Susan Clark: Leading Ladies in Epigenetics Research

Most of us had part-time jobs when we were younger. Pizza delivery, janitor, life guarding, you know name it, but not so many us of managed to develop one of the most practiced techniques in epigenetics research today while doing the part-time gig. Between successful careers in industry and academia, when she wasn’t driving the uptake of bisulphite sequencing one workshop at a time, or amassing key publications highlighting the complexity of DNA methylation in gene regulation, Dr. Clark raised three kiddos.  It’s no surprise trying to pin her down for an interview was as about as easy spotting Nessie, or Sasquatch, but way worth it.

EpiGenie: To kick things off, can you share with us your recent research interests and some of the areas that are really catching the eye of the Clark lab?

Clark: Well, I’ve been working in the field of DNA methylation and gene regulation since the early ‘90s, and the fundamental questions really haven’t changed very much. The major questions still to be resolved concern the role and relationship between DNA methylation, histone modifications, coding and non-coding RNA in controlling gene regulation in normal cells and how this is disrupted in cancer cells.  What has changed considerabley though in the last 18 years is the fact that we now understand more about the complexity of “epigenetic” players that are involved – meaning its a lot more complicated than we ever anticipated. So our labs focus has moved from looking at changes that occur in individual genes in cancer, such as tumor suppressors, to what’s happening at a more global level.

In this endeavor, we were really surprised to find that epigenetic changes in cancer weren’t just limited to discrete foci but could actually span large megabase regions. Now we’re trying to understand how this works, because this is a new and really exciting finding as it has potential for new cancer markers and changing how we may approach epigenetic therapy . These large domains suppressed by epigenetic changes can be equivalent to genetic changes such as loss of heterozygosity in cancer but unlike genetic deletions have the potential to be re-activated. We have found that epigenetic suppression of large domains occur through epigenetic remodeling of the chromatin, which has major consequences because the process can encompass not just critical genes but also bystanding genes, noncoding RNA, and microRNA.

Epigenetic therapy we would expect to impact on the bystander genes as well as the critical genes in these domains.  We are currently investigating the role of noncoding RNA expression and the role of polycomb modification in directing regional suppression. We’re finding that in our system DNA hypermethylation seems to be a consequence of gene silencing and/ or normal tissue specific gene suppression and is exquisitely linked to the formation of heterochromatin. I think all of our knowledge to date is converging, especially with recent genome-wide tools that we now have available. We in the field can look at the genome as a whole rather than as discrete regions, and it’s telling us a lot more rich stories than we had ever appreciated.

In the last 50 odd years, which is pretty much my age (laughs), the field has gone from just understanding the structure of DNA double helix to now the completion of the Human Genome Project. So, I’ve felt that I’ve been lucky to be around at one of the best times in molecular biology as far as understanding—or trying to understand—the intricacies of how gene regulation occurs.  But in those 50 plus years, I think we’ve generated more questions than answers.

I finished my undergraduate degree just when gene cloning started in 1974, DNA sequencing was in ’75, and recombinant DNA technology in ’82 and PCR in 1985.  It was PCR that provided that extra leap in technology that enabled us to kickstart new studies in DNA methylation and has revolutionized most of our work direction since then—the fact that we can now look at very small amounts of DNA in great detail means we can unravel the complexities of this remarkable modification in different cell types during development, differentiation and disease.

EpiGenie: In the Summer Olympics, the Australians picked up several golds. We know that you were intimately involved with the development of the gold standard for methylation analysis—bisulphite sequencing. Back when you were working on that method, did you have any idea of how much of an impact it was going to have?

Clark: We did, actually, because many of us had spent a number of years trying to find ways to sequence methylated DNA. So back in the early days, even when we were doing Maxim-Gilbert sequencing, we could see the methylated cytosines migrating differently with a less intense band from the unmethylated cytosines. My colleagues, in particular Peter Molloy and Marianne Frommer and I kept pursuing this cause as we could all see how uninformative restriction enzymes were for DNA methylation analysis, as you could only get such a small snap shot of the methylation profile. So it was clear how understanding which specific sites were methylated was going to totally change how we perceived the role of DNA methylation in gene regulation.

