RNA Polymerase II control in the changing transcriptional landscape

Abstract

Eukaryotic RNA polymerase II (Pol II) is a 12 subunit complex responsible for transcribing all protein coding genes, as well as producing many other RNA species. Regulation of gene expression is essential for the viability of any organism, and it is primarily achieved at the transcriptional level. Transcriptional control is essential as cells adapt to changing conditions, ranging from undergoing differentiation, reacting to signalling cascades, or responding to stimuli. Traditionally, transcription regulation was studied at a steady-state, there is however a growing need to better understand how the system works from a dynamic perspective. New approaches, possible thanks to technological advancements, allow us to gain a better understanding of the mechanistic intricacies of transcription regulation. Early on, as molecular biology was establishing itself, it was noted that transcription is regulated at the initiation stage through the action of transcription factors. These proteins bind to specific motifs in DNA, can recruit Pol II, and activate or repress transcription. In recent years, it has been increasingly understood that regulation also occurs at later stages of the transcription cycle, altering the dynamics of paused Pol II complexes immediately after initiation, and modifying the processivity and speed of Pol II elongation. This thesis explores the role of Elongin, a complex originally linked to transcription elongation, in modulating transcription dynamics at a steady-state level and, as was subsequently discovered, more importantly in response to stimuli. Factors involved in the process are characterized, and the role of Elongin in transcriptional regulation in embryonic development is investigated. The largest subunit of Pol II, RPB1, has a conserved C-terminal repeat domain (CTD) that contains 52 heptapeptide repeats and functions as a scaffold where transcription regulators bind. This unstructured 'tail' undergoes phosphorylation changes during the transcription cycle, modifying its interaction with associated proteins thereby affecting the dynamics of transcription as well as processing of the synthesized RNA. The second part of this thesis describes how a modification in the CTD, phosphorylation of Serine 7, affects transcriptional dynamics. Its role in response to transcriptional stalling is determined, and a novel pathway is identified, the GSK3-Ser7P transcriptional stalling response pathway. Finally, work towards investigating the role of RECQL5 as a possible effector protein in this pathway is explored. Another way in which transcription is regulated is by effectively modulating the quantity of Pol II available for transcription. In response to DNA damage, for example, Pol II ubiquitylation and degradation is essential to the damage response. The third part of this work describes the discovery of a new Pol II ubiquitylation pathway in human cells that targets excessive and defective complexes at the initial stages of the transcription cycle. We study the effect of dysregulating this basal mechanism, in particular at the transcriptional level, in relation with complementary processes that control transcription at early stages of the cycle. Finally, the transcriptional response in cancer cells to the host defences was studied. We investigate the role of a subunit of Mediator in basal transcription and in the dynamic context of the interferon response, finding it to be critical for immune evasion in cancer cells. In summary, this thesis describes multiple investigations done across varying transcriptional landscapes with a common motif: the dynamic control of transcription. The work explores how transcription is controlled through changes in the conformation of the transcriptional machinery and through modifications to Pol II itself.