The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny

Abstract

Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This "O-GlcNAcylation" of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.

Figure 1

OGT mutagenesis in ES cells and mice. A mouse OGT genomic clone bearing five exons (filled boxes) was used to construct two types of gene-targeting vectors. (A) The first vector would be expected to produce a null mutation by deletion of two OGT exons with insertion of a neomycin phosphotransferase cistron (neo). (B) The second vector incorporates loxP sites that initially would generate the OGT^F[tkneo] allele. (C) ES cells bearing the OGT^F[tkneo] allele that are subjected to transient Cre expression should yield both OGT^Δ (type I) and OGT^F (type II) alleles. (D) Homologous recombinants were obtained with the loxP-containing vector shown in B and were characterized by Southern blotting with probes of OGT genomic (Left) or loxP (Right) sequences. (E) Only OGT^F alleles are present among subclones, as represented, in over 200 analyzed specimens. (F) The OGT^Δ allele is detectable by PCR in polyclonal ES cell extracts only at early times after Cre expression. (G) OGT alleles in offspring of chimeric male (♂) mice bred to C57BL/6 females (♀) were analyzed by Southern blotting. Female offspring are either homozygous wild type or heterozygous for the OGT^F allele, whereas male offspring are either homozygous wild type or appear homozygous for the OGT^F allele. wt, wild type.

Figure 2

Regional localization of the OGT gene on the X chromosome in mice and humans. (A–C) Fluorescence in situ hybridization using both OGT cDNA (A) and genomic probes (B and C) localizes the OGT gene to the long arm of human X chromosome near the centromere (arrows). (D) Radiation-hybrid analysis of OGT gene localization in the mouse placed OGT on the X chromosome at the indicated centirads (cR) from the markers temp016, DXMit4, and DXMit95. (E) Statistical analysis placed OGT on the mouse X chromosome between markers DXMit41 and DXMit95 and in a region syntenic with human Xq13. LOD, logarithm of odds; MGD, mouse genome database.

Figure 3

OGT RNA expression among normal mouse tissues. (A and B) Total RNA (6 μg) was probed for OGT expression among ES cells and normal tissues of male mice either streptozotocin-sensitive (C57BL/6J) (A) or streptozotocin-resistant (C3H/HeJ) (B). Lower gels in A and B show that RNA is intact and comparable levels are present as detected by Sybergreen II staining.