O-GlcNAc signalling during embryonic stem cell differentiation

My lab is studying the signalling mechanisms governing the onset of differentiation of pluripotent embryonic stem (ES) cells. Work from this and other labs (e.g. 1, 2) has revealed a critical role for autocrine FGF signalling and consequent sustained phosphorylation of the kinase Erk during the differentiation process. Although essential to the differentiation process, Erk activation is not the only signalling event regulating the decision between self-renewal and differentiation of these cells (3). Cellular signalling is most commonly associated with post-translational modification of proteins by addition of a phosphate to serine, threonine and tyrosine residues by the large family of kinase enzymes. However, there are other protein post-translational modifications with increasingly recognised very important roles in protein control. One of these is the addition of β-O-linked N-acetylglucosamine (O-GlcNAc) to serine or threonine residues, first described over 25 years ago. This modification (O-GlcNAcylation) involves the covalent addition of a single sugar to aminoacids via O-glycosidic linkage and occurs with similar time scales, dynamics and stoichiometry as protein phosphorylation.

Compared to the body of work accumulated around the study of phosphorylation, O-GlcNAcylation is much less understood, and relatively little is known about the types of extracellular signals controlling it. However, as these two modifications can occur (mutually exclusively) on the same residue or (in an antagonistic or synergistic fashion) on neighbouring ones, it is easy to see how O-GlcNAcylation can modulate the phosphorylation downstream of a large number of signal transduction cascades (4).

In recent years evidence has been accumulating for a critical role played by O-GlcNAcylation in ES cells, although its precise function(s) and the mechanisms operating are still poorly defined. This project aims to study in detail the role of O-GlcNAcylation on cell signalling, using ES cells as a model system. ES cell differentiation is a complex process, governed by the interaction of multiple signalling pathways (e.g. ERK, Gsk3/Wnt, BMPs etc.) Work in our lab has identified an important and novel role for O-GlcNAc during mouse ESC differentiation, and we have generated preliminary data showing how alteration of O-GlcNAc levels (using a specific inhibitor of the O-GlcNAc hydrolase, GlcNAcstatin, abbreviated GNS) affects cell signalling, gene expression and the self-renewal/differentiation balance. Other labs have recently reported that O-GlcNAc modification of the ESC transcription factors Oct4 and Sox2 controls their function suggesting further mechanisms by which O-GlcNAc profoundly affects cell behaviour (5,6)

This project will build on these preliminary findings and define the mechanisms by which O-GlcNAc affects ES cell function using cell biological, biochemical and proteomic approaches.

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