Basic Medical Research Award
Interview by James Darnell
Darnell: It's my very distinct pleasure this morning to have a conversation with Ronald M. Evans. He is one of the three winners of the Lasker Basic Science Award for 2004 and a very old friend. Ron was a postdoctoral fellow in my laboratory in the 1970s when we had lots of fun in the lab—that was about the time I quit doing experiments, but Ron has now, for the last 30 years, done brilliant experiments, which has brought him this prize.
I will start to ask him to tell us about his career by going back to the early days in southern California, where he grew up and went to undergraduate school in Los Angeles. Ron, let's hear from you about how you became interested in science. Did it happen early? What were the influences as you began your career?
Evans: Well thanks, Jim, it's a real pleasure actually to be here and have you give the interview, after a long association. Dipping back into those memories back in California—probably in middle school or high school, I was clear I was going to go into science of some sort. I didn't really have any long-term plans. I was mostly interested in tennis at that stage and mostly math, a little bit of chemistry. But we were not a very sophisticated high school where I was at. Only a half-a-dozen or so kids that were slotted to go off to college. And so, it was like my career track wasn't clear, other than going into biology or some type of science.
Darnell: Your father was a physician, I think. Did that play a role?
Evans: My father was a physician—he was becoming a little bit disenchanted with medicine at that stage and thought that I should go into business because that was his desire. In fact, in filling out the forms for college, which he filled out, he enlisted me as a business major, which I dutifully went in and quickly changed after one semester—and went into more or less a biology track. In fact, it was microbiology, along with another classmate of mine at the time, Randy Schekman, who was a previous recipient of this award, actually.
Darnell: I see. That was at UCLA?
Evans: It was at UCLA. I was an undergraduate and a graduate student at UCLA—and mostly enjoyed the initial freedom. I was a pretty young student at that time and had not really been away from home, and was tremendously enjoying being at this big, active campus. Somehow, I managed to find my way into a lab to do undergraduate thesis project with Eli Serkaz, who was a young and up-and-coming immunologist at the time. I spent many days and nights in a laboratory working on T cells and the thymus. That was my first attraction into a serious research project that led to an actual publication from that study. My first publication.
Darnell: I see. You had just learned to tie your shoes when you had your first publication?
Evans: (Laughs) Basically. Sandals. Remember, California.
Darnell: Sandals? Oh yes, right, in those years, sandals. Yes. Now, what year did you get your undergraduate degree?
Darnell: Right. And then, you went directly into graduate school at UCLA?
Evans: Directly into graduate school at UCLA.
Darnell: Tell us about the projects that interested you there.
Evans: You know, I joined the lab of Marcel Baluda, who was a rumored hot and up-and-coming scientist. He had recently left the lab of Renato Dulbecco of Cal Tech. He was there with Howard Temin. Renato had recently left Cal Tech, actually to go to the Salk Institute, where he recruited David Baltimore. Baluda and Temin, while fellows with Renato, had observed that in cells infected with an RNA tumor virus, they believed they had evidence for a DNA form of the RNA genome. That meant that somehow RNA had to be transformed into a DNA intermediate that presumably was integrated into the genome. This was a big and controversial idea. For that conversion to take place, as you know, there would have to be an enzyme that converted RNA to DNA.
There was a big race, which I have to admit I did not understand how big it was at the time. Our lab was searching for an enzyme that would make this conversion. Temin was searching for it. And this unknown, who Renato helped to put onto this interest, was David Baltimore, who began searching with his enzymology expertise from polio—he began looking for reverse transcript as well. We were soundly beaten in that project, which created a tremendous upheaval in our lab. Temin and Baltimore, of course, did isolate reverse transcriptases, which was a transforming event not only for retroviruses, but for all of future molecular biology.
I learned at that point that those who get the discovery first have a tremendous advantage and a tremendous leverage in moving onto the next step. They basically rolled over us at that stage. I still had an exciting time as a student and learned a tremendous amount. But that was one of my learning experiences was how fast fields can move once a discovery is made.
Darnell: What did you actually do, Ron, for your thesis work?
