Molecular Mechanisms Regulating Rna Polymerase Ii Pausing During Gene Activation

RNA Polymerase II (Pol II), the enzyme that transcribes all messenger RNAs (mRNAs) has another activity by pausing at gene promoters. The paused Pol II generates short 5’-capped RNAs of about 50 nucleotides in length. Much is known about Pol II pausing including its prevalence across the genome. But, the molecular mechanisms that are involved in regulating Pol II pausing and its roles in gene regulation are yet to be fully explored. In this study, I have investigated the molecular mechanisms that regulate dynamics of Pol II pausing in response to gene activation. The main goal of this study was to determine how cells respond to stimuli by altering Pol II pausing states. To this end, we characterized changes in Pol II pausing in MCF-7 human breast cancer cells, using two distinct stimuli, heat shock and Trichostatin A (TSA), a histone deacetylase inhibitor. Two genes SNAI1, a master regulator of epithelial to mesenchymal transition, and HSPA1B (heat shock protein 70), a master regulator of heat shock response, show mRNA upregulation upon treatment with both stimuli. To determine changes in Pol II pausing and its dynamics in response to gene activation, I have used permanganate footprinting for single gene analysis, and short-capped RNA sequencing (scRNA-seq) for genome-wide analysis. I have shown that, upon activation, paused genes can retain Pol II pausing and non-paused genes can acquire Pol II pausing. Further, I observe that genes such as HSPA1B, undergo pause release during heat shock activation but SNAI1 does not. In addition, I have shown that the turnover of the paused Pol II complex changes in a stimulus-specific manner, indicating that the release of paused Pol II is sensitive to the nature of the stimulus. To investigate the rate of turnover of the paused complex, I used a specific inhibitor of a general transcription factor TFIIH, Triptolide and measured the residence time of the Pol II complex at the paused site. Genome-wide analysis of Pol II turnover demonstrates that not all genes respond the same way to heat shock. This project describes a novel mechanism for regulation of transcription during gene activation in human cells during responses to environmental stresses