Life with two X chromosomes | 91TV
Transcript
- It's a huge honour to be awarded this medal. I suppose what I'm going to do today is take
- you through, what life does mean with two X chromosomes. I know it's an intriguing
- title. So some of you might be wondering, is she going to talk about her career, herself?
- I'm lucky enough to actually work on the process that involves dealing with two X chromosomes.
- I thought I would start just by giving you a short tour of my recent past. So I spent
- much of my career having studied in the UK. I was brought up in the UK, in London actually,
- and studied at Cambridge. I moved to Paris in 1990, following the person who's very important
- in my life and who's sitting here, Vincent Coelho, and I moved to the Pasteur Institute initially,
- where I did a postdoc, and that's where I fell in love with X inactivation,
- the process that I've worked on for pretty much the rest of my career. I spent many
- years after that at the Curie Institute, which was an incredible institute in terms of doing
- basic research in an environment that allows you to apply it to human disease and cancer,
- as it's linked to a cancer hospital in a very interdisciplinary way. It was during that period
- that I actually became elected as a professor at the College de France, which is a very unique
- institution, not everyone has heard of it, but I really emphasise that it's probably unique in
- the sense that it was set up in 1530 by a King to sort of challenge the church and provide education
- to people outside of a religious context. It's a place that really does seek to transmit knowledge
- to the general public. So every year, I have to give lectures in French to the general public,
- and they have to be different every year. So I have to admit that I think that's actually been
- a huge factor in my career, because it's really forced me to constantly think about different
- things and challenge myself. I always say that it was because of that, that probably I decided to
- move to EMBL and become the director general there and come up with a programme that David mentioned,
- molecules to ecosystems. It was very much thanks to the fact that I had to do this teaching at the
- at the College de France. So I moved from Paris to Heidelberg in 2019, and my last
- day as director general was actually just last Monday. So it's been an amazing journey. I think
- it's an incredible organisation and in these times of geopolitical anxiety and distress,
- I think infrastructures and intergovernmental organisations like EMBL are even more precious
- than ever before. They're there to deal with the challenges that are thrown at us,
- and as scientists, we have a duty to make sure that things continue forward, and EMBL is really,
- I would say, unique in Europe in terms of molecular life sciences in doing that. Indeed,
- as was mentioned, I will be moving in September to be the new director of the Francis Crick
- Institute, an institute that's close to my heart because in fact, my PhD was at the ICRF, which
- is one of the three institutes that led to the creation of the Crick. I'm very excited because
- not only does it mean that I'll be coming back to London, but also that I will be surrounded by
- many, many important and cherished colleagues and scientists here in the UK. I want to say one word
- about a programme that I was involved in, that I helped to set up when I was at the College de
- France in 2017. It's called Pause. I mention it because when I was awarded or I was informed that
- I was getting the Croonian medal, I was asked, what would your message be? At the time, this
- was actually last year, we didn't realise what was going to happen in the world with the arrival of
- Trump. President Trump has had an impact that I think highlights how important it is to preserve
- the freedom of science. So this organisation was set up actually to help scientists in exile,
- refugees, people who have to flee their countries because of political distress, but also war,
- and indeed Pause, I think, is exemplary of what we need to do to make sure that science without
- frontiers can continue despite the challenges that we're facing right now in the world.
- So before I move to life with two X chromosomes, I also wanted to just express my deep thanks to
- my lab. Now, this isn't my lab, actually, you'll be pleased to hear, this is actually a farewell
- that happened at the Curie Institute when I moved, in 2019, when I moved to EMBL. Actually in here,
- there are many of my lab members, alumni, but also many of my mentors, the people who've inspired me.
- Some of them are actually sitting also here in this audience, and some of them are actually in
- the photo. I have to say that as a scientist, I think success has to be measured by the
- degree to which one can transmit knowledge and enthusiasm and inspiration to others,
- but also this is how one becomes a scientist, and I really want to give my deep thanks to all those
- people who've helped me in my career, and also to the many institutes that have hosted me and
- allowed me to thrive, and of course, my funders as well. So now to sex chromosomes, and maybe I
- should just start by saying that sex really does matter. It's important. It's important because
- sexual reproduction allows for the generation of genetic diversity, allows for the loss or
- elimination of mutations, it allows species to evolve. Organisms that do not sexually reproduce
- are usually evolutionary dead ends, and in the case of many organisms, sex is defined by sex
- chromosomes. I'm particularly interested with one of the sex chromosomes in mammals, the X, but I
- wanted to take this opportunity to just highlight how if you do have sexes, the differences between
- them can be incredibly striking. So here on the left - let me see if I can use this - you can see
- how extremely dimorphic sexes can be. In this case you can see the flamboyance of the male and
- the rather less flamboyant, rather small and dull looking female. But then in other species, you can
- see the female is huge, the male is tiny, and this can go to an extreme. Here you can see these are
- deep sea water fish where the female is actually totally the giver to the male, the male is almost
- a parasite, has to hang on to the female for food. The only thing the male produces, of course,
- is the sperm. That's it. So you have incredible differences in some cases, and in the case of
- mammals, we're actually not that dimorphic. We're not that different, as you can see,
- but there are some differences, and in particular the differences that one finds in females, due to
- the fact that females have two X chromosomes and that this actually is something that has
- to be dealt with by shutting down one of the two X chromosomes, does lead to sexual dimorphism that,
- again, can be quite striking. As you can see here in the case of a calico cat. So a female carrying
- an X-linked coat colour gene that gives you orange or black fur, you can see these beautiful patches
- of orange and black. Whereas a male will either be black or orange, he cannot be both. I'll come
- back to this in a moment, but in any case, I want to just highlight that although sexual dimorphism
- is striking, as we can see it at the level of the whole organism, we're now starting to understand,
- because we have the tools as scientists, that this kind of dimorphism is present at the molecular and
- cellular level. If you go in and you look at the cells that make up a male or a female, they are
- different. They're different in the genes they express. They're different in the way they can
- deal with certain changes such as hormonal stress, etc. So basically, the differences that are due to
- sex are way beyond what one sees on the surface, so to speak. I want to just take a moment to
- dwell on this, because in fact, when we think about female biology, human female biology, human
- health, in fact females have been tremendously understudied. This is due to centuries of, for
- historical and societal reasons, but centuries of misconceptions and actually misinformation, books
- have been written on this. The reality is that we've only started to understand female biology
