| Summary: | <p>Transcription by RNA polymerase II (RNAPII) is a central process in gene expression and so subject to many layers of regulation: post-translational modifications of the RNAPII C-terminal domain (CTD), transcription factor association, chromatin compaction and the control of polymerase access to DNA. Transcription can also be altered when DNA damage is encountered.</p>
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<p>My thesis addressed different aspects of these regulatory mechanisms. Firstly I studied the role of RNA:DNA hybrids (R-loops) in pause-type termination, and assessed whether data based on the <em>β-actin</em> gene could be extended to further genes. I also investigated whether DNA damage repair (DDR) factor Rad51 is present over R-loop associated pause regions. Secondly I studied the roles of tyrosine and threonine phosphorylation (Y1P and T4P) of the RNAPII CTD, by testing their association with DDR and transcription termination respectively. I also developed a modified MNase-seq protocol using mNET-seq isolation conditions to allow close comparison between chromatin configuration and the nascent RNA signal.</p>
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<p>The outcome of my research was that I found the <em>PKM</em> gene exhibits the same basic characteristics of pause-type termination, including R-loop formation. However, little evidence for heterochromatin formation as observed for <em>β-actin</em> termination was identified. I also showed that Rad51 accumulates at R-loop regions in pause-type termination, but does not appear to induce R-loops. More likely Rad51 is recruited by downstream DDR factors. Y1P is associated with factors involved in UV DDR. My studies showed that UV damage causes a global RNAPII elongation defect with altered chromatin associated histone marks and defective recruitment of elongation factors including the PAF1 complex and SPT6 to the polymerase. Finally my studies on T4P-associated termination cis-elements indicated that positions of cleavage-independent termination are formed relative to mRNA 3’ ends but not based on specific DNA sequence. Also cleavage factor depletion increased mononucleosome signals in these termination regions.</p>
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