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

描述（由申请人提供）：本项目的目的是表征蛋白质O-GlcNAc酰化在干细胞生物学关键领域的功能作用。O-GlcNAc化是一种翻译后修饰，其中单个单糖（O-GlcNAc）被安装在胞质和核蛋白的Ser或Thr残基上。这种修饰已经在包括转录因子在内的多种蛋白质中发现。尽管人们对O-GlcNAc化在细胞过程中的关键作用了解很多，但这种修饰尚未在再生医学的背景下进行研究。本文中，我提议表征O-GlcNAc化在干细胞生物学的两个关键领域中的功能作用：神经元分化（Aim 1）和多梳组蛋白对基因组转录物的抑制（Aim 2）。在目标1中，我建议使用代谢寡糖工程（莫伊）和糖蛋白质组学分析，以确定在神经元分化的各个阶段存在的O-GlcNAc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。目的2：研究O-GlcNAc化在多梳组蛋白（PcGs）维持人胚胎干细胞和成体干细胞中的作用。最近在果蝇中的研究表明，O-GlcNAc化在发育过程中控制PcG介导的基因沉默。在此，我们将通过莫伊促进的染色质免疫沉淀（ChIP）型测定来鉴定O-GlcNAc酰化蛋白和受其调节的基因。这项工作可能会扩大目前的理解的自我更新和多能性的hESC的分子机制。总之，本文提出的研究的成功完成将构成hESC中蛋白质O-GlcNAc化的首次全面研究，并将极大地扩展目前对hESC生物学中控制神经元分化、多能性和转录控制的分子机制的理解。 公共卫生相关性：人类胚胎干细胞几乎可以转变为成人体内的任何细胞类型，因此有可能治愈绝大多数现有的人类疾病。该项目中提出的研究结果可能会大大增加对干细胞如何保持其转变为其他细胞类型的能力的理解，以及干细胞的命运如何决定分化。这两个领域都是需要探索的关键领域，以使现代再生医学能够充分发挥其作为治疗人类疾病（如阿尔茨海默病，帕金森病和巴滕病）的工具的潜力。

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.