Global Predictions and Tests of Hematopoietic Regulation

描述（由申请人提供）： 定义造血细胞的调控结构以阐明谱系决定和分化可以产生对发育生物学的见解，并且可以帮助识别具有潜在应用于人类疾病（如白血病和贫血）的靶点。小鼠造血是一个多功能的系统，用于研究基因调控分化过程中，因为我们可以纯化祖细胞和分化的细胞群体的转录本和调控序列的全基因组映射，我们可以遗传操纵的关键蛋白质和顺式调控模块（CRM）的研究机制的监管。该申请旨在更新多名研究人员之间的长期有效合作，这些研究人员在造血细胞分化，基因调控，基因组学，生物信息学和统计学方面具有互补的专业知识。我们以前的工作奠定了全基因组数据集的基础，转录组，转录因子占用率和染色质状态在培养的细胞模型中用于红系分化和红系和巨核细胞谱系中的成熟原代细胞，这导致了关于调控的关键新见解。我们现在提出（目的1）产生全基因组数据的转录组和信息表观遗传特征的纯化细胞从小鼠造血干细胞分化的每个阶段的成熟细胞的红系和髓系。对于所有的细胞类型，包括多系祖细胞，只有在少量，我们建议确定转录组，DNA甲基化，和染色质的可及性（使用一种新的方法在体外转座的基础上）。在更丰富的细胞类型中，我们将使用ChIP-seq来定位转录因子和组蛋白修饰，并使用染色体构象捕获方法Hi-C来构建远端调控区与靶基因的相互作用图谱。然后，我们将（目标2）进行综合，定量 建模以发现在不同谱系中差异表达和具有不同转录因子结合模式的基因;在该集合内是参与细胞谱系选择的基因的候选者。假设驱动的贝叶斯网络模型将学习包括表达水平在内的特征之间的定量关系，并预测转录因子和CRM扰动后系统的行为。然后，我们将（目标3）进行遗传操作，以检验目标2中综合分析产生的假设。通过将干扰或强制表达构建体转导入小鼠胎肝祖细胞和双能细胞中，将对涉及谱系选择的基因的具体假设进行测试。 细胞培养。将测试来自表达水平决定因素的定量建模的假设，靶向特定蛋白质（使用具有或不具有GATA 1的细胞转染）和CRM（通过Cas9-CRISPR指导的基因组编辑）。这项拟议工作的结果将是深入的，广泛传播的数据在多个造血谱系的监管景观和敏锐的洞察力如何在调节蛋白和染色质的变化导致谱系选择和渐进分化。

DESCRIPTION (provided by applicant): Defining the regulatory architecture of hematopoietic cells to elucidate lineage determination and differentiation can produce insights into developmental biology and can help identify targets with potential application to human diseases such as leukemias and anemias. Mouse hematopoiesis is a versatile system for studying gene regulation during differentiation because we can purify populations of progenitor and differentiated cells for genome-wide mapping of transcripts and regulatory sequences, and we can genetically manipulate critical proteins and cis-regulatory modules (CRMs) to study mechanisms of regulation. This application is for a renewal of a long-standing, productive collaboration among multiple investigators with complementary expertise in hematopoietic cell differentiation, gene regulation, genomics, bioinformatics and statistics. Our previous work laid a foundation of genome-wide data sets for transcriptomes, transcription factor occupancy and chromatin states in a cultured cell model for erythroid differentiation and in maturing primary cells in the erythroid and megakaryocytic lineages, which led to key new insights about regulation. We now propose to (Aim 1) generate genome-wide data on transcriptomes and informative epigenetic features in purified cells from each stage of differentiation from mouse hematopoietic stem cells to mature cells of the erythroid and myeloid lineages. For all cell types, including multilineage progenitor cells available only in small numbers, we propose to determine transcriptomes, DNA methylation, and chromatin accessibility (using a new method based on in vitro transposition). In more abundant cell types, we will use ChIP-seq to map transcription factors and histone modifications and also the chromosome conformation capture method Hi-C to build an interaction map of distal regulatory regions with target genes. We will then (Aim 2) conduct integrative, quantitative modeling to find genes differentially expressed and with different transcription factor binding patterns in the distinct lineages; within this set are candidates for genes involved in choice of cell lineage. A hypothesis-driven Bayesian network model will learn quantitative relationships between features, including expression level, and make predictions about how the system would behave after perturbation of both transcription factors and CRMs. We will then (Aim 3) conduct genetic manipulations to test hypotheses arising from integrative analysis in Aim 2. Specific hypotheses about genes involved in lineage choice will be tested by transduction of interfering or forced expression constructs into mouse fetal liver progenitor cells and bipotential cells in culture. Hypotheses from the quantitative modeling of determinants of levels of expression will be tested, targeting specific proteins (using transfections of cells with or withou GATA1) and CRMs (by Cas9-CRISPR-guided genome editing). The result of this proposed work will be deep, widely disseminated data on the regulatory landscape in multiple hematopoietic lineages and keener insights into how changes in regulatory proteins and chromatin lead to lineage choice and progressive differentiation.