Investigating the role of the O-GlcNAc modification during the neural differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) have the ability of propagating indefinitely and can be induced to differentiate into specialized cell types; however, the molecular mechanisms that govern the self-renewal and conversion of hESCs into a variety of cell types are not well understood. In order to understand the molecular mechanisms involved in the modulation of these processes it is necessary to look beyond what is encoded in the genome, and look into other forms of cellular regulation such as post-translational modifications. The addition of a single monosaccharide, Beta-N-acetylglucosamine (O-GlcNAc), to the hydroxy side chain of serine or threonine residues of protein is an intracellular, post-translational modification that shares qualities with phosphorylation. The enzymes involved in the addition and removal of O-GlcNAc, O-GlcNAc transferase and O-GlcNAcase, respectively, have been shown to target key transcriptional and epigenetic regulators. O-GlcNAc is believed to play a very important role in ESC biology, as this modification is required for cell viability and perturbations to the regulation of O-GlcNAc have been associated with abnormal development./nA great deal of interest is currently devoted towards deciphering the functional role of O-GlcNAc in stem cell maintenance and development. Chapter 1 summarizes the state of knowledge in the area of O-GlcNAc as it pertains to transcriptional regulation and development while also addressing how these processes might be regulated by the interplay between O-GlcNAcylation and phosphorylation. Moreover, it explains how chemical and biochemical tools have advanced our understanding of the functional significance of the O-GlcNAc modification. Chapter 2 describes the application of one of these chemical tools, known as metabolic labeling, in the identification of O-GlcNAcylated proteins in undifferentiated hESCs. The use of this tool coupled with biotin affinity purification and mass spectrometry analysis allowed for the identification of different O-GlcNAcylated proteins, including transcription factors, metabolic enzymes, and histones. The results presented in this chapter represent the first comprehensive proteomic characterization of protein O-GlcNAcylation in hESCs./nSimilar to metabolic labeling, lectin weak affinity chromatography (LWAC) is another tool used for the enrichment of O-GlcNAcylated proteins. The O-GlcNAc modification has been found on proteins important for neuronal plasticity and brain development, and the enzymes responsible for the modification are expressed highest in the brain. Chapter 3 focuses on characterizing the different O-GlcNAcylated proteins present during the neural differentiation of hESCs using mass spectrometry analysis and LWAC. The results discussed here provide fundamental knowledge of stage-dependent protein modification by O-GlcNAc and will help further elucidate the roles of O-GlcNAc in the development of the nervous system./nFinally, chapter 4 describes the effects of perturbing O-GlcNAcylation during the neural induction of hESCs. Inhibition of OGT induced the early expression of neuronal proteins and accelerated the conversion of hESCs into neural stem cells, suggesting a regulatory role of O-GlcNAc in maintaining proper brain development. The results presented in this chapter will help define the molecular behavior of stem cells during neuronal development so that they can be used effectively and reliably for the treatment of neurodegenerative disorders.

Advisor: Bertozzi, Carolyn R