Epigenetic regulation of pluripotency and lineage commitment in the early mouse embryo

Lead Research Organisation: Imperial College London
Department Name: Surgery and Cancer

Abstract

In the last few years the genetic code or 'blueprint' for man has become available. Although this information is nearly complete, we still do not know how to translate the code, how to use it to rebuild the cells of damaged tissues or how to understand the remarkable development of an individual from a single fertilised egg. What we do know now is that this code is interpreted in each cell in the body by a group of agents that are collectively called 'chromatin-modifiers'. Our laboratory looks at the chromatin structure of embryonic stem (ES) cells. These remarkable cells are pluripotent, meaning they have the potential to differentiate into any cell type found in the body. Twenty-five years ago the first ES cell lines were derived from early mouse embryos into culture. The regulatory mechanisms, which define ES cell 'pluripotency' or differentiation, were not understood, although potential application for regenerative medicine was immediately foreseen. Deciphering how pluripotency is achieved and how it can be harnessed and maintained in culture are key questions for understanding normal development and successfully applying the knowledge to stem cell-based therapies. We intend to unravel the unique chromatin 'make-up' of pluripotent ES cells and their counterparts in the embryo that confers them flexibility and unlimited cell fate options. We also explore how this can be dynamically changed in a manner that is predictable when a stem cell decides to become a specific cell type (e.g. nerve, muscle or blood cell). This information will help us to understand how the genetic code is used, and help us to design better strategies for turning stem cells into cells that are useful for treating degenerative diseases and other human diseases.

Technical Summary

Pluripotent cells develop within the inner cell mass (ICM) of early-stage blastocyst embryos, a cell population surrounded by an extraembryonic layer, the trophectoderm (TE). Here, we use both embryo-derived stem cells and in vivo studies to investigate the epigenetic mechanisms leading to blastocyst lineage segregation and pluripotency safeguarding. Recent studies in ES cells showed that many silent genes that are required later on during development are simultaneously marked with Polycomb repressor (PRC)-mediated H3K27 methylation, and marks normally associated with gene activity. Moreover, these so-called bivalent genes assemble RNA Polymerase (RNAP) complexes and are transcribed at low level, specifying a 'primed' state in pluripotent cells. We have begun to compare the chromatin profiles of ES and TE-derived trophoblast stem (TS) cells. Though many genes are bivalently marked in TS cells as in ES cells, they are not normally expressed in this lineage. To explore this further, we will examine the chromatin environment of ES and TS cells at bivalent promoters by assessing PRC1 and PRC2 binding, RNAP occupancy and conformation and, additional 'lock in' mechanisms in TS cells that may operate to avoid reprogramming. We also address the role of key stem cell transcription factors in targeting chromatin changes using ES cell de-differentiation models upon Oct4 loss or gain of Cdx2. In order to confirm the biological significance of 'primed' chromatin in the embryo we will use carrier ChIP assay to investigate the epigenetic relationship between ES and TS cells and in vivo ICM and TE cells. In particular, we intend to explore how and when specific differences in epigenetic programming are specified, at the locus level, between embryonic and extraembryonic tissues. This will lead to in vivo lineage tracing studies where single blastomere knock-down (RNA interference), jointly labelled with a dye, can reveal the effects of epigenetic program failure on cell allocation.
 
Description Balancing cell potency and specialisation is an inherent challenge for all developing organisms. In the context of the early mammalian embryo this implies promoting the formation of two essential extra-embryonic tissues for embryo survival (e.g. the trophectoderm and primitive endoderm), while critically maintaining a pool of pluripotent cells with abilities to generate all somatic cell types found in the body. The isolation and propagation in culture of pluripotent cells from the early embryo was a pivotal achievement in the field of developmental and stem cell biology. These remarkable embryonic stem (ES) cells can indeed self-renew for unlimited periods of time whilst retaining their full developmental potency, hence offering a valuable model system to decipher how pluripotency is achieved/balanced and subsequently lost as the embryo develops.

In the past few decades, it has become clear that chromatin and epigenetic factors can play a key role in a large array of biological processes, including development. The modification of chromatin and its histone components adds an additional layer of regulation that ultimately extends the information content of the underlying genome sequence. This project investigated the specific chromatin 'make-up' of pluripotent ES cells and their in vivo counterparts, and furthermore identified novel epigenetic pathways that underlie cell specialisation while preserving pluripotency during early embryonic development.

