Aire creates immune tolerance via an unconventional transcriptional mechanism

by Dina Danso-Abeam

The white blood cells of our immune system defend us from infection, a function which is coordinated by T cells. Immature T cells are formed with an ability to attack random targets (an adaptation to the rapid evolution of microbes), which means that by chance some targets are "self-targets" (normal proteins part of a healthy body). As a consequence, these "self-reactive" T cells can atatck the body, so it is critical to prevent them from causing autoimmune disease. To prevent autoimmunity, the immature T cells are screened in an organ called the thymus (located just above the heart), in order to ensure that all self-reactive T cells are eliminated.

The Aire gene plays an important part in eliminating self-raective T cells, by expressing genes that are normally restricted to specific organs (eg, insulin in the pancreas) in the thymus, providing full coverage for screening against self-reactivity. In patients that have mutations in the gene Aire, the thymus cannot provide full coverage of self-targets and fatal autoimmune disease develops. One of the mysteries of how Aire functions is its ability to express thousands of genes like insulin in the thymus. 

It has previously been shown that Aire is a transcription factor (meaning, it can bind DNA to activate genes and cause the expression of proteins) which can activate the expression of other transcription factors. We hypothesized that these secondary transcription factors might mediate the expression of the thousands of Aire-dependent genes like insulin; in effect, we predicted that Aire creates a cascade of transcription factors that results in the expression of thousands of genes.

In order to test this hypothesis, we investigated whether such a cascade regulates the transcription and tolerance of pancreas-specific antigens (e.g. insulin and glucagon) in the thymus. In the pancreas, Pdx1 is the key transcription factor which drives the expression of insulin. Interestingly both Pdx1 and insulin have been shown to be Aire-dependent in the thymus, so it was possible that Pdx1 was acting as a secondary transcription factor in the cascade by which Aire expresses insulin. Therefore we generated mice that specifically lack Pdx1 in the thymus.

By generating these mice, we found that expression of pancreatic-specific antigens such as insulin, needed Aire expression in the thymus, but did not need the transcription factor Pdx1. These results suggest that the broad tolerance that Aire creates in the thymus is not mediated by a conventional cascade of transcription factors, but rather relies on an unconventional transcriptional mechanism.  

This work will be published in a forthcoming issue of The European Journal of Immunology as:

Aire mediates thymic expression and tolerance of pancreatic antigens via an unconventional transcriptional mechanism

by Dina Danso-Abeam, Kim A Staats, Dean Franckaert, Ludo Van Den Bosch, Adrian Liston, Daniel H D Gray* and James Dooley*

If birds are baby dinosaurs and humans are baby apes...

A recent publication in Nature suggests that in many ways, birds are baby dinosaurs. The finding is less unusual than it might seem, afterall it is well established that humans have many traits of baby apes and dogs are in some ways baby wolves. The process is known as paedomorphosis or neoteny - the retention of juvenile traits in the adult form. This can take the form of enlarged eyes (birds), larger brains (humans) or retention of juvenile behaviour (dogs).

The reason why paedomorphosis works is that the basic body plan has much deeper evolutionary roots than the species-specific add-ons. Think of it this way, all mammals pretty much have the genetic program to make a nose, but only the elephant has evolved an additional genetic plan to turn that nose into a trunk. Deep in the genetic code of the elephant there is still the "standard nose" code (and indeed, the foetus has a relatively normal nose), it just has added lines of code that upgrade the standard nose into a trunk. This means that in theory, the elephant could evolve away from the trunk by just ditching the upgrade code, letting it default into the standard nose code. This is true for most of development - new code is never optimally created for the organ, rather it is always adding a bit of extra code to change the outcome. For a software engineer it would be the hight of laziness, creating bloated useless code, with every problem solved by kludge. 

Despite being inefficient and inelegant, the system of "generic code" plus "species specific" is very useful for evolution. This is because species evolve to be adapted to a specific environment. The flamingo beak is fantastic for a filter-feeder, but it has lost the generic functions that a sparrow could use its beak for.

Imagine an island with brine lakes that is populated only by flamingoes. If those brine lakes dried up, the flamingoes would go extinct. But what if new niches opened up? The ordinary "forward" process of evolving a generalist beak is quite slow, because you need to generate new code, but the "backwards" process of paedomorphosis could be quite fast, because it is just the process of deleting the species-specific code, defaulting back to the generic beak (as in anything else, destruction is faster and easier than generation). It is not difficult to imagine a relatively small set of genetic deletions that would mean the adult flamingo retained the juvenile generic beak, and then these "de-evolved" generalist birds could take advantage of the new habitat, and indeed start to evolve specific changes to specialise towards that new habitat.

