Architectural Epigenetics of Embryonic and Induced Pluripotent Stem Cells

描述（由申请人提供）：本申请涉及广泛的挑战领域（14）干细胞和特定的挑战主题14-AG-104：描述控制多能干细胞分化的因子。将成人体细胞转化为诱导多能干细胞（iPS）的能力与人类胚胎干细胞（hES）的特性无法区分，代表了再生医学的重大进展。在拟议的研究中，我们将检查iPS和hES细胞中（重新）编程的保真度，该保真度与控制自我更新期间多能性（Aim 1）和分化期间细胞命运决定（Aim 2）的表观遗传机制有关。我们将结构表观遗传学描述为有丝分裂期间子代细胞对染色质结构信息的遗传，包括（i）谱系特异性和多能性相关基因调控因子，（ii）变异核心（H2 A，H2 B，H3和H4）组蛋白，以及（iii）有丝分裂期间变异连接体组蛋白（H1）蛋白与特异性靶基因启动子的关联。我们将通过实验来解决中心假设，即与有丝分裂染色体中的基因相关的蛋白质的互补是iPS和hES细胞的多能性的基础，并且在该有丝分裂蛋白质/DNA相互作用组中的修饰对于谱系定型是至关重要的，并且在机械上与多能性的丧失相关联。此外，将对间期期间染色质微环境的有丝分裂后组织进行功能分析，以诊断多能和谱系定型细胞中自我更新和细胞周期进程的保真度。我们的方法将建立从建筑表观遗传学的角度多能性和（重）编程的根本基础。因此，我们将确定主要的基因调控蛋白，有丝分裂细胞分裂完成后的基因表达的影响，并定义干细胞的自我更新和谱系特异性编程的能力。在拟议的研究中确定的调节参数和因素可以作为支持老年患者组织工程和再生医学的生物学策略的目标。公共卫生相关性：许多与年龄相关的疾病可以通过将来自患者的正常细胞转化为具有成为任何其他细胞类型以再生恶化的组织或器官（例如，骨、脑、肌肉或心血管细胞）。诱导细胞达到这种所谓的“多能状态”是可能的，但这一过程产生真正干细胞的保真度仍然不确定。我们的实验室已经表明，转录因子可以保持绑定到有丝分裂染色体，以定义一种新的机制，可以传递可遗传的调控信息（“建筑表观遗传学”），细胞分裂后的后代。我们将使用先进的和最先进的生化，分子和细胞的方法来定义在幼稚和编程干细胞的细胞周期过程中与有丝分裂染色体结合的调节蛋白的机制作用。此外，我们将研究这些因素如何有助于细胞核内介导基因表达的微观结构域的形成。我们的研究将从结构表观遗传学的角度建立细胞如何保持多能性或成为特化细胞。我们的方法将确定控制基因在细胞完成一轮细胞分裂后立即使用的主要因素。由于这些因子调节通过自我更新或转化为专门细胞的细胞增殖指令，它们可能特别适合于支持老年患者组织工程和再生医学的生物学策略。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells, and the specific Challenge topic, 14-AG-104: Delineate factors that control the differentiation of pluripotent stem cells. The ability to convert adult somatic cells into induced pluripotent stem (iPS) cells with properties indistinguishable from human embryonic stem (hES) cells represents a major advance in regenerative medicine. In the proposed studies, we will examine the fidelity of (re-)programming in iPS and hES cells that is linked to epigenetic mechanisms controlling pluripotency during self-renewal (Aim 1) and cell fate determination during differentiation (Aim 2). We will characterize architectural epigenetics as the inheritance of chromatin structural information by progeny cells during mitosis that includes the association of (i) lineage-specific and pluripotency-related gene regulatory factors, (ii) variant core (H2A, H2B, H3 and H4) histone proteins, as well as (iii) variant linker histone (H1) proteins with specific target gene promoters during mitosis. We will experimentally address the central hypothesis that the complement of proteins associated with genes in mitotic chromosomes is fundamental to the pluripotency of both iPS and hES cells and that modifications in this mitotic protein/DNA interactome are critical for lineage commitment and are mechanistically coupled with loss of pluripotency. Also, the post-mitotic organization of chromatin micro-environments during interphase will be functionally analyzed to diagnose fidelity of self-renewal and cell cycle progression in pluripotent and lineage-committed cells. Our approach will establish the fundamental basis of pluripotency and (re-)programming from the perspective of architectural epigenetics. We will thus identify principal gene regulatory proteins that influence gene expression following completion of mitotic cell division and define the ability of stem cells for self-renewal and lineage-specific programming. The regulatory parameters and factors identified in the proposed studies can be targeted for biological strategies supporting tissueengineering and regenerative medicine in elderly patients. PUBLIC HEALTH RELEVANCE: Many age-related diseases may be curable by converting normal cells from patients into cells that have the potential to become any other cell type to regenerate a deteriorating tissue or organ (e.g., bone, brain, muscle or cardiovascular cells). It is possible to induce cells to reach this so-called 'pluripotent state', but the fidelity by which this process produces genuine stem cells remains undefined. Our laboratory has shown that transcription factors can remain bound to mitotic chromosomes to define a novel mechanism that can transmit heritable regulatory information ('architectural epigenetics') to progeny after cell division. We will use sophisticated and state-of-the art biochemical, molecular and cellular approaches to define the mechanistic roles of regulatory proteins that are bound to mitotic chromosomes during the cell cycle in na¿ve and programmed stem cells. In addition, we will investigate how these factors contribute to the formation of microscopic domains within the nucleus that mediate gene expression. Our studies will establish how cells can stay pluripotent or become specialized cells from the perspective of architectural epigenetics. Our approaches will identify major factors that control how genes are used immediately after cells complete a round of cell division. Because these factors regulate instructions for cell multiplication through self-renewal or for conversion into specialized cells, they may be particularly suitable for biological strategies supporting tissue-engineering and regenerative medicine in elderly patients.