Transcription initiation is highly regulated by genomic elements in promoters and enhancers that bind transcription factors and recruit RNA polymerase II (Pol II). After Pol II escapes from the promoter, many regulatory steps occur during transcription elongation that control Pol II progression, RNA processing, and finally release of the RNA from chromatin for nuclear export. These steps are tightly controlled by genomic and epigenomic features, such as histone modifications, nucleosome positions, and cis-elements that bind transcription factors and RNA-binding proteins (RBPs). Current functional genomic technologies tend to provide a static, averaged view of genomic function that obscure the transient roles that many genomic elements play in shaping the transcriptome. To identify and characterize all functional genomic elements, we need to develop approaches that quantitatively monitor the many layers of gene expression regulation, including transient interactions and short-lived events. In the proposed research, we will create a suite of genome-wide tools to map Pol II across single chromatin fibers, track the association of RBPs, and follow RNAs from transcription initiation to nuclear export. These techniques will highlight unappreciated connections between genomic elements, molecular events on chromatin, and post-initiation regulatory events. Our rationale is that by creating tools to quantify short-lived steps in mRNA production, unappreciated functional roles for genomic elements can be revealed. To demonstrate how these technologies reveal unappreciated gene regulatory mechanisms, we will apply them to the heat shock response (HSR), a stress response that has served as a model to study the many regulatory layers in gene expression. In Aim 1 we will develop a strategy that reveals Pol II locations across single long chromatin fibers. Fiber-seq is a method that identifies regions of accessible DNA across long (up to ~20 kb) chromatin fibers one molecule at a time. We will extend this approach to identify Pol II footprints, analyze the distributions of Pol II complexes across gene bodies, and characterize the interactions between Pol II complexes and nucleosomes. In Aim 2 we will measure the timing of binding of proteins across long pre-mRNA molecules. RBP binding will be marked by RNA editing and long- read direct RNA sequencing of nascent RNA (nano-COP) will enable us to resolve transient binding events that impact pre-mRNA processing and nuclear export. These events are transient, as many RBPs bind to introns and are released from RNA in the cytoplasm immediately after nuclear export. In Aim 3, we will create an approach to measure kinetics of RNA production, chromatin release, and nuclear export that couples 4sU metabolic labeling of newly synthesized RNAs with biochemical purification of RNAs from chromatin, nucleoplasm, and cytoplasm. The data from this technique will allow us to estimate the rates of release of RNA from chromatin, nuc...