Evans: So I stayed—my goal was to study the nature of this putative DNA intermediate. And I used a shearing technique based on sonication to fragment DNA into various sizes and tried to measure the size of the integrated DNA genome. The shearing technique was able to demonstrate—at least my thesis was—that there was a single copy of a single genome integrated at a single site. Now that might not sound like a big deal, but for me it proved the nature of this integration event, and the conversion to DNA was there. It was a specific identifiable molecule. We used renaturation kinetics. There were no restriction enzymes. So we're looking for a specific gene signature and found it. That was really what I did my thesis on.
Darnell: Right. Now, I know very well your next stop in life because it was to come to New York and to join our laboratory. And although I recognized the importance of the integration event with the RNA tumor viruses, our interest was not at all in tumor viruses as such, but rather in the manufacturer of messenger RNA molecules. So had you picked up on that? Exactly what was it that caused your interest in coming to New York? Tell me again.
Evans: Gene control was a looming big feature of molecular biology. It had been for a long time. And the history of that goes back a long way to Thomas Hunt Morgan, and possibly even before that. But certainly, there's a long history of the importance of gene control. In identifying DNA sequences that can direct—that was not really possible in those early days. But having worked on integrated genes whose regulatory sequences were somewhat identified in what are called long-term repeats of the retrovirus, it seemed that there was something at the ends of these viruses that could direct gene expression. So I was interested in this mechanism.
Randy Wall, a former postdoc in your lab, had landed not only at UCLA but was recruited by my boss, Marcel Baluda, who was the chairman of the department. His temporary space was next to my lab bench while his labs were being built. After many, many discussions with Randy, I decided to come to your lab—at least to apply to your lab. I remember that process very well—[I was] very excited about leaving California for the first time and joining what was, you know, in my view, a very big time high-powered research lab. So this was an exciting step for me.
Darnell: Well, we can't leave out from this interview, Ron, something that we've recalled to our friends many times about your arrival in New York. I was then at Columbia and just before moving to Rockefeller where we're sitting today, and I have to describe to the fans your California appearance ...
Evans: You don't have to…
Darnell: When you first appeared in the office with your very wide-brimmed hat. But the hat didn't hide the intelligence, and I was very, very pleased that you decided to come to New York and join us. I, of course, have fond memories of the years you were in New York, not only on occasional tennis games but all the experiments that we did—some of which we actually did together. And quite a number of which have recorded successes, if success is to have a paper that comes out of it. So why don't you take a few minutes to tell us about your years in New York? Some of the last part of which was important in your starting your independent work. So tell us about time in New York, whatever you'd like to say about it.
Evans: Well, it was without a doubt the major turning point in my career—being in New York City, being in the lab, working with you, who had a long history of working with other outstanding people, and the thought process that was imparted by osmosis and also directly from you changed my approach to science. I was trying to absorb all that when I got here. I loved being in the city. It was a real exciting environment for me. We had excellent colleagues, many of whom were much better trained than I was, I felt, coming in. And the lab was really at sort of the molten core of a very exciting area, which is RNA processing and gene transcription. You had chosen adenovirus as a model because there were no cloned genes at that time to study. Viral genomes were the single best way to model this process, and virologists had a clear advantage on thinking through the problem. You're a trained virologist.
I landed, I feel, at the right lab probably better than I had realized, at the right time. And I did not realize there was going to be an explosion as there was in this field of our understanding of virtually all events that were somewhat mysterious and confusing that would change our thinking and launch a new age. So this was an exciting time for me. I wanted to focus on the gene initiation or transcription start sites. Much of the lab was focusing on poly-adenylation and RNA processing. So I had a little bit of a free reign with some of the techniques that were being explored to try to map the start site for the adenovirus major late promoter, as it came to be known. Those papers were incredibly challenging, fun, and I would say re-crafted my approach to science. And so, in those few years—I came in somewhat naive, but I'm sure I left naive. But with a different approach to thinking about the problems.
Darnell: During the latter part of your time here, we went back to Randy Wall's lab. I remember you and Michael Harpold and I all going to California to learn how to clone. Tell us about what you did when you came back with Carter Bancroft and the latter part of your time in New York.