Most significant achievements of this grant 1- Alder, Lavial, et al. Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment. Development 2010. These findings provided the first in vivo evidence that Polycomb-mediated repression and bivalent histone marking operate at silent, key developmental genes in the mouse early embryo and furthermore proposed a novel epigenetic pathway by which extra-embryonic lineage commitment can be reinforced and pluripotency safeguarded upon blastocyst formation. 2- Lavial et al. Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development. Genes & Development 2012. These findings uncovered a novel and early developmental role for the Polycomb member Bmi1 as a key post-transcriptional regulator of Gata factors. Notably, this study revealed how Bmi1 action on Gata6 stability can alter the balance between Nanog and Gata6 protein levels in individual blastomeres to introduce a bias towards a primitive endoderm identity, and more generally how mechanisms regulating protein stability/activity can directly impact on cell fate decisions. 3- Rute Tomaz et al. Jmjd2c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation. Development 2017. Further funding were successfully secured based on the above outcomes (point 1) and preliminary data generated by F. Lavial (Research Associate funded on this grant). This allowed us to identify a novel role for the epigenetic regulator Jmjd2c/Kdm4c in embryonic versus extra-embryonic stem cells independent of its canonical histone demethylase activity. Notably, we uncovered that Jmjd2c is strictly required for somatic multi-lineage differentiation by promoting the assembly of multi-protein complexes at poised enhancers in embryonic stem cells.
Exploitation Route Understanding how differentiation occurs in nature is vital to recapitulating it in the clinic, for the benefit of patients. This work, though very basic in its focus, has great potential to be exploited for therapeutic and economic good. The research will shed light on how the correct balance is struck between generating/maintaining stem cells and directing their differentiation. This understanding will naturally flow into the clinic, in the form of better protocols for directing the differentiation of pluripotent and other stem cells. It will also inform regenerative medicine approaches that aim to reactivate endogenous stem cell pools in the body, by providing insights into which molecular pathways should be targeted. Increased understanding of how the formation of two essential extra-embryonic tissues (e.g. the trophectoderm and primitive endoderm) is promoted while safeguarding pluripotency in pre-implantation embryos will also greatly help identification of likely candidate factors that determine embryo implantation and its subsequent development and therefore impact on reproduction.
Sectors Education,Healthcare,Other
 
