Delineating the roles of NSun proteins at the onset of mouse embryogenesis

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

Fertilisation transforms two cells that are destined to die - the gametes, sperm and egg - into one that engenders an entire individual. The transformation integrates complex intracellular events in ways that we hardly understand. Our ignorance of this is remarkable given the fundamental nature of the gamete-to-embryo transition and its implications for health and disease - including stem cells and cancer - and compels us to develop a comprehensive model explaining how the transition occurs.

In ongoing MRC-funded work, we made the unexpected discovery that mammalian (porcine and mouse) sperm introduce NSun1 into oocytes during fertilisation. This preliminary finding is remarkable. NSun1 is the prototype of a small protein family that includes RNA methyl-transferases and although the general importance of NSun family members is only recently becoming appreciated, it is clear that they are involved in multiple developmental pathways, regulating cell division and proliferation. These are key events in the gamete-to-embryo transition and strongly imply a link to NSuns that would strengthen the over-arching hypothesis in the applicant laboratory, that carcinogenesis and embryogenesis share mechanisms of initiation.

New unpublished evidence shows that most members of the NSun family are up-regulated immediately after fertilisation in the mouse. The present proposal seeks to investigate this in an integrated embryological and biochemical approach to produce a model of NSun function in reprogramming to totipotency during the mammalian gamete-to-embryo transition.

Our output will include careful characterisation of NSun transcripts and proteins during and immediately after mouse fertilisation and will show how interfering with NSun expression during this period affects embryo development. NSuns have a highly-conserved RNA methyl-transferase catalytic domain, enabling us to employ a technique recently developed by our collaborator to capture and characterise NSun RNA substrates. This will reveal the RNA substrates of different NSuns at single-base resolution and allow us to begin an analysis of early embryonic roles played by NSun targets. Our work identified NSun1 by its DNA binding ability, which we have confirmed in living oocytes, but DNA binding by NSuns has not so far been reported. We shall therefore characterise the binding of NSuns in oocytes and embryos to DNA and RNA using a novel in vivo microbead assay; the assay will also identify the domains in NSuns responsible for binding. Together, this work is expected to indicate how maternal mRNA are regulated immediately after fertilisation and whether regulatory mechanisms are shared by cellular potency changes in other contexts.

These experiments will reveal the phenotypic consequences of NSun disruption and target RNA networks at the onset of mammalian development. A better understanding of NSuns in the emergence of totipotency has the potential to impact the controlled induction of pluripotency with two clear additional translational applications that we will start to investigate. First, because of their roles promoting proliferation, NSuns may be oncogenic. We will work with clinicians to investigate whether there is a link that can be used in the diagnosis and treatment of cancer, particularly breast cancer. Secondly, altered NSun activity may impair fertility. Our collaboration with a major IVF clinic will determine whether NSuns represent (a) novel markers, and (b) treatment targets of impaired fertility.

Technical Summary

Our objective is to understand how NSuns regulate the mammalian gamete-to-embryo transition at fertilisation. This phase governs balanced chromosome segregation, transgenerational inheritance and the establishment of totipotency. NSun function will be dissected in living mouse eggs using a unique integrated molecular, cellular and embryological approach established by us since returning to the UK. We will reduce protein levels in oocytes using morpholino- or Cas9-based strategies, or elevate them with cRNA constructs encoding NSun protein fusions to fluorescent tags (eg mCherry, Venus). This approach relies on micromanipulation of mouse oocytes, but because conventional sperm injection typically kills mouse oocytes, we use piezo-actuated microinjection (piezo), which is not widely mastered outside of Asia but is routine in our laboratory. We have generated transgenic lines expressing fluorescent markers in gametes, including H3-mCherry and Venus-tubulin. When combined with piezo, these trangenic tools will allow us to monitor protein dynamics after NSun levels have been altered, thereby mapping each NSun to molecular behaviours and developmental phenotypes, which we can also determine. We have developed a microbead technology to dissect NSun interactions in vivo at the onset of development, which will be extended in a collaboration with Shobbir Hussain; Dr. Hussain has devised a powerful method to identify at single-base resolution NSun RNA substrates. We will start to evaluate the biomedical applications implied by the biology of NSuns. We will determine whether NSuns are dysregulated in infertility and cancer patients following knowledge transfer to our collaborators. These approaches that will reveal the biomedical function and relevance of NSuns.

