Project 2: Mechanism of Chromatin Engagement & Remodeling by Pluripotency Factors

描述 该提案的目标是揭示多能性转录因子获得发育沉默基因的机制，打开染色质，从而启动自我维持的调控网络。只有1/100至1/10，000的表达Oct 4、Sox 2、Klf 4和c-Myc因子的体细胞可以被重编程为多能性，即使使用最有效的蛋白质、mRNA或基因递送方法，并且尽管在初始细胞中这些因子的可证实的共表达。我的实验室已经表明，FoxA转录因子具有内在的生物化学能力，可以结合沉默基因致密染色质中的靶位点。这会产生局部超敏反应，促进其他转录因子的结合和基因激活。我们进一步发现，FoxA“先锋因子”含有不同的蛋白质结构域，这些蛋白质结构域调节因子在染色质中移动并产生超敏反应的能力，并且我们发现限制FoxA参与的染色质修饰状态。基于Oct 4、Sox 2、Klf 4和c-Myc激活分化细胞中沉默多能性网络的深刻能力，沿着Nanog在该过程中的作用，我们假设一种或多种这样的蛋白质具有“先锋”活性，沿着影响染色质功能的相关结构域;这些结构域可以提供对抗性染色质状态的敏感性;并且这些知识将使我们能够设计多能性因子以更有效地启动重编程过程。基于我们对Fox蛋白的研究，我们有能力揭示多能性因子功能的机制基础，并确定如何增强它，目标如下：1）为了评估五种人多能性因子在体外重构为纯化的染色质模板的天然人靶序列上的初始染色质接合和开放性质，与体内相同染色质位点上的因子初始接合相比（与普拉斯和劳里实验室）。2)绘制对染色质接合和开放至关重要的蛋白质结构域，以及相关结构域的作用机制（与普拉斯实验室合作）。3)确定染色质的哪些修饰可以限制多能性因子的接合或染色质开放，以及如何通过修饰因子的适当结构域来克服这些限制（与Lowry和Smale实验室合作）。这些目标中的每一个都涉及与P01提案的其他成员的直接互动和合作。这些研究的见解和方法将适用于不同类型的细胞和重编程治疗环境。 不会有人类或动物实验与拟议的工作。

Description The goal of this proposal is to reveal the mechanisms by which pluripotency transcription factors gain access to developmentally silenced genes, open the chromatin, and thereby initiate self-sustaining regulatory networks. Only 1/100 to 1/10,000 of somatic cells in which the Oct4, Sox2, Klf4, and c-Myc factors are expressed can be reprogrammed to pluripotency, even with the most efficient methods of protein, mRNA, or gene delivery and despite the demonstrable co-expression of the factors in the initial cells. My laboratory has shown that FoxA transcription factors have the intrinsic biochemical capacity to bind target sites in the compacted chromatin of silent genes. This creates local hypersensitivity that facilitates binding by other transcription factors and gene activation. We further found that FoxA "pioneer factors" contain distinct protein domains that modulate the ability of the factor to move in chromatin and to create hypersensitivity, and we found chromatin modification states that restrict FoxA engagement. Based on the profound ability of Oct4, Sox2, Klf4, and c-Myc to activate the silent pluripotency network in differentiated cells, along with role of Nanog in the process, we hypothesize that one or more of such proteins has "pioneer" activity, along with relevant domains that affect chromatin functions; that such domains may provide sensitivity to resistant chromatin states; and that knowledge of such will allow us to engineer pluripotency factors to more efficiently initiate the reprogramming process. Based on our studies of Fox proteins, we are well positioned to unveil the mechanistic basis for pluripotency factor function and to determine how to enhance it, with these Aims: 1) To assess the initial chromatin engagement and opening properties for the five human pluripotency factors on natural human target sequences reconstituted into purified chromatin templates in vitro, in comparison to the factors' initial engagement on the same chromatin sites in vivo (with Plath and Lowry labs). 2) To map protein domains crucial for chromatin engagement and opening, and the mechanism of action of relevant domains (with Plath lab). 3) To determine what modifications of chromatin can limit the engagement or chromatin opening by the pluripotency factors, and how such may be overcome by modifying the appropriate domains of the factors (with Lowry and Smale labs). Each of these aims involves direct interactions and collaborations with other members of the P01 proposal. Insights and approaches from these studies will be applicable to diverse types of cell and therapeutic contexts for reprogramming. There will be no human or animal experimentation with the proposed work.