- and thus female health and medicine in the last century or so, and now we understand that indeed,
- the sex based differences manifest in the clinic, in epidemiological studies, and also in the course
- and therapy of most diseases, there are sex differences, and it's only very recently that
- clinical trials and research have started to be inclusive of both sexes. Up until very recently,
- males were systematically used, not just in clinical trials when it comes to humans,
- but also in models. Even cell models tended to be focused on male cells, not female cells. Again,
- the reasons for this are multiple and complex, but it's a truth, it's a reality. So although
- we know that sex differences manifest at many, many different levels, we still don't actually
- understand what this is about, what the causes are, and we realise that many of these differences
- will be important if we want to try and understand male and female biology. So of course,
- a lot of this is to do with the hormones, males and females do express different hormones or
- different levels of hormones, but also it's due to genetic differences and this brings us back
- to the sex chromosomes. These differences are not just in the gonadal tissues, the tissues that are
- involved in reproduction, they're also important in nongonadal tissues. Of course, there are other
- factors that play into differences both sex and gender, and these can be the societal differences,
- the environmental factors we're exposed to, our lifestyle differences, and all of this deserves
- to be studied and this is an area that I think is going to be burgeoning. I mean, it's already
- become a focus and hopefully some of the science that people like myself and my colleagues do is
- going to become more relevant than ever before. I want to just stress that in disease there are
- many, many diseases that show extreme bias and sex bias. So for example, autoimmune diseases are very
- often female biased. In some cases, such as lupus, it's up to 90 per cent of affected individuals
- are women. Then of course, infectious diseases also show differences, but then some cancers,
- especially the non-reproductive cancers, show a much stronger male bias than in females. So what
- is all this about? As I said, some of it is hormonal for sure, and some of it is genetic,
- but we know that out of the 23,000 genes approximately in humans, up to 6000 of them are
- differentially expressed in men and women. So this is massive. As I already mentioned, most clinical
- trials have actually been performed only in men. So in fact, when women are treated, the doses
- that they're given are usually just adjusted for our size and our weight. So we're being
- treated simply as mini men, and of course this has to change. I should say that in the 1990s,
- early 2000s, eight out of ten drugs taken off the market in the US were due to the side effects that
- they had on women. So it's a huge economic effect as well for drug companies. So there really is an
- increasing interest. So now to move to life with two X chromosomes. What does it entail? It does
- have advantages, and actually impressively the tobacco industry figured this out a long time ago.
- This is actually a poster, I think it's from the 1970s for this make of cigarettes, Virginia Slims,
- which was made specially for women because they are biologically superior to men. That's right,
- superior. The reasons for this, they actually point out women have two X chromosomes,
- men only have a Y, which some experts consider to be the inferior chromosome. They're also inclined
- to things like baldness, improperly developed sweat glands, colour-blindness. So some of this
- is actually accurate. Some of it is clearly not. But in the end, what's important is that
- these slimmer cigarettes are much better for women than the fat cigarettes that men smoke. With rich
- Virginia flavour women like, you've come a long way, baby. Anyway, just so we all realise, life
- with two X chromosomes has clearly been in the radar of the tobacco industry. But more seriously,
- what do we know about life with two X chromosomes? Here, I'm just showing you the chromosomes,
- the typical chromosomes of a human, the karyotype, as we call it. For each autosome,
- and I'm trying to be both scientific and not so scientific in the words I'm going to use,
- but for each of these autosomes, there are two copies. The only chromosomes where this
- is different between males and females are the sex chromosomes, where you have the two Xs in females
- and only one X and one Y in males, and indeed, the Y is much smaller, and it does carry fewer genes.
- That doesn't mean to say it's inferior. It carries fewer genes, and the genes that it carries are
- essential for determining the male sex, so SRY, testis determining factor, and other genes that
- are involved in spermatogenesis, but the X is much bigger. In fact, the sex chromosomes evolved from
- a pair of autosomes initially and through this evolution, the Y progressively becomes smaller and
- smaller, carrying fewer genes and therefore much less homology with the X, and in fact, the big
- problem is that the X is a very big chromosome, carrying over 1000 genes as you can see here,
- or you can't see but you'll have to believe me, and many of these genes are essential for life,
- for cellular life, and the dosage of these genes can be a problem, and having a double dose of some
- of these genes, if you have two X chromosomes is a problem, because if you don't do something about
- it, life with two X chromosomes ends very early. In other words, if you don't somehow compensate
- for this dosage effect between females and males, a female embryo will die very early on.
- So in fact, this amazing process, which shuts down one of the two X chromosomes in mammalian females
- was discovered by mouse geneticist, Mary Lyon, many years ago. She, simply by studying
- the mouse mutants and the genetics of the mice that she was interested in, noted that indeed, in
- female mice carrying X-linked genes that affected coat colour and other features, she noticed that
- they showed these patches or variegated patterns where it seemed as if the mutation, if one copy of
- the gene was mutated and the other one wasn't on the two X chromosomes, it seemed like some
- cells would express the wild type copy, the normal copy, and other cells would not express it at all,
- but would manifest the mutated copy. So this is how you end up with these cells, with either one
- or the other. She proposed that one of the two X chromosomes must be silenced and the choice of
- which X, the paternal or maternal X must happen very early on during embryonic development, and
- one of the two, when it's silenced, then becomes stably propagated in its inactive state. So she
- hypothesised this purely based on genetics. There were also studies using cytogenetics that had
- already started to characterise the X or the two X's and their difference in females. In any case,
- she came up with this hypothesis of X inactivation and actually underlined that this was not just
- specific to mice. It was also found in other mammals, including humans, and of course, mammals
- such as cats. In fact, I skipped this slide, which is the paper that Mary Lyon published back
- in 1961, and in fact, there's a wonderful obituary that was written by Sohaila Rastan, who is here,
- who was actually the student of Mary Lyon. Sadly, Mary passed away in 2014 but she was an incredible
- inspiration and she definitely should have got this medal some time ago, but in any case, this is
- the paper that she published with this hypothesis and the data she had. I think for us scientists,
- this is awesome. It's one page. There are no figures. There are no supplementary figures.
- One page in Nature. Times have changed, but she was absolutely right. So what was a hypothesis
- ended up becoming a law several decades later, the Lyon Law. So this is just to illustrate what I've
- told you about the mosaicism, where female tissues have cells that are expressing either one X or the
- other due to this random X inactivation process. Here, you can see what's been done in mice is just
- to take what we call a housekeeping gene. This is a gene that produces a protein in all cells.