Evans: So the dawn of cloning was upon us. There was initially a moratorium on cloning because of the potential dangers. You served on one of the national committees that helped set the rules for cloning, and we knew it was coming. In fact, we had somewhat of an advantage because you knew what all the rules would be, and we could build laboratory facilities that would match the future requirements, which we did.
We went back to California to work with Randy and others who were developing the early techniques for cloning cDNAs that have copies of messenger RNA. And there was a great deal of excitement about this technology. And at that time, I was beginning to realize that we may no longer need to depend on a viral template, but we could consider working on cellular genes to study their regulation, which was looming larger and larger in my own thought process.
So Michael Harpold and I and you trundled out to the west coast again, back to UCLA, picked up these techniques, and then trundled back. And after a great deal of angst and discussion with many people, my interests centered on the growth hormone gene to try to clone. The reason for that is because that gene is expressed in certain cultured cells, pituitary cells, namely, and could be induced by two different agents: glucocorticoids, a steroid hormone; and thyroids, another hormone not related to steroids produced from the thyroid gland. And these two small molecules independently were shown to induce the secretion of growth hormone from cells. Therefore, in my view, [it] probably had the effect of inducing the transcription of the growth hormone gene, which then led to increase the release of the product from the cell. And so with that, I settled on growth hormone as a model for steroid and thyroid hormone regulation and basically switched from adenovirus to working solely on cellular genes. It was a conscious switch and [I] left the promoter, which I loved, and did not really continue that series when I left the lab.
Darnell: So we did successfully get the cDNA copied and had cloned growth hormone. Do you remember what we did with that?
Evans: I do…it was a little bit humorous in those days. You had to wear these paper gowns and paper hats and you had to go into a special low pressure room so that nothing could escape to do this cloning. And the recombinant library that Michael and I generated had initially 24 clones in it when it first came out and...
Darnell: A bookshelf rather than a library.
Evans: Not a library, right. A pamphlet, I think is ... (Laughter). And by the time, one week later, it was down to 12 that survived. After picking those 12 and being very cautious, ultimately, only two survived of that original so-called library. And one of those two was growth hormone. So there was a little bit of luck that was involved, but in fact there was so much produced in that cell that that's what created, I think, part of the lot. But we're off and running with that cDNA clone. And immediately we tried to use it to look for the transcriptional footprint, and we found that in fact this relatively small cDNA was contained in information in a much larger RNA transcript in the nucleus. Clearly, that demonstrated that the cellular gene, like the viral genes, was undergoing specific RNA processing to produce the mRNA. So we knew that what we were studying with the virus was basically being recapitulated with a true cellular gene.
Darnell: Right. And my recollection is, or I am sure, we proved it was growth hormone by showing that that messenger RNA could be translated and produce a product that could be precipitated by a growth hormone antibody.
Evans: Yes. We did ..exactly.
Darnell: And that at that point satisfied me. It should not have satisfied me and you tell me why.
Evans: I'm not sure what...what you're...what you're getting at there.
Darnell: Well, my...my lack of interest in things practical...
Evans: Oh, yeah. (Laughs) Right.
Darnell: ... turned out to be for the two of us at that point a minor disaster in retrospect. Tell them about it.
Evans: In retrospect, I almost managed to block that out of my mind. Growth hormone turned out to be a very big and important molecule to have cloned. And in that era, the idea of patenting was not prominent on our minds. I believe that we were the first to correctly clone the growth hormone gene, although there was another group in San Francisco that also cloned it basically, within the same few weeks that we did. But they were sharper. They patented and made a handsome sum from that cloning. But we learned from that mistake. It was an expensive training on that, but that's a very good point.
Darnell: If anybody in the Rockefeller hierarchy ever hears this story, I'm going to get run out of town. But at any rate, we didn't. Fortunately, that lesson was not lost on you, and in later days you realized the intersection between our fundamental science of microbiology or molecular genetic biology was going to produce important products. Had you recognized that by the time we got to the end of that trail and by the time you headed for California? Or did that come later in your life?