As a general rule, following a large change in the environment, the generalised (juvenile) body plan is probably going to be more successful than the specialised (adult) body plan. Paedomorphosis in effect provides a default option to revert to in case of catastrophic change, allowing a species to shed its specialised features and start again. One possibility that interests me is that an open niche may drive paedomorphosis by selecting for rapid population growth. Consider the drying up of Africa that occured 5 million years ago. All of the apes that were specialised to live in rainforest would have seen dramatic contraction of their habitat, leaving just a few thousand gorillas left today. But the drying also created a new niche, the savanna, which could be exploited by any ape that was able to adapt. Paedomorphosis probably played a role in human evolution, by shedding the arboreal features required to swing in trees, allowing the pre-humans to venture onto the savana. Now consider the first pre-humans that were suitable for the savana - they has a continent to spread across, with the only limitation being the reproduction rate. We already know that a truly open niche creates an evolutionary pressure to fill it - such as the natural selection of cane toads in Australia with longer legs simply because they can move faster into virgin territory. What if this put selection on humans to reproduce at a younger age? Any variants that became fertile younger (and thus, while still carrying juvenile features) would outcompete the others, creating a population shift. In effect, there would be selection for paedomorphosis simply to increase the reproduction rate, with the retention of other juvenile traits (such as a larger brain) being a side-effect. 

If this model it correct, it would mean that open niches would drive paedomorphosis via two mechanisms - by selecting for the retention of juvenile traits to give a more generalist body plan, and by selecting for sexual maturity at a younger age to give more rapid reproduction. This dual selection force would drive much more rapid evolution, and may be responsible for some of the most remarkable evolutionary shifts, including the evolution of humans. 

AIG laboratory members successful at the FWO

This week we received exciting news that the Autoimmune Genetics laboratory had three successful candidates at the FWO, the premier fellowship program in Belgium. 

Dr Stephanie Humblet-Baron won an FWO Post-doctoral Fellowship award to research a new genetic disease caused by a loss of dendritic cells:

In the immune system, dendritic cells (DCs) are a subset of white blood cells that are specialized to activate lymphocytes when a pathogen is present In the absence of DCs, activation of lymphocytes and clearance of infections is impaired.  A new genetic disease has recently been identified where patients have no DCs, and surprisingly not only do they have poor clearance of infections, but they also have a large expansion of myeloid cells in their blood. For this project we have created a mouse model of this disease, which we will use to try to understand the biology of the myeloid expansion and to test potential therapeutics. 

Dr Susan Schlenner won a Pegasus Post-doctoral Fellowship award to move to the laboratory from Harvard. Here she will use novel genetic approaches to understand the biology of regulatory T cells.

Regulatory T cells are an important subset of white blood cells that have the ability to prevent the immune system from attacking components of the body (“autoimmunity”) and from attacking harmless environmental components (“allergy”). In order to exert this function the regulatory T cells need to be educated as to which components are safe and should be protected from immune attack. The location where this occurs is highly controversial as previously there have not been the correct tools to do functional tests. This project aims to generate a sophisticated set of genetically-altered mouse strains to allow measurement of where regulatory T cells are educated, and then to use these mice in models of autoimmunity and allergy. Having more knowledge about the education process of regulatory T cells may allow the future development of therapeutic interventions in those patients where regulatory T cells fail to prevent autoimmunity or allergy.

Dr Lien Van Eyck won an FWO PhD Fellowship, to move from the clinic to the laboratory to study auto-inflammatory diseases.

Blau Syndrome (BS) and Early Onset Sarcoidosis (EOS) are rare monogenic auto-inflammatory diseases characterized by a clinical triad of granulomatous arthritis, uveitis and rash. Extended manifestations with potentially high morbidity have been reported recently. The pathologic hallmark of BS/EOS is the presence of multinucleated giant cell and epithelioid cell granulomas in affected tissues. Both diseases are associated with gain-of-function mutations in the NOD2 gene. NOD2 is a specialised intracellular protein that plays a critical role in the regulation of the host innate immune response through recognising conserved microbial molecular signatures, thus leading to the induction of pro-inflammatory and anti-microbial responses as well as apoptosis. While the genetic basis of BS/EOS has been characterized, the molecular mechanisms by which NOD2 mutations drive granuloma formation and the development of sarcoidosis remain unclear. A better understanding of these mechanisms is of direct relevance for the development of targeted immunotherapies. The present project aims to determine the mechanisms by which NOD2 gain-of-function mutations lead to immunopathology in BS/EOS by developing a murine model with a gain-of-function mutation in NOD2. This model will allow for a full characterization of the immunopathology of NOD2 associated inflammation, and for the unravelling of molecular and cellular mechanisms involved in disease pathogenesis.

