Role of O-GlcNAc in human embryonic stem cell pluripotency and differentiation

描述（由申请人提供）：本项目的目标是表征蛋白质O- glcn酰化在干细胞生物学关键领域的功能作用。o - glcna酰化是一种翻译后修饰，在细胞质和核蛋白的丝氨酸或苏氨酸残基上安装一个单糖（O-GlcNAc）。在包括转录因子在内的许多蛋白质上都发现了这种修饰。虽然我们对o - glcn酰化在细胞过程中的关键作用了解甚多，但这种修饰尚未在再生医学的背景下进行研究。在此，我建议描述O- glcn酰化在干细胞生物学的两个关键领域的功能作用：神经元分化（目标1）和多梳群蛋白对基因组转录物的抑制（目标2）。在目标1中，我建议使用代谢寡糖工程（MOE）和糖蛋白组学分析来鉴定在神经元分化的各个阶段存在的o - glcn酰化的蛋白质底物。这些研究将有助于阐明神经元分化过程中干细胞命运决定的分子决定因素。在Aim 2中，我将研究o - glcn酰化在多梳蛋白（PcGs）维持hESC和成体干细胞中的假定作用。最近对果蝇的研究表明，o - glcn酰化控制着发育过程中pcg介导的基因沉默。在此，我们将通过MOE辅助的染色质免疫沉淀（ChIP）型测定来鉴定o - glcn酰化蛋白和受其调节的基因。这项工作可能会扩大目前对hESCs自我更新和多能性的分子机制的理解。综上所述，本文所提出的研究的成功完成将构成对hESC中o - glcn酰化蛋白的首次全面研究，并将极大地扩展目前对hESC生物学中神经元分化、多能性和转录控制的分子机制的理解。

DESCRIPTION (provided by applicant): The objective of this project is to characterize the functional role of protein O- GlcNAcylation in crucial areas of stem cell biology. O-GlcNAcylation is a post- translational modification in which a single monosaccharide (O-GlcNAc) is installed on Ser or Thr residues of cytosolic and nuclear proteins. This modification has been found on a wide array of proteins, including transcription factors. Although much is known about the crucial role of O-GlcNAcylation in cellular processes, this modification has yet to be studied in the context of regenerative medicine. I propose herein to characterize the functional role of O- GlcNAcylation in two critical areas of stem cell biology: neuronal differentiation (Aim 1), and the repression of genomic transcripts by polycomb group proteins (Aim 2). In Aim 1, I propose to use Metabolic Oligosaccharide Engineering (MOE) and glycoproteomic analysis to identify the protein substrates of O-GlcNAcylation present during various stages of neuronal differentiation. These studies will aid in the elucidation of the molecular determinants of stem cell fate decisions during neuronal differentiation. In Aim 2 I will study the putative role of O-GlcNAcylation in the maintenance of hESC and adult stem cells by polycomb group proteins (PcGs). Recent work in Drosophila has shown that O-GlcNAcylation governs PcG-mediated gene silencing during development. Herein we will identify both O-GlcNAcylated proteins and the genes that are being modulated by them by performing a chromatin immunoprecipitation (ChIP)-type assay facilitated by MOE. This work may expand the current understanding of the molecular mechanisms underlying self-renewal and pluripotency of hESCs. In summary, the successful completion of the studies proposed herein will constitute the first-ever comprehensive study of protein O-GlcNAcylation in hESCs, and will greatly expand the current understanding of molecular mechanisms governing neuronal differentiation, pluripotency and transcriptional control in hESC biology. PUBLIC HEALTH RELEVANCE: Human embryonic stem cells can be changed into virtually any cell type in the adult body, and thus, have the potential to cure a vast majority of existing human disorders. The results of the studies proposed in this project may lead to a greatly increased understanding of how stem cells retain their ability to be changed into other cell types, and also how the fate of stem cells is decided upon differentiation. Both are critical areas that need to be explored to enable modern regenerative medicine to realize its full potential as tool for the treatment of human diseases, such as Alzheimer's, Parkinson, and Batten disease.