Hello, everyone. I’m Jeanie Lee. I’m a Professor of Genetics at Harvard Medical School, and I’m also a faculty member within the Department of Molecular Biology at Massachusetts General Hospital. What I’d like to tell you about today is a scientific problem that we’ve been working on for the past 22 years or so, and that is the problem of why and how one of our sex chromosomes needs to be inactivated in early development. So, this is a process called X chromosome inactivation. And what I’m going to do is divide this topic into three lectures. So, in the beginning we’ll talk about a general overview of the scientific problem and the medical relevance of X chromosome inactivation. And then we’ll take a deeper dive, in the second and third lectures, and talk about how inactivation initiates with a counting and choosing mechanism. And then end with a very challenging problem of how X inactivation initiates within a tight locus before spreading to the rest of the X chromosome. Okay. So, we’ll start with… by taking a step back and asking this question of, why do we need to inactivate one of our sex chromosomes? And I think the answer lies in the fact that, in mammals, sex is determined by a pair of the so-called unequal sex chromosomes. And so, here what you see is a normal human female karyotype, so it’s 46 XX. And you’ll notice that these chromosomes come in pairs. And so humans have 23 pairs of chromosomes. And the first 22 are so-called autosomes. And we obtain one copy of each autosome from mother and one copy from our father. Okay? And for all intents and purposes, for this lecture, we’ll say that the two chromosomes that we get from mother and father are genetically identical. Now, the 23rd pair is very special, and that’s because these are our sex chromosomes. So, we call them sexually dimorphic because they’re different between males and females. And if we obtain one X chromosome from our mother and one from our father, we develop as genetic females, so 46 XX. But on the other hand, if we obtain one X chromosome from our mother and one Y chromosome from our father, we develop as genetic males. Okay. So, to simplify this scheme, here I’ve drawn two X chromosomes belonging to the female, and to the male one X and one Y chromosome. So, the X chromosome is a very large chromosome. It accounts for about 5% of our genome, and it has approximately 1000 genes. By contrast, the Y chromosome is much, much smaller and only contains a fraction of genes. And so this mechanism of determining sex in mammals produces this genetic inequality between the sexes that needs to be compensated in order for development to proceed normally. And so, to compensate, we mammals do something truly unique, and that is a process called X chromosome inactivation. And what we literally do in early female development is to transcriptionally silence one of the X chromosomes in the female sex. And so what we have, then, is an epigenetic situation in which males and females express only one functioning copy of the X chromosome. So, this is very interesting, because the X chromosome is in fact the only chromosome in mammals that’s capable of undergoing this global inactivation. And so the mechanisms that underlie this process have been intensively investigated for more than half a century, firstly because it is a very nice model by which we can study something called non-coding RNA, and also to study general mechanisms of epigenetic silencing. And the X chromosome, of course, is also very interesting to both basic scientists and to clinicians, because of the numerous disease genes that are associated with the chromosome. So, I’m gonna tell you more about these things in a few minutes. But first, I wanted to just say a few words about the short history of X inactivation. So, the inactive X chromosome was actually first visualized by a pair of Canadian scientists, Barr and Bertram. So, in 1949, what they published was that they could, in almost all female nuclei, observe this structure, which you might be able to see here as a small dot lying next to the nucleolus. Okay. Whereas that structure is not present in these male nuclei. Now, they didn’t actually know what they were observing at the time. They didn’t know that this was an inactivated X chromosome. But what they did mention was that even without looking at these cats, which is… these were the organisms that they were working with… without laying their eyes on the cats, they could predict the sex of that animal simply by the presence or absence of the so-called Barr body, which has been named in honor of the scientist who first visualized the structure. Okay. So, fast-forward 10 years. An American scientist, Susumu Ohno, took this observation one step further. Now, he was looking in rats, but rats are also mammals… and we do some… and mammals do a similar process of X chromosome inactivation, and what he observed is that the number of Barr bodies in the rat very closely parallels the number of X chromosomes in that cell. And he called the Barr body an X chromosome and suggested that it was pyknotic, which is a fancy word for condensed, and potentially biochemically distinct, and maybe even functionally disabled. Okay. So, then… the person who really put all of this together was a British scientist, Mary Lyon, in 1961, who published a seminal paper in the field, in which she calls the Barr body an inactivated X chromosome, and suggests that X chromosome inactivation is the mechanism of dosage compensation that allows males and females to equalize their sex chromosome dosage, and that it would lead to a random inactivation of one X chromosome in the female cell. Now, she deduced all of this by working in mice and looking at coat color variegation, but the same phenomenon holds true in these cats. Okay? So, in cats and in mice, the coat color gene is carried on the X chromosome. And it comes in two flavors: there’s an orange flavor, and then there is a gray flavor. Alright? So, what she noticed in mice — but we’re illustrating here with cats — is that while males can adopt either a solid orange color or a solid gray color, depending on whether the male inherited an orange… here’s the… here’s