Fig 1.
Structure-guided generation of Ogt’s hypomorphic allelic series.
(A) Crystal structure of human OGT isoform 1 (UniProt O15294-1) in complex with UDP-5SGlcNAc (PDB 4GYY). The area in the square is shown as an insert with higher details. Except for two single amino acid substitutions outside the catalytic core, human OGT1 is identical to Mus musculus OGT isoform 1 (UniProt Q8CGY8-1), hence residues’ numbering is the same. The residues mutated in this study are shown in sticks representation in blue and UDP-5SGlcNAc in sticks representation, coloured by heteroatom. T931 and Q849 establish direct interactions with the donor substrate. Y851 interacts with the donor substrate via hydrogen bonds with an intermediate water molecule. H568 is considered to be the catalytic base and in the crystal structure it coordinates different water molecules near the binding site. (B) Scheme of the murine Ogt genomic context with the location of the introduced point mutations. The exons of the longest transcript Ogt-201 are numbered. (C) Catalytic rate measured in vitro for the four studied OGT single amino acid substitutions. Data are from Martinez-Fleites et al. 2008 [46] and for H568A also from Lazarus et al. 2011 [111]. (D) For the four hypomorphic mutants of OGT described in (A-C): number of animals (F0) bearing the correct point mutation after CRISPR-targeted mutagenesis in the zygote (founders); number of mutants (F1) produced by female founders (female germline transmission, i.e. maternal transmission); lethal phenotypes observed from the F1 generation onwards.
Fig 2.
Maternal inheritance of a severe Ogt mutation causes preimplantation sub-lethality and perturbs retrotransposons silencing.
(A) Number and frequency of genotypes for the progeny produced by the mating of female heterozygous OgtT931A/+ with WT males. The left pie chart shows the theoretical Mendelian ratios for an X-linked mutation, the middle and right pie charts show the observed distribution of genotypes at the blastocyst stage and at weaning, respectively. P-values were computed using the chi-squared test for independence. (B) Experimental design for the study of blastocysts with maternal inheritance of a mutation at Ogt-T931. Oocytes from females OgtT931A/+ and seemingly Ogt+/+ female littermates were fertilized in vitro with WT sperm and the embryos grown ex vivo to the blastocyst stage (E4), when they were collected for single embryo mRNA-Seq. For the embryos produced by OgtT931A/+ females, the sex and presence of the T931A mutation was determined by PCR genotyping of the cDNA and Sanger sequencing. For the rest of the embryos, the T931del allele was identified a posteriori by manual inspection of the RNA-Seq reads, and the embryos were genotyped based on sequencing reads mapping to Ogt. The WT genotype was assigned based on the absence of reads bearing a mutation at T931. The total number of embryos analyzed per genotype after the in silico filtering steps (S5 Table) is indicated. Note that the absence of T931del-containing reads cannot formally exclude the presence of this allele within the group of heterozygous females. (C) Transcriptomes of individual male blastocysts produced by the experiment in (B) in the space defined by PC2 and PC3 of their principal component analysis (PCA), which result in a better separation based on embryonic genotype than PC1 and PC2 (shown in S2B Fig). The variance explained by each PC is shown in parentheses. (D) MA-plot from DESeq2 differential expression analysis of single copy genes in OgtT931del/Y versus WT male blastocysts from all maternal genotypes (N = 10 embryos per embryo genotype). All genes with mean DESeq2-normalized gene counts > 10, adj. p-value < 0.05, any log2FC (DEGs) are colored, and their number is indicated. Dashed lines show log2FC = ±0.5. Genes standing out (and with abs(log2FC) ≥ 0.2) are labeled. (E) Gene set enrichment analysis (GSEA) of gene expression change in OgtT931del/Y versus WT male blastocysts. Among significant Biological Process (BP) and Cellular Component (CC) gene ontology (GO) terms, the first 20 based on Normalized Enrichment Score (NES) are shown. Terms are ordered based on gene ratio. The size of dots is proportional to the number of total genes of a GO term. Gene ratio = fraction of total genes of the GO term which are concordantly changing in mutant embryos. (F) Top: expression dynamics of OgtT931del/Y DEGs belonging to the indicated GO terms