Mammalian sperm-borne DNA binding proteins as reprogramming factors

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

Abstract

If ordinary cells could be coaxed to become any designated cell type, they could be used to repair defective tissue in patients; this is a vision of regenerative medicine. Most work on this re-designation uses cells grown artificially and it is still poorly understood. We suggest that a complementary approach is to see how Mother Nature achieves re-designation after fertilisation. Fertilisation unites two unique cells - sperm and egg - and re-designates them to become one cell (a single-cell embryo) that can develop into an entire individual. This is the most dramatic example of re-designation, so we think that by understanding it, we will learn something about all the others.

At present, it is assumed that the egg is the only active player in re-designation following fertilisation - the sperm is overlooked - but we have evidence that the sperm also plays a role. The present proposal requests financial support to build on this observation and determine the mechanisms involved.

A clue to these mechanisms is that sperm contain unusual proteins able to bind to the genetic material, DNA. These proteins are not tightly associated with DNA in the sperm head, because they are released by gentle treatments that mimic conditions inside the egg. The first part of our proposal is to make a complete list of the proteins, termed sperm-borne DNA binding proteins , or sbDBPs.

We will then carefully apply a range of approaches to try to find out whether sbDBPs can bind to DNA inside the egg. We would also like to know what happens if we interfere with sbDBP activity immediately after sperm entry, perhaps by removing it. We are able to do this by injecting sperm into eggs - together with other selected molecules - and watching what happens once inside. These experiments use mice, because there is no other source of sperm and eggs, but if the work is successful it will improve alternative methods and bring forward the day when animal research is obsolete.

The laboratory is relocating to England after an absence representing over 10 years of international research experience which we will bring to the UK. We hope to use this experience to gain a better understanding of the active role sperm play in re-designating their own fate, so that it will be easier to re-designate the fates of other cells for medical applications, including tailor-made treatments of patient-specific diseases.

Technical Summary

The most dramatic shift in cellular potency - from assured death to totipotency in a matter of hours - occurs in the formation of an embryo following fertilisation. This transitory phase, from gamete to embryo, includes exhaustive, replication-independent removal and rebuilding of paternal (sperm) chromatin. The mechanisms responsible for this reprogramming promise to inform the links between structural changes to chromatin and their functional readout that predispose to all increases in cellular potency.

Our over-arching goal is a mechanistic understanding of genomic reprogramming following fertilisation. Current dogma assumes this reprogramming to be entirely mediated by maternal factors in the egg, but there is little supporting evidence for this view and we propose an overlooked, vital role for sperm-derived proteins. Preliminary data are presented to show that mammalian spermatozoa possess reprogramming factors that we propose to characterise primarily in the mouse.

These unpublished data reveal abundant sperm-borne DNA binding proteins (sbDBPs) that enter the oocyte during fertilisation. A unique combination of molecular, cellular and embryological approaches are proposed to generate a complete profile of sbDBPs and to characterise sbDBP function. Novel DNA-bead and sperm head oocyte microinjection assays, transgene-expressing oocytes and anti-sbDBP antibodies will be employed alongside stringent controls to reveal the DNA binding characteristics, expression patterns and sub-cellular localisation in the testis, sperm and early embryo of sbDBPs. Mutagenesis and interaction strategies designed to interfere with function will complement studies on interactions between sbDBPs with each other and other proteins. These studies are expected to delineate sbDBP roles and reveal mechanisms in chromatin remodeling and exogenous (eg sperm) genome reprogramming.

We have a head-start in an unexplored aspect of chromatin remodeling whose broad clinical potential makes it the subject of intense international competition. The work paves the way for novel diagnostic tools for idiopathic infertility and detailed exploration of the possibility that sbDBPs broaden cellular potency in multiple cell types via ectopic chromatin remodeling.

Publications

10 25 50
 
Description Nuffield Council Core Working Party Membership, Platform Report (London, UK)
Geographic Reach Europe 
Policy Influence Type Participation in a national consultation
URL http://nuffieldbioethics.org/news/2015/genome-editing-working-group-announced/
 
Description Presentation at COGEM (Amsterdam, NL)
Geographic Reach Europe 
Policy Influence Type Gave evidence to a government review
URL http://www.cogemsymposium.nl
 
Description Presentation at PET (ICH, London, UK)
Geographic Reach National 
Policy Influence Type Participation in a national consultation
URL http://www.progress.org.uk/conference2015
 
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 genome editing in ICSI 
Description We reported a highly efficient CRISPR-Cas9 method of mammalian genome editing via sperm injection (ICSI). 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact 1. ICSI is the method of choice in two thirds of human assisted reproduction internationally; 65% of all cycles globally use ICSI. Our findings in the mouse therefore suggests that the tools of genome editing can be delivered by the standard method of human assisted reproduction. 2. This delivery system will impact all users of ICSI internationally, by providing a protocol for tractable, efficient genome editing in mammals such as mice. 
URL http://www.egg2embryo.com/
 
