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: 10536610
• 负责人: Chad Brandon Stein
• 金额: $ 1.74万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536610
• 关键词: Address; Affect; Autoimmune Diseases; Biological Models; Bone Marrow; Cell surface; Cells; Chronic; Clinical; Code; Complex; Crohn' s disease; Cues; DNA; Data; Data Set; Development; Disease; Dissociation; 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; Literature; Location; Macrophage; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Nucleotides; Organism; Pathogenesis; Phosphotransferases; Physiological; Physiology; Play; Polymerase; Process; Productivity; Property; Proteins; RNA; RNA Binding; RNA Polymerase II; RNA chemical synthesis; Reaction; 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; cytokine; derepression; embryonic stem cell; endonuclease; experimental study; gene repression; insight; 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.

溃疡性结肠炎和克罗恩病等炎症性疾病是越来越普遍的慢性疾病