Planned Impact

1. The biology of the system
The study of NSuns in mammalian fertilisation is a fundamental area that will initially impact academic and commercial research communities - including post- and under-graduate students - working on epigenetics, the cell cycle, cytokinesis, fertilisation, early development and stem cells. The biology of NSuns make them prime candidate mediators of recently-appreciated epigenetic inheritance through gametes and this work will indicate whether this possibility should be pursued. As the project develops, applications of NSun biology in general and in embryos in particular will become increasingly appreciated, with the result that the audience will become wider locally, nationally and internationally. Outreach to potential stake-holders including academics, policy-makers and the commercial sector will be maximised.

2. Developing imported expertise
Since Dr Perry has worked for 14 years in the US and Japan, there is an opportunity for knowledge transfer to the UK in technology and IP. Technological advances will benefit post-graduate students and post-docs and include piezo-actuated microinjection (piezo), which is not widely established outside Japan and China, who are racing ahead with it. Establishing piezo in the UK will increase our competitiveness and facilitate mouse strain archiving in biomedical research establishments such as MRC Harwell. We have also developed a latex microbead method to interrogate intermolecular interactions as they occur within a cell. We will apply it to NSun but the technology is relevant throughout cell biology. We are extending this work in a trans-disciplinary nanotechnology collaboration with physicists at the Microelectronics Institute of Barcelona IBM-CNM. We anticipate that intracellular machines will be used to measure hitherto inaccessible parameters such as intracellular forces produced during sperm decondensation, establishing a new interface between physics and biology.

3. Clinical impacts: infertility, cancer and regenerative medicine
Infertility. Within the lifetime of the grant, we expect to have evidence that NSuns are determinants of impaired human fertility. This will contribute to assisted reproductive technology (ART) as a diagnostic marker and in treatment, benefiting healthcare workers and many of the 3.5 million people in the UK who have difficulty conceiving. It will impact commercial entities conducting ART in the UK, such as the CARE Fertility, and UK charities such as WellBeing of Women, given that in mice at least, NSun dysregulation results in infertility.

Cancer. NSuns possess oncogenic activity and one might therefore expect them to be up-regulated in gynaecological and certain other cancers. Our work on the parallel proliferative process - embryogenesis - will shed light on which NSuns and how. NSuns could be candidate cancer markers, opening doors to earlier diagnosis - a major factor where death rates have remained stubbornly high as is the case for ovarian cancer - and treatment, to the benefit of clinical oncologists and patients. It is not possible to evaluate these hypotheses other than experimentally.

Regenerative medicine. By making the model of safe cellular potency transitions more complete, NSuns will increase the efficacy of cellular medicine and help move it from bench to bedside. This coheres with Aim One of the MRC strategic plan by enabling safe regenerative therapies and will translate into a competitive advantage for commercial entities in cellular medicine such as the UK Stem Cell Foundation. Work on NSuns will impact pig genome manipulation by knowledge transfer from mice to pigs in a collaboration with Prof. Akira Onishi (National Institute of Agrobiological Science, Japan). Prof. Onishi uses piezo to engineer pigs for xenotransplantation, with potential benefits to organ transplant patients.
 
Description Among the first to raise concerns (quoted in Nature Biotechnology in September, 2017) and elsewhere questioning recent paper in Nature on human genome editing using a method discovered in my laboratory.
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in systematic reviews
 
Description Nuffield Council Core Working Party Membership, Genome Editing and Human Reproduction Report (London, UK)
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
URL http://nuffieldbioethics.org/project/genome-editing/genome-editing-human-reproduction
 
Description Nuffield Council Core Working Party Membership, Platform Report (London, UK)
Geographic Reach Europe 
Policy Influence Type Contribution to a national consultation/review
URL http://nuffieldbioethics.org/news/2015/genome-editing-working-group-announced/
 
Description Presentation at COGEM (Amsterdam, NL)
Geographic Reach Europe 
Policy Influence Type Contribution to a national consultation/review
URL http://www.cogemsymposium.nl
 
Description Presentation at PET (ICH, London, UK)
Geographic Reach National 
Policy Influence Type Contribution to a national consultation/review
URL http://www.progress.org.uk/conference2015
 
Description Presentation to House of Lords
Geographic Reach National 
Policy Influence Type Contribution to a national consultation/review
 
Description Translational Biomedical Research Centre, Board Member (University of Bristol, UK)
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.bristol.ac.uk/health-sciences/research/tbrc/
 
Title Human one-cell embryo transcriptome 
Description Single-cell RNA-seq data for human bipronuclear one-cell embryos. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact The associated paper has a high Altmetric score. 
URL https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(21)00484-7?_returnURL=https%3A%2F%2Flinkingh...
 
