Project Abstract/Summary My research focuses on the precise understanding of the regulation of RNA in living cells and human diseases. Mapping where the regulations take place is critical in identifying factors and elements that alter RNA expression. Further enhancing the genomic resolution and the spatiotemporal resolution of RNA expression have been a strong driver of leading discoveries in molecular and disease mechanisms. My first focus is to define the high-resolution maps of the transcriptional enhancers in peripheral blood immune cells, using enhancer RNA (eRNA) as the guide. eRNA transcription is a novel mechanism of which the RNA is synthesized from enhancer sequences themselves other than their target genes. eRNA has expanded the previous understanding of how enhancers are constructed and allowed mapping important subregions within the enhancers in higher resolution. eRNA based maps of enhancers will guide us to more efficiently discover genomic elements linked to the dysregulation of disease genes, and specifically dissect causal genetic variations or mutations. This will have a widespread impact on major human diseases, and my specific focus is on immunometabolic diseases and the impact of enhancer variations at the eRNA start sites. The second focus is on the subcellular compartments that leads to differential RNA trafficking, processing, and decay. Membraneless organelles, such as stress granules, are suggested to be the novel mechanism of gene expression control through phase separation and sequestration. I will explore the idea that spatial compartmentalization confers specificity of temporal gene expression. In particular, I will dissect the specificity of RNA processing in stress granules in high temporal resolution in immune cell activation and exhaustion and explore their linkage to the dysregulation of disease associated genes. This will lead to the discovery of novel therapeutic targets on the specificity factors during the phase separation process. To address these, my group harnesses the power of high throughput RNA sequencing methods, and further tailor them. We developed novel RNA sequencing methods such as Precision Run-On sequencing, Chromatin Run-On sequencing, Tail End Displacement sequencing, and RNA granule sequencing. These methods elucidate multiple aspects of RNA mechanism in high throughput: nascent transcription, poly(A) tail modification, and RNA sequestration. We will continue to improve and develop novel RNA analysis strategies to dissect eRNA transcription, RNA modifications, and combining these methods with subcellular compartmentalization in high spatial and temporal resolution in cost efficient manners. These innovations will lead to enhancements in refining genetic maps and higher spatiotemporal resolution in analyzing RNA expression. Our mission is to discover critical mechanisms and checkpoints of RNA lifespan, focusing on eRNA and RNA granules. This will provide foundations to apply the ideas and...