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

项目2：染色质结合机制

• 批准号: 8520350
• 负责人: Kenneth Zaret
• 金额: $ 47.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8520350
• 关键词: Affect; Animal Experimentation; Architecture; Binding; Biochemical; Cells; Chromatin; Collaborations; Complex; DNA Sequence; Data; Deoxyribonucleases; Engineering; Foxes; Frequencies; Gene Activation; Gene Delivery; Gene Silencing; Gene Targeting; Genes; Genetic screening method; Genomics; Germ Cells; Goals; Human; Human Experimentation; Hypersensitivity; In Vitro; Intervention; Knowledge; Laboratories; Light; Maps; Messenger RNA; Methodology; Methods; Modification; Molecular; Nucleosomes; Positioning Attribute; Process; Property; Proteins; Publishing; Resistance; Role; Site; Somatic Cell; Staging; Tertiary Protein Structure; Testing; Therapeutic; Time; Transcription factor genes; Work; base; c-myc Genes; cell type; chromatin modification; embryonic stem cell; histone modification; in vitro testing; in vivo; induced pluripotent stem cell; insight; knock-down; member; pluripotency; reconstitution; transcription factor

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.

描述 这一提议的目的是揭示多潜能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。在表达Oct4、Sox2、Klf4和c-Myc因子的体细胞中，只有1/100到1/10,000个细胞可以被重新编程为多能性细胞，即使用最有效的蛋白质、mRNA或基因传递方法，而且尽管这些因子在初始细胞中明显共表达。我的实验室已经证明，FoxA转录因子具有内在的生化能力，可以结合沉默基因紧密染色质中的靶部位。这造成了局部的超敏反应，促进了其他转录因子的结合和基因的激活。我们进一步发现，FoxA“先锋因子”包含不同的蛋白质结构域，这些结构域调节该因子在染色质中移动并产生超敏反应的能力，我们还发现染色质修饰状态限制了FoxA的参与。基于Oct4、Sox2、Klf4和c-Myc激活分化细胞中沉默的多能性网络的深刻能力，以及Nanog在这一过程中的作用，我们假设一个或多个这样的蛋白质具有“先锋”活性，以及影响染色质功能的相关结构域；这些结构域可能提供对染色质抗性状态的敏感性；对这些的了解将使我们能够更有效地设计多能性因子来更有效地启动重新编程过程。基于我们对Fox蛋白质的研究，我们处于有利地位，可以揭示多能性因子功能的机制基础，并确定如何增强它，其目的是：1)评估五种人类多能性因子在体外与重组到纯化的染色质模板中的自然人类靶序列上的初始染色质结合和开放特性，并与这些因子在体内相同染色质部位的初始结合进行比较(Plath和Lowry实验室)。2)绘制染色质结合和开放的关键蛋白质结构域，以及相关结构域的作用机制(与Plath Lab)。3)确定染色质的哪些修饰可以限制多能性因子的结合或染色质的开放，以及如何通过修改因子的适当结构域来克服这一点(与Lowry和Smear实验室)。这些目标中的每一个都涉及与P01提案其他成员的直接互动和协作。这些研究的见解和方法将适用于重新编程的不同类型的细胞和治疗环境。 这项拟议的工作不会进行人体或动物实验。