- So it's important for the life of all cells. So this gene is expressed in all tissues and
- it's been tagged on one X with a green fluorescent protein and on the other X with a red fluorescent
- protein. So females that carry one X, that's GFP and the other that's tomato, show these beautiful,
- variegated phenotypes that I mentioned earlier already. You can see in these pups,
- and these mouse pups are actually genetically identical other than just this modification
- in this gene that tags it with these fluorescent proteins, and you can see how different the pups
- are. This is because X inactivation is random and then the way cells migrate is also not
- completely identical. So you end up with a great deal of variation between female individuals
- who even when they're genetically identical, because of this process of X inactivation. Here,
- you can see these are the left and right retinas of the same mouse and you can see that some cells
- are expressing mum's X chromosomes, some cells are expressing dad's X chromosomes, and this is
- very different. The patterns are very different even in the same individual in the two eyes. So X
- inactivation mosaicism creates immense diversity between and within individuals, which is rather
- unique. There's another level of mosaicism and diversity that we find due to the fact that some
- genes on the X chromosome can actually escape this process. So although most of the 1000 or so genes
- get silenced during development and stay silent, it now transpires that up to 25 per cent of genes
- can actually come back on or stay on right from the start, in other words, don't get silenced and
- therefore expressed in a double dose. So about ten per cent of these escapees, as we call them,
- do this constitutively, in other words, they always escape, and about 15 per cent, and actually
- the percentage is going up the more we look using technologies that allow us to look at single
- cells, but in any case, there are many of these genes that can escape variably. They don't escape
- always, and they don't escape in all tissues, and they escape variably between individuals and the
- reasons for this variation, on the one hand, is due to genetic differences. In other words, there
- must be some sequences in these genes or around them that predisposes them to being inactivated
- or escaping, but there are also stochastic events that clearly take place and environmental events,
- and this is something that's increasingly being looked at, particularly by looking at cohorts of
- twins. Now, this escape from X inactivation has increasingly become of interest because it feeds
- back to what I said earlier about sex differences and sex biases, because as I've already told you,
- females have two X chromosomes, males have only one X and one Y, but of course, if the one of the
- two X's is completely shut down, then in fact the X chromosome that's active can be, if you like,
- the same both in females and males. The fact that there are many genes that can actually come back
- on or stay on shows that there is going to be dosage differences in the levels of protein that
- these genes produce. I just want to point out what these genes could be about. Now in some cases,
- these genes that escape actually have homologues on the Y, here you can see the Y,
- and there are some genes that still remain on the X. So for these, you don't actually need to do X
- inactivation. In fact, we think these genes are highly dosage sensitive and they have to stay on,
- a double dose is really important both in males and in females. Now for these other genes that
- escape, we think that this could be for many different reasons along the X chromosome. Some
- of this might actually be accidental. So in other words, some of these genes might get switched off,
- but then they show what we call epigenetic instability, they come back on. Some of this might
- actually be required. It might be purposeful. You might need a double dose of some products of these
- genes in XX individuals. So in other words, some of these escapees might lead to neutral effects.
- Others might be deleterious and others might be advantageous. The reason why people are getting so
- interested in them is because some of these genes, or many of these genes, are now being implicated
- in disease, and it's thought that they could underlie some of the metabolic diseases, immune
- diseases, neurological and other diseases, and in fact, in the case of lupus, which I mentioned
- earlier, TLR7 is a gene that's involved, a protein that's involved in the innate immune system, and
- it escapes X inactivation and it seemed to escape in females that show lupus. So it's really been
- very tightly correlated with the onset of lupus. So here, we're thinking that indeed many of these
- escapes might actually end up having roles in some of the sex bias diseases that I mentioned earlier.
- So what my lab has been interested in over many decades since I started working on X inactivation
- back in the 1990s, when I joined the lab of Phil Avner at the Pasteur Institute, is when,
- where, how, and why does this process happen? We didn't really know very much. Mary Lyon was very
- visionary when she came up with her hypothesis and actually immediately opened up a whole field
- of exciting research to find out what molecules actually trigger this process, but also how does
- it happen during development and how conserved is it. So just to recap for the non-scientists in
- this room, I just want to point out that early on in development, two X chromosomes, one from mum,
- one from dad are both present, and what happens then during development is the establishment
- of silencing of one of the two X's. As I said, it's usually random in most eutherian mammals,
- and once it's established, it's maintained. So you end up with these clonal populations expressing
- either one X or the other. So here I'm showing you the same gene, but each copy is slightly
- different. This time it's red and blue. So in some cells the red gene is on, the blue copy is off,
- and in other cells the blue allele is on, the red allele is off. So when I say epigenetic silencing,
- these are changes that we believe allow the stable propagation of an off state or an on state, and
- there are many levels at which these epigenetic changes could happen. So trying to understand how
- X inactivation happens is an ongoing adventure, I should say. Many people in this room are still
- doing it, and there's still many, many open questions, but one thing that was clear right
- from the beginning, and this actually preceded Mary Lyon's studies, was that the inactive X
- is special in the way it folds. It becomes more compact. It becomes what we call heterochromatic
- or heteropycnotic. So condensed chromatin. It was actually originally observed back in the 1940s by
- Barr, who was a Canadian geneticist. So how do you get from two chromosomes that are presumably
- both active and on, to switching off one of the two X chromosomes and keeping it switched off all
- through development and into somatic life? This is really a complicated problem, if you think about
- it, because the two X chromosomes are present in the same nucleoplasm, in the same soup of factors
- that could attack either of them, and yet one of them seems to become immune to the factors that
- will switch genes on and the other one continues. So how do you do this? Actually, many scientists
- over several decades mapped a region of the chromosome, genetically mapped a region of
- the X chromosome that is important for this, and actually one of those scientists is also sitting
- in this room, Sohaila. So the region that's key to triggering this process is known as the X
- inactivation centre. It's shown here as a box in red. This is a mouse karyotype and you can see
- a probe that we detect using fluorescent label, and in red here, these are the two X chromosomes
- with the X inactivation centre region detected in red. Within this region, people, including myself
- actually, had been hunting for what could the key gene be that could trigger this, and in fact,
- almost by serendipity, the molecule that triggers X inactivation was discovered, and it's this long
- non-coding RNA known as the X inactive specific transcript that is located within this region,
- and that triggers the whole process. Here, this is work of many, many labs, including the lab
- of Sohaila Rastan, Neil Brockdorff, Phil Avner, Hunt Willard, Caroline Brown, and more recently,
- Anton Wutz and Rudy Jaenisch. So many labs have been working on Xist and what it does. It's
- an amazing gene, actually. It's a long gene. It produces this non-coding RNA that doesn't
- produce a protein. The RNA stays in the nucleus, it is not exported and it coats the chromosome in
- cis. So here actually what you can see is Xist RNA coating the inactive X chromosome and the active X
- chromosome is not coated by Xist. So this molecule gets switched on, this gene gets switched on,
- and somehow it becomes restricted to being expressed only on one of the two X chromosomes and
- it produces this RNA that coats the chromosome in cis, and it was the genetic studies of many labs
- that showed that Xist is essential for triggering X inactivation, and indeed, some of the regions
- that are important are very conserved across mammals and they can be seen here. These repeats,
- A, B, C, D, E, F repeats, these are the sequences that are most conserved, and they're the ones that
- do the business of X inactivation. However, Xist alone is not enough. In order to regulate Xist
- accurately, you need a large region around it, and I'll come to that in a minute. Furthermore,
- although Xist triggers the process, once it's been triggered, it's thought that the state,
- the silent state of the X is maintained by other mechanisms, by epigenetic mechanisms,
- including changes in chromatin. Chromatin being the physiological template of the
- genome. So the genome is wrapped up in proteins into chromatin. So chromatin changes, changes
- in nuclear organisation, changes in when the X chromosome gets copied, the timing of replication,
- it becomes asynchronous, and changes probably in the 3D structure of the chromosome as well. In
- fact, my lab was one of the labs that started to look into this 3D organisation using both
- microscopy techniques and other techniques, but I'll say a little bit about that in a minute.