Evans: You know, that was the year I left. I think the year that Genentech was being founded. I knew the founders of that. Honestly, I didn't believe in that they could make this a commercially viable technology. I was of course wrong with that
I was being a little bit too elitist because they hired some outstanding people and they have done a fabulous job. And indeed, the era has changed and I grew in part with the lesson at Rockefeller to realize the importance of translating basic discoveries into their clinical relevance. That, in fact, is not just an idea—it now happens all the time. I got onto it relatively early. I think it's an important thing for anyone who's working on something that's medically relevant.
Darnell: Even I have come around to understanding that finally. I didn't teach you that, however. So you had the growth hormone clone in your hand, and you were interviewing for jobs around the country as a very prominent, by this time, prominent young postdoc. Tell us about your location and your first independent lab job.
Evans: I interviewed one round of interviews basically, and wound up at the Salk Institute, where many years ago I had trundled down to meet Renato Dulbecco as a graduate with student Marcel Baluda to review some papers. I always had fond memories of the Salk, and I was delighted to go back there and take a position as an assistant professor. My goal was to finish off the growth hormone project. We had the cDNA; I wanted the gene. Because within the gene presumably was a promoter, which I thought I could map based in the techniques with adenovirus. And within that promoter should be the regulatory elements that controlled expression, particularly by glucocorticoids and thyroid hormone. So I needed to get the upstream parts of that gene. And at that time, there were really very few techniques that were in place that allowed that to happen. One person who was exploring that was actually Tom Maniatis at Cal Tech at that time. He was building something that he was calling genomic libraries in phage in bacterial viruses.
About once a week, I would go up to Tom's lab and talk to Dick Lawn and other people that were there. Extremely generous group. They loved to talk. They gave me all the diagrams for the little apparatuses that they were building. I reproduced a mini Tom Maniatis bench in my lab just to clone the growth hormone promoter. That was an all-consuming project for a few years, in which we cloned the response element.
Now that was an era where there were no response elements mapped. There were no such things as enhancers, tissue-specific enhancers. It was a very early day. We believed they existed, but the techniques were just being developed to find them. As an aside, as we were doing this project, I went up to Seattle to visit Richard Palmiter—he and Ralph Brinster were developing a technique that would later be called transgenic, where you inject DNA into fertilized eggs to make transgenic mice.
Richard said with the growth hormone gene that I was talking about at the time, maybe we could inject that with a link to a promoter that he was trying to study, the metallothionein gene, and create a transgenic mouse that expressed growth hormone. We'd discussed this idea and decided to initiate this collaboration, which in 1982 produced a rather spectacular mouse which they called the super mouse. This was a growth hormone-expressing mouse, and that was an exciting project for us because it proved that you could manipulate physiologic systems in predictable ways with molecular techniques. It opened up a whole set of technologies which have transgenic approaches...now become very widely used since then. But it was an important step for us conceptually, being involved at the very first steps of transgenesis. Later on, that would help us map the regulatory elements of the growth hormone gene.
Darnell: I don't remember—has anybody actually studied the stages at which that mouse is enlarged and the effect on embryology of the development of the large mouse? I don't remember it if it's been done.
Evans: Yes. It is mostly postnatal because the metallothionein gene is expressed at its highest levels postnatally and because it's inducible by zinc. We fed the animals on water that contained zinc. Now we did start feeding the mothers on zinc and the pups were a bit larger as well. In fact, Ralph Brinster said he could tell which of the animals were transgenic because he said they're clearly different. And he wrote down a code—I wrote down the code of which animals were positive by a gene hybridization analysis. And the code matched exactly. So all of the animals with the growth hormone gene were growing much more rapidly from a very early age than their litter mates.
Darnell: So there is a normal function for growth hormone early in development, I suppose?
Evans: We did not move it very far back into the embryo. But certainly from postnatal early days, it's very effective.
Darnell: So the regulation of this growth hormone gene became the central interest of the lab. And that's going to lead, I can see, into what's made you, if not very rich, rich and famous. So tell us about the control of that gene and how that got you started on steroids and steroid receptors.
