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，以及具体的挑战主题，14- ag -104：描述控制多能干细胞分化的因素。将成人体细胞转化为具有与人类胚胎干细胞（hES）细胞难以区分的特性的诱导多能干细胞（iPS）的能力是再生医学的一项重大进展。在拟议的研究中，我们将研究iPS和hES细胞中（重）编程的保真度，这与自我更新过程中控制多能性的表观遗传机制（目标1）和分化过程中细胞命运决定（目标2）有关。我们将建筑表观遗传学描述为有丝分裂过程中后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的基因调控因子，（ii）变异核心（H2A, H2B， H3和H4）组蛋白，以及（iii）有丝分裂过程中变异连接组蛋白（H1）与特定靶基因启动子的关联。我们将通过实验解决中心假设，即有丝分裂染色体中与基因相关的蛋白质补体是iPS和he细胞多能性的基础，并且有丝分裂蛋白/DNA相互作用组的修饰对谱系承诺至关重要，并且与多能性的丧失机制相关。此外，间期染色质微环境的有丝分裂后组织将进行功能分析，以诊断多能性和谱系承诺细胞的自我更新和细胞周期进展的保真度。我们的方法将从建筑表观遗传学的角度建立多能性和（重新）编程的基本基础。因此，我们将确定在有丝分裂完成后影响基因表达的主要基因调控蛋白，并定义干细胞自我更新和谱系特异性编程的能力。研究中确定的调控参数和因子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：许多与年龄有关的疾病可以通过将患者的正常细胞转化为有可能成为任何其他细胞类型的细胞来再生恶化的组织或器官（例如骨、脑、肌肉或心血管细胞）的细胞来治愈。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未确定。我们的实验室已经证明，转录因子可以保持与有丝分裂染色体的结合，从而定义了一种新的机制，可以在细胞分裂后将遗传调控信息（“建筑表观遗传学”）传递给后代。我们将使用先进的生物化学，分子和细胞方法来定义在无细胞和程序化干细胞的细胞周期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促进细胞核内介导基因表达的微观结构域的形成。我们的研究将从建筑表观遗传学的角度确定细胞如何保持多能性或成为特化细胞。我们的方法将确定在细胞完成一轮细胞分裂后，控制基因如何立即使用的主要因素。由于这些因子通过自我更新或转化为特化细胞来调节细胞增殖的指令，因此它们可能特别适合于支持老年患者的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.