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

项目2：染色质结合机制

基本信息

批准号：
8382272
负责人：
Kenneth Zaret
金额：
$ 49.4万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8382272
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

项目摘要

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.

描述该提案的目的是揭示多能转录因子能够获取发育沉默的基因，打开染色质并从而启动自我维持的监管网络的机制。只有1/100至1/10,000的体细胞，即即使采用最有效的蛋白质，mRNA或基因递送的方法，也可以将OCT4，SOX2，KLF4和C-MYC因子表达为多能性，并且可以将其重新编程为多能性。我的实验室表明，FOXA转录因子具有固有的生化能力，可以结合静音基因压实的染色质中的靶位点。这会产生局部超敏反应，从而促进其他转录因子和基因激活的结合。我们进一步发现，FOXA“先锋因素”包含不同的蛋白质结构域，这些蛋白质结构域调节了该因子在染色质中移动并产生超敏反应的能力，我们发现染色质修饰状态限制了FOXA参与度。基于OCT4，SOX2，KLF4和C-MYC的深刻能力激活分化细胞中的无声多能网络以及Nanog在此过程中的作用，我们假设一种或多种此类蛋白质具有“先锋”活性，以及影响染色质功能的相关域；这样的结构域可能会对抗性染色质状态提供敏感性；并且对这种知识将使我们能够设计多能因素，从而更有效地启动重编程过程。根据我们对FOX蛋白的研究，我们有好处公布多能性因子功能的机理基础，并确定如何增强其，并以这些目的评估：1）评估自然靶向序列中五个人类多能因素的五个人类多能因素的开放性和开放性。 Plath和Lowry Labs）。 2）绘制蛋白质结构域对于染色质的互动和开放至关重要，以及相关结构域的作用机理（使用PLATH LAB）。 3）确定哪些染色质的修饰可以限制多能因素的参与度或染色质的开放，以及如何通过修改适当的因子（使用Lowry和Smale Labs）来克服这种修饰。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多种类型的细胞和治疗环境，用于重编程。拟议的工作将不会进行人类或动物的实验。