Regulation of R-loops for transcriptional control
Lead Research Organisation:
John Innes Centre
Department Name: Cell and Develop Biology
Abstract
The Dean group study the control of flowering time in the reference plant, thalecress (Arabidopsis thaliana). Several flowering pathways converge to regulate a gene called FLC and variation in expression of this gene contributes enormously to natural variation in flowering time of Arabidopsis types collected from around the world. One pathway regulating FLC involves so called non-coding RNAs - for FLC this is the RNA encoded from the opposite strand to the protein. This RNA has been called COOLAIR because it is up regulated by cold temperature. Analysis of the regulation of COOLAIR has recently led to the identification of a novel regulatory mechanism that involves an opening up of the DNA duplex, invasion by RNA and stabilization of this structure by a protein called AtNDX. This structure is called an R-loop and it inhibits production of RNA initiating in that region of the gene.
This proposal aims to further dissect this mechanism and identify other proteins that play a role. In the last few years similar non-coding RNAs have been found in many organisms but their regulation and function is generally not known. Advancing our understanding from a study such as this can provide concepts important to gene regulation across many genomes.
This proposal aims to further dissect this mechanism and identify other proteins that play a role. In the last few years similar non-coding RNAs have been found in many organisms but their regulation and function is generally not known. Advancing our understanding from a study such as this can provide concepts important to gene regulation across many genomes.
Technical Summary
We are exploring different pathways involving RNA-mediated chromatin regulation of gene expression. The importance of these mechanisms has emerged through our work determining natural variation in flowering, an important adaptive trait in native plants and many crops. Multiple pathways regulate the Arabidopsis floral repressor FLC and these converge on co-transcriptional mechanisms involving antisense transcripts (named COOLAIR) and chromatin pathways. Regulation of COOLAIR transcription has been monitored using a COOLAIR:LUC reporter system. This enabled identification of a repressor of COOLAIR transcription that encoded an atypical homeodomain protein, which bound single-stranded DNA. Investigation of where single-stranded DNA might occur in vivo revealed the existence of an R-loop over the COOLAIR promoter. We intend to exploit this finding to identify and functionally analyse proteins that modulate the R-loop, which in turn influences transcription. We will build on preliminary data that identifies in vivo interactors of the homeodomain protein AtNDX and functionally analyse their action on the R-loop. We will also identify other regulators that repress COOLAIR transcription and investigate how their function intersects with R-loop formation, stabilization and resolution.
The project will have two overall objectives. The goal of the first will be the effective combination of genetic, molecular and proteomic approaches to define in vivo interactors of AtNDX and to understand how their function modulates the R-loop formation/stabilization/resolution thus influencing COOLAIR transcription. The goal of the second will be to clone four additional COOLAIR repressors and fully integrate their activities into the mechanism detailed in Objective 1. A mechanistic understanding generated from this study will detail the links between transcript processing, R-loop stabilization and RNA decay and is likely to be widely relevant.
The project will have two overall objectives. The goal of the first will be the effective combination of genetic, molecular and proteomic approaches to define in vivo interactors of AtNDX and to understand how their function modulates the R-loop formation/stabilization/resolution thus influencing COOLAIR transcription. The goal of the second will be to clone four additional COOLAIR repressors and fully integrate their activities into the mechanism detailed in Objective 1. A mechanistic understanding generated from this study will detail the links between transcript processing, R-loop stabilization and RNA decay and is likely to be widely relevant.
Planned Impact
Dissection of the mechanisms underlying RNA-mediated chromatin regulation is a very active area of biology at the moment. Important concepts relevant to gene expression generally are emerging from very different systems. The Dean lab has identified an excellent system in which to explore co-transcriptional mechanisms and the functioning of one class of non-coding RNAs, antisense transcripts. This has emerged through their work determining natural variation in flowering, an important adaptive trait in native plants and many crops. The chromatin mechanisms involved in flowering time control are likely to inform chromatin mechanisms relevant to many organisms. FLC regulation represents a system where different approaches can be combined to give a comprehensive view of the complexity and plasticity of gene regulation.
The work will also contribute fundamental information that provides a framework of understanding for dissection of flowering time control in less tractable but strategically important plants. The understanding emanating from analysis of the flowering network in Arabidopsis is a good paradigm for how fundamental information informs generally and influences experimental strategies of a wide community of plant biologists. The John Innes Centre is very unusual in combining basic biological research with crop-based studies. Groups interact on a daily basis, so results emerging from model species are quickly applied to crops such as Brassicas and cereals. Prof. Dean, collaboratively with Dr Judith Irwin (Dept. Crop Genetics, John Innes Centre) also has very good links with the plant breeding and biotechnology industries, and there are frequent visits in both directions. A clear understanding of the components regulating flowering will open up many avenues with impact in different areas.