I’d have to say the study of DNA methylation was a very unattractive field of research for most people. Every time I (or others…. it just want me!!) gave a talk on the topic people would roll their eyes in boredom. You’d show Southern blots and the bands that would change after digesting with HpaII, and it was very much, “Ho-hum. What does this really mean?” So DNA methylation and its role in gene regulation was a hard field to get funding for and to generate excitement. Even at the time that Marianne Frommer knew the bisulphite sequencing was going to finally work for generating DNA methylation profiles, she looked at getting patent protection on it, and our institute said, “No, this isn’t really at all interesting. It’s not going to go anywhere. Just publish it.”  This is why the first publication in 1992 ended up being a proof of principal paper in PNAS using the human kininogen gene and this still took 8 months to accept after rejection from Science.

So that’s when I decided we really needed to “develop” the method in more detail to make it more robust, and develop the “nitty gritty” parameters of the technique which was then published in 1994. It took a lot of effort, and basically I seemed trouble shooting world wide with designing bisulphite primers and running DNA methylation workshops and hosting international scientists in my lab to learn how to do it! As with a lot of things in science, especially a challenging method, and at the time reasonably expensive and time consuming, you really do have to sell the significance of embarking on a different technique and how it adds to important scientific questions.

This was especially true with bisulphite sequencing, because restriction enzyme technology was a lot easier to use and to publish as you just got a yes or no answer—your RE site was either cut or not cut. What the methylation bisulphite technique showed us was that biology is not that simple. We found that methylation was heterogeneous, that is not each CpG site was methylated equally or with 100% fidelity, which meant there was an aspect of probability in methylation at any one site and this was probably the biggest finding for us at the time- that is a region could be methylated but not all CpG sites are fully methylated. The dogma was that once a CpG site was methylated it remained methylated because of the efficient maintenance DNA methyltransferase activity. So the bisulphite sequencing showed that that wasn’t true, which meant we really had to revisit the role and mechnism of methylation in the cell—and how is it involved in differentiation and cell-type specificity as it did not seem to be simply a on-off switching mechanism.

EpiGenie: In terms of DNA methylation assays, we now have digestions, immunoprecipitations, methyl-binding proteins, in addition to bisulphite conversion, now coupled with advances in downstream readout technologies such as high-density arrays and sequencing. What do you think is the most promising combination of methods for looking at global patterns of DNA methylation?

Clark: Well, I’m biased. We’ve played around with many of the methods.  Even though we might be using MeDIP-chip [methylated DNA immunoprecipitation–microarray analysis] at the moment to get some sort of flavor of the global methylation pattern, I appreciate that this is limited, as was and is the use of restriction enzyme based approaches.  But the information from global methylation arrays is important as you get a flavor of what’s happening over a kilobase-or-so region.  So where I see the future is doing bisulphite sequencing at the genome level—enriching or capturing your DNA or region of interest, for example, a1- 4-megabase region, and then bisulphite sequencing that. I actually think we need that sort of detail, so we can map in detail what is important- compare what CpG sites remain unmethylated and what sites are methylated and what sites are stochastic in their methylation profile- and see how this relates to genomic location and RNA expression.

The problem with an antibody approach is that it all depends on the antibody people use and the size of DNA template. My concern is that using antibodies to MeC/ MeCP2 and or MBD2 methyl-binding protein will all bias to some extent the methylation profiles detected. We also know that the antibody to methylated cytosine pulls down DNA whether it’s got one methylated site or many, and so it’s quite difficult to discriminate the subtle changes between the low levels of methylation and the high levels of methylation.  It’s not very quantitative. But I do think the technology is improving and becoming more cost effective so that we will be able to do bisulphite sequencing of the human genome very shortly.

EpiGenie: So right now, you’re doing a sort of subtraction or sequence capture prior to bisulphite conversion?

Clark: That’s right.  Because of the cost of DNA sequencing and the complex bioinformatics needed to relate your sequence back to the whole genome, I think the first step, which has proven to work quite well, is to capture your DNA region of interest, and then do bisulphite conversion and sequencing. That allows the bioinformatics to be much easier because you only have to mine the data back to a specific region.  But with technology getting, hopefully, cheaper, I think it will be possible in the near future to look at the whole genome. What’s going to be important to study is the methylation of the repeat sequences, and so, again, capturing means that you’re able to interrogate not just the single-copy regions but some of the repeat regions in your captured DNA.

EpiGenie: With the interplay among non-coding RNA, chromatin, and DNA methylation, has the Clark lab started to interrogate some of these different areas, or do you collaborate with other groups (for instance, looking at non-coding RNAs and their putative role in methylation)?