Thymic involution: how a chance observation saved years of work

An interview with the VIB following the recent publication of our article:

The thymus is an organ crucial for the functioning of our immune system. During aging or infection the thymus can shrink severely, a process called involution. Although the mediators that trigger involution are known, the mechanisms regulating the sensitivity to their presence remained a mystery. Now, Smaragda Papadopoulou from the Bart De Strooper Lab and James Dooley from the Adrian Liston Lab describe in Nature Immunology a microRNA network that plays a key role. A chance observation kick-started the collaboration.

What did you discover about the regulation of thymic involution?

Adrian Liston: The main finding was the tight regulation by miR-29a over sensitivity to thymic involution. miR-29a serves to suppress the involution response, in effect "saving" involution for those situations where we really need it, such as during a major infection. Knowing what drives the reaction of the thymus is important, since it is the only place where T cells can develop. No thymus, no T cells, no infection prevention.

Is there an application side to those results?

For most of us, being born with a healthy thymus, we will generate enough T cells to last a life-time. Thymus involution during an infection is generally not a problem, nor the slow progressive involution that occurs from birth. The major problem is among the very elderly and with radiation/chemotherapy patients. If we could reverse thymic involution in those populations, we could rejuvenate their T cell population, providing them with a younger, more robust, immune system.

How did you go from studying regulatory T-cells to the regulation of thymic involution?

We have been interested in both the thymic epithelium and microRNA for years, so it was natural for us to look at what microRNA does in the thymic epithelium. As for thymic involution in particular, that was observation-driven. When we knocked out microRNA in the thymic epithelium using a Cre-Lox system, the main phenotype was chronic involution. But working out which microRNA is important was an enormous task. The big breakthrough for us was serendipitous. The Bart De Strooper Lab had generated a novel knockout mouse with a defect in one particular microRNA, miR-29a, to look at the neurophenotype. A conversation, a quick look and just by chance this microRNA turned out to be the one we needed for our lead. This enabled us to start a cross-disciplinary collaboration years before anyone else even knew there was a story there.

Did you use or design any new technologies for this research?

Far from it. The most important read-out in this work was the humble cell count. There are still enormous opportunities for high-level research using basic technologies. In this particular case the edge we had was a new mouse strain (the miR-29a knockout) and a new permutation of old mouse strains (Foxn1-Cre and Dicer-flox), but the rest was simply applying old techniques to a new problem. Immunology has so many fascinating questions that remain under-investigated that we spend our time working out which ones to tackle next, rather than designing new technology.

What’s the next step in your microRNA research?

MicroRNA are such interesting molecules. So tiny, they hold only a fraction of the information of a normal gene, yet they are incredibly versatile, affecting multiple completely unrelated targets in every cell type. We pretty much cracked the role of miR-29a in the thymic epithelium, but we are sure it is doing a lot more in other cell types of the immune system.

For the full research results see:

Aikaterini S. Papadopoulou#, James Dooley#*, Michelle A. Linterman, Wim Pierson, Olga Ucar, Bruno Kyewski, Saulius Zuklys, Georg A. Hollander, Patrick Matthys, Daniel H. Gray, Bart De Strooper and Adrian Liston. #Equal first authors. *Co-corresponding authors. 'The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.' 2012. Nature Immunology. 13 p181.  Pubmed | Direct access

Three years as a junior faculty member

In 2010, after one year as a junior faculty member, I wrote up that year in numbers.

Now, three years in and racing towards my five year evaluation mark, I can calculate the first three years in numbers:

227: the number of grants I have reviewed for various foundations
63: the number of articles I have reviewed for different journals

45: the number of grants submitted (32 project grants and 13 fellowship applications)
        20: grants accepted (17 project grants and 3 fellowships)
        16: grants rejected (13 project grants and 4 fellowships)
        9: grants pending (3 project grants and 6 fellowships)
5,513,005: euros given to the lab in project grants
2,842,774: euros spent in research

35: invited talks
13: conferences
6: lectures

45: article submissions and resubmissions
        26: articles published or in press (9 primary papers, 11 reviews, 6 book chapters)
3: number of edited volumes

16: number of lab members
         5: PhD projects ongoing
       2: Masters projects ongoing
       10: number of full-time researchers in the lab 
(17: number of ex-lab members)

0: still the number of days I've spent doing experiments

So an average month for me is reviewing 8 grants or papers, submitting one grant and getting one paper accepted, giving a talk somewhere, having one new person start in the lab or an old person leave, and spending 80,000 euros on research - and I still work less than my PhD students and post-docs!