A clearer understanding of the molecular basis of flowering time will inform strategies as to how to manipulate the timing of the transition to flowering, a key trait in breeding of many crops. The focus in the Dean/Irwin labs is manipulation of vernalization requirement and response - a key process in the production of many vegetable crops, broccoli, cauliflower, parsnips and carrots. Varieties of these vegetables are bred to ensure year round supply but the vagaries of winter temperatures tends to lead to gluts or shortages in production. Development of varieties less influenced by temperature but still producing in different seasons of the year would considerably reduce waste, potentially open up new production areas and increase efficiency of delivery. Ongoing funded collaborations with breeding companies aim to translate this understanding into practical benefits.
The work will also contribute fundamental information that provides a framework of understanding for dissection of flowering time control in less tractable but strategically important plants. The understanding emanating from analysis of the flowering network in Arabidopsis is a good paradigm for how fundamental information informs generally and influences experimental strategies of a wide community of plant biologists. The John Innes Centre is very unusual in combining basic biological research with crop-based studies. Groups interact on a daily basis, so results emerging from model species are quickly applied to crops such as Brassicas and cereals. Prof. Dean, collaboratively with Dr Judith Irwin (Dept. Crop Genetics, John Innes Centre) also has very good links with the plant breeding and biotechnology industries, and there are frequent visits in both directions. A clear understanding of the components regulating flowering will open up many avenues with impact in different areas.
A clearer understanding of the molecular basis of flowering time will inform strategies as to how to manipulate the timing of the transition to flowering, a key trait in breeding of many crops. The focus in the Dean/Irwin labs is manipulation of vernalization requirement and response - a key process in the production of many vegetable crops, broccoli, cauliflower, parsnips and carrots. Varieties of these vegetables are bred to ensure year round supply but the vagaries of winter temperatures tends to lead to gluts or shortages in production. Development of varieties less influenced by temperature but still producing in different seasons of the year would considerably reduce waste, potentially open up new production areas and increase efficiency of delivery. Ongoing funded collaborations with breeding companies aim to translate this understanding into practical benefits.
Organisations
People |
ORCID iD |
Caroline Dean (Principal Investigator) |
Publications
Zhao Y
(2020)
Publisher Correction: Temperature-dependent growth contributes to long-term cold sensing.
in Nature
Description | The project investigated the regulation of one particular R-loop structure (an RNA-DNA hybrid in the chromatin) that had recently been identified as influencing the expression of an Arabidopsis developmental regulator. The work we achieved in this grant showed that this R-loop provides an excellent model for understanding how these structures affect transcriptional output generally in eukaryotic genomes. Indeed, the research we completed has opened many more questions linking transcription and replication and underpinned a successful grant application to the Wellcome Trust. We achieved all the planned aims within the two main objectives: 1) Identification of in vivo interactors of AtNDX and functional analysis of their role in R-loop stabilization/resolution. A list of provisional regulators had been available at the beginning of the work. We replicated these proteomic studies and validated several of the interacting proteins. For example, we confirmed the RNAi component AGO1 is an in vivo interactor of NDX1. We showed that AGO1 affects the R-loop, associates directly with the locus and functions to terminate the transcription of the COOLAIR antisense and thus resolve the R-loop. We completed analysis on other NDX interactors: PRP8, a general splicing component ; FCA, a 3' processing factor; CDKC2. In addition we identified a new interactor, a B3 protein named VAL1 that we had shown influenced the epigenetic switching of FLC expression by cold. This has opened up a whole new avenue of research in the lab identifying a second RNA/DNA hybrid structure at the 5' end of FLC associated with transcriptional regulation of the sense transcript, and epigenetic silencing during winter. We also pursued the interactor XRN3, but due to functional redundancy knock out of this function did not influence the R-loop or FLC expression. 2. Characterization of additional regulators of COOLAIR and analysis of how their function intersects with R-loop induced transcriptional modulation. The genetic mapping of the first mutants showing ectopic high or low COOLAIR expression in the warm and cold had been complicated by linkage to multiple sites in the genome. We realized this was a complication from a large inversion caused by the flc-2 mutant allele in the COOLAIR-LUC transgene background. We therefore retransformed the COOLAIR-LUC transgene into wild-type plants and repeated the mutagenesis and mutant screening. This revealed many new non-allelic mutants and we have currently characterized three new loci: TOC65, a chloroplast import protein - this has opened up an unexpected connection between retrograde signaling and R-loop/chromatin regulation; NAP1, a protein associated with a SCAR-WAVE complex involved in nucleation of the actin cytoskeleton: THO complex, previously shown to be involved in nuclear export of transcripts. These mutants affect COOLAIR expression, both in the transgene context and also on the endogenous locus. |
Exploitation Route | Our work has thus opened up a number of novel aspects to R-loop regulation relevant to gene expression broadly across eukaryotes. The field of molecular biology has focused more and more on R-loop and non-B DNA structure in recent years so this new understanding and regulators will therefore contribute broadly to understanding local chromatin structures that affect a variety of co-transcriptional mechanisms. |
