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

项目摘要 整合子-EGR轴在髓系增强剂调控中的作用 目的：我的研究旨在确定基于增强子的调控如何决定单核细胞的命运。 具体地说，我们试图了解一个新确定的监管轴的功能，该轴由集成商组成 复合体、EGR-1/EGR-2转录因子及其辅助因子NAB2。我们的目标是确定这些 染色质调节剂：a)激活的主要髓系增强剂；b)重塑核小体可及性和 重塑3D基因组构象以实施单核细胞特异性转录组。我们试图刻画出 整合者蛋白复合体的第一个谱系特异性功能，以前被认为服务于 在转录中的一般作用。 建议研究：骨髓生成过程由一个精心设计的基因表达程序控制 起源于骨髓，在外周血液和组织中成熟。这个 从造血干细胞生成成熟髓系细胞的转录过程启动 通过序列特异性转录因子(TF)指示增强子诱导基因激活。然而，如何 这些髓系转录因子参与基本的转录机制，并激活谱系特异性增强子 人们对此知之甚少。我们发现，整合者复合体的INTS13亚单位在 我们认为，整合子具有以前未知的靶向髓系特异性的能力 TFS，并通过增强子调控调节细胞命运。我们将从分子上剖析其功能 整合子及其功能伴侣EGR-1/2和NAB2在单核/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INTS13调制积分器的 单核细胞承诺过程中EGR靶向增强子的活性。因此，我们将分析INTS13和 EGR-1/2在人类细胞中的募集，我们将通过监测确定靶向增强子的状态 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法研究INTS13、EGR-1/2和NAB2缺失对髓系基因转录的影响2)我们会 确定INTS13、EGR-1/2和NAB2介导的染色质结构的变化。我们假设 这些染色质调节剂为激活和执行细胞命运承诺而启动增强剂。我们的数据 提示INTS13、EGR-1/2和NAB2控制增强子的活性/平衡状态并监督基因组 分化过程中的拓扑结构。我们将通过分析核小体重塑来从功能上检验我们的假设 增强子(先锋活性)和确定整合子和EGR-1/2如何调节染色体环 用Hi-C和3C分析进行三维基因组构象分析。最后，我们将对生物化学进行表征 整合器的特性，以确定调节单核细胞中EGR活性的髓系特异性模块。

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.