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

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New unpublished evidence shows that most members of the NSun family are up-regulated immediately after fertilisation in the mouse.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.

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.

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.