Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells - PubMed (original) (raw)

Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells

Samuel A Myers et al. Proc Natl Acad Sci U S A. 2011.

Abstract

The monosaccharide addition of an N-acetylglucosamine to serine and threonine residues of nuclear and cytosolic proteins (O-GlcNAc) is a posttranslational modification emerging as a general regulator of many cellular processes, including signal transduction, cell division, and transcription. The sole mouse O-GlcNAc transferase (OGT) is essential for embryonic development. To understand the role of OGT in mouse development better, we mapped sites of O-GlcNAcylation of nuclear proteins in mouse embryonic stem cells (ESCs). Here, we unambiguously identify over 60 nuclear proteins as O-GlcNAcylated, several of which are crucial for mouse ESC cell maintenance. Furthermore, we extend the connection between OGT and Polycomb group genes from flies to mammals, showing Polycomb repressive complex 2 is necessary to maintain normal levels of OGT and for the correct cellular distribution of O-GlcNAc. Together, these results provide insight into how OGT may regulate transcription in early development, possibly by modifying proteins important to maintain the ESC transcriptional repertoire.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

ETD MS/MS spectra of the two positional isomers of the _O_-GlcNAcylated SOX2 peptide. Adequate c- and z-series ions allow the unambiguous site assignment of the HexNAc mass modification to serine 248 (Upper) and threonine 258 (Lower). Charged reduced species and common neutral losses (55) are not labeled.

Fig. 2.

Fig. 2.

Western blot analysis of the relationship between PRC2 and OGT. (A) Disruption of PRC2 by mutations in either two, eed or suz12, of the three core components abrogates complex formation and methyltransferase activity as seen by EZH2 levels (third core component) and H3K27me3 levels, respectively. However, the pluripotency marker, OCT4, is unaffected. OGT and general _O_-GlcNAc levels are altered by PRC2 disruption. (B) siRNA knockdown of OGT does not significantly affect levels of OCT4, EZH2, or H3K27me3.

Fig. 3.

Fig. 3.

SILAC LWAC liquid chromatography-MS/MS workflow. Nonquantitative samples were prepared similarly, except a single cell type was used. LC, liquid chromatography; LTQ, linear trap quadrupole.

Fig. 4.

Fig. 4.

Diagram for landmarks of HCF-1. A zoomed-in view of the basic region and region of alternative proteolysis shows the extent and location of _O_-GlcNAc sites identified in this study. Numbers indicate landmark residues (Lower) or _O_-GlcNAc sites (Upper). g, glycosylation; NLS, nuclear localization sequence. Adapted from Wysocka et al. (44).

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