- I just want to now say what many labs, including my own, decided to do, was actually really to look
- at when and where does X inactivation happen in life? We knew it must happen early, Mary Lyon had
- already predicted that, but exactly how does that work? So building on the work of many scientists,
- I was lucky enough to host a very talented scientist in my group, Iku Okamoto, who joined me
- just as my lab started back in 2001 at the Curie Institute, and later on, Maud Borenstein. So we
- started to look at the timing of X inactivation and of Xist expression. Here, what I'm showing is
- basically that X inactivation begins very early on, this was already known. Xist is expressed
- early on, even at the 2 to 4 cell stage. It starts to coat the chromosome in cis. But what's unusual
- about this phase of development is that it's always the paternal X that gets silenced, at
- least in the mouse, and of course, the mouse was really the organism that we and others had focused
- on when studying X inactivation. So in the mouse, in fact, X inactivation is initially imprinted.
- It's only the paternal X that gets silenced. The paternal X actually gets reactivated, and this was
- work that both our lab, together with Iku and Neil Brockdorff's lab, showed that the paternal X gets
- reactivated, reprogrammed in the inner cell mass, which actually corresponds to what can be derived
- in vitro as embryonic stem cells. But in the X harmonic tissues that will form the placenta, for
- example, here, the paternal X remains inactive. So we published many papers looking at the events,
- the order of events and how they happen, and I should say that the imprinted paternal X remains
- inactive in the X harmonic tissues, but in the embryo proper, random X inactivation,
- which is the form that Mary Lyon had initially discovered, kicks in at around the time of
- implantation of the embryo, and so it happens very early on and once it's been established,
- is indeed stably maintained, but then is only reprogrammed in the germline. So here, you see
- reactivation of the inactive X chromosome just prior to meiosis, which of course is important
- so that the two X chromosomes can be treated in the same way. One of the questions that came up
- when we were doing these studies was, well, of course this is all in the mouse. Could it be
- that this is indeed the way this happens in other mammals, and in particular, this imprinted form
- of X inactivation was intriguing because it's also found in marsupials. Here, I have an expert,
- James Turner, whose work has actually discovered the non-coding RNA that triggers X inactivation
- in marsupials, RSX. In marsupials, you only have paternal X inactivation. Only the paternal X gets
- inactivated. In mice, you have this imprinted form of paternal X inactivation that then
- gets superseded by random X inactivation. So we wanted to look at what happens in other mammals.
- I should say that across mammals, you see very, very different situations. We focused on looking
- at in vivo in rabbits and humans expecting that maybe rabbits - so lagomorphs are evolutionarily
- more proximal to rodents - and we expected to see very similar effects, and in fact we saw something
- completely different. Actually rabbits and humans are more similar than mice. Some people say mice
- are aliens. Sometimes I believe that. Rodents are incredible. They're very different, and in fact,
- both humans and rabbits show a very different pattern of early X inactivation. In fact, Xist
- is expressed initially from both X chromosomes, if you're a female, and from the single X if you're a
- male, and then somehow later on in development in females, one of the two X's stops expressing Xist,
- and the one that still does will become the fully inactivated one. So there's a kind of backtracking
- that happens that we still don't understand. Ultimately, you still end up with females with
- one inactive X and one active X, but how you get there really is very different. I think this is
- something, it was a real eye opener for us, and I think others in the field that across different
- mammals, you do see very different strategies being used. The sort of tinkering that Francois
- Jacob talked about. Now I should just come back to marsupials, where indeed there, only the paternal
- X gets inactivated. Here, you can see this Xist is fundamentally important we know for mouse X
- inactivation. We think it probably also is for humans and rabbits, but it doesn't exist in
- marsupials. They do it with a completely different RNA, so they've evolved a whole different way of
- doing it, and then monotremes, which are absolutely fascinating but very difficult
- to study, such as the duck billed platypus, they have five X chromosomes and five Y chromosomes,
- and they do something rather different. It doesn't seem that they inactivate a whole chromosome. They
- seem to show stochastic and partial monoallelic expression of X-linked genes. So just to show you
- that evolution clearly presents us with many, many different ways of achieving dosage compensation
- for the sex chromosomes. So having looked at the evolutionary conservation of the process,
- we were also interested in understanding how it's triggered. Now I already told you that
- Xist is the key trigger, but how do you end up activating Xist during development?
- Only in cells that have two X chromosomes, at least in mice, and only on one X, not on both.
- So this is still very much a fundamental question that we and others are interested in,
- and actually, this is what I started my postdoc on way back when. How does the X inactivation centre
- region lead to this very unique expression pattern of the Xist gene? Here, I'm showing
- you several hundred kilobases around Xist. This region is packed with other long non-coding RNAs,
- some of which are regulators of Xist, and also some protein coding genes that are also regulators
- of Xist. So it's a complex landscape, and we and others have been trying for a long time to
- work out how does a cell recognise it has two Xs through this region, and how does it switch off
- the Xist locus on one of the two? I wanted to put this in because it's an important tale to tell as
- a scientist. One of our favourite hypotheses was that the two X inactivation centres come together,
- and that's how they recognise that there are two of them present, and when they come apart,
- they show asymmetric expression of Xist. So in fact, this is actually what we see. We did
- this using many techniques, including live cell imaging. So the two X inactivation centres do
- come together temporarily early on and then they come apart, but then what Tim Pollax, a very brave
- student in my lab did, was actually prevent this from happening, this pairing, as we called it. He
- prevented it and he showed that it doesn't matter at all. So it's not because you see a beautiful
- process happening that that actually means it matters. Beautiful hypotheses and ugly facts.