throughout preimplantation development. The two biological replicates per stage were averaged. For each gene, the TPM value in the E3.5 blastocyst is the highest TPM value between the values in E3.5 ICM and E3.5 trophectoderm. The mean among all genes is drawn, as well as the 95% confidence interval, computed using basic nonparametric bootstrap (R function ‘mean.cl.boot’). Y-axis ticks are in log2 scale. TPM: Transcript Per Million. Bottom: enrichment analysis of up- and downregulated OgtT931del/Y DEGs in the genes changing in unperturbed embryos between the 8-cell stage and the trophectoderm of the E3.5 blastocyst (ranked by -log10(adj. p-value)*sign(log2FC) from E3.5TE-vs-8-cell comparison). Gene ratio = fraction of total up-/downregulated DEGs which are up-downregulated between the two preimplantation stages. Top and bottom: mRNA-Seq data for unperturbed embryos are from GSE66582 and GSE76505. (G) Heatmap of the expression (in Fragments Per Kilobase Per Million, FPKM) of the main families of retrotransposons in the single male blastocysts produced in (B). Values are scaled by rows. Retrotransposon families are clustered based on Euclidean distance and coloured by class. P-values were computed using unpaired Wilcoxon rank sum exact test of FPKM values, and they are indicated when < 0.05. (H) MA-plot from DESeq2 differential expression analysis of retrotransposons in OgtT931del/Y versus WT male blastocysts (N = 9 WT, 10 OgtT931del/Y embryos). All repeats with mean DESeq2-normalized gene counts > 10, adj. p-value < 0.1, any log2FC are colored by their class. Repeats standing out are labeled.
Fig 3.
A mild reduction in OGT’s activity affects placental development in a sexually-dimorphic manner.
(A) Litter size for intercrosses of WT mice and for crosses between mice bearing the Ogt-Y851A mutation. P-values are from unpaired two-sided Student’s t-test, assuming unequal variance. N = 20 WT crosses, 8 OgtY851A/+ x OgtY851A/Y and 6 OgtY851A/Y851A x OgtY851A/Y crosses. (B) Breeding scheme used to produce placentae with the four different genotypes, which were analyzed via western blot and mRNA-Seq. (C) Western blot analysis of OGT, OGA and O-GlcNAc levels in the placenta isolated from E12.5 embryos of the four genotypes produced by the cross described in (B). LF: long form, SF: short form. (D) Normalized optical density for OGT (top), O-GlcNAc entire lane (middle) and ratio OGA long form vs. OGA short form from the western blot in (C). In (C,D), for each genotype, the two placentae belong to embryos from two different litters (the same two litters for all genotypes), except for the hemizygous males which both come from the same litter. (E,F) Separate DESeq2 analyses for female and male placentae, comparing (E) OgtY851A-homozygous versus heterozygous female placentae or (F) hemizygous versus WT male ones. All genes with adj. p-value < 0.05, any log2FC are colored, and their number is indicated. Genes standing out (and with abs(log2FC) ≥ 0.2) are labeled. (G) Scatter plot representing the average DESeq2-normalized counts of upregulated and downregulated DEGs found in OgtY851A/Y (versus WT male) placentae, in single placentae of all four genotypes analyzed. (H) GSEA of gene expression change in hemizygous OgtY851A/Y versus WT male placentae. The first 10 GO terms for the three gene ontologies based on Normalized Enrichment Score (NES) are shown. Terms are ordered based on gene ratio. The size of dots is proportional to the number of total genes of a GO term. Gene ratio = fraction of total genes of the GO term which are concordantly changing in mutant embryos. (I) Enrichment analysis of placental clusters’ marker genes [63] expression changes in OgtY851A/Y versus WT male placentae. All enriched clusters (adj. p-value < 0.05) are shown, ordered by gene ratio. The size of dots is proportional to the number of markers of each placental cluster. Gene ratio = fraction of total markers which are concordantly changing in mutant embryos. JZP: junctional zone precursors; LaTP: labyrinth trophoblast progenitors; SynT-I: syncytiotrophoblast layer I; SynT-II: syncytiotrophoblast layer II; sTGC: sinusoidal trophoblast giant cells; SpT: spongiotrophoblasts. In (E-I), N per genotype = 6 placentae coming from at least two different litters, except female OgtY851A/+ placentae for which one sample was excluded because outlier in unsupervised clustering.
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.