Description The primary beneficiaries and users of the research are members of the academic sector - researchers, teachers and students as well as the general public as described below. Communications and engagement (tailored to specific audiences) 1- Professionals - practicing researchers: • Scientific results have been published in leading peer-reviewed journals. • Unpublished and published results have been presented at scientific conferences and invited lectures - see below specific examples of communications. • All mouse cell lines engineered and reagents/dataset generated in the course of this project are made freely available upon request. 2- Professionals - students: We consciously incorporate much of the primary data emerging from work in our research group into lectures taught to pre-clinical medics and graduate students in molecular genetics, reproductive and developmental biology and cancer biology. Results from this award have been be incorporated into the following lectures that are delivered annually: (i) Pluripotency; (ii) Regulation of stem cell identity, and (iii) Epigenetics. 3- Public/audience: As in the past, we continued to participate in local events that promote science among the public such as 'Open Days' to our laboratories and awareness raising events with Genesis Research Trust (http://genesisresearchtrust.com). Collaboration and co-production The project involved long-lasting and/or novel collaborative links with different UK-based and oversea researchers/groups including Laura O'Neill (University of Birmingham), Ana Pombo (MRC LMS, London), Amanda Fisher (MRC LMS, London), Philippe Arnaud (GReD, Clermont-Ferrand), Claire Chazaud (GReD, Clermont-Ferrand), Takashi Hiiragi (EMBL, Heilderberg), Ian Chambers (MRC Centre for Regenerative Medicine, Edinburgh), Toshinori Nakayama (Chiba University, Japan), Huck-Hui Ng (Genome Institute of Singapore), Jesus Gil (MRC LMS, London) and Malcolm Parker (IRDB, Imperial College London). Application and exploitation Our research lies at the heart of BBSRC's mission to support basic research underpinning human health. This project advanced fundamental understanding of how pluripotency is achieved and maintained, and how cell potency and specification are balanced in stem cells and in the early embryo. Capacity and involvement All members of our research team participate in the various impact and communication activities. Staff/PhD students who contributed to the success of this award received multi-disciplinary training and benefited from a world-leading research environment at Imperial College and through collaborations at other Research Institutes of Excellence. They were active participants in weekly lab and divisional meetings. Attendance to national and international meetings as well as workshops (e.g. Epigenetics, Marie Curie Institute) exposed them to the latest developments in the field and provided career development opportunities by allowing them to present results to their peers and to the leaders in the field. Staff/PhD students also participated in teaching at our divisional MSc programme in Reproductive and Developmental Biology including practical training course as well as co-supervising MSc students within our group. Additional transferrable skill training was ensured within Imperial College London. Altogether, this means that staff has been trained to a very high level, enhancing UK's economic competitiveness and making him a highly employable and sought-after expert. Invited Lectures/presentations at conferences during the course of this award and onwards (selected) Véronique Azuara (PI) - BSDB fall meeting (Oxford, 2018) - Wellcome Trust - MRC Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine (Cambridge, 2017) - BCGB, Institute of Biomedical Research, University of Birmingham (Birmingham, 2017) - IMT, Philipps-University Marburg (Germany, 2016) - Pasteur Institute, Department of Developmental and Stem Cell Biology (France, 2016) - STEMBRYO Symposium (Sitges-Spain, 2015) - Blizard Institute, QMUL, 'London Epigenomics Club' (London, 2015) - ISRMN Meeting, Imperial College London (London, 2014) - GReD, University of Auvergne, Clermont-Ferrand (France, 2013) - Wellcome Trust/ CRUK Gordon Institute (Cambridge, 2012) - Abcam meeting 'Epigenetics and Stem Cells' (Cambridge, 2012) - BACR meeting 'Chasing Cancer Stem-like Cells' (Harrogate, 2012) - University Paris Diderot, Epigenetics and Cell Fate Unit (Paris, 2012 and 2018) - Institute Necker, INSERM Unit U768 (Paris, 2010) - Institute of Molecular Genetics (Montpellier, 2009) FL (Postdoc) - STEMBRYO Network Meeting (Madrid, Oct 2011) - EuroSyStem 2011 Consortium Meeting (Prague, June 2011) AH (PhD student) - Cambridge Stem Cell Symposium 'Pluripotency and Development' (Cambridge, July 2011) RT (PhD student) - ISRMN Meeting, Imperial College London (London, 2016) - STEMBRYO Symposium (Sitges, 2015) Awards and Appointments 1- Associated Member of the EuroSyStem Project coordinated by Professor Austin Smith (2nd Call 2010) European Federation for Systematic Stem Cell Biology funded by EU Framework 7 http://www.eurosystemproject.eu - Véronique Azuara 2- Promotions Véronique Azuara (Imperial College London) was promoted to Senior Lecturer in August 2012. FL received an AVENIR Career Development Award in 2014 to establish his own independent group at ENS Lyon (France). 3- Editorial Boards - Véronique Azuara • Scientific Reports - Nature http://www.nature.com/srep, Member of the Editorial Board, 2015 • Cells - Structure, Function and Systems Biology http://www.mdpi.com/journal/cells-sfsb, Member of the Editorial Board, 2011
First Year Of Impact 2009
Sector Education,Healthcare,Other
Impact Types Societal
 
Description BBSRC New Investigator Award
Amount £483,000 (GBP)
Funding ID BB/G011117/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2009 
End 12/2012
 
Description FCT PhD GABBA programme SP
Amount £100,000 (GBP)
Funding ID SP 
Organisation Government of the Portugese Republic 
Department Foundation of Science and Technology (FCT)
Sector Public
Country Portugal
Start 11/2005 
End 10/2009
 
Description FCT PhD studentship RT
Amount £120,000 (GBP)
Funding ID SFRH/BD/7024/2010 - RT 
Organisation Government of the Portugese Republic 
Department Foundation of Science and Technology (FCT)
Sector Public
Country Portugal
Start 10/2011 
End 09/2015
 
Description Genesis Research Trust - PhD studentship OA
Amount £116,000 (GBP)
Funding ID OA 
Organisation Genesis Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2005 
End 09/2009
 
Description Genesis Research Trust - PhD studentship running cost supplement RT
Amount £30,000 (GBP)
Funding ID P14762: 2012-2013 and P55000: 2014- 2016 
Organisation Genesis Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 09/2016
 