- So we published a paper showing that it doesn't matter, but that didn't answer the question of how
- does it actually work. This whole area of symmetry breaking, how do you go from expressing low levels
- to the two alleles of Xist, to only expressing one? Clearly, there are still many, many things
- to understand. We know that the locus that is needed is very big, and actually Edda Schulz,
- who used to be a postdoc in my lab and now has her own lab, has been actively studying… Oops,
- sorry, I'm running ahead of myself here. Way ahead. Has been looking at how Xist,
- together with it's an antisense transcript known as Tsix, at least in mice, could be involved in
- this regulation. This is a field that many people are interested in. I won't go into the details of
- this for the sake of the non-scientists. It's a complicated process, undoubtedly, but one of
- the angles that my lab took, in fact, just as we were starting to realise that this pairing was not
- underlying the symmetry breaking, we started to get interested in, what are the sequences that are
- located far away from the Xist gene that might be regulating it? Some people call this the
- dark genome regulatory elements. We're realising more and more that genes alone are not sufficient
- to explain, for example, disease. So we need to understand, how do some genes get switched on at
- some steps of development, and how do we see the very refined gene expression patterns that we get
- across the genome, and in the case of Xist, to add to it? How do we end up expressing just one of the
- two? So we actually set out to explore this using a new technology that a wonderful collaborator,
- Job Dekker from UMass in the States had set up called chromosome conformation capture, and a
- student of mine, a very talented student, Elphege Nora, went to his lab to learn this technique. I
- won't go through the details of how it works, but basically it captures all the interactions in the
- genome within cells. So that way, you can capture what are the sequences far away from a gene that
- might meet it and might be important in regulating it. This is what we were after. We also used
- high resolution or super resolution microscopy to try and understand how the X inactivation centre
- region is organised, and this was actually with another wonderful collaborator, John Sedat, who
- actually was the inventor of the microscope that we use, the OMX microscope that allowed us to look
- by super resolution microscopy at how the locus is organised. We combined this actually with physical
- modelling thanks to a very talented postdoc, Luca Giorgetti, a physicist who came to the lab and who
- exploited this chromosome conformation capture data with the microscopy data, to come up with
- physical models of how this locus is organised. Now actually, what we discovered when we did this
- experiment was not the very precise sequences that might exist. In fact, we discovered a whole new
- way, or level of organisation of the genome, which we call topologically associating domains. I don't
- think this pointer is working anymore. So I think I'm going to use my finger, if I dare.
- That doesn't work either. Anyway, so I will… Here it is, yes. So topologically associating
- domains are actually regions of several hundreds of kilobases of the genome that tend to interact
- more frequently than others. This was absolutely not what we were looking for. In fact, Elphege,
- who did the experiments, repeated it about 100 times. It took us about two years to convince
- ourselves that this was actually real, but what we were seeing was that some regions have a higher
- frequency of interaction with each other than with their neighbours, and it turned out that
- the Xist gene actually is located right at the boundary between two of these TADs. These TADs
- are several hundred kilobases, and in fact, within the TAD upstream of the Xist gene, we find many of
- the positive regulators of Xist. Those regions that will actually lead to the activation of
- Xist. On the other side, this TAD, we find many of the negative regulators have exist. So actually,
- we stumbled across a new level of organisation, but also a way to partition this very interesting
- region, the X inactivation centre. It covers at least 800 kilobases, so it is very large, and in
- fact, through the modelling that I mentioned to you, as well as through genetic engineering where
- we could delete precise sequences, we came up with the conclusion that probably this region,
- these two TADs, fluctuate in an asynchronous manner. In other words, they're more open or
- closed in an asynchronous way, which could be the way in which the Xist gene can become activated
- during development only on one allele and not on the other. So without going into the details,
- it was this combination of microscopy, chromosome conformation capture which allows you to look at
- the molecular level, and the physical modelling combined then with genetic engineering that
- allowed us to predict that indeed this kind of partitioning is important and that the way
- chromatin, if you like, breeds during the cell cycle and during development could be important
- in allowing for the asymmetry of expression of Xist that's important early on in order to
- trigger this process of monoallelic expression. Now, because we were working on this region, we
- became interested in applying similar technologies actually to the whole X chromosome, and in fact,
- we then used, together with Job Dekker again, the approach of chromosome conformation capture,
- but applying it genome wide using what's known as Hi-C. Using this genome wide approach, we could
- actually look at the molecular organisation of the active and inactive Xs. I told you earlier that
- the Barr body, so the cytogeneticist Barr had actually seen this heteropycnotic body.
- Now here with this technique, we could actually look at the molecular architecture of it, and to
- our surprise, and so here is the inactive X using this chromosome conformation capture technology,
- and in fact you can see the diagonal. So this is just mapping all of the sequences on both axes,
- and the more frequently sequences interact, the darker the colour. So sequences that are right
- next to each other of course interact very frequently. Sequences that are further away
- interact less frequently, and what we found was that on the active X, you see these domains of
- a few hundred kilobases which correspond to these TADs that I mentioned. So the whole X chromosome,
- just like the rest of the genome, is actually organised in TADs, but the inactive X seems to
- be depleted for TADs, except for some regions that escape X inactivation.
- Moreover, it's organised into these two very large domains that we call mega domains, which actually
- are divided by a very unusual sequence known as the DXZ4 locus, and this DXZ4 locus is still quite
- mysterious. It's conserved. It seems to matter. When you delete it, you lose these mega domains,
- but it's not clear exactly what the impact of that is. In any case, this allowed us to get the first,
- I would say, high resolution picture of what the inactive X chromosome looks like, and it
- actually allowed us to see that indeed, the genes that escape X inactivation actually do show TADs,
- and you can't see them here because of the resolution but there are some small regions that
- show these topologically associated domains, and this is where the escapees lie. So having worked
- on the X inactivation centre for many, many years, my lab actually became interested in
- in understanding what X inactivation actually entails. How do you silence genes? It sounds
- like a very simple question. We know quite a bit about how you switch a gene on, but how do you
- switch a gene off, especially when you do it at a chromosome wide scale. So this is a very cartoony
- figure but just to show that a gene on the X, when it's active, is being transcribed. In other words,
- it produces RNA. The chromatin that it has is in a fairly open state with certain epigenetic
- modifications such as histone modifications, transcription factors binding to it, and this
- active gene becomes rapidly silenced when Xist RNA coats the chromosome. We've known that
- for decades, or for over a decade, but we really didn't know how that worked. Why does Xist or how
- does Xist do this? What actually are the factors that do this? So it was a mystery. We also knew
- that once the genes get silenced, several other chromatin changes in epigenetic modifications come
- in, including DNA methylation. So my lab actually for several years now, and I'm going to summarise
- this quite quickly, has been working or trying to contribute to the field of understanding how genes
- actually get silenced and whether chromatin is involved in this process early on or just is there
- to maintain. In fact, we had a collaboration with Howard Chang's lab about ten years ago where he
- actually applied a technology, Howard Chang is at Stanford University, and he applied a technology
- that allowed one to look at the proteins that bind Xist RNA. Now, there were several labs, actually,
- who simultaneously used different approaches and actually, for once, the field came to the same
- conclusion. Usually in X inactivation, we disagree about many things and eventually we agree.
