Interview How Cool Is That: An Interview with Caroline Dean

Jane Gitschier* Departments of Medicine and Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America

Having grown up in Pennsylvania, I recall that late winter’s delight in spotting a purple crocus piercing through desiccat- ed gray snow, a harbinger for warmer days ahead. The eruption of the crocus, the bloom of the magnolia, and the flowering of winter wheat are all examples of plant processes that are not simply delayed by winter, but indeed require a sustained period of cold to proceed. Today’s inter- view journeys into the molecular under- pinnings of one such cold-dependent process: ‘‘vernalization.’’ Our guide is Caroline Dean from the John Innes Centre in Norwich in the United King- dom, and our destination is an ,10-kb genomic stretch of the humble Arabidopsis thaliana, a small white-flowering bit of greenery also known variously as thale or mouse-ear cress. A little background is in order here, as perhaps you, like me, had never heard the term vernalization. During the develop- ment of flowering plants, shoot growth occurs at the apical meristem, an undif- ferentiated mass of cells that can divide to make more stems, leaves, or flowers. While some plants are ‘‘rapid cyclers’’—i.e., they flower quickly after a short growth peri- od—others require a duration of cold before they can flower, an evolutionary adaptation that allows them to resist flowering until the winter has ended. Often, varieties within a single species can exhibit one behavior or the other, such as spring and winter wheat, only the latter of which requires vernalization. Accessions of Arabidopsis vary in their vernalization requirement too, depending upon whether they grow along the Med- iterranean coast or above the Arctic Circle, for example. A few decades ago, when Arabidopsis Image 1. Caroline Dean. Photograph by Jane Gitschier. emerged as the plant of choice for doi:10.1371/journal.pgen.1003593.g001 molecular geneticists, Dean (Image 1) began to question how vernalization works at the mechanistic level: what genes are required for vernalization and how do vernalizing plants sense and remember the cold? She and her colleagues attacked the Citation: Gitschier J (2013) How Cool Is That: An Interview with Caroline Dean. PLoS Genet 9(6): e1003593. problem via three genetic routes, all of doi:10.1371/journal.pgen.1003593 which ultimately converged on the regu- Published June 27, 2013 lation of a single gene, FLC (FLOWERING Copyright: ß 2013 Gitschier. This is an open-access article distributed under the terms of the Creative LOCUS C), whose gene product is a Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, repressor of downstream flowering genes. provided the original author and source are credited. The ‘‘overwintering’’ accessions respond * E-mail: [email protected]

PLOS Genetics | www.plosgenetics.org 1 June 2013 | Volume 9 | Issue 6 | e1003593 to cold by epigenetically silencing FLC, of a genetically engineered organism. John months of cold. It is an evolutionary enabling flowering to occur. This silencing offered me a position and I grabbed it. adaptation to make sure you don’t fully involves a conserved Polycomb complex Gitschier: Very exciting time. flower until spring. We still don’t know and associated PHD (plant homeodomain) Dean: You know, it was a dream. I really much about all of those processes proteins that modify histones at the FLC arrived in February—[in England] it was except for vernalization. locus. The FLC locus also produces cold, very dark, I had hardly any money. Vernalization is very important for many antisense transcripts known as ‘‘COOL- Then suddenly, here I was in California. crops, and winter and spring varieties have AIR,’’ which differ in their splicing and They loaned me a car. The salary seemed been bred independently. All the cereals— polyadenylation sites, and these assist in enormous. And sunshine! barley, wheat, rye—have winter and spring locking down FLC transcription in the Gitschier: Actually, our paths are varieties that the farmer sows either in cold. Once FLC is silenced, flowering can quite similar because I also did a postdoc October or in March. And a lot of the begin in earnest with the advent of longer in California with a young company at Brassicas, of course, most of the