Mechanisms of miRNA regulation of early embryonic development

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

During the development of an embryo, cells transit through a stage where they possess the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent are stem cells and hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer.
The cells that are in the pluripotent state need to make choices regarding the cell type that they will give rise to and pass strict quality controls, as only those that are not defective will survive. This implies that pluripotent cells are under tight regulation and understanding this regulation is important not only to gain insight into how an embryo forms but as well to be able to use pluripotent cells for regenerative medicine. This project aims to understand the regulation of the cell survival and differentiation programs in pluripotent cells.
MicroRNAs are small non-coding RNAs that inhibit gene expression and have emerged over the past decade as key regulators involved in many processes including development, cell proliferation, apoptosis and in numerous diseases, including cancer. In the preliminary work leading up to this proposal we have found that microRNAs are required for the survival of pluripotent cells and for these cells to differentiate into neural cell types. These observations therefore have directly implicated microRNAs in the regulation of pluripotent cells. Understanding how microRNAs regulate cell survival and differentiation of the pluripotent state is the main focus of this project.
As a first step we will use mouse genetics as well as pluripotent stem cell lines for "in a dish" approaches to delete Dicer, that is the protein that is essential for microRNA generation. By studying the effects of Dicer deletion, and therefore microRNA depletion, in the embryo and in pluripotent stem cell lines we will be able to uncover precisely what the roles of microRNAs are in the regulation of the pluripotent state.
MicroRNAs fall into families according to the genes that they inhibit, those within a family are thought to target the same genes. Our preliminary work to this project identified microRNAs of the miR-291a-3p, miR-17, miR-19 and miR-92 families as strong candidates for being involved in the regulation of the survival and differentiation of pluripotent cells. For these reasons our work will focus on these microRNAs and test which families can rescue the defects caused by the depletion of microRNAs in pluripotent stem cells.
Finally, we aim to identify what genes are targeted by the microRNAs that regulate pluripotent cell survival and differentiation. This will establish the microRNA-target interactions that regulate pluripotency. Given that microRNAs inhibit gene expression, we will study what genes decrease in expression when cells are treated with a given microRNA. Given that many cell types are saturated with microRNAs, we will use Dicer deficient pluripotent stem cells for these studies, as they represent a microRNA free cell type. Bioinformatic programs to predict microRNA targets will be used to generate a list of likely microRNA target genes from those identified experimentally. The best candidate genes will be validated by mutation of the microRNA target site in it. This will establish which interactions are functionally important.

Technical Summary

Understanding the regulatory networks underlying pluripotency and differentiation is essential to effectively use stem cells for therapeutic applications. Two distinct phases of pluripotency have been proposed, a naïve state and a primed state and these states are thought to be regulated by different mechanisms. microRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression and have been shown to be required to for proper proliferation and exit of the naïve pluripotent state. However little is known about their roles in the primed phase of pluripotency. In preliminary work to this proposal we have found that in contrast to the naïve pluripotent state, miRNAs regulate cell survival and neural differentiation of primed pluripotent cells. The main focus of this project is to understand how miRNAs regulate these processes. Dicer is an enzyme that is essential for the production of mature microRNAs. We will first establish the functional requirements of miRNAs in the epiblast by analysing embryos in which Dicer has been specifically deleted in primed pluripotent cells or in derivatives of each of the germ layers. These studies will be complemented with the analysis of the effects that Dicer deletion has on the survival and differentiation of epiblast stem cells (EpiSCs). Next we will identify what microRNAs regulate the primed pluripotent state by determining which miRNAs can rescue the defects of Dicer deleted EpiSCs. Finally we will establish the miRNA-mRNA target interactions that regulate primed epiblast pluripotency. For this we will transfect single miRNAs into Dicer deleted EpiSCs and study what genes become downregulated. These genes will be compared to those obtained by bioinformatic prediction of microRNA targets. The best candidate genes will be validated by mutation of the microRNA target site in them to establish which interactions are functionally important.

