Interplay between RNA Pol II transcription and DNA replication
Lead Research Organisation:
University of Birmingham
Department Name: Institute of Cancer and Genomic Sciences
Abstract
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.
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.
Technical Summary
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.
Planned Impact
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.
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.
Publications
Bayley R
(2022)
H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ.
in Molecular cell
De S
(2022)
Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe.
in Nucleic acids research
Muste Sadurni M
(2023)
Deregulations of RNA Pol II Subunits in Cancer
in Applied Biosciences
Saponaro M
(2022)
Transcription-Replication Coordination.
in Life (Basel, Switzerland)
Scaramuzza S
(2023)
TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells.
in Nature communications
Wang J
(2021)
Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M.
in Cell reports
Wang J
(2023)
RNAPII response to transcription-blocking DNA lesions in mammalian cells.
in The FEBS journal
Wang J
(2021)
Protocol for analysis of G2/M DNA synthesis in human cells.
in STAR protocols
Description | We have identified a new process that is important to complete the replication of the genome during the cell cycle. Perturbations to this process are associated with increased genome instability, and could be a potential driver of DNA damage in disease related contexts. |
Exploitation Route | We are aiming to identify the components and factors involved in this novel process, as these could provide a novel window for treatment for specific drugs. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | We have established a collaboration with AstraZeneca to determine the impact of their pre-clinical drugs on G2/M DNA synthesis, aiming to identify factors regulating G2/M DNA synthesis and/or biomarkers for the clinical use of these compounds |
First Year Of Impact | 2022 |
Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal,Economic,Policy & public services |
Title | Identification of replication fork stalling sites genome-wide |
Description | We have developed a pipeline for the identification of hotspot sites where DNA replication forks stall/pause, with genome wide information, and with an analysis of the persistence of the pausing |
Type Of Material | Model of mechanisms or symptoms - human |
Year Produced | 2020 |
Provided To Others? | No |
Impact | This pipeline will allow identifying and characterise the role of replication factors and/or treatments in reducing/increasing the frequency of replication forks stalling/pausing. |
Title | Identification of sites of G2/M DNA synthesis |
Description | We have set up an assay that allows identification of the sites of DNA synthesis in G2/M. For this, we have been invited to publish the protocol for free on Star Protocols. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | We have been invited to submit a protocol paper on STAR Protocols of our procedure, as this tool would be useful for a broad range of scientists. Because this is an invited submission, the publication charges have been waved from the publishing group |
Title | Deposit of RNA-Seq and Repli-Seq like data |
Description | We have deposited on the GEO database all the raw data and the metadata from the analysis of the Che-RNA-Seq and Brad-Seq datasets generated so far, as in the process of publishing our findings. |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | It contains a series of genomic data about how transcription and replication affect each other during the S-phase |
Description | Changes on RNAPII transcription elongation rates following hypoxia treatment |
Organisation | University of Oxford |
Department | Department of Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | In collaboration with the group of Prof Ester Hammond, Department of Oncology at the University of Oxford, we are assessing the impact of hypoxia on RNAPII transcription, with a focus in particular to changes in its elongation rates. |
Collaborator Contribution | We have provided a series of tools and assays for the analysis of RNAPII transcription elongation, as well as providing expertise in the data analysis collected. |
Impact | At this stage, the collaboration is at a very early stage of development and therefore the outcomes are so far only experimental and not yet integral part of a publication and/or future grant application. |
Start Year | 2019 |
Description | Characterisation of the role of SETD1A in DNA double strand break repair |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have provided expertise in the analysis of a series of genome wide datasets that have contributed to determine the role of SETD1A in DNA double strand break repair. A manuscript has been generated in collaboration with the Higgs lab at the University of Birmingham. This manuscript has just been accepted for publication on the journal Molecular Cell. |
Collaborator Contribution | No direct contribution to our project |
Impact | A manuscript has been just accepted for publication on Molecular Cell, although at this stage there is not yet a doi address for it. |
Start Year | 2020 |
