Regulation of Transcription Through RNA Polymerase II Promoter-Proximal Pausing

Cells need to respond to environmental signals in order to adapt to changing stimuli, maintain cell viability and establish fate decisions. One way the cell accomplishes this adaptation is via altering gene expression of critical genes by influencing transcription of their mRNAs. mRNA Transcription is carried out by the enzyme RNA Polymerase II (Pol II). Regulation of RNA Pol II occurs at every step of transcription, the most well studied one being transcription initiation. However, in many metazoan genes, after transcription initiates, RNA Pol II experiences a pause 20-60 nucleotides downstream of transcription start site. This is mediated by two pausing complexes negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF). In order to pursue productive elongation, Pol II needs to be relieved from this promoter-proximal pause by recruitment of P-TEFb. Recent studies have demonstrated that regulation of transcription pausing, and elongation is necessary for cells to respond to external stimuli and for mammalian development, and that dysregulation of this network has been seen in developmental and immunological disorders, heart disease and a variety of cancers. However, what are the molecular and functional roles of Pol II pausing during normal mammalian development are yet to be elucidated. Here, using mouse embryonic stem cell differentiation as my major model system of mammalian development, I asked whether disruptions in RNA Pol II pausing would have an effect on embryonic development. I utilized CRISPR/Cas9 based genome editing to mutate pausing complex subunits nelfe (NELF) and spt5 (DSIF) in mouse embryonic stem cells. Using genomic approaches such as global run-on sequencing, I validated that promoter-proximal pausing is perturbed in these mESCs. By utilizing monolayer and three-dimensional differentiation protocols, I have observed disruption of lineage differentiation in pausing deficient mESCs. Finally, I established CRISPR/Cas9 genome editing protocols to mutate chromatin regulators and pausing factors in order to study their roles in developing organisms.