Transitions in development – an interview with Evgeny Kvon

Let's start at the beginning, when did you first become interested in science?

My dad is a professor of quantum physics, so I was exposed to science from a very early age. In high school I had an excellent biology teacher, Grigory Dymshits, who was also a university professor. He taught us about the molecular biology experiments that led to the discovery of DNA as the carrier of genetic information. I was amazed by the simplicity and genius of those experiments. That was the moment when I realised I wanted to study molecular biology and genetics.

Although it's very difficult for high-school students to get first-hand research experience, I got lucky. Thanks to my biology teacher and his connections I managed to join the lab of Igor Zhimulev who was doing Drosophila genetic research. I went to the lab every week after class and did simple tasks like preparing media and pushing fly stocks. The lab was studying a classical genetic model, ‘polytene chromosomes’, gigantic chromosomes consisting of thousands of DNA strands found in the salivary glands of fly larvae. You can see them under the microscope at low magnification, and these chromosomes have a very characteristic band structure, so one can identify which part of the chromosome corresponds to which genetic region. I stayed in the same lab during college and wrote my diploma thesis on the molecular characterisation of these bands and interbands.

You moved from Russia to Austria to begin your PhD at the University of Vienna. What was that experience like for you?

I ended up in Vienna by chance. Initially, I wanted to apply to the European Molecular Biology Laboratory (EMBL), but then a friend of mine sent me a post for a PhD programme in Italy. When I clicked on the link, I realised it wasn't in Italy; it was in Vienna Austria. I applied and got invited for an interview.

Moving to Vienna was my first culture shock. The lab I worked in during high school and college was one of the top molecular genetics labs in Russia, but we still had to prepare our own LB media, make miniprep buffers, and even autoclave and reuse pipette tips. When I came to the Research Institute of Molecular Pathology (IMP) in Vienna to start my PhD, all the buffers and plates were prepared for us by the media kitchen. The institute had amazing facilities, all of which were available to students and postdocs. There was even a vending machine with free enzymes. When I started, I didn't realise that IMP was one of the leading molecular biology institutes in Europe with generous core funding from Boehringer Ingelheim. Suddenly, I could do whatever experiments I wanted and focus on science. The downside was that it puts a lot of pressure on you to produce good science because you couldn't say you didn't have enough resources to do these experiments!

Can you tell me about what you worked on during your PhD?

I started in the lab of Alex Stark. He was a new group leader and I was one of his first graduate students, which was an interesting experience. The big question, which Alex is still addressing in his lab, was to understand the so-called ‘regulatory code’, by which the information about where and when genes are expressed in development is encoded in a DNA sequence. We know that the DNA sequence contains all necessary regulatory information because if you take one of the regulatory DNA regions that turns on a gene, called an enhancer, and you put it in the embryo upstream of a reporter, in most cases, it exactly recapitulates endogenous activity. However, in contrast to ‘genetic code’, which we understand in great detail, we can't predict enhancer activity from the DNA sequence because we don't understand the ‘regulatory code’.

One of the approaches that Alex proposed was to use an embryo as a sensor of DNA's regulatory activity by introducing a large library of candidate regulatory sequences into flies using a transgenic reporter assay. We could then use the expression of the reporter gene as a readout of the DNA's regulatory activity and then use machine learning to learn some rules about regulatory code. One would need to create an unrealistic number of transgenic fly lines to have a sufficient training set, but we were lucky because we were next to Barry Dickson's lab. At that time, members of his lab were taking random non-coding pieces of fly DNA and placing them upstream of the Gal4 reporter gene, with the goal of labelling individual neurons in the brain so they can genetically manipulate them. During my PhD I used thousands of these transgenic fly lines to look at the activity of the regulatory regions in the embryos to learn about developmental enhancers. What was quite a surprise is that almost half of these non-coding regions had some kind of activity in the embryo, suggesting that animal genomes are packed with functional regulatory elements (Kvon et al., 2012, 2014).

In 2014, you moved to California, USA, to start your postdoc, where you've remained since. What attracted you there initially and what's influenced your decision to stay?