Description Genesis Research Trust - Project Research Grant FL
Amount £82,000 (GBP)
Organisation Genesis Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2008 
End 02/2010
 
Description Genesis Research Trust - Supplement Studentship Funding 2005 OA and SP
Amount £64,000 (GBP)
Funding ID OA and SP 
Organisation Genesis Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2005 
End 09/2009
 
Description Genesis Research Trust - Supplement Studentship Funding 2007
Amount £8,000 (GBP)
Funding ID AH 
Organisation Genesis Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2007 
End 09/2008
 
Description MRC DTA PhD studentship AH
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2007 
End 04/2011
 
Title A role for Jmjd2c/Kdm4c in promoting somatic multi-lineage differentiation. 
Description 1- Generation of converted wild-type and Jmjd2c-knockout EpiSC and XEN cell lines 2- Generation of "rescued" Jmjd2c-knockout ESCs via re-introduction of Jmjd2c expression 3- Generation of ESC clones that express Flag-tagged wild-type and mutant Jmjd2c forms lacking both Tudor domains 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact * Publication (PMID: 28087629) and scientific communications (meetings, guest lectures). * Knowledge used in taught lectures in various courses. * in vitro model systems for studying the role of Jmjd2c/Kdm4c in the early developing embryo (Replacement method). 
 
Title A role for Ncoa3 as an essential Esrrb co-activator in embryonic stem cells 
Description Generation of ESCs overexpressing (1) Ncoa3, (2) Esrrb and (3) Ncoa3 and Esrrb together. 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact *Publications (PMID: 23019124; PMID: 23287469) and scientific communications (conferences, guest lectures). * Knowledge used in taught lectures in various courses. * in vitro model systems for studying the role of Esrrb and Ncoa3 at the exit of naive pluripotency -i.e. the transition from pre- to post-implantation embryonic stages in vivo (Replacement method). 
 
Title A role for the Polycomb member Bmi1 in promoting primitive endoderm differentiation 
Description Generation of stably knocked-down ESC and XEN cell lines for Bmi1 and associated control lines 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact * Publication (PMID: 20573702) and scientific communications (conferences, lectures). * Knowledge used in taught lectures in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes. * in vitro model systems for studying an early developmental role for Bmi1 during blastocyst lineage formation and epiblast-derived lineage specification (Replacement method) 
 
Title Protein-protein interactions - mapping of functional domains 
Description Generation of truncated Gata6 mutants lacking the C-terminal domain alone (dCT) or including its Zinc finger region (dCTZF), where putative lysine ubiquitination sites are preferentially mapped. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact * Publication (PMID: 22713603) and scientific communications (conferences, guest lectures). * Knowledge used in taught lectures given in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes. 
 
Title Requirement for Ncoa3 recruitment to mediate Esrrb function in embryonic stem cells 
Description Generation of truncated DBD, AF1 and AF2 as well as point-mutated AF2 Esrrb mutants 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact Publications (PMID: 23019124; PMID: 23287469; PMID: 32111830) and scientific communications (conferences, guest lectures). 
 
Title Mapping of Jmjd2c DNA-binding sites in naive (2i/LIF) and primed (serum/LIF) embryonic stem cells 
Description Generation of Flag-Jmjd2c ChIP-sequencing datasets in embryonic stem cells cultured under serum LIF (primed) and 2i LIF (naive) conditions 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact * Publication (PMID: 28087629) and scientific communications (meetings, guest lectures). * Knowledge used in taught lectures in various courses. 
 
Title Mapping of Ncoa3 functional DNA-binding sites in embryonic stem cells 
Description Generation of Ncoa3 ChiP-sequencing datasets in embryonic stem cells ad well as microarray datasets upon shRNA-mediated Ncoa3 depletion. 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact * Publications (PMID: 23019124; PMID: 23287469) and scientific communications (conferences, guest lectures). * Knowledge used in taught lectures in various courses. 
 
