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Discussing the Past, Present, and Future of Epigenetics with Active Motif Founder Joe Fernandez

September 13, 2019

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Joe Fernandez is the founder of Active Motif. He founded the company in 1999 to focus on nuclear events in cell biology. That started as developing new and innovative tools to study transcription factors and quickly evolved to include products and services to enable and simplify epigenetics research. Joe guided Active Motif to launch the first ever chromatin immunoprecipitation (ChIP) kit in 2003, and the company has been pushing the boundaries of what’s possible in epigenetics research ever since.

A couple of notable developments over the past few years include developing the AbFlex® line of recombinant antibodies, which can be efficiently and specifically labeled using the Sortag-IT™ labeling kit, and TAM-ChIP™ technology, which was the first antibody-guided transposase tagmentation method developed as an alternative to traditional ChIP to analyze histone modifications or other DNA-binding factors.

Before starting Active Motif, Joe was a co-founder of Invitrogen (which is now part of Thermo Fisher Scientific). At Invitrogen, Joe saw a need for a better way to clone pieces of DNA for expression in mammalian systems, which led to the company developing the first molecular cloning kits. The TOPO TA cloning system revolutionized how scientists did cloning, making the process much easier, faster, and more efficient.

In his recent interview on Active Motif’s Epigenetics Podcast, Joe Fernandez talked about how he became interested in science, his views on the role of microbes in biotechnology, and shared his thoughts on what will come next in the field of epigenetics research.

Listen to the full interview with Joe Fernandez on Active Motif’s Epigenetics Podcast.

Joe Fernandez on How He Got Started in Science

Dr. Stefan Dillinger: A question I like to ask every guest in the beginning of our podcast, and you're no exception, is how did you become interested in biology?

Joe Fernandez: I've always been interested in science, but during my O- and A-levels I got interested in molecular biology when I read The Double Helix by James Watson. That gave me a fascination with DNA that is still with me today. Then, in college, I became more interested in molecular biology after a seminar where they turned the lights off and they made me visualize mRNA transcription, ribosome and protein production, in a three-dimensional, visual way. From there on I started looking at molecular biology as molecules and interactions, and that really excited me and motivated me to continue studying molecular interactions.

Stefan: Biology and the biotechnology industry was very different at the time when you were getting started. What was the atmosphere like in the field at that time?

Joe: When I first started, molecular biology was very, very, very young. As an example, my thesis in grad school was looking at how the chemotherapy drug cisplatinum bound to DNA. I was just cutting pieces of DNA, treating with cisplatinum, and then sequencing it through Maxam-Gilbert sequencing. I used to sequence 20, 30 bases, 40 bases. Then Sanger sequencing came around, I started sequencing maybe 200 bases. The industry was very new, so everything that you did was new, unique, new vectors, new systems. Since then, it has matured a lot, and it's now a mature field.

Invitrogen and the Early Days of the Biotech Industry

Stefan: Then you went on and co-founded Invitrogen. The biotech industry, also, not just the field of molecular biology, was very different then than now. You were the first ones to sell cloning kits. How was the industry different back then?

Joe: Well, back then there was the need to clone mRNAs and express them in mammalian systems. We took advantage of that need and started developing cloning systems for mammalian expression. That’s actually something that hasn't matured that much over the years, the actual expression of these cloned vectors. You have the baculovirus and Pichia pastoris systems, but not much else new in recent years.

Back then, before the human genome was sequenced, we actually did think that was going to be the answer to biology: cloning a gene, expressing it, and understanding it. It wasn't until after the human genome was sequenced that we realize that there was way more complexity to molecular biology than a linear DNA to RNA, RNA to protein expression.

Stefan: What you basically did was reuse biological tools that were already there, right? These were enzymes that were expressed in bacteria, and you harnessed them for different purposes, creating a toolbox to use in mammalian systems.

Joe: Yes. I feel very strongly that the field of molecular biology is the field of microbial genetics. We just used the microbial genetic tools that were there to make molecular biology for mammalian cells a little more efficient, better, and move from the study of genes in E. coli to study the genes in mammalian cells.

And the exciting thing is that I think there's a ton more still to discover. I think microbial genetics will always be the basis of molecular biology. Microbes and phages have to be so much more efficient on the evolutionary pathway that you're going to find an incredible amount of tools still to come. As molecular biologists, we use the Tn5 transposase, the T7 RNA polymerase, the T4 DNA polymerase. Almost everything that we use today for common molecular biology approaches comes from bacteriophages or bacteria.

Active Motif: Focused on Enabling Epigenetics Research

Stefan: Active Motif was founded 20 years ago now, and in the past 20 years many other companies have been acquired or had to close their doors. What do you think is it about Active Motif that has allowed the company to continue growing and stay independent so long?

