group on media lacking Ac4GlcNAz. We then allowed the eggs to develop and harvested resulting larvae from both groups. This provided the control group and the 0 h time point larvae that were labeled with O-GlcNAz. Additional time points were generated by transferring larvae to control media at a range of different time times, which were then all allowed to grow for a total of 36 h after which they were similarly harvested (Figure 3b). We recognized that loss of O-GlcNAz from proteins bound to chromatin could be driven by either cleavage of O-GlcNAc from the genome by OGA or dissociation of O-GlcNAz Figure 3. Validation of TC Ac4GalNAz strategy a Visual representation of TC Ac4GalNAz b Timeline of Ac4GalNAz metabolic labeling performed in WT and OGA-null Drosophila larvae. Synchronized parents were given a 2 h window to lay eggs on Ac4GalNAz-containing media and non-Ac4GalNAz media for the no feed control. After 36 h of growth, larvae were collected (0 h and no feed control) or transferred onto non-Ac4GalNAz media. Figure reproduced from reference8). ChIP–seq tracks of the Ubx–Abd-B locus shown in descending order were obtained using anti-Pho antibody (Pho, top track), Ac4GalNAz (GalNAz) feeding, WGA pulldown (WGA), GalT labeling (GalT), and non-enriched genomic DNA (Control, bottom track). Genes are labeled under tracks and show exons as darker boxes, introns as thin lines, and the transcriptional start site indicated by an arrow. Figure reproduced from reference7). feeding workflow. WT and OGA-null Drosophila embryos were incubated on Ac4GalNAz media or DMSO control for 36 h. The resulting larvae were collected from the Ac4GalNAz media and transferred to Ac4GalNAz free media (DMSO). The larvae were grown on Ac4GalNAz free media for 0, 4, 8, 12, 16, 20, 24, and 36 h post transfer and snap frozen and ued for downstream analyses. In vivo genome wide dynamics of O-GlcNAcylated chromatin-associated proteins brainDavid J. VocadloFigure 2. Metabolic feeding combined with antibody-free genome-wide chromatin precipitation and sequencing reveals O-GlcNAcylated proteins at discrete loci in Drosophila S2 cells were interested in understanding dynamic changes to the levels and distribution of O-GlcNAc on the genome as a function of time and reasoned that we could using such a pulse-chase experiment in combination with ChIP-seq performed at different time points to monitor O-GlcNAc levels in a genome wide manner (Figure 3a). To do this we cultivated two groups of synchronized Drosophila and constrained the time of their egg laying to be within a 2 h period. We cultivated the experimental group on Ac4GalNAz containing media, using O-GlcNAz as a readily monitored surrogate for O-GlcNAc, and the control 68
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