Role of an Integrator-EGR axis in the regulation of myeloid enhancers

Integrator-EGR 轴在骨髓增强子调节中的作用

• 批准号: 10536618
• 负责人: Alessandro Gardini
• 金额: $ 44.65万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536618
• 关键词: 3-Dimensional; Acute Myelocytic Leukemia; Architecture; Automobile Driving; Biochemical; Biochemistry; Biological Assay; Bone Marrow; CD34 gene; Cells; ChIP-seq; Chromatin; Code; Complex; Data; Development; Enhancers; Foundations; Future; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Hematopoietic; Hematopoietic stem cells; Hi-C; Histone Acetylation; Human; Knowledge; Macrophage; Measures; Mediating; Mediator; Molecular; Molecular Conformation; Monitor; Multipotent Stem Cells; Myelogenous; Myeloid Cells; Myelopoiesis; Myeloproliferative disease; Nucleosomes; Physiology; Play; Positioning Attribute; Process; Property; RNA; Regulation; Research; Role; Shapes; Testing; Therapeutic; Time; Tissues; Transcription Process; Transcriptional Activation; Untranslated RNA; Work; active control; cofactor; functional genomics; genome-wide; global run on sequencing; histone methylation; insight; monocyte; novel; peripheral blood; programs; promoter; protein complex; recruit; stem cells; targeted treatment; transcription factor; transcriptome; transcriptome sequencing

Project Summary Role of an Integrator-EGR axis in the regulation of myeloid enhancers Objective: My research aims to identify how enhancer-based regulation determines monocytic cell fate. Specifically, we seek to understand the function of a newly identified regulatory axis, comprising the Integrator complex, the EGR-1/EGR-2 transcription factors and their co-factor NAB2. We aim at determining how these chromatin regulators: a) prime myeloid enhancers for activation; b) remodel nucleosome accessibility and reshape 3D genome conformation to impose a monocytic-specific transcriptome. We seek to characterize the first lineage-specific function of the Integrator protein complex, which was previously believed to serve a general role in transcription. Proposed research: The process of myelopoiesis is governed by an elaborate gene expression program that originates in the bone marrow, and is brought to maturity in the peripheral blood and tissues. The transcriptional process that generates mature myeloid cells from hematopoietic stem cells (HSCs) is initiated by sequence-specific transcription factors (TFs) that instruct enhancers to elicit gene activation. However, how these myeloid TFs engage the basal transcriptional machinery and activate lineage-specific enhancers is poorly understood. We uncovered that the INTS13 subunit of the Integrator complex plays an essential role in myelopoiesis and we propose that Integrator carries the previously unidentified ability to target myeloid-specific TFs and modulate cell fate determination via enhancer regulation. We will molecularly dissect the function of Integrator, and its functional partners EGR-1/2 and NAB2, in monocytic/macrophagic differentiation by leveraging our expertise in biochemistry and functional genomics through the following aims. 1) We will define the enhancer network that determines myelopoiesis. We hypothesize that INTS13 modulates Integrator’s activity at EGR-targeted enhancers during monocytic commitment. Therefore, we will profile INTS13 and EGR-1/2 recruitment in human cells, and we will determine the status of targeted enhancers by monitoring their histone methylation and acetylation levels (H3K27Ac and H3K4Me1). Further, we will evaluate the transcriptional effect of INTS13, EGR-1/2 and NAB2 depletion on myeloid genes by RNA-seq. 2) We will determine the changes in chromatin architecture mediated by INTS13, EGR-1/2, and NAB2. We hypothesize that these chromatin regulators prime enhancers for activation and execute cell fate commitment. Our data suggest that INTS13, EGR-1/2, and NAB2 control the active/poised status of enhancers and oversee genome topology during differentiation. We will functionally test our hypothesis by analyzing nucleosome remodeling at enhancers (pioneer activity) and by determining how Integrator and EGR-1/2 modulate chromosomal looping and 3D genome conformation using Hi-C and 3C assays. Lastly, we will characterize the biochemical properties of Integrator to identify the myeloid-specific module that regulates EGR-activity in monocytes.

项目摘要 集成符轴的作用在调节髓样增强子中 目的：我的研究旨在确定基于增强子的调节如何决定单核细胞命运。 具体而言，我们试图了解新确定的监管轴的功能，完成集成器 复合物，EGR-1/EGR-2转录因子及其共同因素NAB2。我们旨在确定这些 染色质调节剂：a）激活的主要髓样增强剂； b）重塑核病室的可访问性和 重塑3D基因组会议强加单核细胞特异性转录组。我们试图表征 积分蛋白复合物的第一个谱系特异性功能，以前认为它可以使用 在转录中的一般作用。 拟议的研究：骨髓虫的过程受到精心制作的基因表达程序的约束 起源于骨髓，在外周血和组织中成熟。 从造血干细胞（HSC）产生成熟的髓样细胞的转录过程已开始 通过指示增强子引起基因激活的序列特异性转录因子（TFS）。但是，如何 这些髓样TF参与基本的转录机械和激活谱系特异性增强剂是 理解不佳。我们发现，集成子复合物的INTS13亚基在 骨髓卵皮，我们建议集成剂具有先前未鉴定的靶向髓样特异性的能力 通过增强子调节调节细胞脂肪的测定。我们将分子剖析 集成剂及其功能合作伙伴Egr-1/2和Nab2，在单核细胞/巨型分化中通过 通过以下目标利用我们在生物化学和功能基因组学方面的专业知识。 1）我们将定义 确定脊髓虫的增强剂网络。我们假设INTS13调节了集成器的 在单核细胞承诺过程中，在EGR靶向增强子的活动。因此，我们将介绍INTS13和 人类细胞中的EGR-1/2募集，我们将通过监视靶向增强剂的状态 它们的组蛋白甲基化和乙酰化水平（H3K27AC和H3K4ME1）。此外，我们将评估 RNA-seq对INTS13，EGR-1/2和Nab2耗竭对髓样基因的转录作用。 2）我们会的 确定由INTS13，EGR-1/2和NAB2介导的染色质结构的变化。我们假设 这些染色质调节剂的主要增强剂用于激活和执行细胞脂肪承诺。我们的数据 建议INTS13，EGR-1/2和NAB2控制增强剂和海外基因组的主动/固定状态 分化过程中的拓扑。我们将通过分析核小体重塑在功能上检验我们的假设 增强剂（先驱活动）以及通过确定积分器和EGR-1/2如何调节染色体环 使用HI-C和3C分析的3D基因组组成。最后，我们将表征生化 积分器的性能识别调节单核细胞中的Egr活性的髓样特异性模块。