Investigating the Integrator Complex's Role in Regulating Inflammatory Transcription

研究整合复合体在调节炎症转录中的作用

• 批准号: 10380245
• 负责人: Chad Brandon Stein
• 金额: $ 3.46万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380245
• 关键词: Address; Affect; Autoimmune Diseases; Autoimmunity; Biological Models; Bone Marrow; Cell surface; Cells; Chronic; Clinical; Code; Complex; Crohn' s disease; Cues; DNA; Data; Data Set; Disease; Drosophila genus; Endoribonucleases; Equilibrium; Gene Activation; Genes; Genetic Transcription; Genomics; Homeostasis; Hour; Human; Human Genetics; Immune; Immune response; Immune system; Immunoprecipitation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Innate Immune System; Interphase Cell; Invaded; Lead; Light; Literature; Location; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Nucleotides; Organism; Pathogenesis; Phosphotransferases; Physiological; Physiology; Play; Polymerase; Process; Property; Proteins; RNA; RNA Binding; RNA Polymerase II; RNA chemical synthesis; Regulation; Repression; Rest; Ribonucleosides; Risk; Role; Route; Signal Transduction; Site; Stimulus; Structure; Systemic infection; Therapeutic; Transcript; Transcription Initiation Site; Transcriptional Regulation; Transgenes; Ulcerative Colitis; Up-Regulation; Work; cell type; crosslink; derepression; embryonic stem cell; endonuclease; experimental study; insight; macrophage; monocyte; pathogen; prevent; programs; promoter; protein complex; recruit; response; small molecule; therapeutic target; transcription termination; transcriptome sequencing

Inflammatory diseases like Ulcerative Colitis and Crohn’s Disease are increasingly prevalent chronic conditions with significant morbidity if untreated. While the exact molecular underpinnings of these diseases are unknown, it is clear that misregulation of the innate immune system plays a key role in their pathogenesis. Importantly, precise control of mRNA synthesis by RNA Polymerase II (RNAPII) is essential for immune homeostasis and responses to environmental cues like invading pathogens. In metazoans, RNAPII pauses shortly downstream of transcription start sites, where the polymerase can remain in a poised state until transcriptional kinases release it into productive elongation. Promoter-proximal pausing is widespread and is critical for proper responses to stimuli like pro-inflammatory molecules. Recently, it has become appreciated that another fate for paused RNAPII is common: promoter-proximal termination. Here, instead of paused polymerase being released into productive elongation, it is dissociated from its DNA template and releases a short, non-functional mRNA. Promoter-proximal pausing is critical for proper regulation of pro-inflammatory genes, so understanding this process is important for human physiology and disease states. Our group and others recently showed that the Integrator complex is responsible for inducing promoter- proximal termination at a subset of protein-coding genes. The Integrator complex is essential for viability, comprised of at least 14 subunits, and induces termination through cleavage of nascent RNA. Specifically, the catalytically active Integrator Subunit 11 (INTS11) induces RNA cleavage. However, the regulation of Integrator’s catalytic activity is poorly understood. A growing body of work shows that Integrator regulates responses to pro- inflammatory cues, and human genetics suggests its involvement in inflammatory bowel disease pathogenesis. Here, I aim to uncover how Integrator activity is regulated, and how this complex affects transcription of inflammatory genes. Because of Integrator’s emerging role in regulating inflammatory transcription, I will use mouse embryonic stem cell-derived macrophages (ESDMs) as a model system. I will first determine Integrator’s role in inflammatory transcription by rapidly depleting INTS11 and monitoring cells’ ability to mount an immune response. Using rapid INTS11 depletion paired with nascent RNA sequencing, I will precisely define the targets of Integrator in this physiologically relevant immune cell type. I will also determine where Integrator is targeted and explore whether its genomic localization changes in response to immune challenge. Next, I will characterize the RNA-binding properties of Integrator in an effort to understand how its catalytic activity is regulated. Finally, I will probe Integrator protein partners, which will shed light on how it is targeted and how it interacts with the transcription machinery. Together, this work will provide insight into this understudied complex and may allow for Integrator to be therapeutically targeted in inflammatory disease.

炎症性疾病如溃疡性结肠炎和克罗恩病是越来越普遍的慢性疾病 如果不治疗，发病率很高。虽然这些疾病的确切分子基础尚不清楚， 很明显，先天免疫系统的失调在其发病机制中起关键作用。重要的是， 通过RNA聚合酶II（RNAPII）精确控制mRNA合成对于免疫稳态是必需的， 对环境线索的反应，比如入侵的病原体。在后生动物中，RNAPII在 转录起始位点，在那里聚合酶可以保持在平衡状态，直到转录激酶释放 转化为生产性延伸。启动子近端停顿是普遍的，并且对于正确的反应是至关重要的。 刺激物如促炎分子。最近，人们开始意识到，另一种命运暂停 RNAPII很常见：启动子近端终止。在这里，不是暂停的聚合酶被释放到 在生产性延伸中，它与其DNA模板解离并释放短的非功能性mRNA。 启动子近端停顿对于促炎基因的正确调节至关重要，因此了解这一点 这一过程对人体生理和疾病状态很重要。 我们的小组和其他人最近表明，整合剂复合物负责诱导启动子- 在蛋白质编码基因的子集处的近端终止。整合者复合体对生存能力至关重要， 由至少14个亚基组成，并通过切割新生RNA诱导终止。具体而言是 具有催化活性的整合子亚基11（INTS 11）诱导RNA切割。然而，集成商的监管 催化活性知之甚少。越来越多的研究表明，整合者调节对亲- 人类遗传学提示其参与炎症性肠病的发病机制。 在这里，我的目标是揭示整合剂活性是如何调节的，以及这种复合物是如何影响转录的。 炎症基因 由于Integrator在调节炎性转录中的作用，我将使用小鼠胚胎干细胞。 干细胞衍生的巨噬细胞（ESDM）作为模型系统。我将首先确定集成商的角色， 通过快速消耗INTS 11和监测细胞的免疫能力来检测炎症转录 反应使用快速的INTS 11缺失和新生RNA测序，我将精确地定义靶点， 在这种生理相关的免疫细胞类型中的整合剂。我还将确定Integrator的目标位置 并探索其基因组定位是否会因免疫挑战而发生变化。接下来，我将描述 Integrator的RNA结合特性，以了解其催化活性是如何调节的。最后， 我将探测Integrator蛋白伴侣，这将揭示它是如何被靶向的，以及它如何与蛋白质相互作用。 转录机器总之，这项工作将提供深入了解这一研究不足的复杂，并可能允许 使Integrator成为炎症性疾病的治疗靶点。