vegetables, and warmer days. These epigenetic chang- about the same time. It was the best four actually have a vernalization requirement. es are maintained throughout future cell years of my life, without a doubt. And what breeders have done is to breed divisions and even persist in cuttings Dean: Yes. Coming from a small plant for the need for different lengths of cold, so regenerated from the vernalized plants so lab in York, suddenly being exposed to this that you then have a nice supply for the that they do not need to be re-vernalized. amazing amount of information and supermarket year-round. However, when a seed is being produced, technology and ‘‘can-do’’ attitude, I was Gitschier: Well, the old-fashioned FLC silencing is removed so that the next just transformed bang! into a new way of cauliflower, before anybody bred in any- generation of plants also requires vernal- doing science. thing, when would that have appeared? ization. We had five successful years establishing The spring? I learned about Dean’s work just as I the fundamentals behind genetic engineer- Dean: Have you ever left a cauliflower was heading to the UK and quickly ing. And then, slowly but surely, financial or broccoli too long and then it’s sprouted? arranged to visit her on a Wednesday reality set in for these biotech companies. You actually get a beautiful flower coming afternoon in November. It was certainly The venture capital money ran out, and out of the top. my most active interview to date, as we research contracts with very specific goals Gitschier: No, but I’m keen to do that scooted out to the ‘‘barns’’ to see Arabi- replaced it. At that point it was a ‘‘we’’ experiment! dopsis and its Brassicaceae cousins—broccoli, rather than a ‘‘me,’’ so we looked around Dean: So, it’s an arrested meristem, the rapeseed, cauliflower—in all manner of for academic positions, and we took two bit we eat. leafing and flowering. This involved some here in Norwich. It was home, but also the Gitschier: The bit we eat is an robing and disrobing of bright red lab Sainsbury Lab had just been set up. arrested meristem! coats and hairnets with much humor along Jonathan [Jones, Dean’s husband] got Dean: Cauliflower is an earlier arrest the way. It has taken me a little while to the position there, and I came back to than broccoli, sort of little stubs, and if you digest this complex subject, but following the position at the John Innes. leave them longer, each one of the many its own ‘‘overwinter,’’ the interview has Gitschier: For me, going back to buds turns into a proper flower. now gone on to flower. academia from industry was appealing Gitschier: Cool. Gitschier: Let’s start with your choice because it was a new kind of a challenge. Dean: For the last couple of years, we’ve of a postdoc and how you got involved in In industry, I felt I knew what was had very extreme winters, but not always at the question of vernalization. expected of me, whereas in academia, it the same time. Last year after Christmas we Dean: I started off doing plant biology was a question of how creative I might be. had very, very cold temperatures. The year at York University. I did my PhD work on Dean: Yes, that’s right. I started to plan before, there was a famous satellite picture chloroplasts and then I wondered what on what to do in my own lab, and the answer of the UK all under snow before Christmas. earth I would do as a postdoc. My came from a sort of bizarre experience in Too much cold at any point can mess up supervisor had been on a sabbatical to California. I wanted to have a few bulbs in the scheduling of the broccolis and the Riverside [at the University of California], my apartment. So I bought some tulips, cauliflowers because they vernalize way too and she raved about it. I remember and the sales chap said to me, ‘‘Now don’t quickly and come into heading at about the thinking gosh, it sounds really exciting forget to put them in the fridge for six same time. So suddenly there was a glut of over there. weeks.’’ Of course, in England, you sow cauliflower and then months when there It was just at the time—1982—when it them in the ground in September, Octo- was no production in the UK after that. was discovered that a piece of the DNA ber, but you never think ‘‘why.’’ This was Gitschier: Ah, so it’s not just the from the plasmid in Agrobacterium [tumefa- how I learned about the requirement for duration of the cold but also the actual ciens] could be inserted into the plant prolonged cold for many plant processes: temperature. This is very complex. chromosome. So, genetic engineering was flower emergence in plants like tulips, Dean: It is. The current thinking born. A lot of money went in from venture breaking bud dormancy in trees, and describes vernalization efficiency in terms capital to start up new biotech companies, vernalization in many plant species. of the ‘‘day degree’’ exposure, but this is and John Bedbrook from New Zealand— Now vernalization is not quite the same poorly understood. We also don’t know the person who had cloned the first plant process as the need for cold in tulips. In the temperature optima for the different gene—was persuaded to start one of the the [tulip] bulb, the flower has already varieties—they are probably different. new companies in Berkeley: Advanced initiated before the cold, but its growth is Gitschier: Well that triggers a pressing Genetic Sciences. AGS is famous for arrested; the low temperature is important question, but before I ask it, let’s discuss putting engineered bacteria on strawber- for stalk elongation. A similar break in late the molecular definition of vernalization. ries as an ice-protection measure called developmental arrest by prolonged cold Dean: For Arabidopsis, vernalization is ‘‘Frostban.’’ It created a big stir in also occurs in the case of spring blossoms the process by which FLC—a floral California because it was the first release in trees. In each case you need a couple of repressor gene—becomes epigenetically

PLOS Genetics | www.plosgenetics.org 2 June 2013 | Volume 9 | Issue 6 | e1003593 silenced. FLC is one of many factors that Gitschier: Are people working on from plants from Northern Sweden. With- regulate when a plant flowers. that? Are there tulip molecular biologists? out cold, it is such a strong brake; they just A good analogy is to think of FLC as a Dean: There are people looking at a stay vegetative without cold until we car brake. You’re in a car, you have your similar question in trees. Many buds in vernalize them. foot hard on the brake, you won’t move— trees are arrested in autumn, and they Gitschier: This is great. And ‘‘we you won’t flower—even if you are pressing have gone in and said, ‘‘If I knock out the vernalize them’’ how? the accelerator with long day photoperiods Polycomb, do I change bud dormancy?’’ Dean: I’ll show you the vernalization and warmth, you’re not going to flower. And yes they do. So it is clearly following room. Butoverwinter,slowlybutsurely,youtake the Polycomb paradigm. They don’t know Gitschier: Now what is going on with your foot off the brake. It takes a long time. the target, but they know the mechanism. these? They look like vines. When you come into spring, if you haven’t OK, it’s getting dark, time to go to the Dean: When Arabidopsis flowers, it got warm days, you don’t actually flower, but barn! produces an inflorescence from which the you have the ability to flower because you are [My pressing question will have to wait! flowers emerge. They self-fertilize and now not havingafootonthebrake.Nowyou We take a brisk walk to the barn. Upon seed is produced. So what someone has wait for the photoperiod to get longer, you arriving things get animated.] done here is stake them up because they have temperatures increase, now you are Dean: We have to put on… are trying to collect as much seed as putting your foot on the accelerator. Gitschier: Oh, we have to suit up to possible. Vernalization, then, is to epigenetically see the plants! Gitschier: How did you get interested silence FLC. Dean: We do! This is for your head [as in Arabidopsis [as we meander]? Gitschier: Equivalent to taking your Dean hands me a hairnet]. We had a Dean: I heard Chris Somerville’s foot off the brake to flowering. terrible time with a virus which is carried postdoc, Jose Martinez-Zapater, give a Dean: Yes. And the interesting thing is by a little insect called a thrip, which of talk in 1987 about late-flowering mutants that it is a quantitative effect. How can one course gets in your hair. in Arabidopsis. But actually, if you vernalize week of cold not be as good as two weeks Gitschier: Oh God, I don’t want that. these late-flowering mutants, they flower or three weeks? That has been a mys- Dean: Well, we’re preventing the thrip early again! As soon as I heard this, I tery—how can you quantitatively silence getting in! We’re protecting the plants! thought, ‘‘This is how I can dissect something? It’s because of progressive [Next we meet George.] vernalization. Here is genetic variation switching of bistable epigenetic states, This is Jane from San Francisco and that will be my entry point.’’ That from one that promotes expression to we’ve come to see the plants. This is recessive vernalization requirement turns George Lomonossoff. out to identify the ‘‘autonomous’’ path- another associated with silencing after cold Now George, once we get way: Loss-of-function of a whole set of exposure. Gitschier: suited up, will you take a picture of the two genes—FCA, FPA—causes late-flowering We showed that the cold causes a slow of us together? through de-repression of FLC. This path- accumulation of PHD proteins at the FLC Dean: You have to promise me—there way involves alternative processing of the locus. These proteins associate at one site is no way we can have this published! FLC antisense transcripts. in the FLC locus with the Polycomb Gitschier: I promise you. [This is so This mechanism contrasts with the repressor complex 2 [PRC2], which is much fun!] dominant vernalization requirement that already poised at the locus. We think the George: OK, smile. we identified through analysis of natural PHD proteins somehow supercharge the [Click. We enter the barn.] variation in flowering time in Arabidopsis. activity of PRC2, just as Juerg Muller, Gitschier: Oh my God this is beauti- This involves a protein called FRIGIDA working on an analogous complex in ful! I want to work here! Now, does the that upregulates FLC. The rapid-cycling in vitro Drosophila, finds increased activity barn house your research only? Arabidopsis types have mainly arisen of the methyltransferase if a PHD protein Dean: No, as we go round we’ll see lots through independent loss-of-function mu- associates with PRC2. And the longer the of other things. We have petunias because tations at FRIGIDA. plants stay in the cold, we see more they are very, very sensitive to the virus. And then our third tack was to muta- methylation of lysine 27 on histone 3 They will come down before the Arabidop- genize vernalization-dependent Arabidopsis [H3K27me3, the classic histone modifica- sis. They are sentinel plants. and look for mutants that no longer tion associated with Polycomb silencing] at Gitschier: Oh, I love that! OK, let me vernalize. These were the VRN mutants, that site. This probably explains why just take a picture of a sentinel petunia. and that’s what got us into the Polycomb vernalization is so slow and why six or [Click.] world. eight weeks of cold gives more H3K27me3 Dean: These are Arabidopsis. There are Gitschier: It’s amazing, actually, that at that site compared to say two weeks. winter and spring types, you see. Some of it all points to this one gene, FLC. Once plants begin to feel warm again, them are very late flowering, some are Dean: It is! Slight changes in FLC the PHD proteins zipper along the whole early flowering. Look at these for late expression affect the flowering time, and length of the gene, increasing H3K27me3 flowering: they are so late! They keep any change in flowering time affects seed along the whole gene. This is needed for making more leaves! Instead of just yield or fitness, so the trait will be under mitotic stability of the silencing through making five or six leaves and then enormous selection. the rest of development. flowering, they make over a hundred [We leave the Arabidopsis part of the Gitschier: So, for the tulip bulb, say, leaves before flowering. They are so barn, crashing into a science review panel you’re slowing down not necessarily this squished. who are having entirely too much fun.] FLC switch, but something else. Gitschier: Now what is the gene that Gitschier: There is a party out here! Dean: Yes, it would be a different is wrong with them? Dean: This is Jane from San Francisco, regulator, but probably via a similar Dean: FLC! These are transgenic from PLOS Genetics. This is half the science mechanism. plants where we added the FLC allele review panel from BBSRC!