During my PhD, I was reading papers on gene regulation and many of the most exciting papers came from labs in the USA. It seemed logical that if I wanted to continue working on gene regulation, I should move to the USA. There was also an unwritten rule that if you want to become a group leader in Europe, you need to do a postdoc in the USA, which is not true anymore. Moving to the USA was my second culture shock. Vienna is a very nice place to live as an expat because the quality of life is among the highest in the world, it's safe and there is good public transportation. Shortly after I arrived in California, I needed to buy some household items, so I took a bus to the nearest Walmart but didn't realise that the bus would go through a city with the highest crime rate in America. Eventually, I fell in love with California because of its diversity, amazing food and, of course, sunny weather.

Your postdoc work was carried out at Lawrence Berkeley National Laboratory. Could you tell me about the projects you were involved with and what you discovered?

The lab I joined at Berkeley was run by four people, Eddy Rubin, Len Pennacchio, Axel Visel and Diane Dickel. It was a highly collaborative, unique place with multiple PIs working together. I wanted to continue to work on enhancers, but I switched the model organism from Drosophila to mice. I was initially worried because making a knockout mouse could take 1-2 years. In 2012, the first CRISPR genome editing paper was published and suddenly it took just a couple of months to make a knockout mouse – it was a game-changer.

The idea for my project was to use CRISPR to study the evolution of vertebrate enhancers. One approach was to do an interspecies regulatory element swap. Eric Davidson, Sean Carroll and many others proposed that the differences between species are driven not by changes in genes but by changes in the non-coding genome. You can test this hypothesis by swapping a regulatory region from one animal to another, and the mouse is a good model for that.

We demonstrated this concept in our ‘serpentised’ mouse paper (Kvon et al., 2016). By looking at sequenced snake genomes, we found that they've retained a crucial limb enhancer of the sonic hedgehog gene, called ZRS, which was a little surprising to us because snakes don't have limbs – so why do they need that enhancer? We then replaced the mouse enhancer with the snake version and showed that, although 80% of the enhancer sequence is preserved in python, there are some microdeletions that have led to the loss of enhancer activity and resulted in limbless ‘serpentised’ mice. These deletions potentially caused or contributed to the loss of limbs in snakes. It was such a visually appealing experiment demonstrating the power of CRISPR to study evolution that it is already included in college textbooks, and many colleagues are using this paper for teaching.

The second paper was about how we can use mice to study human enhancer variants implicated in congenital disorders. There are many potentially causal variants discovered from genetic studies that do not affect genes but seem to affect regulatory regions, but the challenge is how do you characterise and test them in vivo? Traditionally, scientists have relied on mouse transgenesis based on random genomic integration, which was very inefficient and expensive. Based on my experience working with Drosophila, I had an idea to develop a site-specific transgenic reporter assay in the mouse. We found a previously characterised ‘safe-harbour’ locus, which we used to develop a highly efficient transgenic reporter assay for testing rare human enhancer variants (Kvon et al., 2020).

When did you start looking for independent positions?

I started pretty late, not until the fourth year of my postdoc because I was focused on the projects I was doing. In hindsight, maybe I should have started to think about it earlier.

What were your most important considerations when looking for group leader positions?

There are a lot of criteria, such as environment, start-up amount, teaching load, salary, collaborative opportunities, etc. Because it's impossible to consider all the different factors, I think in the end it's down to your gut feeling and which place will have the best fit for your research programme. Of course, it shouldn't all be just work; you also have to consider the lifestyle you want for your family and your spouse's career opportunities. I feel like these factors will be unique for every person.

What factors contributed to your decision to go to UCI to start your group?

When I visited UCI, I noticed a very collaborative and welcoming culture with scientists working together, not only within the same department, but also across different disciplines and schools. There is also a lot of support for junior faculty, including mentoring and grant-writing bootcamps. It was clear that the school and the department were investing in each new faculty long-term and ‘plan A’ was to keep all hires beyond tenure – that was nice. UCI has one of the best transgenic animal cores in the country, which was important for my research programme. It also helped that my wife was from Orange County and still has family and friends close by!

How was the transition to becoming a group leader?

I started my lab in April 2020, right after the first COVID-19 lockdown was announced in California. When I arrived at UCI, the school was empty because everybody was working remotely. I was on my own. I couldn't talk to colleagues; there were maybe one or two other PIs on the floor, but no students or postdocs – they were also remote. Of course, there were other challenges as well, as I had two young kids at home. However, the silver lining was, because there were no conferences, travel or seminars, there were no distractions and I could fully focus on setting up the lab, hiring people and thinking about science.