Evans: Well, at that stage, Bill Rutter and others were studying the insulin gene, looking for what became known as enhancers that targeted gene expression to individual tissues. And about the time he was doing that, we—and along with Geoff Rosenfeld, who at that time was on sabbatical with me in studying prolactin—we kind of joined our forces on growth hormone and prolactin—and found a pituitary-directing type signature in the growth hormone promoter. Marcia Barinaga, my very first student ...
Darnell: She's become an important science writer...right, right.
Evans: She became a science writer in California working for Nature and then Science magazine. She deconstructed the growth hormone promoter and found a glucocorticoid response element and thyroid hormone response element, which became a detailed part of our work. Her thesis was in the work that we were doing at the time. It was at that point that we had the promoter mapped, a number of regulatory elements in place, that I realized we were running into a little bit of a conceptual brick wall. What we really wanted was to get the proteins, the presumptive proteins that actually bound to these sequences and controlled it. Our ideas were pretty much directed by lac operon, that gene control was mediated by simple molecules, possibly in a simple molecule, such as the glucocorticoid receptor. They would simply magnetically attract RNA polymerase.
Darnell: Were these the first hormone response elements, do you think, that people recognized?
Evans: No. They weren't the first...so in the preceding, actually several years, Keith Yamamoto, Miguel Beato and a number of other people had focused on identifying in the mouse mammary tumor virus long-term repeat—clear glucocorticoid responsiveness in the retrovirus. And based on their studies, it seems very clear that the ability to endow a gene to respond to glucocorticoids was mediated by a relatively small set of sequences. The key to be known was the glucocorticoid response element, or GRE. And we found such a sequence in the growth hormone promoter.
Darnell: At that point, it was not clear what the sequence consisted of, as I recall.
Evans: This was a very active area, trying to map hormone response clones. Because these were one of the few sets of sequences that clearly allowed genes to be controlled through a signaling pathway. There was a consensus that was being built by Beato and by Yamamoto and others. It was very hard to test, however, because it did not have mutational techniques at that point, site-directed mutations. We didn't have ways to reinsert these punitive response elements back into reporter promoters.
Darnell: So this was '83, '84?
Evans: Eighty-two, '83, '84. At that time, in my renewal of my NIH grant, I just decided the next step is to actually try to clone the glucocorticoid and thyroid hormone receptor. We would not spend our time characterizing their response elements because enough people were doing that. We had to go to the next step. And that was a rather bold move at the time because there were no transcript factors that had been cloned. So setting out the strategy for that was one challenge and actually doing it was an even bigger challenge.
Darnell: So integrate what you knew of Elwood Jensen's work at that time with your thinking about trying to get this protein.
Evans: Well, (Laughs) I didn't really...honestly, I did not know much about Elwood Jensen at that time. Because Elwood, co-recipient of this prize, was an endocrinologist. He was approaching the problem of steroid signaling from the endocrine point of view. And he had laid out the concept that perhaps the mechanism of action of steroids was through gene control. It was controversial. There were many people who felt that steroids acted in the cytoplasm to increase protein synthesis directly. Elwood said he thought it acted in the nucleus. And so, it was still a bit of a confusion.
The studies by Yamamoto and others in the mid-70s redirected our thinking towards transcriptional control, and I was convinced that it was a transcriptional based process. I knew about Elwood Jensen, but it wasn't highly influential because I was coming at it from a pure transcription RNA initiation template-driven molecular biology point of view. The importance of the physiologic pathways would later play a very big role in my research. And even at that time with the growth hormone work on creating the super mice, I was trying to integrate the physiology of these hormones with the molecular process of transcription. But it was all focus—I was really staying on message at that point in the lab. And this was a lesson from Joe Goldstein, who was a visiting faculty member at the Salk for many years—every time he would visit, he basically would say stay on message. Stay focused. Don't try to do too many things. And one of the things that we were trying to do was clone this transcription factor.
Darnell: Did you actually ever try to purify the protein that you were ultimately going to try to clone?
Darnell: Gustafsson is in my memory as the one who—Jan Ake Gustafsson from Sweden, maybe he did the first purifications that took it considerably beyond Jensen's work. Is that right?