Description Bmi1 protein expression in early embryos. 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Country France 
Sector Academic/University 
PI Contribution Uncovered an early developmental role for the Polycomb member Bmi1 as a key post-transcriptional regulator of GATA factors in stem cells and during early development.
Collaborator Contribution Performed immunofluorescence using anti-Bmi1, anti-gata6 and anti-Nanog antibodies in pre-implantation embryos.
Impact * Publication (PMID: 22713603) and scientific communications (conferences, guest lectures) • Highlighted on issue cover. • These findings discovered an early developmental role for the Polycomb group member Bmi1 as a key posttranslational co-regulator of Gata factors, and proposed a novel regulatory pathway by which Bmi1 action on Gata6 protein stability/activity directly impacts on cell fate allocations between extra-embryonic and pluripotent identities. * Generation of key reagents (ES and XEN cell models, lentivirus vector containing short-hairpin RNA (shRNA) targeting mouse Bmi1 mRNA to assess the effects of knocking-down this epigenetic factor in TS cells, optimisation of shRNA-mediated targeting of Bmi1 in individual blastomeres and thus assess the impact of these epigenetic regulators, or lack of, on cell fate allocation in vivo). * Knowledge used in taught lectures given in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes.
Start Year 2010
 
Description Bmi1 transcript segregation event in individual blastomeres of early embryos. 
Organisation European Molecular Biology Laboratory
Department European Molecular Biology Laboratory Heidelberg
Country Germany 
Sector Academic/University 
PI Contribution Uncovered an early developmental role for the Polycomb member Bmi1 as a key post-transcriptional regulator of GATA factors in stem cells and during early development.
Collaborator Contribution Performed single cell RT-qPCR analysis of blastomeres dissected from early embryos.
Impact * Publications (PMID: 22713603) and scientific communications (conferences, guest lectures). • Highlighted on issue cover. • These findings discovered an early developmental role for the Polycomb group member Bmi1 as a key posttranslational co-regulator of Gata factors, and proposed a novel regulatory pathway by which Bmi1 action on Gata6 protein stability/activity directly impacts on cell fate allocations between extra-embryonic and pluripotent identities. * Knowledge used in taught lectures in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes.
Start Year 2010
 
Description Carrier ChIP on small samples of dissected embryos 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Olivia Alder (team member) visited the university of Birmingham to be trained on using a newly designed Carrier ChIP technology. This allowed us to establish and adapt this assay to epigenetically analyse small pools of cell populations derived from pre-implantation embryo (cleavage-stage, morula and dissected ICM/TE cell samples).
Collaborator Contribution Provided training in Carrier ChIP on small samples of dissected embryos.
Impact * We have established and adapted a specially devised Carrier ChiP assay to epigenetically analyse small pools of cell populations derived from pre-implantation embryo (cleavage-stage, morula and dissected ICM/TE tissues). This allowed us to provide the first in vivo evidence that bivalent histone marking (i.e. H3K27me3/H3K4me2) operates in the early mouse embryos at silent, key developmental genes (PMID: 20573702). We furthermore demonstrated that a set of somatic lineage regulators that carry bivalent signatures is selectively targeted by Suv39h1-mediated H3K9 methylation (and de novo DNA methylation) upon TE lineage formation. In contrast, H3K9me3 marks were not detected in ICM as well as in morula cells at these loci, further suggesting that the acquisition of additional repressive marks at bivalent, lineage-inappropriate genes might be a late event linked with trophoblast cell fate consolidation and loss of lineage plasticity. * Knowledge used in taught lectures given in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes.
Start Year 2009
 
Description Cdx2 gene expression regulation in stem cells and in vivo 
Organisation Spanish National Centre for Cardiovascular Research
Country Spain 
Sector Public 
PI Contribution Provided training in ChIP in embryonic and trophoblast stem cells and in Carrier ChIP in small samples of dissected embryos through a 6-month placement; data interpretation; exchange of reagents, methods and idea.
Collaborator Contribution Joint publication in Scientific Reports 2016
Impact * Publication PMID: 27256674 - These findings report the existence of distinct mechanisms to regulate Cdx2 expression in the blastocyst and in trophoblast stem cells. * Associated members of the European STEMBRYO Network of stem cell and developmental biologists.
Start Year 2010
 
Description Gata6 protein activity 
Organisation Yale University
Country United States 
Sector Academic/University 
PI Contribution Generation and provision upon collaboration of truncated Gata6 mutants lacking the C-terminal domain alone (dCT) or including its Zinc finger region (dCTZF) as described in Lavial et al. Genes and Development, 2012.
Collaborator Contribution request of reagents upon collaboration.
Impact No resultant outcome yet.
Start Year 2016
 