Clark: At the moment, our lab does have a number of collaborations in this particular field. We are developing new technology to look at the role of sense and antisense transcription on tiling arrays, so that we can interrogate more exquisitely the role of transcription, be it transcription of small molecules or transcription of large ones, and how double-stranded RNA plays a role. This is something that we’re doing in-house because we’re trying to develop more tools so that we can actually look at this at a more sensitive level.

EpiGenie: Interesting. Now we’re going to switch gears a bit and touch on some things related to your career. We’ve heard a lot about some of the challenges that women scientists in America face. In the course of your career, have you seen any challenges that are unique to women in research in Australia?

Clark: Well, from my perspective, it’s changed a lot. When I graduated there were much fewer women studying in science in Australia, and the ones that did graduate, had a difficult choice of getting married and therefore leaving career science or remaining single and pursuing a scientific career pathway.  The expected procedure in Australia for any respected scientific researcher was to go abroad for your phD or after your PhD to America or England or Europe for postdoctoral studies. And you could not take easily take your a partner unless you were married, and if you were married, your spouse could not work (visa problems) and so there were very few men at that time who would travel for their wives/girlfriends career. It tended to be the other way around which meant many more men pursued research science careers in Australia.

As for me, I did get married early while studying my phD and therefore I made the decision not do a traditional research post-doc as my husband already had work in Australia.  Also, and I don’t know if it was like this in America, but in Australia at that time in the late 70s early 80s, married couples couldn’t always work at the same research or government institute. So I’d say things have changed dramatically over the last 28 years. Obviously, these issues are no longer so much of a problem. Women are a lot more independent and have more choices and men are a lot more flexible and considerate in their attitudes to work and family.

EpiGenie: In the United States, there is now much more representation of women at the graduate level, but women are still very much a minority at the PI level. What do you think could be done to decrease the attrition rate?

Clark: I think that’s also very true in Australia, but it’s changing.  So definitely at the graduate or undergraduate level, women are now the majority in the biological sciences, but as you said, the field is still dominated by male directors and PIs. I think the shortage of women at the top has a lot to do with the difficulty of combining a career with having a family. Women are having babies later, but how long do you wait? When you’ve finished your 1st or 2nd post-doc or when you get tenure or after the next grant or fellowship is awarded- there’s no actual opportune time you feel you can stop and take a bit of time to start a family. I think this is the reason that many women have not continued up the scale on to be PIs and to run their own labs, as it often becomes too difficult to do it all and do it all as well! One thing however that is changing is shared family responsibilities with more partners taking time to care for the children. And many of the women I know who have been successful in balancing family life and a scientific career,  including myself,  have been lucky enough to have a partner who shares the housework and family responsibility.

There’s also, in Australia anyway, a lot more flexibility in having time off for children and then being able to apply for grants after a break in your career. So a system in which you can still be competitive for grants if you’ve had maternity leave and have time off for early childrearing is making it more practical for women. There’s also childcare in most of the larger institutes and universities now, whereas when I had my children in the late 80s, there were limited childcare options available. So I think there’s a lot more infrastructure in place that wasn’t there before, and women are having a lot more say and are deserving respect for their role in science. Having women leaders in science means that they really do understand the complexities of these issues—well, many of them anyway—that it really is a juggling act to successfully manage a career and a young family at the same time.

EpiGenie: As a mother of three, do you have any advice for women scientists who are trying to do this juggling act?

Clark: Well, the first thing I always say is that there’s no right time for having a baby. So I wouldn’t be saying, “Put it off till I have got the next grant,” or “Put it off till I get my next paper published or tenure ” because it’s not always easy to have children. So when you think you finally established in your career and can fit in a baby, it doesn’t necessarily happen as easy as that especially the older you get. I’m very much a person that can juggle many things at once, like most women, always seem to have a touch of chaos in my life and I know if I had thought too much about exactly when to have had my first or subsequent children we would never had any.  Interestingly children often make you more organized and focused in your work and give you an even stronger reason to succeed and enjoy the many good things of life.  Societies perception have changed over my career and will continue to change Im sure, so that women can more easily succeed in a scientific research career and enjoy the benefits of a family at the same time.

EpiGenie: Lucky for the community you did! Thanks for chatting with us.