Interplay between RNA Pol II transcription and DNA replication

RNA Pol II transcription is the process that generates copies of the genes in our genome, that are mainly used as a template for the production of proteins in cells, allowing them to express the factors that characterise their cellular identity; DNA replication is the process through which a cell duplicates its genome, generating two identical copies to pass on to daughter cells. They are therefore by definition both essential processes that any living cell at some point in their life will find doing together. However, we know that transcription and replication can interfere with each other affecting their functions, and ultimately inducing DNA damage and genome instability. This is a fundamental biological question in cell biology, for which we have no clear understanding, and that has direct consequences for cell growth as well as human health. Nevertheless, the complete interplay between RNA Pol II transcription and DNA replication is actually so far unknown. In order therefore to determine how transcription and replication are organised in the genome to avoid negative consequences for genome stability, we have analysed both processes at the same time during cell cycle progression. We have so uncovered how and where transcription and replication affect each other. We found that when replication passes through a transcribed gene it reduces temporally transcription activity; transcription vice versa affects replication progression through genes; we also found that difficult to replicate regions in transcribed genes have their replication delayed compared to the surrounding area, postponed to later stages in cell cycle progression. All together we have uncovered so far, the first description on how these two processes are coordinated and how exactly they affect each other, at the genome wide level, identifying key molecular processes that are crucial for this relationship.
In our proposal we are now going to characterise in greater detail the molecular mechanisms regulating the interplay between transcription and replication. We will determine how transcription regulates DNA replication progression inside genes but also how it affects the choice of where DNA replication starts, called DNA replication origins. We will determine how maintaining the RNA Pol II around transcription start sites might be important to keep a signpost of where these sites are in the vastity of the genome, although this means delaying DNA synthesis across them to a later moment in the cell cycle; we will also determine which pathways regulate this DNA synthesis. Moreover, we will analyse the impact on genome stability that comes as cost of this interplay. Altogether, we intend dissecting the interplay between RNA Pol II transcription and DNA replication to a level so far never achieved.

Transcription and replication are the two processes that use the genome as substrate, but importantly, the DNA can be used only by one of them at any given time. As a consequence, clashes between transcription and replication lead to reciprocal interference driving genome instability because of replication stress and DNA damage. As currently we do not understand how these two processes are organised to reduce the negative impacts from their interference, we have analysed transcription and replication together throughout S-phase progression. We uncovered how transcription and replication appear co-existing on genes, with replication progression controlled by transcription's one. We also found that transcription start sites of transcribed genes are difficult to replicate regions, with preliminary evidences that DNA replication across these regions might be deferred at a later stage in the cell cycle, when cells are entering mitosis. We have already presented the first systemic analysis of how transcription and replication affect and impact on each other, and are proposing now to dissect in finer details the molecular mechanisms and pathways behind our findings. We have identified that the accumulation of the RNA Pol II in proximity of the transcription start sites, in a process dependent on promoter proximal pausing, induces the formation of gaps during DNA replication, that may be filled when cells are entering mitosis. We will now identify which are these G2/M DNA synthesised regions and determine the pathways behind this DNA synthesis. We will understand how halting the RNA Pol II during transcription at the promoter proximal pausing is important for this late G2/M DNA synthesis, but also how it is required to preserve the nucleosome free region around the transcription start sites. Finally, we will determine how transcription progression and regulation determines multiple DNA replication parameters like replication forks speed and replication origins activation.

Our research proposal will broadly benefit directly other genome biologists in the UK, as well as worldwide. The proposal aims to investigate a biological problem that connects many different research areas, with the intention of linking these different fields together. I am in a unique position to achieve this goal, as evidenced from my publication record and by the data already present in the grant proposal, proving our expertise in characterising, correlating and understanding together RNA Pol transcription and DNA replication progressions using a combination of genome wide, cellular and molecular approaches. This proposal will provide practical tools and biological insights that will be used by other researchers interested in RNA Pol II transcription, DNA replication and/or chromatin biology. Our data will characterise crosstalk and define regulatory steps that define the interplay between these processes. We will share our findings with the scientific community in order to make them accessible to the largest audience possible using a broad range of means. We will publish at least two open access research papers, one focused on the biological findings from our studies and another methodological, describing laboratory procedures and computational pipelines used for our analysis. We will take part to a series of local, national and international scientific meetings and conferences to share our findings with the rest of the scientific community. As our research is not specifically restricted to one specific research field, so also the choice of the meetings will be wide-ranging, covering research topics such as transcription, chromatin biology and genome instability. Finally, we are organising a workshop involving other researchers here at the University of Birmingham that are interested in RNA Pol II transcription or DNA replication progressions, sharing with them our assays and tools, to discuss with them about the possibility of implementing these in their projects.
Indirectly however, and in a way not yet fully predictable, we expect that our findings might have a broader impact also on the development of new drugs and a better definition of patients' stratification strategies. Recently, it has attracted great interest the development of inhibitors targeting kinases involved in the regulation of the loading and the initiation-to-elongation transition stages of RNA Pol II transcription (CDK7 and CDK9), ultimately reducing RNA Pol II levels over sets of specific genes. These compounds showed great efficacy in in vitro models and represent promising alternatives in the treatment of leukaemias, lung and breast cancers. All these tumours appear as "transcriptionally" driven, meaning that they are promoted by oncogenic signalling that alter the regulation of the transcription process. Our data will allow understanding what happens, in particular in actively replicating cells, when we remove the RNA Pol II from genes and more specifically, from the transcription start site. Would cells lose their open chromatin conformations around the transcription start sites leading to a shutdown of gene expression? And if so, is this altering also DNA replication progression and DNA replication origin activation across genes? Can we predict which other deregulations, or combinatorial treatments, would increment the efficacy of these drugs? These are some of the questions that we will intend answering as well, with the intention of developing future partnerships with pharmaceutical companies for the development of new and novel therapeutic treatment options.