Description ICL collaborative links - epigenetic regulation of embryonic and extra-embryonic stem cells. 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of ideas, reagents, technologies and joint publications and grant applications.
Collaborator Contribution Exchange of ideas, reagents, technologies.
Impact * Publications (PMID: 20157423,PMID: 20573702, PMID: 22713603) and scientific communications (conferences, lectures). * Joint grant applications. * Knowledge used in taught lectures in various courses.
Start Year 2009
 
Description ICL collaborative links - epigenetic regulation of embryonic and extra-embryonic stem cells. 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of ideas, reagents, technologies and joint publications and grant applications.
Collaborator Contribution Exchange of ideas, reagents, technologies.
Impact * Publications (PMID: 20157423,PMID: 20573702, PMID: 22713603) and scientific communications (conferences, lectures). * Joint grant applications. * Knowledge used in taught lectures in various courses.
Start Year 2009
 
Description ICL collaborative links - epigenetic regulation of embryonic and extra-embryonic stem cells. 
Organisation Medical Research Council (MRC)
Department MRC/UVRI and LSHTM Research Unit Uganda
Country Uganda 
Sector Academic/University 
PI Contribution Exchange of ideas, reagents, technologies and joint publications and grant applications.
Collaborator Contribution Exchange of ideas, reagents, technologies.
Impact * Publications (PMID: 20157423,PMID: 20573702, PMID: 22713603) and scientific communications (conferences, lectures). * Joint grant applications. * Knowledge used in taught lectures in various courses.
Start Year 2009
 
Description Identification of Ncoa3 as an essential Esrrb coactivator in embryonic stem cells 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Uncovering a key role for Ncoa3 as an essential Esrrb coactivator in embryonic stem cells. Supervision of a joint PhD student (Michelle Percharde - BBSRC funded) together with Professor Malcolm Parker; expertise in pluripotent stem cells and gene regulation; exchange of ideas, reagent, technologies, joint publication and grant application.
Collaborator Contribution Co-supervision of a joint PhD student (Michelle Percharde - BBSRC funded); expertise in nuclear receptors in somatic cells; exchange of ideas, reagent, technologies, joint publication and grant application.
Impact *Publications (PMID: 23019124; PMID: 23287469) and scientific communications (conferences, guest lectures). • Commented in Cell Cycle. 2013 Jan 15; 12(2):195-6. • Commented in EMBO J. 2012 Nov 14; 31(22):4255-7. • These findings uncovered novel and essential roles for the co-activator Ncoa3 in pluripotency, and proposed an integrated model of transcriptional and co-activator control, mediated by Ncoa3, for the maintenance of embryonic stem cell self-renewal and reprogramming. * Generation of truncated DBD, AF1 and AF2 as well as point-mutated AF2 Esrrb mutants and ESC models. This allowed us to identify Esrrb AF2 domain as being critical for its function as self-renewal enhancer in embryonic stem cells. * Knowledge used in taught lectures in various courses.
Start Year 2009
 
Description Jmjd2c/Kdm4c binding site mapping in pluripotent stem cells 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Uncovering a novel function for Jmjd2c/Kdm4c at the exit of ESC pluripotency; joint publication in Development 2017.
Collaborator Contribution Provide expertise in embryonic stem cell conversion into extra-embryonic stem cells; exchange of reagent and technologies; access to sequencing facilities and bioinformatics support.
Impact * Successful applications for further funding (FCT and Genesis Research Trust) based on outcomes from BBSRC grant BB/G011117/1 and preliminary data generated by Fabrice Lavial (BBSRC-funded Research Associate) to fund and recruit a new PhD student in the group (Rute Tomaz). * Publication (PMID: 28087629) and scientific communications (meetings, guest lectures). • Highlighted in the NODE- the community site for and by developmental biology - see http://thenode.biologists.com/people-behind-papers-14/interview/ • This study stems from previous BBSRC-funded research (PMID: 20573702) identifying an imbalance between the expression and activity of H3K9 methyltransferases (Suv39h1) and demethylases (Jmjd2c) between extra-embryonic and embryonic stem cells and in vivo. Here, we uncovered a novel mechanism by which the H3K9 demethylase Jmjd2c regulates multi-lineage priming via Jmjd2c-mediated stabilisation of essential protein complexes at enhancers in embryonic stem cells. * Generation of Flag-Jmjd2c ChIP-sequencing datasets in embryonic stem cells cultured under serum+ LIF (primed) and 2i+LIF (naive) conditions. * Generation of Flag-tagged wild-type and Tutor domain mutant Jmjd2c expressing embryonic stem cells. * Generations of converted wild-type and Jmjd2c-knockout XEN cell lines * Knowledge used in taught lectures in various courses.
Start Year 2015
 
