Investigating the Integrator Complex's Role in Regulating Inflammatory Transcription

像溃疡性结肠炎和克罗恩病这样的炎症性疾病是越来越普遍的慢性疾病 如果不治疗，会有很大的发病率。虽然这些疾病的确切分子基础尚不清楚， 很明显，先天性免疫系统的失调在其发病机制中起着关键作用。重要的是 通过RNA聚合酶II(RNAPII)精确控制mRNA的合成对于维持免疫内稳态和 对入侵病原体等环境线索的反应。在后生动物中，RNAPII在下游不久暂停 转录起始点，在那里聚合酶可以保持稳定状态，直到转录激酶释放 它变成了生产性的伸长。启动子-近端停顿是普遍存在的，对于正确反应 刺激物就像促炎分子。最近，人们开始意识到，另一个暂停的命运 RNAPII是常见的：启动子-近端终止。在这里，不是暂停的聚合酶被释放到 在生产性伸长过程中，它从DNA模板中解离出来，释放出一个短的、无功能的信使核糖核酸。 启动子-近端停顿对于适当调节促炎基因至关重要，因此理解这一点 过程对人类的生理和疾病状态很重要。 我们的团队和其他人最近发现，整合子复合体负责诱导启动子- 在蛋白质编码基因的子集上的近端终止。集成商复合体对于生存是必不可少的， 由至少14个亚基组成，通过裂解新生RNA诱导终止。具体地说， 催化活性整合子亚单位11(INTS11)诱导RNA裂解。然而，对积分商的监管 人们对其催化活性知之甚少。越来越多的工作表明，整合者调节对亲- 炎性线索，人类遗传学表明它参与了炎症性肠病的发病机制。 在这里，我的目标是揭示整合子活性是如何调节的，以及这个复合体是如何影响转录的 炎症基因。 由于整合者在调节炎症转录方面的新角色，我将使用小鼠胚胎 干细胞来源的巨噬细胞(ESDM)作为模型系统。我将首先确定集成商在 通过快速耗尽INTS11和监测细胞启动免疫的能力进行炎症转录 回应。利用INTS11的快速耗尽和新生的RNA测序，我将准确地定义靶标 在这种生理上相关的免疫细胞类型中。我还将确定集成商的目标位置 并探讨其基因组定位是否因免疫攻击而改变。接下来，我将描述 研究整合子的RNA结合特性，以了解其催化活性是如何调节的。最后， 我将调查Integrator Protein Partners，这将阐明它是如何被定位的，以及它是如何与 转录机器。总之，这项工作将提供对这一未被研究的复杂情况的洞察，并可能允许 作为炎症性疾病的治疗靶点。

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.