- In this case, most of us all agreed, and the key proteins that bind to Xist were identified. Here,
- I'm showing you, again, Xist RNA coating the X chromosome. This is an interphase nucleus.
- So in blue, you see the DNA. This is the genome. In green, you see RNA. But what are the proteins
- that bind to this? Using different technologies, Howard Chang was able to pull out several proteins
- and in fact with my lab, Simao Rosha, who was a postdoc at the time, he actually tested whether
- some of these proteins were actually involved in X inactivation genetically,
- and here now I'm summarising several years of work by several very talented people. So in fact,
- Francois Dawson, who was a student in the lab, actually dissected the role of this SPEN protein
- and it seems to be one of the master factors that leads to silencing of genes across the
- X. SPEN binds to Xist through a very conserved region, known as the A repeat region, and this
- triggers gene silencing chromosome wide. Now how SPEN does this, we started to explore. Again,
- Francois's work actually pulled out some of the factors that SPEN interacts with. It could
- interfere directly with transcription of genes. It could also bring in factors that change chromatin,
- and I'm not going to go through all the different things that SPEN might do. It's a fascinating
- protein. But in another study, we'd actually showed that some of the proteins that change
- histone modifications, change chromatin, such as histone deacetylase three, are involved in
- the initiation of X inactivation, and that was the work of Yann Gilles and Aurelie Broussard,
- two postdocs in the lab. Now, several groups also showed that Xist does bring in polycomb,
- and in fact, Neil Brockdorff, who's here, showed that through H&R and PK, the PLC-1 complex, which
- is one of the polycomb complexes, which is one of the key complexes involved in helping to maintain
- chromatin states, is brought in via Xist. So how one recruits these polycomb proteins was also
- found, and it's a different part of Xist. So Xist is clearly a very important multitasking molecule,
- and then some of the later changes that come in, including DNA methylation, but also some of the
- other histone modifications and the proteins associated with them are shown here. So many
- different steps in this process. We now start to see, we understand their importance. Putting it
- all together is still, I would say, an ongoing project or many projects, many labs. I just want
- to highlight one point though, because now I'm going to tell you a few things about escape and
- then finish. The dogma for many years was that Xist is only needed early on. You only need this
- RNA, this beautiful, long non-coding, multitasking RNA. You only need it to sort of switch off genes
- and then other factors, epigenetic factors take over. So already just by looking at the factors
- that are brought in by Xist, this was giving us an indication that Xist doesn't just silence genes,
- it also actually brings in the epigenetic modifiers, the chromatin modifiers. I just
- want you to bear that in mind with what I'm going to tell you in a minute. More recently, my lab has
- also been interested in the genes that can escape. I told you why we think they're important, and we
- stumbled across them almost by accident in the studies we were doing during early development,
- and we realised that indeed, many genes can escape. The inactive X was traditionally thought
- to be remarkably stable in its inactive state. The frequency at which some X-linked genes reactivate
- in a somatic cell is extremely low, 1 in 10 to the 9, which is even more rare than the mutation rate.
- In the field, we thought that indeed the inactive X, genes on the inactive X stay off very,
- very stably, thanks to all of the epigenetic modifications that I mentioned that keep them
- locked in that state. But then we, and others, have discovered increasing numbers of genes that
- can escape. On the one hand, there are the genes that escape constitutively that I also mentioned,
- and these are often the genes that have a Y-linked homologue. So they have to escape somehow. A
- double dose is important in males and females. But then there are these other genes, these variable
- escapees, the facultative escapees, and these were really a mystery because they do get switched off,
- for some of the ones we've looked at, they get switched off early on in development and then
- they come back on. One of the genes that caught our eye when we were starting to investigate
- this was indeed the MeCP2 gene, and now MeCP2, I'm sure all of you or many of you have heard
- of Rett syndrome. So a mutation in one of the two copies of MeCP2 in females leads to Rett syndrome,
- which is a severe neurological disorder that leads to severe autism and epileptic attacks.
- Young girls who are affected are very severely affected, and they only have one X mutated for it,
- not both. A male foetus that has a mutation in MeCP2 will die. So there are no male mutants for
- MeCP2. So MeCP2 females survive because they have two X chromosomes and because of X inactivation,
- half of their cells will express the normal copy of MeCP2, the wild type copy. So half of the cells
- are okay and the other half cannot express or express the mutated copy. So basically the dogma
- was that indeed the 50 per cent approximately of cells that show this aberrant MeCP2 form or lack
- of expression of MeCP2 are there. So probably, MeCP2 should never escape, and in fact, my lab
- and others had spent years trying to re-activate MeCP2, but of course, there's tremendous interest
- in using epigenetic drugs that can reprogramme epigenetic marks to maybe wake up genes like
- MeCP2, because then of course you could perhaps rescue the effect. So all of this to say that we
- didn't imagine that MeCP2 could escape, but then we looked both in clonal cell lines and in vivo,
- and this is in a mouse, early postnatal mouse brain, and we could see that MeCP2, which you can
- see here is labelled at the RNA level in cells in the brain, is expressed not just from the active
- X but from the inactive X in a subset of cells, and this is in about 20 per cent of cells. It's
- not many cells and it actually varies during life, and in fact, together with Adrian Bird, with whom
- we collaborate, we have mice that are expressing a green fluorescent and a red fluorescent form of
- MeCP2. So in most cells, you're either green or red, as I've already told you, but in some cells,
- if there is MeCP2 escape, you should see yellow, both green and red being expressed simultaneously,
- and this is indeed what we see. So we know that this does happen in vivo, and we can even sort
- these cells and look at what's happening. Now, does it matter? We don't know. It definitely
- happens. It provides hope that maybe there could be epigenetic therapies that could awaken MeCP2 in
- the in the context of Rett syndrome, but it really does beg the question of what this is telling us,
- because escape from X inactivation leads to a double dose. It could be useful in some contexts,
- but it could be actually very unuseful, and in fact, in the case of MeCP2, a duplication
- of MeCP2 on the single X chromosome in males also leads to severe mental retardation and
- neurological effects. So a double dose of MeCP2 early on can really be a problem. No MeCP2 is
- also a problem. So you've got to get dosage right. So when you start to see this escape, what is it
- actually telling us? Is it something that could be useful in this context in the brain or is it
- actually a mistake? Is it an aberration? In fact, maybe these cells actually die. So these are the
- open questions we're interested in. Around this period, when we were starting to look at escapees,
- several groups started to realise that there were situations where escape seemed to be changing,
- and these were often linked to situations where Xist itself was changing, and now we're talking
- about thematic tissues. We're not talking about the embryo. So there were several publications
- that came out, one of which we were also involved in, this one here, where it was
- shown that in human mammary stem cells, the loss of Xist actually leads to a block in mammary stem
- cell differentiation, and this actually leads to increased tumorigenicity. The gene that was shown
- to be key for this was a gene called MED14, which is mediator 14, it's part of a big complex which
- is very important for gene regulation, genome wide. So just a little bit more of this gene
- when you got rid of Xist, and this is what was shown in this paper, a slightly higher dose of
- this gene which escapes, and escapes even more in the context of the loss of Xist, can lead to this
- block in mammary stem cell differentiation and can potentially contribute to breast cancer. Other
- studies had shown that if you knock out Xist, or if it becomes delocalised from the inactive X,
- this also leads to haematological cancers. More recently, it was also shown that in human B cells,
- Xist actually seems to prevent escape of X-linked genes that have less DNA methylation and so have
- a tendency to escape. So Xist can actually prevent this. So this actually opened up a question which
- is does Xist really directly influence escape, and what are the implications of this in diseases like
- cancer? The reason why we're so interested in cancer is going back to Barr. In fact,
- even before we knew about Xist or X inactivation, Barr himself had published papers and was already
- using, applying the loss of the Barr body. So this is a normal cell. So these are cervical cells. So
- this is a normal cell and you can see the Barr body here and here, this is malignant tumour,
- so this is a cervical tumour, and you can actually see completely disrupted heterochromatin and an
- apparent loss of the inactive X or of the Barr body at that point. So already, it was being used
- actually clinically to detect the more aggressive forms of cancer, the loss of the Barr body.