Evans: Jan Ake Gustafsson had a very sophisticated purification effort going to attempt to purify enough of the receptor, which could be labeled with a radioactive steroid, enough to characterize biochemically. He was able to do a bit of that by proteolysis, show that the hormone binding domain was resistant to enzymes when the hormone was there and was a smaller part of the whole protein. He thought there would be a domain that was specific for ligand and binding, which was correct. But there wasn't enough in the material that he was purifying to actually microsequence and characterize. There was enough to show that his material could bind to DNA. Demonstrating that this parent pure factor that he had contained two linked domains: a DNA binding domain and a ligand binding domain. That was the implication from the study.
Darnell: And so, this is the mid-80s, mid-70s, when...
Evans: This is about the mid-70s. And he was one of the real leaders in that field, as was Keith Yamamoto for helping the map to response elements.
Darnell: So bringing us back to the mid-80s, when you were starting your attempt to do the cloning, what made it successful? Tell us about that.
Evans: There were a group of people who had other probes and these were immunologic probes to the glucocorticoid receptor. Brad Thompson and John Cidlowski independently had some very good antibodies.
Darnell: How did they know they had an antibody to the group of glucocorticoid receptor?
Evans: Well, they had used purified receptors, they were using radiolabeled glucocorticoids to purify the label that was bound to the protein. It's pretty much known that the receptor was a 90 kilodalton molecule based on covalent linkage of a ligand that could chemically react with the protein. So they purified and made an antibody to it. What we were doing is creating phage expression libraries that would express snippets of protein. And screening those libraries with the antibodies from these two labs hoping to find that one portion of the protein that matched the antibody and hoping that the antibody was correct—and using that to ...
Darnell: It was a bit of circularity in this which...
Evans: There's a lot of circularity...
Darnell: ...which disappears upon completion of the project, but it is...
Evans: Right. A lot of assumptions in many different groups sort of fell on their own pitard on the circularity of these arguments. A lot depended on the quality of the reagents, and also the quality of the people that were committed to doing this. At that time, it was very hard, and I had another individual that you know very well, Cary Weinberger, who I first met in your lab as a technician and actually went to graduate school at Stony Brook, joined my lab as a postdoc. His job, which he naively accepted, was to try to clone the glucocorticoid receptor. He and another student that I had, Stan Hollenberg—tremendous workers—made libraries and screened, and I think on our fourth or fifth library, about ready to give up, we screened a few more million clones and Carey found one film with a dot on it that he said, "This is it."
I should say, at that time I was still working in the lab. I was still in the cold room trying to purify mRNA for these libraries, and there was a real team effort. Cary was right. The faint signal was in fact the first signature we had of the glucocorticoid receptor. We knew it because he took that clone as pure DNA and we hybridized RNA to it. We asked what it picked out. And we took that RNA and translated it into the protein and then used our antibody and the protein was the right size—it reacted to the antibody. So we thought we were on the way.
But the hybridization technique showed us it was a very large mRNA. The cloning task was going to be arduous, but we accomplished it actually with some help from Paul Berg at that time and a fellow in his lab named Okayama, who's had secret techniques to make big clones. And we worked with them and managed to eke out a nearly full-length clone.
Darnell: From which cells?
Evans: Cells were...were called IM-90 cells. It's a human leukemia cell line.
Darnell: It makes glucocorticoid. Right.
Evans: It's responsive to glucocorticoids. And so we got the full-length human receptor out of this effort and we began sequencing it. Stan Hollenberg had set up DNA sequencing and we're off and running.
Darnell: This is '86? Or ... ?
Evans: ...'85. And in the summer of that year, as the sequence was pouring out, you have to remember, there were no databases at that time. In fact, I'm not even sure we had a computer in the lab yet. Everything was being done by hand. Gels were read by hand. Each base one at a time written down by hand and the rechecked secret—you know, in parallel blindly by others to work everything out. Russ Doolittle at UCSD was in the beginnings and he did have computers. He was in the beginning of developing databases.
At that time, the only way to develop a database was to read the literature by subscription to journals and copy out any sequence that someone was publishing. He had a full-time administrative assistant who was transcribing oncogene sequences. He was keeping his own oncogene database. We gave Russ our glucocorticoid receptor sequence and he quickly came back to us by saying it's clearly related to an oncogene called v-erbA.