Joe: I guess I would characterize our success as the result of three different things. First, the people that we have. I think we have some incredible people, and very committed to our success and growth. Second, the focus. We focus on epigenetics and try not to move away from that. Then, thirdly, good science is good business, and we try to drive the company through good science.

Prefer listening over reading? Check out the full interview with Joe Fernandez on Active Motif’s Epigenetics Podcast.

Stefan: When you started Active Motif, the focus was not yet quite on epigenetics, right?

Joe: Right, when we started it wasn't epigenetics, but I was trying really hard to stay inside the nucleus. Like I said, after the human genome was sequenced, and we found out there were probably only 25,000 genes or so, we knew that the answer wasn't the gene.

So now it was time to study cell biology, what was going inside the nucleus. Almost everything that we started with was looking at transcription factors or things like that, and then the buzz became epigenetics, so we started moving more into epigenetics.

The Current State of Epigenetics and Its Promising Future

Stefan: In the early 2000s, with the rise of chromosome conformation capture methods, the view changed. It was first the linear genome that was sequenced as part of the Human Genome Project, and then focus turned to the 3-D genome and epigenetics, which essentially opened a new field. How did you see this change?

Joe: Well, it's always fascinating if you look at two meters of DNA and 23 pairs of chromosomes having to fit into a nucleus of 90 microns. The way that it does it is actually like DNA being wrapped around a bead, which are nucleosomes containing histones H2A, H2B, H3, and H4. The DNA condenses about 10,000-fold from the linear form to the form in the nucleus.

The ability of DNA to fold in such a way that it can then control what genes are expressed, which determines what cell type it is, and still be able to be replicated at around 500 nucleotides per second, 50 revolutions per second, such an incredible speed, and still form the same structure of the chromosome that it had from mother to daughter and be able to express the same genes, to me has been fascinating.

That is all controlled through mechanisms like modification of histone tails, through microRNAs, through lincRNAs, and more.

What surprises me even more is how accurate it is, every single time, and disease states rarely happen. The most common event is that things happen normally. With the complexity of epigenetics and the chromosomal folding, of histone marks and all that, to me, it's just surprising how efficient the system is.

Stefan: What are your thoughts on the more general “open or closed” chromatin assays like ATAC-Seq?

Joe: These methods are a good entry point to epigenetics for many researchers, but I'm a believer that ATAC-Seq is just a rough way of looking at open chromatin. There are better ways to study the details of epigenetic mechanisms, such as ChIP-based methods for histone marks. You're going to be able to then interpret that open reading frame a little more; is it an enhancer? What is it? I think that you're going to start seeing ChIP and related technologies becoming more utilized in the coming years.

We ultimately need to study the details DNA-protein interactions, so specific methods like ChIP-Seq will be needed. These methods might not always be called “ChIP”, it might be TAM-ChIP or CUT&RUN, but the ability to understand the precise details of protein-DNA interactions, and the roles of histone modifications in DNA interactions and transcription factor binding, is important.

I think that we still have a long way to go. We're beginning to understand very clearly that naked DNA means very little. So now we have to understand how the 3-D structure of non-naked DNA on the chromosome, and the encoded proteins, affect transcription, translation, and differentiation.

Clinical Applications of Epigenetics Research

Stefan: You already mentioned that epigenetics does have vast implications in disease states, and the field of epigenetics is now expanding into clinical applications and is also important in aging. Where do you think epigenetics is going in the clinic?

Joe: Well, for me, epigenetics is like the canary in the coal mine. Epigenetic modifications can be biomarkers, signals of something going wrong or something going right. As long as we can start better understanding these signals and reading them in the liquid biopsies, signals like histone H3 mutations or histone tail modifications or mutations in blood, or microRNAs in saliva.

All these things are telling you something's happening, even though it may not have happened yet. For example, you may not have cancer yet, but epigenetic biomarkers can tell you that you're about to have cancer. You may not have a neurological disorder yet, but the saliva microRNAs may tell you will have a neurological disorder in the future. So, I think that you're going to see a lot of new diagnostics coming forward are epigenetics-based.

There are many different epigenetic modifications, hundreds of different histone tail marks, for example, and I'm a believer that, through evolution, all these marks have a function. We know very little right now, so the correlations we're putting together are based on a few marks. Once we understand them all, we understand the language, we understand how they fit with refolding proteins, we understand how they fit with RNA structures, then we'll be able to then interrupt them before they can cause diseases like cancer. Now you can start building cures that are specific to that individual and to that epigenetic effect.

What Will Be the Next Big Breakthrough in Epigenetics?