Title Mouse one-cell embryo transcriptome time course 
Description Single-cell RNA-seq data for one-cell mouse embryos at different times after sperm injection. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Too soon to say. 
URL https://www.cell.com/cell-reports/fulltext/S2211-1247(23)00034-7?_returnURL=https%3A%2F%2Flinkinghub...
 
Description Derivation of atypical embryonic stem cells 
Organisation Wellcome Trust
Department Wellcome - MRC Cambridge Stem Cell Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation of starting material.
Collaborator Contribution Derivation from starting material.
Impact Work in progress.
Start Year 2012
 
Description Development of intracellular nano devices 
Organisation Spanish National Research Council (CSIC)
Department Barcelona Institute of Microelectronics
Country Spain 
Sector Public 
PI Contribution I initiated this collaboration and co-wrote a grant proposal (submitted in Sept 2012)
Collaborator Contribution They visited us and co-wrote the grant proposal
Impact An FP7 grant proposal was submitted in Sept 2012
Start Year 2011
 
Description Transcriptomic characterisation of mouse early embryos 
Organisation University of Regensburg
Department Department of Pathology
Country Germany 
Sector Academic/University 
PI Contribution Subcellular mouse embryo microsurgery and transcriptome analysis
Collaborator Contribution Microarray analysis
Impact VerMilyea, M.D., Maneck, M., Yoshida, N., Blochberger, I., Suzuki, E., Suzuki, T., Spang, R., Klein, C.A. and Perry, A.C.F. (2011). Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres. EMBO J. 30, 1841-1851.
 
Description Xenotransplantation 
Organisation National Institute of Agrobiological Science, Japan
Country Japan 
Sector Academic/University 
PI Contribution Molecular analysis and intellectual imput; I visited the NIAI in March, 2012 and met Akira Onishi in September, 2013 in Tokyo.
Collaborator Contribution Pig cloning and targeting; the work is based at the NIAI. Our contribution is (a) to test in the mouse new technologies that can, when successful, be applied to the pig, and (b) to bring our collaborators to the UK with a view to setting up pig-to-human xenotransplantation here.
Impact Suzuki, S., Iwamoto, M., Saito, Y., Fuchimoto, D., Sembon, S., Suzuki, M., Mikawa, S., Hashimoto, M., Aoki, Y., Najima, Y., Takagi, S., Suzuki, N., Suzuki, E., Kubo, M., Mimuro, J., Kashiwakura, Y., Madoiwa, S., Sakata, Y., Perry, A.C.F., Ishikawa, F. and Onishi, A. (2012). Il2rg gene-targeted severe combined immunodeficiency (SCID) pigs. Cell Stem Cell 10, 753-758.
 
Description Asami, M., Lam, B.Y.H., VerMilyea, M.D., Yeo, G.S.H., Perry, A.C.F., poster, Cell Cycle Conference, London, UK, May 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of interesting new data to emerge from work support by this and other awards.
Year(s) Of Engagement Activity 2023
 
Description BBC Radio 1 
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Results and Impact An interview about the promise and pitfalls of human genome editing.
BBC Radio 1 Stories, "DNA+: Beauty", BBC iPlayer
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URL https://www.bbc.co.uk/iplayer/episode/p06gx2kf/radio-1-stories-dna-beauty#
 
Description BBSRC Stem cell fate choice: mechanism and modelling workshop 
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Results and Impact There was considerable interest in the work, which had been partly supported by the Award.
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Results and Impact Talk entitled "Genome editing and its implications", Bath Royal Literary and Scientific Institution, Bath, February 23, 2017
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URL http://www.egg2embryo.com/
 
Description Bath University Biosciences Society 
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Results and Impact "What is human genome editing and will it happen?", Bath University Biosciences Society, Bath, February 22, 2017
Year(s) Of Engagement Activity 2017
URL http://www.egg2embryo.com/
 
Description COGI, Amsterdam 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Speaker on human genome editing at the 24th World Congress on Controversies in Obstetrics, Gynecology & Infertility (COGI) Amsterdam, Netherlands - November 10-13, 2016
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URL http://www.egg2embryo.com/
 
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URL http://www.egg2embryo.com/
 
Description European Society of Human Reproduction and Embryology, Copenhagen, Denmark: "The good egg: on the onset of transcription in mouse and human embryos" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A summary of recent data enabled by this and other awards.
Year(s) Of Engagement Activity 2023
URL https://www.eshre.eu/Annual-Meeting/ESHRE-2023
 
Description Festival of Genomics 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chair of the Festival of Genomics Genomics & Editing all-day session on Genomes and Editing and concluding panel discussion, Excel, London, UK, Feb 1, 2017.
Year(s) Of Engagement Activity 2017
URL http://www.egg2embryo.com/
 