Hints for potential students: Writing an introduction letter

I get about 200 emails a year from students requesting a PhD position in my laboratory. I pride myself in answering each one, but actually most deserve to be immediately trashed. This is a typical letter I will receive:

Dear Sir or Madam,

I am interest in a PhD position at your institute, the VIB. Please find attached my motivation letter, CV and a scan of every certificate I had ever received. 

Regards.

Before we get to writing a good letter, let's start out by pointing out the worst mistakes of this letter. 

1) "Sir or Madam" is terrible. It shows you didn't research me in the slightest before sending your email. "Professor Liston" or "Dr Liston" is fine, actually "Adrian" is fine for me but I would advise against it in first emails, "Dr Adrian" is weird and makes me feel like a talk-show host.

2) English. Okay, it is not your first language, and you don't need perfect English to be a scientist. But it does demonstrate carelessness that you didn't bother to get your introductory letter right. If you are this sloppy on first impression, how careless would you be in the lab? Get a native English reader to proof read your letter before you send it or use an AI program to clean it up.

3) As if I didn't have enough proof already that this was a bulk email sent out to thousands of scientists, the way "the VIB" is in another font clearly shows cut and paste at work. Anyway, it is a redundant thing to write, I know where I am based, and if you are looking at institutes rather than labs you already have your priorities wrong.

4) The attachments. *sigh*. Don't attach your letter of introduction, put that in your email. Attaching a CV is fine, but that is it, don't annoy me with a lot of extra attachments that mean nothing. One single pdf, nothing more.

So how do you write a good introduction letter? There are a few simple rules:

1) Research the laboratory and the PI beforehand. You need to know who I am and what I do. Yes, this takes a lot more time than having a standard letter that you send to every email address you can find, but it is much more effective.

2) Specify why you are interested in my lab. Not why you are interested in doing a PhD, but specifically why you want to do a PhD in my lab. It is best if this connects your previous experience with the research of the laboratory. For example, when I wrote to my future PhD supervisor (Chris Goodnow) I said I was very interested in working on the issue of genetic variation in T cell tolerance due to my Honours research indicating that the SJL mouse had a defect in tolerance. As he had just published a paper in JEM on defective negative selection in the NOD mouse, could I discuss a PhD project with him? It is only two sentences but it indicates that I know his research, I have relevant experience and I have a specific scientific interest in his laboratory.

3) Don't be aggressive or sycophantic. It is a polite letter of interest, not a last ditch effort to get overseas. Even if it is a last ditch effort to leave your country, don't let that show.

4) Be brief. One or two short paragraphs should be plenty to establish first contact. A good first letter leads to follow-up letters, so there is no need to put everything in there.

5) Have a single attachment, just a pdf of your CV. Like the introduction letter, this should be brief. Keep personal details to a minimum, your age and nationality is useful (for assessing scholarship eligibility) but I really don't need to know your marital status, the names of your children or your blood group. Keep your qualifications and awards to the important stuff - no driver's licence or half-day radiation safety course, just your degrees, marks and the important awards that show real achievement. Don't add copies of these awards. Mostly what I am looking for are your publications, a first-author paper in an international journal tends to be my minimum cut-off for seriously considering a cold call. Language skills are useful, and if you want to have a few sentences on extracurricular activities that is fine (although I only tend to be impressed at volunteer work). 2-3 pages really should be plenty, with no English errors and nice clean formatting. 

Last week I got back a letter from a PhD applicant I had rejected and sent this advice to. He told me that he had sent out hundreds of letters with no reply, but after taking my advice he made carefully written three letters to the labs he was most interested in and within a month he had got back two offers to start a PhD in Germany.  