Stefan: There are a lot of great methods out there to study epigenetics, methods like ATAC-Seq and ChIP-Seq. What do you think will be the big epigenetics breakthrough?

Joe: It is really hard to know what the next breakthrough will be. It seems like after the human genome was sequenced, breakthroughs came through brute force and a lot of work. In other words, doing a lot of ChIP-Seqs, doing a lot of RNA-Seqs, doing them on both populations of cells and at the single-cell level. And also doing mass spec assays, looking at protein levels.

Now, how can we get the bioinformatics to look at all these things and try to figure out what the data are telling us? How does the secondary structure of a chromosome fit with your transcription factor ChIP-Seq data? How does it fit with your RNA transcription data? How does it fit with your protein expression data? I think that bioinformatics is a limiting factor right now, to be able to unite all these databases in a smart manner will be a huge breakthrough. Some people call it artificial intelligence, but to me, it’s just efficiently analyzing interactions within and between databases.

Stefan: 20 years ago, it was possible to put one Ph.D. student on one topic and have them do that for five years, and it's worked out fine because they could focus on that one thing and end up answering one big question. Do you think that's still the case of those days? Because research now needs such specialized methods, so much complex bioinformatics, is it still possible for one Ph.D. student to do projects on their own? Or is a more complex approach needed, where collaborators or companies do parts of experiments or the informatics, and the student does other parts of experiments but not everything?

Joe: No, I think you're right – things have changed. I think that as the industry has matured, many projects require sequencing a billion bases a day to get enough data, and then they have to analyze all that data. Then they have to incorporate the data with different systems.

Science has to be done at both the single-cell level and with populations of cells to answer many complex questions. So, yeah, I don’t it’s possible anymore to sequence a gene and get a paper. Now it's going to be how understanding all these things come together to answer a question. But there are so many important and fascinating questions still waiting to be answered.

In the future, and it has already started to happen, there are going to be people that only do wet lab with a little analysis, and there is going to be enough data in databases that people are going to be able to do some great science without ever touching a wet lab.

However, there's still so much more data that needs to be generated. I don't think we understand transcription factors well enough. I don't think we understand chromatin-remodeling factors well enough. We definitely don't understand histone tails well enough. We're beginning to understand microRNAs a little bit. It still is the very, very beginning of what we can call 3-D biology: understanding biology through all different interactions inside a nucleus.

Stefan: The field of epigenetics historically focused on DNA modifications because that area was well understood. Then histone modifications came into play, and now this whole new field of RNA modification is opening up. Where do you think the epigenetics field is moving next?

Joe: Well, they're all relevant. I am a believer in DNA modifications, 5-hydroxy, 5-methyl, and trying to understand those a little better as they relate to chromatin structure and nucleosome positioning. Then, for the RNA modifications, how do they affect finding the right ribosome and how much time the ribosome spends on that RNA to control protein levels inside a cell?

The more we go into the biology of what's going inside a cell, the more we understand that there is regulation at every single step. I think that the reason why a cell doesn't make too many mistakes is that there's a lot of backup systems to everything. I believe that you have the ribosomes as a backup system, and the mRNA modifications are another backup system. Then you have DNA modifications, and then you have histone tail modifications, and so on. The cell seems to have multiple backup systems for everything.

Stefan: During this conversation, we have taken a journey through your history and also the history of epigenetic discoveries. If you look back, what is your highlight in this span and this journey?

Joe: I think that it's been a lot of fun to see how molecular biology and genomics has matured. I think it's been a lot of fun to see, in our lab, in our Epigenetic Services group, how we can now sequence billions of bases a day, do hundreds of ChIP-Seq assays at a time, be able to now begin to analyze these data by combining ChIP-Seq and RNA-Seq datasets. We even look at mass spec data at the end to compare pathways and transcription factors.

I think the way genomics has evolved has just been fascinating. We have a long way to go, but there is a new generation out there ready to take those next steps. The next generation of researchers will start thinking of biology as a complex interactive system with multiple different components. They will not be thinking in terms of linear biology like the previous generation was taught to do. They will be able to analyze more and more data and better interpret what's going on inside a nucleus, and gain a deeper understanding of the structure of chromosomes and its meaning to transcription, translation, and differentiation way more than we could before.

Stefan: You're really passionate about discussing epigenetics and the future potential of this field. It must be more fun doing it now than it was back then when you just had one vector with one protein and analyzing that.

Joe: I actually don't think that there's a better time to be in science than right now. I would recommend anybody in college to go to grad school and to continue in sciences because there has never been a more exciting time.

Check out the full interview with Joe Fernandez on Active Motif’s Epigenetics Podcast to learn more about his thoughts on the past, present, and future of epigenetics.

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