Description Ncoa3 binding-site mapping and transcriptional impact of depleting Ncoa3 in embryonic stem cells. 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Genome Institute of Singapore
Country Singapore 
Sector Academic/University 
PI Contribution Uncovering a key role for Ncoa3 as an essential Esrrb coactivator in embryonic stem cells; joint publication in Genes and Development 2012.
Collaborator Contribution Performed Ncoa3 ChIP-sequencing in embryonic stem cells, microarray experiments together with providing bioinformatics support; exchange of reagent and technologies; joint publication.
Impact *Publications (PMID: 23019124; PMID: 23287469) and scientific communications (conferences, guest lectures). • Commented in Cell Cycle. 2013 Jan 15; 12(2):195-6. • Commented in EMBO J. 2012 Nov 14; 31(22):4255-7. • These findings uncovered novel and essential roles for the co-activator Ncoa3 in pluripotency, and proposed an integrated model of transcriptional and co-activator control, mediated by Ncoa3, for the maintenance of embryonic stem cell self-renewal and reprogramming. * Generation of Ncoa3 ChiP-sequencing datasets in embryonic stem cells ad well as microarray datasets upon Ncoa3 depletion. * Knowledge used in taught lectures in various courses.
Start Year 2010
 
Description Protein-protein interactions - mapping of domains 
Organisation Chiba University
Country Japan 
Sector Academic/University 
PI Contribution Uncovered a key role for the polycomb Bmi1 in conferring protection against Gata6 ubiquitination and degradation in embryo-derived extra-embryonic stem cells.
Collaborator Contribution Provided a series of Bmi1 mutant plasmids.
Impact * Publication (PMID: 22713603) and scientific communications (conferences, guest lectures). • Highlighted on issue cover. • These findings discovered an early developmental role for the Polycomb group member Bmi1 as a key posttranslational co-regulator of Gata factors,and proposed a novel regulatory pathway by which Bmi1 action on Gata6 protein stability/activity directly impacts on cell fate allocations between extra-embryonic and pluripotent identities. * Generation of truncated Gata6 mutants lacking the C-terminal domain alone (dCT) or including its Zinc finger region (dCTZF), where putative lysine ubiquitination sites are preferentially mapped. This allowed us to identify Gata6 C-terminal domain as being critical for Gata6 ubiquitination/degradation as well as for its interaction with Bmi1 in a ring domain dependent manner, providing further insights into how Bmi1 might stabilise Gata6 protein expression in primitive endoderm derivatives. * Knowledge used in taught lectures given in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes.
Start Year 2012
 
Description RNA polymerase activity and conformation. 
Organisation Imperial College London
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Uncovered how mutually exclusive repressive pathways delineate distinct chromatin states at key developmental genes in stem cells and during early development.
Collaborator Contribution Provided training in RNA Polymerase ChIP using available antibodies.
Impact * Publication (PMID: 20573702) and scientific communications (conferences, lectures). • Recommended as new finding in Faculty of 1000 (f.4671971.4550076). • These findings provided first in vivo evidence that Polycomb-mediated repression and bivalent histone marking operate at silent developmental genes from cleavage-stage embryo onwards. Moreover, this study revealed novel epigenetic pathways by which lineage specification can be reinforced in the developing embryo. * Generation of key reagents (a lentivirus vector containing short-hairpin RNA (shRNA) targeting mouse Suv39h1 mRNA to assess the effects of knocking-down this epigenetic factor in TS cells). * Knowledge used in taught lectures in the context of undergraduate BSc and postgraduate MSc/MRes teaching programmes.
Start Year 2009
 