Generation of a family-specific virus through repeated human passage

Generation of a family-specific virus through repeated human passage

Hayden A M Liston¹, Lydia E Makaroff¹ and Adrian Liston ^1,2*
1 Sleepytown University, Brussels 1060, Belgium
² VIB, Leuven 3000, Belgium
*send correspondance to adrian.liston@gmail.com

Nature Junior 8(2) 103-7 

Background. Effective control over viral infection relies on the host carrying appropriate HLA alleles for viral antigen presentation. The explosive expansion of viruses like small-pox into previously isolated human populations demonstrates the potential for certain viral strains to have a disproportionate effect on particular racial groups. As yet, however, a virus with pathogenic potential restricted to the family level has not been identified. Objective. To generate a family-specific virus in an experimental setting, in order to test the feasibility of this occurrence in nature. Methods. A common cold virus was repeatedly passaged between two related individuals for six months. Mechanisms of transmission included frequent kisses, the placement of hands and feet into the mouth and in one instance direct vomiting into the mouth. Results. A single viral strain was propagated with the capacity to chronically infect both members of this family, while having seemingly non-pathological consequences upon exposure to unrelated individuals. The pathogenic loci are predicted to be a dominant HLA carried by both family members, as the experimental inoculation of a third individual, related to one family member but not the other, did not result in pathology. Conclusions. Generation of a family-specific virus is feasible through repeated experimental transfer between family members. A natural situation analogous to the experimental set-up used here would be the transmission that can occur between parents and young children with low levels of personal hygiene. The dominant activity of the HLA cluster in this infection suggests the generation of a regulatory T cell population which inhibits effective immunity against the family-specific virus.

Key Words: virus, horizontal transfer, HLA, human genetics, regulatory T cell.

Advice on applying for an ERC Start Grant (part 3)

I was asked to give some advice on ERC Start Grant applicants, as a current grant holder. As this has come up several times I thought I would write a series of blog posts covering my hints and tips. Partly, this advice is specific to the ERC grant system, although most points are valid across any grant. In a previous posts I gave advice about the written application - Part B1 and Part B2. In this final post I will deal the interview portion of the grant.

The Interview

The interview is not simply an oral version of your written application. There is a panel of around 15 panel members, each of these panel members will be experts on maybe 5 applications and more-or-less bystanders on the other 15 applications.

• Experts. Your chance to impress the experts was your written application, and if you made it to the interview stage than you already succeeded here. The experts are familiar with your work from reading your 30 page dossier; they do not expect to learn anything new from the talk. Instead they will be waiting for the question time to hit you with any issues they have.
• Additional panel members. These are people who are within your general area of research, but outside your specific discipline. They only glossed over your proposal, if they looked at it at all. Design your talk as if they haven’t read your application and focus on importance and strategy. Don’t get bogged down in experimental details and don’t think they really care too much about your discipline – explain to them the advantage in the knowledge that you propose to generate. Focus on the importance and novelty, and why your approach will succeed while others have failed.

Question Time

The questions you get asked will vary based on your project and your application. Have you been wildly ambitious? Expect to get a lot of questions on feasibility. Have you stuck very close by your existing research? Expect to get questions about competitiveness. The experts should ask most of the questions, any technological or methodological concerns they have will be raised here. Generally these will be along the lines of “X is risky, what will you do if it doesn’t work?” or “this is a highly competitive field, how will you compete?” If there is enough time you may get some standard questions from chair or other panel members, such as questions about your long-term career plan and so forth. A few general points apply across the different questions you will get:

• Listen politely to the full question, never assume where it is going or interrupt to answer
• Your tone and attitude matter as much as your words – a grant application is a sales pitch!
• Being right is less important than having a clear articulate message and sounding competent. Even if the expert is wrong there is little benefit in arguing – it certainly comes off badly to the rest of the panel. That said, you can still disagree – “based on my experience the approach is feasible, but in case we do hit a roadblock there is an alternative strategy that we can take...” is completely reasonable response.
• Don’t waffle. It wastes time and it makes it look like you have not thought about the question before. A clear and concise answer reassures the entire panel that you are aware of the issue and have already got a strategy in place. You don’t need an answer for everything, but you need to look like you are capable with dealing with anything.
• Sometimes this involves thinking quickly on your feet and bluffing

On the Day

• Talk clearly and smoothly
• Do not waste time
• Know what you are going to say
• Make every sentence count
• Look at the panel
• Be calm and confident
• Exude gravitas
• Be polite rather than adversarial

On the day of the interview you will arrive at the ERC building, show your passport and be given a visitors badge to enter. You then need to go and upload your talk and deliver ~15 copies of a printed version of your talk before being shown to the waiting room. The room will be full of the other candidates that are being interviewed that day and the wait can be several hours. When your interview is approaching you will be shown up to second waiting room where you will be alone, at this point there is only 10 minutes or so. You will then be led into the interview room. There will be no introductions of the panel members, your talk will already be on the screen and you will be expected to essentially go straight into your presentation.  

