Genomic site binding rules and regulatory factor function in developing T cells

发育中 T 细胞的基因组位点结合规则和调节因子功能

• 批准号: 8560062
• 负责人: ELLEN V. ROTHENBERG
• 金额: $ 33.3万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-05 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8560062
• 关键词: Acute; Adult; Adverse effects; Affect; Binding; Binding Sites; Bioinformatics; Biological Assay; Blood Cells; Cell Line; Cells; Cereals; ChIP-on-chip; ChIP-seq; Chromatin; Commit; DNA; DNA Binding; Development; Disadvantaged; Disease; Dominant-Negative Mutation; Elements; Embryo; Endogenous Factors; Enhancers; Etiology; Explosion; Factor Analysis; Family; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Hematopoietic; Hematopoietic stem cells; Life; Link; Lymphoid; Maps; Measurement; Measures; Mediating; Methods; Molecular; Mus; Pattern; Play; Process; Proteins; Regulation; Regulator Genes; Repression; Resolution; Role; SPI1 gene; Site; Staging; Stem cells; System; Systems Biology; T-Cell Development; T-Lymphocyte; Testing; Time; Transcription factor genes; Transcriptional Regulation; Work; base; genome-wide; histone modification; improved; in vivo; loss of function; member; preconditioning; progenitor; programs; proto-oncogene protein Spi-1; public health relevance; response; retroviral transduction; tool; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Genome-wide methods for mapping transcription factor binding sites in vivo, by ChIP-seq and ChIP-chip methods, have led to an explosion of information about the deployment of regulatory factors across the genome in living cells. The "parts list" for developmental systems biology now includes not only regulatory factors but also many candidate cis-regulatory target sites. Still, a major problem for understanding transcriptional regulation in development is how to match transcription factor site occupancy with sites of actual functional impact. Static transcription factor binding "snapshots" derived fro cell lines or terminally differentiated states may indicate sites where a factor is working in thos cells. However, in fine-scale examination of actual developmental systems, dynamic changes in factor occupancy occur in parallel with changes in RNA expression. The functional impact of factor binding at many sites is obscure, and many sites of factor occupancy may be superfluous for transcriptional control. This underlines the urgency of finding better methods to predict functionality of transcription factor engagement at specific sites. This project is based on a global strategy for elucidating relationships between genome-wide factor binding and transcriptional function, in a well-established developmental system in which multipotent blood-cell precursors become committed to develop as T cells. Our previous work has defined the transitions in this process at both cellular and molecular levels, including a recent global analysis of stepwise transcriptome changes, changes in transcription factor gene expression, local chromatin marking changes, and changes in magnitude and sites of binding occupancies across the genome by specific transcription factors. The multistage information about regulatory components of this process makes it possible to analyze transcription factor action dynamically, not only in terms of where binding is seen, but also in terms of the changes over time in binding occupancy at each site, which can then be compared to expression changes in the linked genes. To evaluate whether the transcription factor binding changes actually cause these gene expression changes, functional perturbations are required. Here, we propose to apply a systematic program of fast, stage-specific perturbations to determine the mechanisms of action of two essential transcription factors, PU.1 and Runx1. An important set of tools will be obligate repressor constructs used as competitors for endogenous factors to distinguish direct vs. indirect activation and repression. Quick gain and loss of function can thus be applied at particular stages and the effects analyzed by RNA-seq to identify affected genes system-wide, even if effects of a factor change magnitude or sign between developmental stages. The sites and site combinations thus suggested to have functional impact will be examined for distinctive co-clustered sites, histone modification rules, and transcription factor exchange rates. These features will then be tested for generality by functional assays of new, specifically predicted cis regulatory site modules within the gene network.

描述（由申请人提供）：通过 ChIP-seq 和 ChIP-chip 方法绘制体内转录因子结合位点的全基因组方法，已经导致有关活细胞中基因组中调节因子的部署的信息激增。发育系统生物学的“部分列表”现在不仅包括调节因子，还包括许多候选顺式调节靶位点。尽管如此，理解发育中转录调控的一个主要问题是如何将转录因子位点占用与实际功能影响的位点相匹配。来自细胞系或终末分化状态的静态转录因子结合“快照”可能表明因子在这些细胞中起作用的位点。然而，在实际发育系统的精细检查中，因子占据的动态变化与 RNA 表达的变化同时发生。许多位点的因子结合的功能影响是模糊的，并且许多因子占据的位点对于转录控制来说可能是多余的。这强调了寻找更好的方法来预测特定位点转录因子参与功能的紧迫性。 该项目基于一项全球战略，旨在阐明全基因组因子结合与转录功能之间的关系，在一个完善的发育系统中，多能血细胞前体致力于发育为 T 细胞。我们之前的工作在细胞和分子水平上定义了这一过程的转变，包括最近对逐步转录组变化、转录因子基因表达的变化、局部染色质标记变化以及特定转录因子在整个基因组中的结合占据的大小和位点的变化进行的全局分析。有关该过程的调控组件的多阶段信息使得动态分析转录因子的作用成为可能，不仅可以观察到结合的位置，还可以分析每个位点的结合占用随时间的变化，然后可以将其与连锁基因的表达变化进行比较。为了评估转录因子结合变化是否确实导致这些基因表达变化，需要进行功能扰动。在这里，我们建议应用快速、特定阶段扰动的系统程序来确定两个重要转录因子 PU.1 和 Runx1 的作用机制。一组重要的工具将是专性阻遏物构建体，用作内源性因子的竞争者，以区分直接与间接激活和抑制。因此，可以在特定阶段应用功能的快速获得和丧失，并通过 RNA-seq 分析其影响，以识别整个系统范围内受影响的基因，即使某个因素的影响在发育阶段之间改变幅度或符号。因此，将检查具有功能影响的位点和位点组合的独特共簇位点、组蛋白修饰规则和转录因子交换率。然后，将通过新的、专门预测的顺式功能分析来测试这些特征的普遍性 基因网络内的调控位点模块。