Planned Impact

The large promise that pluripotent stem cells hold for regenerative medicine, where stem cell are induced to differentiate towards specialised cells that are then transplanted into patients, has carried with it a large degree of interest both from the academic and medical communities Furthermore, with the advances in reprogramming there is an enormous scope for applying in vitro differentiation of patient-derived induced pluripotent stem (iPS) cells to study diseases. However, increased knowledge about how specific cell types are induced in an irreversible way as well as how to differentiate cells down specific lineages is vital to control that the correct cell types are generated before these stem cell therapy becomes meaningful clinically. Our work aims to unravel the roles of microRNAs in the control of pluripotency and neuronal differentiation and therefore is at the core of this interest. Answering the questions of how do the roles of microRNAs differ in the naïve and primed pluripotent states, how do microRNAs control stem cell survival and neuronal differentiation of pluripotent stem cells, what microRNAs have roles in these processes and what their key targets are, will give fundamental insights into the regulatory networks that control pluripotency.
In the academic environment, those working in the UK and abroad, in the fields of developmental biology and stem cell biology as well as those interested in the diseases that are potential targets for stem cell therapy (such as neurodegenerative diseases) as well as those that possibly have a stem cell origin or where the microRNAs families we study have been involved, such as cancer, are potential beneficiaries of our work. The ability of microRNAs to reprogram differentiated cells to the pluripotent state indicates that our work will also be of relevance to those with an interest in reprogramming, as we will identify those microRNAs that are required for pluripotent stem cell survival.
MicroRNAs have a big potential as therapeutic agents/drug targets and given the importance of their role in pluripotency, they will undoubtedly have an impact in regenerative medicine. Therefore the results obtained in this study will be of direct relevance to translational research and have a potential commercial interest. Furthermore, given the role we have identified fore microRNAs in neural differentiation it is likely that our findings will also impact by improving the protocols designed for the differentiation of stem cells towards neuronal subtypes that are of interest to regenerative medicine. We will exploit these potential commercial interests through Imperial College Innovations so as to identify the most suitable partners.
A further beneficiary of our work will be the lay public. Given the medical relevance and ethical implications of stem cells, our work will provide factual input and therefore benefit the public discussion about the advantages and risks of stem cell therapy. We take seriously the responsibility of scientists to engage with the lay public, to raise awareness among them of the results of publically funded research, to openly debate ethical issues relating to our research so that public opinion may be formed in an informed manner and to take the excitement of our research to the children of today, who will be the scientists of tomorrow. For these reasons we engage through the university but as well as individuals in activities aimed at the public diffusion of science.
 
Description Marie Curie Fellowship
Amount € 189,174 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 01/2012 
End 12/2014
 
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 Med1 and H3K27ac ChIP-sequencing datasets collected from ESRRB-depleted and control embryonic stem cells in serum/LIF. 
Description Mapping and analysis of Med1 (Mediator component) and H3K27ac occupancy at ESC-specific super-enhancers in ESRRB-depleted and control embryonic stem cells in serum/LIF. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact * Publication (PMID: 32111830) and scientific communications (meetings, guest lectures) - Clusters of enhancers, called super-enhancers (SEs), control the expression of cell identity genes. Here, we identified two types of enhancer units within SEs in ESCs, characterised by distinctive CpG methylation dynamics, and find that ESRRB regulates the enhancer units decommissioned at the exit from naïve pluripotency. * Knowledge used in taught lectures in various courses. 
 
Description Internal Collaborators 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution Exchange of ideas, reagents and joint publications
Collaborator Contribution Exchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publications
Impact Joint publications
Start Year 2007
 
Description Internal Collaborators 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution Exchange of ideas, reagents and joint publications
Collaborator Contribution Exchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publications
Impact Joint publications
Start Year 2007
 
Description Internal Collaborators 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution Exchange of ideas, reagents and joint publications
Collaborator Contribution Exchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publications
Impact Joint publications
Start Year 2007
 
Description Internal Collaborators 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution Exchange of ideas, reagents and joint publications
Collaborator Contribution Exchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publicationsExchange of ideas, reagents and joint publications
Impact Joint publications
Start Year 2007
 
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 MED1 and H3K27ac enrichment profiles in ESRRB-depleted and control ESCS 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation and analysis of MED1 and H3K27ac ChIP-sequencing libraries collected from ESRRB-depleted and control ESCs in serum/LIF.
Collaborator Contribution Sequencing of the above ChIP-seq libraries at the sequencing facility of Radboud Institute for molecular life sciences; bioinformatics support.
Impact * Publication (PMID: 32111830) and scientific communications (meetings, guest lectures) - Clusters of enhancers, called super-enhancers (SEs), control the expression of cell identity genes. Here, we identified two types of enhancer units within SEs in ESCs, characterised by distinctive CpG methylation dynamics, and find that ESRRB regulates the enhancer units decommissioned at the exit from naïve pluripotency. * Generation of MED1 and H3K27ac ChIP-sequencing datasets collected from ESRRB-depleted and control ESCs grown in serum/LIF. * Knowledge used in taught lectures in various courses.
Start Year 2014
 
