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

 描述（由申请人提供）：我们提出在模型系统中使用创新的多重荧光原位方法（seqFISH）来确定来自相同细胞中多个基因的转录本数量，以解决转录因子效应如何在动态发育系统中对不同类别的靶基因施加的一般问题。该提案是对现有资助项目“发育T细胞中的基因组位点结合规则和调节因子功能”（R 01 HD 076915）的修订，它增加了一个关键的单细胞分析组件，以澄清解释并扩展项目的见解。同时，生物系统提供了许多优点，以提高功率和证明seqFISH技术的实用性。 我们利用发育事件的良好表征的框架，通过该框架，多能造血祖细胞经历承诺成为T细胞前体，该系统可以在体内和体外平行研究，并且在细胞水平和基因表达模式方面高度定义。该项目侧重于转录因子PU.1的关键但神秘的作用，这是支持T细胞发育早期所需的，但显然是以维持与另一组发育命运（即巨噬细胞、粒细胞和树突细胞命运）的调节桥梁为代价的。PU.1在不同造血前体中的基因组占据模式具有谱系特异性差异，但其许多结合位点似乎与功能无关。母公司的提案结合了急性扰动测定，全基因组RNA-seq和诊断ChIP-seq方法，以确定在这种早期T细胞背景下将PU.1占用与PU.1调节功能相关的规则。然而，重要的是要确定每个基线状态的均匀程度，以及在给定的早期T细胞阶段所有细胞上的PU.1作用的均匀程度，以解决哪些PU.1靶基因实际上在相同的调节状态背景下响应PU.1，哪些在不同的调节状态背景下响应。 为了回答这个问题，并详细揭示不同的靶基因如何“处理”同一调节因子的活性变化，我们提出了Ellen Rothenberg和Long Cai小组之间的新合作。具体目标1：使用seqFISH来表征T细胞前体中PU.1表达的变化以及已知和疑似PU.1靶基因水平的变化，相对于可以调节PU.1效应的基因的表达。具体目标2：优化seqFISH技术，用于检测单个细胞中每个细胞>20个不同基因的转录物。具体目标3：分析PU.1缺失和PU.1拮抗对单细胞中不同类别候选靶基因表达的影响，并确定其与这些细胞中不同调节因子水平的相关性。

 DESCRIPTION (provided by applicant): We propose to use an innovative multiplex fluorescent in situ method for determining transcript numbers from multiple genes in the same cells, seqFISH, in a model system to address the general problem of how transcription factor effects are exerted on different classes of target genes in a dynamic developmental system. The proposal is a Revision for the existing, funded project, "Genomic site binding rules and regulatory factor function in developing T cells" (R01HD076915), and it adds a crucial single-cell analysis component to clarify the interpretation and extend the insights from the project. In parallel, the biological system offers many advantages for enhancing the power and demonstrating the utility of the seqFISH technique. We exploit a well-characterized framework of developmental events through which multipotent hematopoietic progenitors undergo commitment to become T-cell precursors, a system that can be studied in parallel in vivo and in vitro and which is highly defined at the cellular level and in terms of patterns of gene expression The project focuses on the crucial but enigmatic role of the transcription factor PU.1, which is needed to support the early stages in T-cell development but does so apparently at the price of maintaining a regulatory bridge to an alternative set of developmental fates, i.e. macrophage, granulocyte, and dendritic cell fates. PU.1 has lineage-specific differences in its patterns of genomic occupancy in different hematopoietic precursors, but many of its binding sites do not appear to be linked with function. The parent proposal combines acute perturbation assays, genome-wide RNA-seq, and diagnostic ChIP-seq approaches to determine the rules that relate PU.1 occupancy to PU.1 regulatory functions in this early T-cell context. However, it is important to determine how homogeneous each baseline state is, and how uniform PU.1 actions are on all the cells at a given early T-cell stage, to resolve which PU.1 target genes are actually responding to PU.1 in the same regulatory-state context and which are responding in a different one. To answer this question and reveal in detail how different target genes "process" changes in activity of the same regulator, we propose a new collaboration between the Ellen Rothenberg and Long Cai groups. Specific aim 1: Use seqFISH to characterize the variation in PU.1 expression in T-cell precursors and variation in levels of known and suspected PU.1 target genes, relative to expression of genes that can modulate PU.1 effects. Specific aim 2: Optimize seqFISH technology for detection of transcripts from >20 different genes per cell in individual cells. Specific aim 3: Analyze the effects of PU.1 deletion and PU.1 antagonism on expression of different classes of candidate target genes in single cells, and determine their correlation wit levels of different modulating factors in those cells.