Abstract An essential part of being a cell is being able to respond to changing environmental conditions. Extracellular stimuli include developmental morphogens, hormones, growth factors, and disease markers. For a cell to continue functioning during these changes, it often needs to rapidly change its gene expression networks. Different environmental conditions often regulate gene expression via a master regulating protein that is activated upon a stimulus, then binds to specific gene enhancers and promoters. These master regulators may initiate new transcription by recruiting Pol II molecules. In one well studied stress, heat shock, the genes that respond have undergone transcription initiation, but the Pol II molecules have not entered productive elongation. Instead, they pause near the promoter until the master regulator signals that Pol II should be released into the gene body. Understanding how transcription responds to a number of acute stimuli will provide needed information about how cells adapt to changing extracellular conditions. This project will examine how Pol II recruitment and pause release are regulated prior to and during serum response and osmotic stress. The first aim will identify genes and enhancers that are induced or repressed after heat shock, serum response, and osmotic stress in human HCT-116 cells. These experiments will use an approach, PRO- seq, that measures where transcription is occurring on a gene. This approach allows the determination of assessment of transcriptional mechanisms used by different stresses to alter gene regulation. The second aim will degrade the master regulators of these stimuli in minutes, resulting in understanding if these factors set up and/or release polymerase pausing. The primary effects of losing these transcription factors will be identified because degrons will be used to inducibly and quickly degrade these specific proteins. Finally, the third aim will examine how polymerase pausing is established. The Lis lab has identified transcription factors that are enriched at genes with high pausing indices and thus are hypothesized to prepare cells to respond to future stresses via polymerase pausing. These proteins, GABPA and SP1, will be rapidly degraded and changes in polymerase pausing will be measured genome-wide. Together, this project will answer critical questions about how cells are able to respond to their environments and how different stages of transcription regulate stimulus- induced genes.