Title intracellular interactions probe 
Description We have developed a powerful new method for interrogating molecular interactions within cells; it enables us to gauge under physiological conditions protein-protein and protein-DNA interactions. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The method is expected to have broad applicability and we have already used it to dissect multiple interactions that will be described in detail in upcoming publications. 
URL http://www.egg2embryo.com
 
Title transgenic 129 reporter 
Description We have generated transgenic reporters on a 129 background. Such mice are not commonly available but will be helpful to researchers with a variety of interests. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact 129 reporter mice are not commonly available but will be helpful to researchers with a variety of interests. 
URL http://www.egg2embryo.com
 
Title transgenic tools 
Description We have generated transgenic mouse models to study chromatin modification in vivo. We have also generated transgenic models linking fertilisation to tumourigenesis and are hopeful of receiving support to augment the lab (from 3 at present) to characterise them. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The models will be of considerable interest to researchers studying nuclear reprogramming. 
URL http://www.egg2embryo.com
 
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 Proteomic analysis of gametes 
Organisation RIKEN
Department RIKEN Center for Developmental Biology
Country Japan 
Sector Public 
PI Contribution Provision and cell biological analysis of all samples.
Collaborator Contribution Mass spectrometric analysis of samples
Impact Work in progress
 
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 Public 
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 Desborough 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 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 2015
URL http://www.egg2embryo.com/
 
Description Gordon Conference (NH, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact There was lively discussion among the ~150 attendees, who included experimental scientists, journalists and policy makers.

Lively discussions.
Year(s) Of Engagement Activity 2011
URL http://www.egg2embryo.com/
 
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 Nuffield Council 
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 was made, New tools for genome engineering, as part of a workshop during which there was lively debate about genome editing.
Year(s) Of Engagement Activity 2015
 
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 Talk (CIRB College de France, Paris, France) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact 30 attendees with lively discussion.

A new collaborative avenue is planned.
Year(s) Of Engagement Activity 2011
URL http://www.egg2embryo.com/
 
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 (MRC Harwell) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Following the talk, there was interest in the work that has lead to ongoing discussions and a follow-up visit in January, 2016.
Year(s) Of Engagement Activity 2015
URL http://www.egg2embryo.com/
 
Description Talk (Nagoya, Japan) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The talk, Mouseglish and Piganese: translating pig and mouse biomedical research into the language of human clinical medicine, enabled contact renewal in Japan as part of our ongoing translational collaboration in xenotransplantation.
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 Talk (Shady Grove Fertility Center, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The talk was a focal point for 30-40 clinicians and scientists to discuss emergent problems in assisted reproductive techniques.

The interaction strengthened a potential collaboration in clinical reproductive biology. (Shady Grove is the largest IVF centre in the US.)
Year(s) Of Engagement Activity 2011
URL http://www.egg2embryo.com/
 
Description Talk (University of Bristol) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact The audience contained a high proportion of students and there was discussion after the talk.

The meeting established potential collaborations.
Year(s) Of Engagement Activity 2012
URL http://www.egg2embryo.com/
 
Description Talk (University of Cardiff) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact There was considerable interest among the students as judged by the questions afterwards.

The visit consolidated research links.
Year(s) Of Engagement Activity 2012
URL http://www.egg2embryo.com/
 
Description Talk (University of Hawaii, HI, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The attendees were from different disciplines, triggering an interesting discussion.

This lead to some alternative views on old ideas.
Year(s) Of Engagement Activity 2012
URL http://www.egg2embryo.com/
 
Description Talk (University of Newcastle, NSW, Australia) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A mixture of post- and under-graduate pupils attended, with academic staff.

Appointment as Visiting Professor.
Year(s) Of Engagement Activity 2011
URL http://www.egg2embryo.com/
 
Description Talk at CNM, Barcelona, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact The presentation, Nanotechnology and sub-cellular biology: arranged marriage or love at first sight?, at the National Center for Microelectronics, Barcelona, Spain, was attended by around 40 and triggered thoughtful discussion.

A group from the institute visited my lab and we performed preliminary experiments that are expected to contribute to a grant proposal in 2014.
Year(s) Of Engagement Activity 2013
URL http://www.egg2embryo.com
 
Description Talk at IRDB, Imperial College, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact The talk, Dedifferentiation and reprogramming for proliferation: fertilisation, was at the IRDB, Imperial College, University of London and elicited a somewhat muted response.

None that I am aware of.
Year(s) Of Engagement Activity 2013
URL http://www.egg2embryo.com/