PROJECT SUMMARY/ABSTRACT Transcription regulation underlies the diversity of cell types and functions. The process of transcription itself has multiple steps that may be regulated separately. Promoter-proximal RNA Pol II pausing has recently been described as a widespread rate-limiting step of transcription and a possible point of gene regulation. Studies disrupting the critical pausing factor Negative elongation factor-B (Nelf-b) in mouse and mouse embryonic stem cells (mES) highlighted its essential role in development and a cross-talk with FGF/ERK signaling transduction. However, it is unclear what role pausing plays in mammalian development. Furthermore, it is unclear how pausing may affect specific targets of FGF/ERK signaling, and whether this cross-talk is relevant in vivo. The long-term goal of this proposal is to establish a molecular understanding of how gene regulation can be achieved at the level of promoter-proximal pausing during transcription. The objective of the present proposal is to mechanistically determine the role of pausing during epiblast pluripotency transitions in early mammalian development. This objective will be achieved by a detailed phenotypic and transcriptomic analysis of Nelf-b-/- embryos at several early developmental stages to analyze pluripotency transitions (Aim 1). To expand on these results and interrogate pausing at a high molecular and temporal resolution, a Nelf-b-degron mES cell line has been established to test the immediate and direct effects of pausing-loss in mES cells under different signaling conditions. I will utilize nascent RNA-seq techniques and directed differentiation approaches to build a mechanistic link between pausing, FGF/ERK transcriptional activation, and pluripotency transitions in embryos and mES cells (Aim 2). My central hypothesis is that pausing is required for epiblast pluripotency transitions in mouse development via direct regulation of FGF/ERK transcriptional targets. The expected outcome of this project is to uncover the mechanistic link between pausing and signal transduction, explain the role of pausing in mammalian development. It promises to yield novel insights into gene regulation at the pausing level. The link between pausing and signaling is of high importance to other biological contexts considering the prevalence of pausing and necessity of signal transduction to perform fundamental cellular functions beyond responses to FGF/ERK in development. Furthermore, given that FGF/ERK signaling is amongst the top dysregulated pathways in developmental diseases and malignancies, insights into the molecular mechanisms of this pathway will yield novel insights to therapeutically target it.

PROJECT NARRATIVE Regulation of transcription dictates cells’ fates, functions, and potential in development and disease. Promoter- proximal RNA Polymerase II Pausing has been shown to be a critical rate-limiting step of transcription that can control transcription’s outcome and response to extracellular signals in certain scenarios. This study aims to understand the mechanisms of Pausing influence on gene regulation and signal response in early mammalian development in vivo and in vitro, which will give insights into how this process may affect cellular response to extracellular signals and open the door to manipulating this process for therapeutic purposes.