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

描述（由申请人提供）：通过ChIP-seq和ChIP-chip方法，用于体内定位转录因子结合位点的全基因组方法已经导致了关于活细胞中整个基因组中调控因子部署的信息爆炸。发育系统生物学的“部件清单”现在不仅包括调节因子，而且还包括许多候选的顺式调节靶位点。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有率与实际功能影响位点相匹配。来自细胞系或终末分化状态的静态转录因子结合“快照”可以指示因子在细胞中起作用的位点。然而，在实际发育系统的精细尺度检查中，因子占用的动态变化与RNA表达的变化平行发生。因子结合在许多位点的功能影响是模糊的，并且因子占据的许多位点对于转录控制可能是多余的。这强调了寻找更好的方法来预测转录因子在特定位点参与的功能的紧迫性。 该项目基于一项全球战略，用于阐明全基因组因子结合和转录功能之间的关系，在一个完善的发育系统中，多能血细胞前体致力于发展为T细胞。我们以前的工作已经确定了在这一过程中的过渡在细胞和分子水平上，包括最近的全球分析逐步转录组的变化，转录因子基因表达的变化，局部染色质标记的变化，并在整个基因组的特定转录因子的结合occupancy的幅度和网站的变化。关于这个过程的调节成分的多阶段信息使得有可能动态地分析转录因子的作用，不仅在哪里看到结合方面，而且在每个位点的结合占有率随时间的变化方面，然后可以将其与连接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真的导致这些基因表达变化，需要功能扰动。在这里，我们建议应用一个系统的快速，特定阶段的扰动程序，以确定两个基本的转录因子，PU.1和Runx 1的作用机制。一组重要的工具将是专性阻遏物构建体，其用作内源性因子的竞争者以区分直接与间接激活和阻遏。因此，可以在特定阶段应用功能的快速获得和丧失，并且通过RNA-seq分析影响以在全系统范围内鉴定受影响的基因，即使因子的影响在发育阶段之间改变幅度或符号。因此，网站和网站组合建议有功能的影响，将检查独特的共聚集的网站，组蛋白修饰规则，转录因子交换率。然后，通过新的、特异性预测的顺式异构体的功能测定来测试这些特征的一般性。 基因网络中的调控位点模块。

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.