What has been the best moment so far?

I think the best part was honestly seeing the success of my mentees; when they obtain a fellowship, or they get a cool result on their own. These are truly the best moments of being a mentor.

Any other challenges aside from the pandemic?

Dealing with rejection. In academia, it's important to be resilient because there'll always be failures and rejections, and you just have to be able to keep going, despite all the negative stuff.

Can you summarise the research themes of your group at the moment?

I continue to work on gene regulation because I love the topic. Broadly speaking, we are working on two questions. An unanswered question in the field is how remote enhancers can specifically activate their target genes, which are sometimes located very far, often millions of base pairs, away.

Second, I already mentioned that many enhancer mutations cause human disease, so we are trying to understand how exactly this happens at a mechanistic level. How can a point mutation in an enhancer lead to a big phenotypic impact?

What has been your approach to hiring new team members?

When I was starting my group, I was reading advice on Twitter. A lot wasn't very useful, but somebody asked, ‘What is the single most important advice to a new PI?’ Somebody answered that it was to ‘grow slowly’, and I couldn't agree more. I tried to take the same approach: I will only hire someone in my lab if I'm absolutely sure they'll be a good fit. I think that has worked well so far.

Similarly, what advice would you give to people starting their own labs?

Focus on hiring the right people. One thing that I didn't appreciate before starting a lab was that the most important element in a successful lab is the people who will be doing the work. I was lucky to have exceptional mentors and that's why I want to also cultivate a great mentoring culture. The other important thing is, of course, focusing on good science because this is why we're here.

In your opinion, what are the exciting areas in your field?

There's a lot of exciting research happening with new emerging technologies. Now, you can visualise chromatin at unprecedented resolution using super-resolution and live imaging. I think there'll be a lot of exciting insights learned about gene regulation using those methods. In terms of importance for developmental gene regulation, single-cell technology is booming. Today, you can not only profile transcriptomes from single cells, but also get simultaneous information about histone modifications and chromatin, and you can do it in situ in embryo sections.

There are also challenging questions remaining. We know that most of the variation that leads to differences between humans, and human disease, is in the non-coding genome. But we don't know which of the tens of thousands of variants are functional and how they affect the phenotypes. Another related question is what makes us human compared with chimps, given that we are obviously very different but 99% of our genome is identical. Pinpointing those genetic changes that make us unique is difficult and we're only beginning to understand the mechanism.

You posted a preprint a few months ago. Why did you decide to preprint your work and how has the experience been for you?

When bioRxiv first started to rise, I was sceptical, partly because it was not familiar to me. But I've gradually changed my opinion. Lots of my colleagues posted preprints, and it worked to their advantage because they shared their work earlier and got useful feedback. There was also a study saying that the earlier you share your work, the more citations you get. When I posted my first preprint as a PI (Chen et al., 2022 preprint), I was a bit resistant, but based on this positive experience I'm now confident that preprints are the way to go.

You've received several awards, grants and fellowships during your career. Do you have any advice for others looking to apply for similar schemes?

As a junior PI, it's extremely important to have a track record of funding, especially in the USA. I was proactive about opportunities and knowing their deadlines to maximise my chances. For many postdoc fellowships, there's an eligibility time window in which you can apply. It's the same for early-career faculty awards and early-stage investigator status. A lot of these awards are highly competitive and the selection process involves a lot of factors that we cannot control, so you shouldn't be discouraged by failure. You must keep trying. It's also important to seek feedback early to have enough time to get critical feedback and implement it.

Did you ever consider an alternative/non-academic career path?

I trained in a traditional Russian academic culture, which had this archaic perception that if you don't make it in academia, then you've failed. Being raised in that environment, I did consider alternative careers, but only during moments of failure. I don't share this view anymore and believe scientists should consider whichever path fits them to pursue their interests and career goals.

Finally, is there anything Development readers would be surprised to learn about you?

I was born in Russia, but I'm actually of Korean heritage. People wouldn't be able to guess from my first and last name. My first name is Russian, but when I moved to Vienna and had to get a travel passport, they had to translate my Korean last name written in Cyrillic into Latin. Instead of spelling it ‘Kwon’ they spelled it ‘Kvon’. People think it's a Russian last name. It worked out well – there are lots of ‘Kwons’ on PubMed, but I'm the only ‘Kvon’!