Behind the scenes of a panel discussion

In a typical panel, such as the ERC, only a fraction of the applications are read by each panel member. All the panel members are active scientists and all want to support good science. Typically, when going into a panel meeting, each member has a handful of application that they are really keen to push forward – and invariably there is not enough money available to cover all of these applications. In the discussion the experts will take up 90% of the time talking about each grant, but the decision making is split evenly between the panel members. It is not unusual to see an expert trying to convince the rest of the panel that their favourite project is more deserving than your favourite project. In the ERC you have a unique chance to help out the experts on your side, by pitching your talk to the non-experts. If it is dry and technical they will basically ignore it. As an immunologist who regularly sits on an immunology-biochemistry panel I almost fall asleep when there is an application by a structural biologist to find the structure of protein X. So if you are a structural biologist don’t waste your time describing purification strategies to the experts who already read your application – instead use this opportunity to tell the non-structural biologists why this gene is important and what you will be able to do with the structural information (eg, the role of the gene in disease, solid examples of how structural knowledge can be used for rational drug design – perhaps you have a collaboration with chemists?).

Click here for a download of my full set of ERC Start Grant hints and tips.

Advice on applying for an ERC Start Grant (part 2)

I was asked to give some advice on ERC Start Grant applicants, as a current grant holder. As this has come up several times I thought I would write a series of blog posts covering my hints and tips. Partly, this advice is specific to the ERC grant system, although most points are valid across any grant. In a previous post I gave advice for Part B1, in this second post I will deal the written application Part B2.

ERC Start Grant - Part B2

• As a rough length guide, think of ~4 pages for state-of-the-art and objectives, ~2 pages for progress beyond state-of-the-art, ~8 pages for methodology, ~1 page for budget. Adapt to your particular project.
•  You need to be ambitious. Prove that you are thinking as future PI, not as a post-doc. This is not a conservative FWO or IWT grant application, where they pick solid projects. The ERC sees itself more like a MacArthur or Howard Hughes “genius award”, to fund the best and brightest. You can definitely go too far (for example, see the reviewers’ comments that I got from my application), but the panel is generally much more forgiving on over-ambition than under-ambition. The criticism I had on feasibility and over-ambition would have been fatal in an FWO application, but at the ERC the project was approved.

“This is a ground breaking project that interconnects genetic studies, cohort studies and biological studies… It is an extremely ambitious proposal with important and broad objectives and diverse perspectives.”

“Some of the research directions could be difficult to accomplish during the project time, in particular some of the objectives of RT3. Perhaps, the PI should have planned them more realistically.”

“The proposal goes beyond the current state of the art, but its major problem is the over-ambition.”

“The proposed research involves an innovative and ambitious study design, but the risk is justified by the potential impact in the field.”

•  Refer to your unique edge on this project. Is this a direct continuation of your post-doc work? If so, describe how this builds off some technique or tool that you pioneered, giving you an edge over the competition (and either here or in Part B1 make it very clear that you will not be competing with your former PI). Is this a meld of the skills you picked up in your different training periods? Then work in references to strategies you have used in the past. Is this possible due to a unique combination of institute resources or collaborations? Then work in the network you created. Be relatively subtle, the place for direct marketing of your work is Part B1, but references like “using the strategy that I previously designed for gene Y (Jones, Science 2010)” show that you are highly capable of getting this to work.
• The application needs to have an accurate assessment of risk – do you have a back-up plan in case that approach doesn’t work? Why is it that you have a shot of getting this to work while no one else does? (if it is due to your training or past successes, this should be the focus on Part B1). It is not enough to have a grand idea; you need to show that you will have a decent change at success.
• You need to show a future career path. The ERC is not just funding a project, it is funding the start of a new elite laboratory. You need to have tangible outcomes during the 5 year period, but there should also be a sense of how you will build on this after the grant has finished.
• Ethical issues need to show that you have a realistic idea of what is involved, but you do not need to have approval at the time of application (you will need to before you get money from the ERC, however). If it just involves mice a simple referral to an animal ethics committee should be sufficient, if it involves humans or primates you need to demonstrate that you have sufficient knowledge of the ethical and legal framework to make your project practical. 
  

More hints and tips - the interview.