In the avian erythroblastosis retrovirus...I was away at a Gordon conference and I got this call and the idea that the receptor was related to a known oncogene really transformed us at this point. First it said that the oncogene was probably a receptor; it told us there's probably a family of genes, it told us that these genes were potentially proto-oncogenes. So the hormone signaling in oncogenesis might be related to each other. We were very excited about this. I left the meeting immediately...
Darnell: There was plenty of reason, antecedent reason in clinical oncology, to be tuned into that notion.
Evans: Quite a bit of evidence suggests that hormone sealing could relate to cell growth in oncogenesis.
Darnell: In both breast cancer and prostate cancer, it was well-recognized by that time?
Evans: Particularly in breast cancer and prostate cancer. Both driven in large part by estrogen and androgens, respectively.
Darnell: Before going any further, we insert a 30-second lesson for any young people whoever see this. That was only 19 years ago. The mechanization of molecular genetics that has taken place in the last 19 years—breathtaking doesn't begin to capture it. It's amazing that you're telling us the story of writing sequences down and finally identifying it with the help of the sole person in the world that could help you identify it. And boy, how we have graduated in our understanding of things at this point. So much for the commercial for modernity.
So you had the glucocorticoid and you had already from Russ Doolittle's first suggestion the possibility that there was a family. Tell us about the advance of that notion and its importance.
Evans: Well, two things accelerated our thinking there. What Russ identified turned out to be the most conserved region of the future family, which was the DNA binding domain. It was cystine-rich. The cystines were in a signature distribution. Aaron Klug had just published a paper about the RNA binding protein, TFIIIA, [showing] that it contained something called zinc fingers. That's an interesting protein because it also binds DNA, and we suggest maybe the steroid receptors contained a type of zinc finger, which in fact ultimately was correct. These were zinc organized DNA binding domains that turned out to be unique to nuclear receptors. No other class of protein has this particular type of DNA binding domain. And that homology quickly became a hammer to open up the door onto the gene family.
Using the DNA binding domain, many bands started lighting up. It's a little bit like the Hubble Telescope, which if you use this as a probe, you can look back and back into homology and see faint bands. Sort of like the faint universes that are out there. And faint galaxies that are out there. In fact, this produced the superfamily, the nuclear receptor superfamily that was collected by us and others initially based on homology. I should say that Pierre Chambon had isolated the estrogen receptor shortly after we did the glucocorticoid receptor. And the estrogen and glucocorticoid receptors were fairly closely related, whereas the v-erbA product was clearly more distant. That suggested that it was not a steroid receptor but maybe a receptor for some other hormone.
Darnell: Just to recapitulate quickly, the v-erbA sequences, which Doolittle told you, partially matched your original GR sequence. And v-erbA, at that point, was not related to any known mammalian protein. But now you're going to tell us it turned out to be related. Go ahead.
Evans: It was not related to any known mammalian protein. There was a paper about that time in Nature that said it was related to carbonic anhydrases. Maybe the enzyme was oncogenic because it changed the activity of this enzymatic pathway, which was incorrect, obviously. It was oncogenic because we thought it was a mutated version of some type of signaling pathway. It was a very interesting challenge to figure out what this receptor would be. Because of our interest in thyroid hormone, we had ready labeled thyroid hormone in the lab.
Cary Weinberger, taking the lead on the v-erbA clone—the cellular homologue, c-erbA—had completely sequenced it and had for quite some time come actually to one of our data clubs, his data club, and said I think I know what c-erbA is. And he asked everyone in the lab to guess. We were throwing out every idea that we had that could be a possible ligand. No one said the thyroid hormone. He lifted up the screen and on the back of that was drawn the skeletal structure of a thyroid hormone.
And there was just silence. There were no boos or hisses, but he tried to justify why he thought it was that. A few weeks later, Cary went to a meeting in Europe where Bjorn Vennstrom was attending that meeting. And Bjorn was the first person to isolate the original avian erythroblastosis virus and characterize the v-erbA oncogene and the v-erbA oncogene, which incidentally, is the EGF receptor variant, with Mike Bishop.