Description Role of Jmjd2c/Kdm4c and protein partners in pluripotent stem cells 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Institute of Chemistry
Country France 
Sector Public 
PI Contribution Uncovering a novel function for Jmjd2c/Kdm4c at the exit of ESC pluripotency; joint publication in Development 2017.
Collaborator Contribution Exchange of reagents and technologies; performing G9a complex purification experiments and endogenous co-immunoprecipitation assays in wild-type and Jmjd2c-knockout ESCs.
Impact * Successful applications for further funding (FCT and Genesis Research Trust) based on outcomes from BBSRC grant BB/G011117/1 and preliminary data generated by Fabrice Lavial (BBSRC-funded Research Associate) to fund and recruit a new PhD student in the group (Rute Tomaz). * Publication (PMID: 28087629) and scientific communications (meetings, guest lectures). • Highlighted in the NODE- the community site for and by developmental biology - see http://thenode.biologists.com/people-behind-papers-14/interview/ • This study stems from previous BBSRC-funded research (PMID: 20573702) identifying an imbalance between the expression and activity of H3K9 methyltransferases (Suv39h1) and demethylases (Jmjd2c) between extra-embryonic and embryonic stem cells and in vivo. Here, we uncovered a novel mechanism by which the H3K9 demethylase Jmjd2c regulates multi-lineage priming via Jmjd2c-mediated stabilisation of essential protein complexes at enhancers in embryonic stem cells. * Knowledge used in taught lectures in various courses.
Start Year 2015
 
Description Role of Jmjd2c/Kdm4c at the onset of pluripotent stem cell differentiation 
Organisation University of Cambridge
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Uncovering a novel function for Jmjd2c/Kdm4c at the exit of ESC pluripotency; joint publication in Development 2017.
Collaborator Contribution Expertise in epiblast stem cell differentiation towards mesodermal progenitors; exchange of idea, reagents and/or technologies.
Impact * Successful applications for further funding (FCT and Genesis Research Trust) based on outcomes from BBSRC grant BB/G011117/1 and preliminary data generated by Fabrice Lavial (BBSRC-funded Research Associate) to fund and recruit a new PhD student in the group (Rute Tomaz). * Publication (PMID: 28087629) and scientific communications (meetings, guest lectures). • Highlighted in the NODE- the community site for and by developmental biology - see http://thenode.biologists.com/people-behind-papers-14/interview/ • This study stems from previous BBSRC-funded research (PMID: 20573702) identifying an imbalance between the expression and activity of H3K9 methyltransferases (Suv39h1) and demethylases (Jmjd2c) between extra-embryonic and embryonic stem cells and in vivo. Here, we uncovered a novel mechanism by which the H3K9 demethylase Jmjd2c regulates multi-lineage priming via Jmjd2c-mediated stabilisation of essential protein complexes at enhancers in embryonic stem cells. * Generation of converted wild-type and Jmjd2c-knockout EpiSC lines. * Setting up of mouse EpiSC mesodermal (paraxial and lateral plate) and differentiation methods. * Knowledge used in taught lectures in various courses.
Start Year 2015
 
Description Switch of Polycomb orthologs during embryonic stem cell differentiation 
Organisation Imperial College London
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution expertise in embryonic stem cell manipulation and differentiation
Collaborator Contribution joint publication in Cell Stem Cell 2012
Impact Joint Publication (PMID: 22226354). • Commented in Cell Stem Cell. 2012 Jan 6; 10(1):4-6. • These findings provided novel evidence that dynamic interchange between Polycomb Repressive Complex subunits can modulate the balance between ESC self-renewal and differentiation, and highlighted a key role for miRNA regulation in timely down-regulating Cbx7 at the onset of differentiation.
Start Year 2011
 
Description The role of Nanog in regulating the expression of Polycomb members in embryonic stem (ES) cells. 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Uncovered a repressive role for Nanog in controlling the expression of Bmi1 in pluripotent stem cells.
Collaborator Contribution Provided various ES cell model systems.
Impact * Publication (PMID: 22713603) and scientific communications (conferences, guest lectures) • Highlighted on issue cover. • These findings discovered an early developmental role for the Polycomb group member Bmi1 as a key posttranslational co-regulator of Gata factors, and proposed a novel regulatory pathway by which Bmi1 action on Gata6 protein stability/activity directly impacts on cell fate allocations between extra-embryonic and pluripotent identities. * Knowledge used in taught lectures given in the context of our MSc programme in Reproductive and Developmental Biology.
Start Year 2009
 
Description Genesis Research Trust 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talks/presentations sparked questions and discussion afterwards; information provided on GRT website; acknowledgment of the GRT in scientific publications.

Increased awareness of our research efforts and impact.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019