feeding strategy enables robust ChIP-seq-like analysis of O-GlcNAcylated proteins and can be applied to quantify dynamics of O-GlcNAc on those proteins bound to the genome. The metabolic labeling strategies we have developed for application to ChIP-seq analysis of O-GlcNAc are now starting to be applied by others10) to gain an improved understanding of the roles played by O-GlcNAc. One recent example where researchers have exploited this strategy has been applied to examining the functions of O-GlcNAc modification in regulating stress responses and cell homeostasis. This same metabolic feeding strategy was used to analyze O-GlcNAc in human breast cancer cells treated with adriamycin, a genotoxic agent. Many chromatin-associated transcription factors and cofactors that (chr3R:12,589,000-12,591,000) is shown in the left box and charlatan (chn) (chr2R:11,015,000-11,017,000) is to the right. Time points are labeled to the left in hours, and NF indicates the no feed control. null Drosophila using TDCA.reference8).69
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