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

基本信息

批准号：
9256523
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-05 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9256523
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 DNA DNA Binding Dangerousness Development Disadvantaged Disease Dominant-Negative Mutation Elements Embryo Endogenous Factors Enhancers Etiology Explosion Factor Analysis Family Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription Genome Genomics Guanine Nucleotide Exchange Factors Hematopoietic stem cells Link Lymphoid Measurement Measures Mediating Methods Molecular Mus Pattern Play Process RUNX1 gene Regulation Regulator Genes Repression Resolution Role Site Stem cells System Systems Biology T-Cell Development T-Lymphocyte Testing Time Transcriptional Regulation Work base genome-wide histone modification improved in vivo loss of function member preconditioning programs protein expression public health relevance response retroviral transduction tool transcription factor transcriptome transcriptome sequencing

项目摘要

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）：通过芯片seq和chip-chip方法在体内映射转录因子结合位点的全基因组方法，导致有关在活细胞中基因组跨基因组部署调节因子的信息的爆炸爆炸。现在，开发系统生物学的“零件列表”现在不仅包括监管因素，还包括许多候选顺便调节目标站点。尽管如此，了解开发中转录调节的一个主要问题是如何将转录因子位点占用率与实际功能影响的站点匹配。静态转录因子结合“快照”从细胞系或末端分化状态得出的“快照”可能表明一个因子在Thos细胞中起作用的位置。但是，在对实际发育系统的精细检查中，因子占用率的动态变化与RNA表达的变化同时发生。因子结合在许多地点的功能影响是晦涩的，许多因素占用位点对于转录控制可能是多余的。这强调了找到更好的方法来预测特定位点转录因子参与功能的紧迫性。该项目基于一种全球策略，用于阐明全基因组因子结合和转录功能之间的关系，在一个公认的发育系统中，多能血细胞前体致力于作为T细胞发展。我们以前的工作已经在细胞和分子水平上定义了这一过程中的转变，包括对逐步转录组变化的最新全局分析，转录因子基因表达的变化，局部染色质标记变化以及幅度的变化以及跨整个占领的位点的变化特定转录因子的基因组。有关此过程的调节组件的多阶段信息使得可以动态分析转录因子动作，不仅在看到绑定的位置，而且还可以在每个站点的绑定占用中随着时间的时间变化而言，然后可以进行比较链接基因的表达变化。为了评估转录因子结合的变化是否实际引起这些基因表达变化，需要功能扰动。在这里，我们建议应用一个系统的快速，特异性扰动的系统程序，以确定两个基本转录因子PU.1和RUNX1的作用机理。一组重要的工具将是用作内源性因素的竞争者的强制性阻遏构建体，以区分直接激活和抑制。因此，即使因子的影响改变幅度或发育阶段之间的迹象，也可以在特定阶段应用快速增益和功能丧失，并在特定阶段和RNA-Seq分析的效果以识别受影响的基因。因此，将检查具有独特的共聚类地点，组蛋白修饰规则和转录因子汇率率的站点和现场组合功能影响。然后，这些功能将通过新的，特别是预测顺式的功能测定来测试通用性基因网络中的监管站点模块。