PLOS Genetics | www.plosgenetics.org 3 June 2013 | Volume 9 | Issue 6 | e1003593 One of the panel: And you’re lovely! antisense RNAs from the FLC locus, and I function in the autonomous pathway, [It’s the hairnets, I’m sure. We move on to think in the end, when we really get down independently of vernalization, target cauliflowers and strip away our barn to the ‘‘what is it that perceives the COOLAIR too. That was a total eureka accoutrements.] temperature,’’ it may well be an RNA day. Gitschier: We don’t need red coats for thermosensor. Gitschier: When was that eureka day? the cauliflowers? Gitschier: That would be something! Dean: About three years ago. Dean: No, just for the Arabidopsis. Here RNAs altering their secondary structure in Gitschier: It is really heating up! we’ve got rapeseed, Brassica napus, which response to temperature and these changes Dean: It is really heating up! And gives you those wonderful yellow fields. It having some mechanistic effect on the suddenly noncoding RNA is everywhere. I is a very close relative of Arabidopsis. locus. think that’s why FLC regulation is intrigu- Gitschier: What about these guys? Dean: There are clearly several differ- ing to other scientists, because it’s provid- Dean: These are Antirrhinum—snap- ent temperature steps that directly target ing some important concepts for con- dragon. FLC. So another question is how do these served mechanisms. We have lots of And here is broccoli. The breeders have different thermosensors integrate different genetic tools and also have the natural made hybrids and selected ones with aspects of that temperature? variation that allows for molecular and varying requirements for cold. We are Gitschier: How did you discover these biochemical dissection. now figuring out that certain combinations antisense transcripts? Gitschier: So, this temperature ther- of genes have been selected. By knowing Dean: We wanted to identify an early mometer then involves specific sequences all this, we can develop varieties with cold-induced step that occurs before the in these noncoding RNAs? particular cold requirements. And the induction of PHD proteins. Given the Dean: Well, that’s my favorite hypoth- same for cauliflower. excitement of small RNAs in chromatin esis. We are exploiting natural accessions And this is the vernalization room. regulation, we asked whether there is a which are adapted to northern Sweden [Ah! The mecca. Like any cold room, role for noncoding RNA in vernalization and need twelve weeks cold compared to but very cold and very dark.] too. So we made a custom array of FLC,a only four weeks needed by accessions Gitschier: Yikes! How do people work high density, single-nucleotide resolution further to the south. Big, big difference! in here? custom tiling array that included FLC and We’ve done the genetics and mapped the Dean: Well, they don’t really. The neighboring genes, and probed that with lights are on only until 4 o’clock. different RNAs extracted from plants at determinants of the time difference down Gitschier: How do these things even different stages of vernalization and of to the FLC locus. In fact, it’s down to four exist over the winter anyway? different genotypes. noncoding polymorphisms close to the Dean: They are amazingly healthy Gitschier: And you tiled both strands? region that this PHD-PRC2 binds to in under the snow for months. I am fortunate Dean: Yes, because we didn’t really the cold. We’re now looking into whether to be part of a big field experiment in know what to expect. these polymorphisms affect COOLAIR Sweden, and each year, I wonder whether Gitschier: But how did you know secondary structure. Could that then these plants are going to survive under there was even going to be a noncoding somehow influence the association of these snow for four or five months. But they do. RNA in there? different Polycomb complexes? Gitschier: Now, that is one cold room! Dean: We didn’t! But it just jumps We have a lot of experiments going on [As we exit and head back to the lab.] straight out. There is a massive upregula- in Sweden, where we’ve asked: if you plant Dean: You don’t want to be in there tion in antisense transcripts in plants with in the wrong place, can you really show for very long. two weeks’ cold. [She shows me the data.] that all of the variation, say from those Gitschier: And now for my pressing Gitschier: Wow! four polymorphisms, is adaptively impor- question! What is it about the cold? How Dean: And we called them ‘‘COOL- tant? And I think the answer will be does the cell recognize cold? AIR’’ transcripts, based on the ‘‘HO- ‘‘Yes.’’ We can get at the really detailed Dean: Well, that’s still one of the big TAIR’’ transcripts [‘‘homeobox transcript mechanism that is underlying the little questions. There are many different, from intergenic regions’’], which were changes that have allowed these things to independent temperature steps involved named in Howard Chang’s lab at Stanford adapt to very different places. in FLC silencing and we’d very much like and are involved in mammalian Polycomb To go out and actually see it in the field, to figure it all out at the molecular level. If silencing. We thought, ‘‘How funny!’’ to understand the arrangement of the I had to guess, it could be secondary It turns out the COOLAIR transcripts are alleles with the environmental conditions structure of RNA, which is involved in the also important in regulating FLC in the and their fitness consequences: that’s what core chromatin mechanism. There are warm. And the 39 processing factors that I love.

PLOS Genetics | www.plosgenetics.org 4 June 2013 | Volume 9 | Issue 6 | e1003593