Analyzing pioneer factor dynamics and function during differentiation and reprogramming

分析分化和重编程过程中先锋因子的动态和功能

• 批准号: 9911897
• 负责人: Michael P Meers
• 金额: $ 6.53万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911897
• 关键词: Automobile Driving; Binding; Binding Sites; Biology; Cell Differentiation process; Cell model; Cells; ChIP-seq; Chromatin; DNA; DNA Binding; Data; Dependence; Development; Digestion; Dimensions; Disease; Endoderm; Enhancers; Eukaryota; Event; Fibroblasts; Frequencies; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic study; Genome; Genomics; Health; Human; In Situ; Individual; Malignant Neoplasms; Measures; Mediating; Methods; Micrococcal Nuclease; Mission; Modeling; Molecular; Molecular Computations; National Institute of General Medical Sciences; Nucleosome Binding Domain; Nucleosomes; Pathogenicity; Pattern; Play; Population; Proteins; Regulation; Reporter; Research; Role; Site; Specific qualifier value; Specificity; Structure; System; Techniques; Testing; Time; Transcriptional Regulation; base; cell fate specification; cell type; cofactor; embryonic stem cell; epigenome; experimental study; genome-wide; human embryonic stem cell; in vivo; in vivo evaluation; induced pluripotent stem cell; insight; novel; nuclease; overexpression; particle; pluripotency; programs; spatiotemporal; stem cell differentiation; transcription factor

Project Summary/Abstract The long-term objectives of this proposal include the following: 1) to determine the frequency and necessity of pioneer factor-nucleosome interactions genome wide during cell differentiation, and 2) to characterize the individual and cooperative pioneer factor-nucleosome interactions that occur during induced pluripotency. Cell fate decisions require exquisite spatiotemporal regulation of transcription factor (TF) binding to enhancers to mediate gene regulation, which is frequently co-opted in a variety of diseases. It is well established that active enhancers are locally depleted of nucleosomes, and that TF binding is coincident with, and often required for, local chromatin accessibility. However, it remains unclear how TFs might play an active role in displacing nucleosomes at enhancers. One hypothesis is that “pioneer” TFs, so called because they are expressed early in cell differentiation and bind to their enhancer targets before they are rendered strongly accessible, directly bind and displace nucleosomes to facilitate accessibility. However, such a mechanism has never been validated on individual templates in vivo, chiefly due to the difficulty in testing for the presence of the putative intermediate TF-nucleosome particle. Lacking a direct test for in vivo pioneer activity, putative mechanisms for nucleosome eviction by pioneer TFs remain untested, and the locus specificity of pioneer activity is largely unknown. Moreover, the frequency and importance of TF pioneer activity in either natural differentiation systems or TF overexpression contexts during cellular reprogramming remain uncharacterized. To directly test the pioneer TF model, I will leverage an in situ, micrococcal nuclease (MNase) digestion-based alternative to ChIP-seq known as CUT&RUN, in which DNA fragment size is directly informative of the minimal protein-protected sequence, and therefore can distinguish direct TF binding from binding through a nucleosomal intermediate. I will use this information to determine the genome- wide landscape of pioneer factor-nucleosome interactions in embryonic stem cell differentiation and during induced pluripotency, and test the necessity of such interactions using functional genetic experiments. These insights will help us to understand how pioneer factors function to activate pathogenic gene expression in diseases such as cancer. I feel that my proposed project fits the mission of NIGMS to fund research in fundamental biology that uncovers new insights that could positively impact human health. !

项目摘要/摘要 该提案的长期目标无需以下内容：1） 确定先锋因子核体相互作用的频率和必要性 细胞分化过程中的基因组宽，2）表征个体 以及合作的先驱因子因子核体相互作用 诱导的多能性命运决定需要精致的时空 调节转录因子（TF）与增强子的结合以介导基因 在各种疾病中自由选择的法规 确定活性增强子是核小体局部深处的，并且 TF结合与局部染色质相吻合，并且经常需要 但是，尚不清楚TF在 在增强子处的核小体位移。 之所以被称为，是因为它们在细胞分化早期表达并与它们的结合 在增强靶标在将其启用，直接绑定和 但是，核小体可促进可及性。 从未在体内得到验证的跨个性模板，这主要是由于困难 在测试假定的TF核体颗粒的存在时。 缺乏对体内先驱活动的直接测试，用于推定的机制 先锋tfs的核小体驱逐仍未测试，并且 先锋活动是更大的未知数。 天然分化系统或TF过表达中的TF先驱活动 细胞复印过程中的上下文仍然没有表征 先驱TF模型，我将利用原位的微局核（MNase） 基于消化的基于芯片序列的替代品，称为切割和运行，其中DNA 片段大小是最小蛋白质保护序列的直接信息， 因此可以区分直接TF结合与通过A结合的结合 核小体中间有核心。 胚胎干细胞中先锋因子核体相互作用的宽阔景观 分化和诱导的多能性，并测试这种必要性 使用功能性实验的相互作用。 了解先锋因素如何激活致病基因表达 在癌症等疾病中。 诺格姆斯资助基本生物学研究的研究，以发现新见解的新见解 可以积极影响人类健康。 呢