- So when now we fast forward several decades and all sorts of new beautiful techniques, we
- can detect both the X chromosome and the Xist RNA and some of the chromatin marks and genes using in
- situ approaches, and here, I'm just showing you one example where this is a breast cancer cell.
- In green are the X chromosomes. We can paint the X chromosomes using probes, fluorescent probes.
- So you can see here, there are three copies of the X. It's a breast cancer cell. Then in red is
- Xist RNA which you can see is not at all coating the chromosome. It's floating away. So in fact,
- our lab and others have been very interested in working out to what extent in cancer, do you
- actually lose the organisation of the inactive X, the coating by Xist, and to what extent does
- this lead to reactivation of X-linked genes? This is during the time I was at the Curie Institute,
- we published some papers where we showed that indeed, genes do start to get re-expressed more
- frequently in breast cancer. I'm almost finished now. I just want to emphasise that this epigenetic
- instability that I'm showing here in the context of cancer can lead to aberrant escape. I told
- you about this example that we showed in mammary stem cells, where the slightly higher expression
- of even just one gene can contribute. So this might actually facilitate or promote cancer, but
- I also told you earlier on that many cancers show a male bias. So males have a higher tendency to
- have many of the non-reproductive cancers, and in fact, there's a hypothesis, which is that because
- some genes escape, this actually may protect women from cancer, because if you have a mutation in a
- gene that's important in cancer onset, either in a tumour suppressor or an oncogene, this actually
- would be present in two copies in females, only one copy in male, and of course, if you
- only have one copy, you immediately will see the effect, but if you have two copies and one of them
- is escaping, you would somehow mask this effect. So in fact, we have two completely contradictory
- hypotheses but we believe that both of these are true. We believe that in some cases, escape can
- actually be protective, and in other cases, it can actually be a promoter of cancer in females. So my
- lab currently, some of which will be moving to the Crick, we've been very, very interested in working
- out how and why genes escape and the effect that this has. So these are the very talented people
- who have been working on these projects. I'm not going to go through any more. I just want to say
- that we've set up a system now that allows us to vary the levels of Xist on the X chromosome,
- on the inactive X chromosome, we can actually induce more or less of Xist, and this is actually
- allowing us now to silence escapees at will. So in a normal situation where Xist is not necessarily
- very high, you see a lot of escape, and so this is just supposed to illustrate in a mouse brain,
- there are many, many regions where you do see escape, but if you overexpress Xist, we've shown
- that you can actually switch off these escapes, and so does this have an effect? Do we see
- phenotypes? In other words, do we see any changes at the level of development, disease of course,
- or behaviour? So this is for us what the future holds. I'll end with one of the slides I showed
- earlier, which is if we really want to understand about sex bias in disease, we need a molecular
- understanding of what's going on, and I hope that some of the work that I described might contribute
- to this. Maybe a final word about this is that, of course, if we're all talking about precision
- medicine and personalised medicine, but in my opinion, if we really want to be able to have
- an effective treatment for all individuals in this era of precision medicine, men and women actually
- do have to be treated differently in order to be protected equally. This is a very EDI comment to
- make in this era where across the Atlantic, probably this wouldn't go down very well,
- but I believe very firmly that this is what we should all be focusing on if we're interested in
- medicine and biomedical science. So I'm going to stop there and just thank not only the many, many
- colleagues, collaborators, and friends that I've had the chance to work with and my lab, who were
- a huge community of scientists who keep together across the world, and we're going to celebrate,
- we're going to have our first reunion on the 9th June. I'm really looking forward to it. It'll be
- an era of hard lab science before I move back to London, back to where I came from. So thank you.
- Thank you so much for an absolutely wonderful lecture. I hope you'll be happy to take some
- questions because I'm sure there will be many. I'll open the floor to questions,
- and I'll leave it to you to identify who you would like to respond to.
- I can see a hand over there.
- That was a wonderful lecture, but I'm curious about something. You studied these genes across
- species, but as I understand it, cancer researchers use HeLa cells and that most
- of the research is based around - I know Henrietta Lacks is not the only immortal cells used in this
- research, why not do research across populations around the world instead of across species?
- Absolutely. Well, thank you for the question. First of all,
- it raises the topic of the cell lines that people traditionally use and HeLa cells.
- We've never used HeLa cells in my lab because they are so genetically changed from normal that it's,
- in my opinion, very difficult to work out whether dosage matters, and in fact, although of course,
- they were derived from Helen Lane, a woman, they don't have an inactive X chromosome
- anymore. Some cell lines that scientists work on can be very useful for understanding certain
- biological molecular processes, but in fact, for processes such as the one that we work on,
- absolutely are not useful or not relevant. Now, I am a very firm proponent of understanding what
- happens across populations, and studying a process such as dosage compensation across species gives
- us an indication of where one should be looking in terms of molecular mechanisms. It's been very
- helpful for us to look across species in order to identify, well, what are the most important
- molecules, what are the things that are conserved, and once you've identified them, then you want to
- look across a population, and although much of the work that my lab does is on mice,
- we work on mouse population, we're very interested in the diversity of different species of mice,
- we're also very interested in what happens across human populations. Although I am not necessarily
- an expert, I think this is an area where indeed, if you want to understand human biology, you've
- got to understand it across the world, and this is, I think, something that the organisation that
- I worked for until a few days ago, EMBL, is a firm proponent of, gathering the data that comes
- from human populations across the planet. I saw a hand right at the back and then another one here.