Description Godalming College (STEM Ambassador) 
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Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to sixth-formers considering studying a biomedical subject at university.
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Godalming College (STEM Ambassador) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to sixth-formers considering studying a biomedical subject at university.
Year(s) Of Engagement Activity 2016,2017
URL http://www.egg2embryo.com/
 
Description House of Lords 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact A presentation at the House of Lords on the future trajectory of genome editing. We are using genome editing technology to interfere experimentally with NSuns.
Year(s) Of Engagement Activity 2016
URL http://www.egg2embryo.com/
 
Description Interviews 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
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Primary Audience Media (as a channel to the public)
Results and Impact Interviews were as follows: 01.09.17: New Scientist Short Sharp Science, 'We still don't really know what CRISPR does to human embryos'; 28.06.17: Voice of Islam; 08.06.17: BBC Tomorrow's World (radio).
Year(s) Of Engagement Activity 2017
 
Description MHRA invited participant 
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Results and Impact Invited participant at the Medicines and Hospital products Regulatory Authority (MHRA) workshop on the regulation of genomic testing
Year(s) Of Engagement Activity 2016
URL http://www.egg2embryo.com/
 
Description MRC Animals Forum 
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Primary Audience Professional Practitioners
Results and Impact Selected participant at the MRC Animals Forum
Year(s) Of Engagement Activity 2016
 
Description Poster at the 2023 ISSCR meeting in Boston 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The poster generated considerable interest and was visited by over 100 people.
Year(s) Of Engagement Activity 2023
URL https://www.isscr.org/upcoming-programs/isscr-2023
 
Description Presentation at COGEM (Amsterdam, NL) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presentation at COGEM workshop on human genome editing.
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Presentation at IdeaCity, Toronto, 14.06.17 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A public lecture in Toronto about the prospects for heritable human genome editing.
Year(s) Of Engagement Activity 2017
URL https://www.youtube.com/watch?v=N0D9SlVptDE
 
Description Presentation at PET (ICH, London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Presentation to Progress Education Trust during workshop on human genome editing.
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Presentation at TEDx, Thessaloniki, 24.04.17 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A presentation about the prospects of human heritable genome editing.
Year(s) Of Engagement Activity 2017
URL https://www.youtube.com/watch?v=0HsFA5350Rw
 
Description Presentation at the Advanced Medicine Symposium, Royal College of Physicians, 07.02.18 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A presentation about the state and promise of genome editing, with particular emphasis on human heritable genome editing.
Year(s) Of Engagement Activity 2018
URL https://www.rcplondon.ac.uk/events/advanced-medicine-2
 
Description Presentation at the Festival of Genomics, ExCeL, 31.01.18 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A public lecture about the state and promise of genome editing, with particular emphasis on human heritable genome editing.
Year(s) Of Engagement Activity 2018
URL http://www.festivalofgenomicslondon.com/speakers-2018
 
Description Reuters interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The interview by Reuters included extensive footage of the laboratory. The segment was entered by U. Bath as a contribution to the Concordat on Openness in animal research.
Year(s) Of Engagement Activity 2017
URL http://reut.rs/2xUJc43
 
Description Russian science TV programme 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview about genome editing for Russian TV Channel The Science, which is the only science-dedicated TV channel in Russia.
Year(s) Of Engagement Activity 2018
URL https://www.naukatv.ru/video
 
Description Science Media Centre 
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 Media (as a channel to the public)
Results and Impact "The 'fertilisation' of embryos", talk and media panel session, Science Media Centre, London, Sept 12, 2016
Year(s) Of Engagement Activity 2016
URL http://www.egg2embryo.com/
 
Description Sciencewise participant 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Invited participant at the Sciencewise-Nuffield Council Workshop on Genome Editing
Year(s) Of Engagement Activity 2016
 
Description Talk (CafĂ© Scientifique, Bath) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk on human genome editing, to a packed house comprising the general public, followed by lengthy Q&A.
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Talk (Science Media Council) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Talk was part of a Press conference arranged by the Science Media Centre
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Visit to University of Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact A hybrid presentation at the Department of Women's and Reproductive Health at the John Radcliffe Hospital, University of Oxford.
Year(s) Of Engagement Activity 2023
 
Description de Snoo van 't Hoogerhuijs Foundation Conference 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Speaker, 2017 de Snoo van 't Hoogerhuijs Foundation Conference, Amsterdam, NL
Year(s) Of Engagement Activity 2017
URL http://www.egg2embryo.com/