Fig. 2

O-GlcNAc is required to sustain the expression of a set of target genes. A Western blot detection of OGT, pan-O-GlcNAc (RL2 antibody), and histone H3 (loading control) in total protein extracts from WT ESCs transfected with control siRNA (non-targeting) or with siRNA against Ogt. The blots are representative of two independent experiments. B Volcano plot showing differential gene expression between ESCs transfected with siRNA control and siRNA anti-Ogt. Differentially expressed genes included 836 downregulated (dark blue) and 592 upregulated (dark red) genes (adj. p-value < 0.05, Wald Test, any log2FC). Among these, 44 downregulated and 12 upregulated genes have a fold-change higher than two. Thirty-one downregulated (light blue) and two upregulated (orange) genes have an overlapping O-GlcNAc peak in WT cells. C Representative genomic tracks of O-GlcNAc regulated promoters as defined by high occupancy of O-GlcNAc-modified proteins in WT cells (top CUT&RUN track) and downregulated upon global removal of O-GlcNAc (bottom RNA-seq track). D Gene set enrichment analysis of the up-, down-, and down-O-GlcNAc regulated genes shown in panel B. The number of genes enriched in gene ontology (GO) molecular function (MF) are 1461, 1324, and 31, respectively. The gene ratio reflected by the size of dots indicates the proportion of genes matching a GO set