- So this is a question from our online audience. Has X chromosome inactivation been studied in
- individuals with Klinefelter syndrome and are DSDs helpful in this area of study?
- So indeed, X inactivation has been studied in Klinefelter's individuals. Klinefelter's
- individuals have two X chromosomes and a Y. One of the two Xs is inactive. I forgot to mention I
- should have mentioned it, but anything more than one X chromosome in a normal diploid cell will
- be inactivated. In other words, individuals with two X chromosomes or three X chromosomes or four
- X chromosomes, you will only keep one X active. All other X chromosomes will be inactivated.
- In the case of Klinefelter's, indeed, you have two Xs and a Y. Now, this is clearly a very particular
- context, but it seems to be that the double dose of some of the genes on the X,
- the genes I was mentioning, the X-linked escapes, as well as the presence of a Y, some of which also
- are homologous, so we have three copies of these genes being expressed, may well contribute to
- the features of Klinefelter's individuals. So this is an area which I think has been understudied,
- this, and DSDs in general, and this is an area that I think there's
- an increasing interest and some of my colleagues in the room are actually very interested in trying
- to dive into this. Having the tools to actually understand what changes at the molecular and
- cellular level was only recently possible, in fact, and so now I think in particular,
- also given that the UK Biobank is an incredibly rich source of information and material, I think
- there's going to be a renewed interest in trying to understand what happens in Klinefelter's.
- You mentioned at some point that environmental factors might be
- involved in these processes. What is known about how that works?
- Good question. Thanks for picking that up. I mentioned it in the context of escape,
- escapees that vary, and actually it's recent studies looking at identical twins, females,
- where it's clear that early on, the genes that escape are actually quite similar but become
- increasingly divergent with age. So this would imply that perhaps there are either stochastic
- differences or environmental changes that happen. What the nature of those environmental changes
- are, is completely unknown, and I do hope that this will be an area in the future that maybe
- some of us could explore. In fact, I'm looking at Caroline Relton over here who's very interested
- in epigenetics and environmental exposures. I would love for that to be an area that could be
- explored more deeply. I think it will tell us not only about how escapees might matter over ageing,
- but more importantly, what kinds of environmental exposures trigger what
- kinds of changes in gene expression, which I think is something that many of us are interested in.
- Back there, not sure how to describe people, the person with the glasses and the white hair.
- The thing I never understand is why did it stick
- to that one chromosome? Why doesn't the Xist, like most RNAs, just diffuse away?
- Well, sometimes it does diffuse away and then that can create problems as well. So there are
- proteins that clearly help it stick. In fact, Neil Brockdorff's lab and others have identified some
- of them. There's a protein called CIS1. There's another protein called SAF-A. So we think there
- are a number of proteins that act like a kind of glue and when Xist starts to become upregulated,
- initially it doesn't stick as much. Initially it does look rather fuzzy, but then very rapidly,
- after one or two cell divisions, you can really see that it very much sticks to the chromosome
- that it's expressed from, and so there must be some kind of feedback. So first of all, when it
- gets expressed, it recruits some of the factors that probably help it to stick. It probably does
- stick elsewhere initially, but not in a stable way because every cell cycle, actually Xist probably
- uncoats the chromosome before it recoats it again, and there must be changes that are induced by Xist
- that help it to stick better once it's been there but we don't know what they are. You can shout.
- I was very intrigued by the finding that you presented at the end there, if you put more
- Xist in, you end up overriding… So you actually can X in activate everything, right? I guess
- the question is how plastic are overall Xist levels and whether there are any environmental
- factors or otherwise that overall can increase or reduce Xist levels to end up with more or less?
- That was a really nice question, which helped me to say something that I should have said
- at the end, but I didn't, but you're right. Absolutely. We think that there's much variation
- in Xist levels, even between different tissues. There are some tissues where there are very low
- Xist levels. Thymus is one of them. There are other tissues where it seems Xist is higher,
- and clearly there are going to be individual differences. So there is genetic variation that
- will lead to more or less Xist levels, that we know as well, and there must be
- environmental influences as well. Now we ourselves haven't yet looked at them, but
- we know because we spent many years trying to use in vivo live cell imaging of Xist, which we did,
- but we realised that every time the cells would get stressed, Xist would uncoat the chromosome.
- So we think there's an awful lot of different things feeding into Xist expression levels and
- the coating process, and this is exactly why we think that playing around with Xist levels at
- will, because this is using a drug inducible promoter of Xist. So we can really say okay,
- well if you overexpress Xist at this stage or in this tissue, what happens? In other words, do you
- silence genes that are starting to escape or that have already escaped, and does it matter? How does
- the tissue look? So I hope this will be a new era of understanding the genetic and environmental
- influences on Xist. One more question. Okay. I'll choose the person over here at the back.
- First of all, thank you so much for an absolutely brilliant lecture. My question is, I suppose, you
- mentioned that even identical twins don't have the same pattern of X inactivation. So could that have
- any confounding effects on the concordance that you measure for different traits in twin studies?
- I should ask Caroline that.
- I'm sure it could. Caroline being not only an expert in cohort studies, but a twin herself.
- I'm sure it does. I mean, now that we know that there is so much escape in particular,
- I think it definitely is something that should be taken into account. As I said,
- I think this is a new era of trying to understand how much variation there is in X linked gene
- dosage across life between individuals, and yes, females are complicated. That's a good way to end.
Join us for the Croonian Prize Lecture delivered by Professor Edith Heard FRS.
The Croonian Medal and Lecture 2025 is awarded to Professor Edith Heard for being a leading figure in X-chromosome biology.
X-chromosome inactivation during early female development is an essential process that is required to achieve appropriate dosage between XX females and XY males, for X-linked gene products. The Heard lab is interested in understanding how the differential treatment of the two X chromosomes is established during development, how the X chromosome becomes silenced and how this is maintained stably, or reversed in certain circumstances, either normally or in a disease context such as cancer. The establishment of X-chromosome inactivation involves a long non-coding Xist RNA that triggers chromosome-wide chromatin re-organisation and gene silencing. Studying this process has revealed general principles of gene regulation, chromosome architecture and epigenetic mechanisms. The Heard lab’s insights into the nature of the chromosome-wide changes that affect the whole X chromosome. In particular we have studied the role of the Xist-recruited SPEN protein, that triggers gene silencing and dampens expression of genes that escape XCI. The loss of topologically associating domains (TADs) are also early events during XCI2. Our recent insights into the relationship between chromatin states, 3D chromosome organisation and the events underlying X-linked gene silencing and escape from XCI will be presented.
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