Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo
Fig 4
Kinetics of differential gene expression after rapid degradation of endogenous OGT in MEFs.
(A) The ROSA26OsTIR allele was created by insertion of the coding sequence of Oryza sativa TIR1 gene (OsTIR1; fused to C-terminal Myc and HA adjacent tags) downstream the ubiquitous ROSA26 promoter. The transgene is integrated at the splicing acceptor (SA) site of ROSA26, resulting in ROSA26’s interruption and expression of OsTIR1 from the endogenous promoter. (B) The OgtAID allele consists of the targeted insertion of the minimal AID peptide’s sequence (44 amino acids) immediately downstream the initiating ATG codon of the longest Ogt isoform (Ogt-201 or ENSMUST00000044475 or ncOgt), in frame with two Myc and one FLAG epitope tags. (C) Western blot of endogenous OGT (ab177941 antibody) in whole cell protein extracts from male primary MEFs, untreated or treated with auxin for different amounts of time. From the same litter, an OsTIR,OgtAID clone and a control OsTIR,OgtWT clone were treated in parallel. Lamin A/C was probed as a loading control. (D) Volcano plots from DESeq2 analysis of gene expression changes in OsTIR,OgtAID clones after 24, 48 and 96 hours of auxin treatment compared to untreated OsTIR,OgtAID clones grown in parallel. Differentially expressed genes with p-value < 10−5 and absolute log2FC > 0.3 (i.e. 1.2-fold increase or decrease in expression) are colored in red and their number is indicated. (E) Gene ontology (GO) over-representation analysis of DEGs (adj. p-value < 0.05, any log2FC) from the same comparisons as in (D), plus untreated OsTIR,OgtAID versus untreated OsTIR,OgtWT clones (effect of the hypomorphic genotype), and OsTIR,OgtWT control clones treated with auxin for 96 hours versus grown untreated (effect of the auxin drug). The first 25 most enriched Biological Process GO terms are shown, based on p-value across all comparisons. Terms are ordered by gene ratio. Gene ratio = genes belonging to the GO term / total number of deregulated DEGs for that comparison. UP = upregulated DEGs, DOWN = downregulated DEGs. Terms enriched due to auxin treatment on control clones are written in gray. (F) Analysis of enrichment for Hallmark gene sets of the Molecular Signatures Database (MSigDB) among DEGs after 96 hours of acute OGT depletion. For (E-F), DEGs after acute OGT depletion which were also differentially expressed in auxin-treated control cells were excluded before enrichment analyses of auxin-treated OgtAID clones; DEGs with mean DESeq2-normalized counts < 10 across all samples were excluded from all enrichment analyses.