Global Predictions and Tests of Hematopoietic Regulation

造血调节的整体预测和测试

• 批准号: 8912612
• 负责人: DAVID M. BODINE
• 金额: $ 22.43万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912612
• 关键词: Anemia; Architecture; Bayesian Modeling; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Models; Blood Cells; Candidate Disease Gene; Categories; Cell Count; Cell Differentiation process; Cell Lineage; Cell Separation; Cell model; Cells; ChIP-seq; Chromatin; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Communities; Cultured Cells; DNA; DNA Methylation; Data; Data Set; Developmental Biology; Diamond-Blackfan anemia; Disease; Disease susceptibility; Distal; Epigenetic Process; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Fetal Liver; Foundations; GATA1 gene; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genome; Genomics; Goals; Health; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; In Vitro; Knock-out; Lead; Learning; MEIS1 gene; Maps; Massive Parallel Sequencing; Megakaryocytes; Methods; Modeling; Molecular Conformation; Molecular Profiling; Mus; Mutation; Myelogenous; Myeloid Cells; Nucleic Acid Regulatory Sequences; Pattern; Phase; Population; Production; Proteins; Regulation; Research; Research Personnel; Resources; Role; Sequence Alignment; Sickle Cell Anemia; Site; Staging; Statistical Models; Stem cells; System; TAL1 gene; Testing; Therapeutic Intervention; Time; Training; Transcript; Transfection; Work; XCL1 gene; base; cell type; computer based statistical methods; erythroid differentiation; genetic manipulation; genetic regulatory protein; genetic variant; genome-wide; histone modification; human disease; innovation; insight; leukemia; methylome; progenitor; statistics; transcription factor; transcriptome sequencing

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.

描述（由申请人提供）： 定义造血细胞的调节结构以阐明谱系的确定和分化可以产生对发育生物学的见解，并可以帮助识别具有潜在应用人类疾病（例如白血病和贫血）的靶标。小鼠造血是一种用于研究分化过程中基因调节的多功能系统，因为我们可以纯化祖细胞和分化细胞的种群，以用于转录本和调节序列的全基因组映射，并且我们可以通过遗传操纵关键的临界蛋白质和顺式调控模块（CRMS）来研究调节机制。该应用是为了在多个研究人员之间续签造血细胞分化，基因调节，基因组学，生物信息学和统计学的多个研究人员之间的长期富有成效的合作。我们以前的工作为培养的细胞模型中的转录因子，转录因子占用率和染色质状态奠定了培养细胞模型的基础，用于红斑分化以及在红细胞和巨核细胞谱系中成熟的原代细胞中，这导致了有关调节的关键新见解。现在，我们建议（AIM 1）从分化从小鼠造血干细胞到红细胞和髓样谱系成熟细胞的每个阶段的纯化细胞中的转录组和信息表观遗传特征产生全基因组数据。对于所有细胞类型，包括仅以少量数量可用的多列元素祖细胞，我们建议确定转录组，DNA甲基化和染色质的可及性（使用基于体外转座的新方法）。在更丰富的细胞类型中，我们将使用ChIP-Seq绘制转录因子和组蛋白修饰，以及染色体构象捕获方法HI-C构建与靶基因的远端调节区域的相互作用图。然后，我们将（AIM 2）进行综合，定量 建模以找到差异表达的基因，并具有不同的转录因子结合模式。在此组中，是参与选择细胞谱系的基因的候选物。假设驱动的贝叶斯网络模型将学习特征之间的定量关系，包括表达水平，并预测系统在两种转录因子和CRM的扰动后如何行为。然后，我们将（AIM 3）进行遗传操作，以测试AIM 2中综合分析引起的假设。将通过转导干扰或强制表达构建体向小鼠胎儿肝脏祖细胞和双原位来测试有关谱系选择基因的特定假设。 培养细胞。将测试来自表达水平的定量模型的假设，以靶向特定的蛋白质（使用带有或withou gata1的细胞转染）和CRMS（通过CAS9-CRISPR指导的基因组编辑）。这项拟议工作的结果将是深层，广泛传播有关多种造血谱系中调节景观的数据，以及对调节蛋白和染色质的变化如何导致谱系选择和渐进式分化的敏锐见解。

项目成果

Dynamic CTCF binding directly mediates interactions among cis-regulatory elements essential for hematopoiesis.

• DOI: 10.1182/blood.2020005780
• 发表时间: 2020-12
• 期刊: Blood
• 影响因子: 20.3
• 作者: Qian Qi;Li Cheng;Xing Tang;Yanghua He;Yichao Li;Tiffany Yee;Dewan Shrestha;Ruopeng Feng;Peng Xu;Xin Zhou;Shondra M. Pruett-Miller;R. Hardison;M. Weiss;Yong Cheng

A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

• DOI: 10.1038/nm.4170
• 发表时间: 2016-09
• 期刊: Nature medicine
• 影响因子: 82.9
• 作者: Traxler EA;Yao Y;Wang YD;Woodard KJ;Kurita R;Nakamura Y;Hughes JR;Hardison RC;Blobel GA;Li C;Weiss MJ
• 通讯作者: Weiss MJ