Bjorn had of course seen the glucocorticoid receptor paper, and he also believed that the v-erbA gene product would be some potential hormone. He began looking for it. I really didn't know him at the time, but when he and Cary got together, he asked Cary what we thought it was and Cary said he thinks it's the thyroid hormone. And Bjorn stuck out his hand and shook it and said "You're right." (Laughter) Cary immediately was stunned. I think he called me in 30 seconds. He could barely talk. He wanted to get right back and I called up Bjorn, told him what was going on with us, and we agreed—he had a chicken gene, we had the human. We compared sequences. They were quite different. Ultimately, one became alpha and the other became beta because they were two thyroid hormone receptor genes, we now know. And the resulting papers were co-published in Nature later that year, one year after the glucocorticoid receptor.
Darnell: So you said something a couple of minutes ago that will take us to another aspect of this. The surprise was that the thyroid hormone, which is not a steroid, was binding to a member of the family which had the conserved DNA binding domain that you spoke of. And this must have opened up immediately the idea that there would be other kinds of ligands—that is, small molecules that bind to structures of this sort. And maybe you could just give us a brief discussion of that. Since we need to speed along a little bit here, tell us where that has all led with respect to the physiology of the different members that you have discovered and how they interact with each other.
Evans: Well, keeping this moving, it was an electric change for us because we no longer were looking at just a steroid signaling pathway; this began to create a universal signaling mechanism. I had not gone into the original project thinking that glucocorticoids and thyroid hormones acted through a common family, regulatory family, but in fact this proved that they did. That was very unifying. It changed the problem and brought the thyroid and steroid signaling together under one roof, which is why we called this a steroid/thyroid receptor superfamily. That led into considering other small molecules; the next thing was going to be retinoids might also signal through this pathway.
And probably the next big step which occurred in the next year was our isolation of the vitamin A receptor, the receptor for retinoic acid. Something that we did virtually the same week that Pierre Chambon did it as well. I remember when Vincent Giguere in my lab first did this, and he was literally walking on the ceiling with this discovery. Little did we know that Pierre Chambon was popping champagne as well in Strasbourg. And when we learned that, Vincent was right back down on the ground and back in the lab trying to get this paper out.
Darnell: Right. Eventually, it led to many other kinds of ligands, and it's been obvious to those attuned medically that we're talking about things that have a big impact on disease. The steroids themselves are physiological regulators, and abnormal events in these pathways cause cancer. So there has been a tremendous impact of the discovery of all of these factors, only a few of 50 of which we have talked about. But I'd like you to tell us what's happened in your own thinking and in your own laboratory that has brought you closer to medicine after studying all of these things for the last 20 years?
Evans: I think two events happened. One is that the superfamily emerged sort out of the ooze and declared itself as a real series of related proteins that are important signaling factors—not only for the steroids, thyroids and vitamin A—but as we discovered through the orphan receptors and through a process that we call reverse endocrinology, that is, using the receptor to find the ligand. We discovered new receptors for cholesterol, for bile acids, for fatty acids and for something called xenobiotics. In other words, this became a general family to control broad aspects of metabolism from sugars to salts to fatty acids and to cholesterol. It's our belief from all of that that the superfamily of genes are not only related to each other, but their functions are related. And that, in fact, physiology operates not simply at a transcriptional level, but that there's a higher level of integration that we need to understand about systems physiology—essentially, not each individual pathway but how the multiple pathways integrate. And I think that that logic is somewhere hidden in this entire family. There are a series of additional levels we need to understand about this control process. But fundamentally, it opened up a whole new world of medicine and physiology to us, and we began to explore that in earnest.
Darnell: So it's certainly fair to say that this group of transcription factors has reached the stage of being identified with respect to purpose, I suppose, but better than any other set of transcription factors. And those purposes, as you've just begun to state is that they're intimately connected with our every moment of existence. The balances that go on in the body that keep only homeostasis and normally are in the hands of the steroid receptor superfamily more than any other single family. Your career has been an amazingly productive one, Ron. I congratulate you on all of these last 25 years of great success and on the 2004 Lasker Award. Congratulations.
Evans: Thank you.