Description MED1 and H3K27ac enrichment profiles in ESRRB-depleted and control ESCS 
Organisation Radboud University Nijmegen
Department Radboud Institute for Molecular Life Sciences (RIMLS)
Country Netherlands 
Sector Academic/University 
PI Contribution Generation and analysis of MED1 and H3K27ac ChIP-sequencing libraries collected from ESRRB-depleted and control ESCs in serum/LIF.
Collaborator Contribution Sequencing of the above ChIP-seq libraries at the sequencing facility of Radboud Institute for molecular life sciences; bioinformatics support.
Impact * Publication (PMID: 32111830) and scientific communications (meetings, guest lectures) - Clusters of enhancers, called super-enhancers (SEs), control the expression of cell identity genes. Here, we identified two types of enhancer units within SEs in ESCs, characterised by distinctive CpG methylation dynamics, and find that ESRRB regulates the enhancer units decommissioned at the exit from naïve pluripotency. * Generation of MED1 and H3K27ac ChIP-sequencing datasets collected from ESRRB-depleted and control ESCs grown in serum/LIF. * Knowledge used in taught lectures in various courses.
Start Year 2014
 
Description MicroRNA regulation of embryonic differentiation 
Organisation Imperial College London
Department Institute of Reproductive and Developmental Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Addressed the role of microRNAs in differentiation and cell survival
Collaborator Contribution Analysis of mechanism of microRNA regulation of epigenetic processes. Analysis of mechanism of microRNA regulation of cellular stress.
Impact We have identified a role for microRNAs in the regulation of cell stress.
Start Year 2010
 
Description MicroRNA regulation of embryonic differentiation 
Organisation Royal Veterinary College (RVC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Addressed the role of microRNAs in differentiation and cell survival
Collaborator Contribution Analysis of mechanism of microRNA regulation of epigenetic processes. Analysis of mechanism of microRNA regulation of cellular stress.
Impact We have identified a role for microRNAs in the regulation of cell stress.
Start Year 2010
 
Description Role of necroptosis during embryonic development 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We are jointly analysing the role of genes regulating necroptosis and apoptosis during early development
Collaborator Contribution We are sharing mouse strains, reagents and expertise
Impact We have identified how the crosstalk of necroptosis and apoptosis is regulated during embryonic development
Start Year 2013
 
Description miRNA regulation of pluripotency 
Organisation Imperial College London
Department National Heart & Lung Institute (NHLI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of key co-repressor/co-activator expression and activity during the transition from naive to primed pluripotency
Collaborator Contribution provision of conditional Dicer-null embryonic stem cells
Impact Investigating functional link between miR-based regulation and the expression of specific nuclear receptor co-repressors in pluripotent stem cells.
Start Year 2013
 
Description miRNA regulation of pluripotency 
Organisation Weizmann Institute of Science
Department Department of Molecular Genetics
Country Israel 
Sector Academic/University 
PI Contribution Analysis of key co-repressor/co-activator expression and activity during the transition from naive to primed pluripotency
Collaborator Contribution Provision of miR-290-null embryonic stem cells
Impact Investigating functional link between miR-based regulation and the expression of specific nuclear receptor co-repressors in pluripotent stem cells.
Start Year 2013
 
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 2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019,2020
 
Description Participation in the Heart and Lung Repair Shop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Engaged with the general public during two days and this sparked numerous discussions afterwards

Numerous members of the public sought a further understanding of the research carried out by Imperial College
Year(s) Of Engagement Activity 2014
URL http://www1.imperial.ac.uk/nhli/public_engagement/the_curious_act/heart_and_lung_repair_shops/
 
Description Pint of Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 50 members of the public attended my talk and asked questions

High interest from the general public in stem cell related issues
Year(s) Of Engagement Activity 2013
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/eventssummary/event_26-4-2013-9-29-36