Description | Characterising the role of ATR in unperturbed replication |
Organisation | University of Pittsburgh |
Department | School of Medicine Pittsburgh |
Country | United States |
Sector | Academic/University |
PI Contribution | We have been contacted by Prof Bakkenist at the University of Pittsburgh School of Medicine (www.bakkenistlab.com) to help them in understanding and characterising the role of ATR in unperturbed replication. In particular, we have asked to provide support to characterise a series of genomic datasets that the Bakkenist has produced recently and for which we have specific expertise. |
Collaborator Contribution | There has been so far no direct contribution from the Bakkenist group to our project. |
Impact | This collaboration started one month ago, so there are no outputs so far generated. |
Start Year | 2022 |
Description | Determining the impact of TRAIP in genome stability maintenance |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have collaborated with the Gambus lab at the University of Birmingham to determine and characterise the role and impact of TRAIP in genome stability maintenance. This collaboration has involved 3 lab members of the Saponaro lab, and a manuscript is being prepared for submission |
Collaborator Contribution | No direct impact on our project |
Impact | Not yet available |
Start Year | 2021 |
Description | Positive and Negative Impact of RNA Pol II Transcription on DNA Replication Progression |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have collaborated with the Spill lab from the School of Mathematics for some in depth statistical analysis for a manuscript that has been prepared by our group |
Collaborator Contribution | The Spill lab has provided an in depth statistical analysis for the overlap between replication fork stalling/pausing sites we have identified with breakpoints of rearrangements found in cancer cells downloaded from the Cosmic database |
Impact | https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3827411 |
Start Year | 2020 |
Description | RPRD proteins control transcription in human cells |
Organisation | University of Manchester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have collaborated with the Grzechnik lab (University of Birmingham moving to the University of Manchester) to analyse any cell cycle progression defect arising in RPRD protein mutants, involving two lab members of the Saponaro lab, |
Collaborator Contribution | No direct contribution to our project |
Impact | https://www.biorxiv.org/content/10.1101/2021.06.20.449126v1 |
Start Year | 2020 |
Description | Department of Medical and Molecular Genetics at King's College seminar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I got invited to give a seminar as an invited speaker at the Department of Medical and Molecular Genetics at King's College (UK) |
Year(s) Of Engagement Activity | 2021 |
Description | Genomics Birmingham Spring Forum |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Industry/Business |
Results and Impact | Genomics Birmingham organised an open day inviting all the major companies involved in genomic platforms/tools to provide an overview of the different technologies and applications available on the market. We got invited to present in this occasion as we have developed a series of ad hoc applications, that could be applied for other applications too. |
Year(s) Of Engagement Activity | 2019 |
Description | Online seminar for an Alumni event at The FIRC Institute of Molecular Oncology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present my ongoing research for a special IFOM Alumni, that was attended by >70 participants online and that was registered and advertised on the IFOM LinkedIn website. The aim of this seminar was to provide an up to date description of how RNA Pol II transcription and DNA replication affect and impact on each other. |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.linkedin.com/posts/ifom_ifom-alumni-talk-saponaro-activity-6793432129024860160-4UJP |
Description | Selected talk at the EMBO | EMBL Symposium: Friend or Foe: Transcription & RNA meet DNA Replication & Repair |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | My abstract was selected for the 2021 EMBO | EMBL Symposium: Friend or Foe: Transcription & RNA meet DNA Replication & Repair. This has allowed a broader diffusion of the findings of our research |
Year(s) Of Engagement Activity | 2021 |
Description | Video interview as an international opinion leader at IFOM |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I was interviewed by the press office of the FIRC Institute of Molecular Oncology (IFOM, Milan, Italy) as an international opinion leader on genome instability. The interview was an online 5 minutes chat for a webzine (web-magazine) about our research line and the potential translational implications from our findings, addressed to the general public, therefore very lay. |
Year(s) Of Engagement Activity | 2020 |
Description | •Genome Damage and Stability Centre seminar presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I got invited for a formal presentation of the research ongoing in my lab from the Genome Damage and Stability Centre of the University of Brighton (UK). |
Year(s) Of Engagement Activity | 2020 |