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: 10310455
• 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 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10310455
• 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; Measures; Mediating; Mediator of activation protein; Molecular; Molecular Conformation; Monitor; Multipotent Stem Cells; Myelogenous; Myeloid Cells; Myelopoiesis; Myeloproliferative disease; Nucleosomes; Physiology; Play; Positioning Attribute; Process; Property; RNA; Regulation; Research; Role; Testing; Therapeutic; Time; Tissues; Transcription Process; Transcriptional Activation; Untranslated RNA; Work; active control; base; functional genomics; genome-wide; global run on sequencing; histone methylation; insight; macrophage; 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 整合子-EGR轴在髓系增强子调控中的作用 目的：本研究旨在确定增强子调控如何决定单核细胞的命运。 具体来说，我们试图了解一个新确定的调控轴的功能，包括集成 EGR-1/EGR-2转录因子及其辅因子NAB 2。我们的目标是确定这些 染色质调节剂：a）用于活化的初始髓样增强剂; B）重塑核小体可及性， 重塑3D基因组构象以施加单核细胞特异性转录组。我们试图描述 整合蛋白复合物的第一个谱系特异性功能，以前被认为是 在转录中的一般作用。 拟议的研究：骨髓生成的过程是由一个精心设计的基因表达程序， 起源于骨髓，并在外周血和组织中成熟。的 启动从造血干细胞（HSC）产生成熟骨髓细胞的转录过程 通过序列特异性转录因子（TF）指导增强子引发基因激活。但如何 这些髓样转录因子参与基础转录机制并激活谱系特异性增强子， 不太了解。我们发现整合子复合物的INTS 13亚基在以下方面起着重要作用： 我们认为Integrator具有以前未鉴定的靶向骨髓特异性 转录因子通过增强子调控来调节细胞命运。我们将从分子上剖析 整合剂及其功能性伴侣EGR-1/2和NAB 2在单核细胞/巨噬细胞分化中的作用 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 决定骨髓生成的增强子网络我们假设INTS 13调节积分器的 在单核细胞定型过程中EGFR靶向增强子的活性。因此，我们将分析INTS 13， EGR-1/2在人类细胞中的募集，我们将通过监测来确定靶向增强子的状态。 组蛋白甲基化和乙酰化水平（H3 K27 Ac和H3 K4 Me 1）。此外，我们将评估 通过RNA-seq的INTS 13、EGR-1/2和NAB 2消耗对骨髓基因的转录作用。2)我们将 确定由INTS 13、EGR-1/2和NAB 2介导的染色质结构的变化。我们假设 这些染色质调节因子为激活和执行细胞命运定型的增强子做好准备。我们的数据 提示INTS 13、EGR-1/2和NAB 2控制增强子活性/平衡状态并监督基因组 分化过程中的拓扑结构我们将在功能上通过分析核小体重塑来验证我们的假设。 增强子（先锋活性），并通过确定整合子和EGR-1/2如何调节染色体循环 以及使用Hi-C和3C测定的3D基因组构象。最后，我们将描述生物化学 Integrator的特性，以鉴定调节单核细胞中EGFR活性的骨髓特异性模块。