Sectors | Agriculture, Food and Drink,Environment |
URL | http://www.jic.ac.uk/staff/caroline-dean/ |
Description | R-loops are important in genome damage. This means any biotech development of drugs for cancer prevention and other human diseases include assays for R-loop stability and occurrence. |
First Year Of Impact | 2013 |
Sector | Agriculture, Food and Drink,Environment |
Impact Types | Societal,Economic |
Description | GCRF databases and Resources phase 2 |
Amount | £98,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2017 |
End | 07/2018 |
Description | Mechanistic basis of nucleation and spreading underlying a Polycomb-mediated epigenetic switch (EPISWITCH) |
Amount | € 2,100,000 (EUR) |
Funding ID | 833254 |
Organisation | European Research Council (ERC) |
Sector | Public |
Country | Belgium |
Start | 09/2019 |
End | 09/2024 |
Description | R-loop coupled chromatin regulation |
Amount | £1,449,786 (GBP) |
Funding ID | 210654/Z/18/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2018 |
End | 10/2024 |
Description | Invited seminar in Suzhou, China |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Prof caroline Dean was invited to give a seminar in Suzhou, China. Her talk was entitled: "Antisense-mediated chromatin silencing at FLC". |
Year(s) Of Engagement Activity | 2016 |
Description | Invited speaker at Abcam Meeting, Copenhagen |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Prof Caroline Dean was invited speaker at an Abcam meeting in Copenhagen. She delivered a talk entitled: "Antisense-mediated chromatin silencing at FLC". |
Year(s) Of Engagement Activity | 2016 |
Description | Invited speaker at Imperial College |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | Prof Caroline Dean was invited speaker at Imperial College, Hammersmith Campus Institute of Clinical Sciences. She delivered a talk entitled: "Epigenetic switching in seasonal timing". |
Year(s) Of Engagement Activity | 2016 |
Description | Invited speaker at Peking University, Beijing, China |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Caroline Dean was invited to speak at Peking University, Beijing, China, on the 1st June. Her seminar was entitled: 'Epigenetic switching and antisense transcription'. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited speaker at Sustech Shenzhei, China. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Caroline Dean was invited to speak at Sustech Shenzhei, China on the 28th May. Her seminar was entitled: 'Epigenetic switching and antisense transcription'. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited speaker at the EMBO Non-coding RNA meeting, Heidelberg. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Prof Caroline Dean was invited speaker at the EMBO Non-coding RNA meeting in Heidelberg. Talk title: "Antisense-mediated chromatin silencing at FLC". |
Year(s) Of Engagement Activity | 2016 |
Description | Keynote Lecture at International Congress of Genetics, Iguassu, Brazil |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | ON the 11th September 2018 Caroline Dean gave the Keynote Lecture at the International Congress of Genetics, Iguassu, Brazil, entitled: 'Epigenetic switching and antisense transcription'. |
Year(s) Of Engagement Activity | 2018 |
Description | Keynote Lecture on epigenetics at the Netherlands Cancer Institute (NKI). |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Prof Dean gave the Keynote lecture at the Dutch Chromatin meeting at the Netherlands Cancer Institute (NKI) on the 2nd November 2018. Translational talks (outside the field of plant science) increase the impact and application of GEN Science. Her talk was entitled: 'Epigenetic switching and antisense transcription'. |
Year(s) Of Engagement Activity | 2018 |
Description | McClintock Lecture Cold Spring Harbor Laboratory |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Caroline Dean gave the Barbara McClintock Lecture at Cold Spring Harbor Laboratory NY on chromatin regulation and evolutionary adaptation. The talk stimulated discussion and questions. |
Year(s) Of Engagement Activity | 2018 |
URL | http://cshlwise.org/institutional-events/mcclintock-lectures/ |
Description | Plenary Lecture at Keystone Symposia on Molecular and Cellular Biology 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Caroline Dean gave the Plenary Lecture at the Keystone Symposia on Molecular and Cellular Biology "Antisense-Mediated Chromatin Silencing at FLC". The talk triggered lots of discussion and questions throughout the rest of the meeting. |
Year(s) Of Engagement Activity | 2018 |
URL | http://www.keystonesymposia.org/18C2 |
Description | Seminar as part of the Tsinghua University Seminar in Frontiers in Biology special series. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Prof Caroline Dean gave a seminar as part of the Tsinghua University Seminar in Frontiers in Biology special series entitled: "Antisense-mediated chromatin silencing at FLC". |
Year(s) Of Engagement Activity | 2016 |
Description | Seminar at EMBL Epigenetics & Chromatin conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This was a virtual presentation to a diverse audience on the mechanistic basis of epigenetic switching |
Year(s) Of Engagement Activity | 2021 |
Description | Seminar at Int Congress of Arabidopsis Research workshop |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | The virtual International Arabidopsis meeting was well attended and as well as plenary sessions they had focused workshops of which I was part of. |
Year(s) Of Engagement Activity | 2021 |
Description | Seminar to LANGEBIO Cinvestav Mexico |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | This is the largest plant bioscience centre in Mexico. They wanted to learn about epigenetic regulation in plants |
Year(s) Of Engagement Activity | 2021 |
Description | Seminar to national Dutch PhD student programme |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | All the current PhD students meet annually in Holland and invite an external speaker and this year it was me |
Year(s) Of Engagement Activity | 2021 |