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.

In order to protect against invading pathogens, the immune system must react in a rapid, yet balanced manner: overactivity can lead to autoimmune diseases, whereas insufficient responses risk systemic infection. This equilibrium is governed by a highly choreographed cascade of gene activity that is elicited in response to challenge. This proposal addresses how the Integrator protein complex contributes to inflammatory responses through its control of messenger RNA synthesis.