an orange coat color gene or a gray coat color gene… females can not only be solid orange or solid gray, but could have this very interesting pattern of variegation that you can see in this beautiful calico cat, okay?, if she inherited one copy of the gray and one copy of the orange gene on the X chromosomes. So, the idea here is that if a cell in this heterozygous female inactivated the orange-color chromosome, then that cell would manifest as a gray cell. But if a cell lying next to that gray cell chose instead to inactivate the gray gene, or the gray chromosome, then that cell would manifest as an orange cell. And so, in this manner, you can see this wonderful coat color variegation in the female calico cat. Okay. So, the important point to make here is that X inactivation is random. This is a choice step, which is random, and that is made by every single cell in the female body, and therefore every female mammal is a mosaic of cells that either inactivate the paternal chromosome or the maternal chromosome. Okay. So, we and the X inactivation field like to think about X inactivation as something that happens once, early in female development. But in fact, X inactivation happens in both sexes. And it happens as part of a much more complex life cycle of the X chromosome. So, here we are looking at the life cycle of X inactivation in the mouse, and we’re going to start in the germline, here, with the female germline. You see that the two X chromosomes are active, and then one of the two X chromosomes gets passed on to the next generation, to daughter and to sons. Okay. So, now a very interesting process happens in the male germline, shown up here. So, during the first stage of male meiosis — so, this is during spermatogenesis — the X and Y chromosomes physically pair, at least partially, and then they undergo a sex chromosome inactivation, of both the X and the Y chromosome. And then… the… we believe that that silencing remains in effect until the… until the end of spermatogenesis, at which time either the X or the Y chromosome gets passed on to the next generation. Okay. So, early pioneers in this field proposed the very interesting hypothesis that the X and Y chromosomes may be getting inactivated in the germline not only to suppress homologous recombination, which would be detrimental to the sex chromosomes, especially if we wanted to preserve the identities of the sex chromosomes, but that it is also a mechanism by which the sex chromosomes could be imprinted. Okay? And be predisposed to silencing in the next generation, at least in daughters. So, we like that idea. And in fact, in the very first stages of development in the next generation, what we observe is an imprinted form of X inactivation, in which the father’s X chromosome is always inactivated. Okay. And that persists until the blastocyst stage. You see here in the… this is a peri-implantation embryo, where the extraembryonic tissues will maintain… so, these are the placental… the tissues that will go to make the placenta. Okay. They maintain this pattern of paternal X inactivation. And the placenta will keep that until the time of birth, at which point of course the placenta is discarded. Now, on the other hand, in the embryo proper… so, we call it the epiblast lineage, and that is the lineage which will go to make all of the somatic cells in our body. Now, in the epiblast lineage, which is shown here, inside the blastocyst, the cells will erase the paternal imprint and undergo a reactivation so that the embryo itself can decide who… which of the two X chromosomes will become the inactive chromosome. So, there’s a reactivation of the paternal X, and then a re-inactivation, except that this time inactivation proceeds in a random fashion, where either of the maternal or paternal chromosome could be inactivated. And that is the form of X inactivation that I’ll be talking about today, and it is the form which persists throughout the rest of development and throughout the rest of adult female life. Okay. So, I’ll briefly mention that in the germline of a female embryo, the two X chromosomes… well, of course, one is active and the other one is inactive… the inactive one will also undergo a reactivation event, so that in female meiosis the two X chromosomes would have an equal chance of being passed on to the next generation. And then of course the whole cycle reinitiates again. Okay. So, that is the life cycle of X inactivation in the mouse. And now we’re going to address the question of why the X chromosome and X inactivation might be important to study. Okay. So, firstly, the X chromosome, as I mentioned before, is a very large chromosome, and it carries something like 1000 genes and makes up about 5% of our genome. And equally importantly, along with the Y chromosome, the X chromosome is responsible for so-called sexual dimorphism. So, that’s a fancy term to say that males and females look different, and that we might even behave differently as a result of our sex chromosomes. Okay. And the X chromosome is enriched for genes that not only go to determine reproduction but also genes that influence brain development, behavior, and cognition. So, in fact, on the X chromosome there are more than 200 disease genes that are presently known. And many of these genes are responsible for neurodevelopmental disorders, autism, and other X-linked intellectual disabilities. Here I just list two known examples, so, Rett syndrome, which mostly affects girls, and Fragile X syndrome, which can affect both men and women. And then there’s a muscular dystrophy that’s fairly common, called Duchenne’s muscular dystrophy, which mostly affects men. And then there are even non-disease traits, like red-green color blindness, which affects about 10% of all men across the world. Okay. So, then… you might also ask, what happens if X inactivation doesn’t actually happen during development, right? So, this experiment was done more than 20 years ago in Rudolf Jaenisch’s lab, in which they removed one of the critical factors for X inactivation called Xist, and observed that no female embryos were born. In fact, they all perished shortly after the time of implantation. Now then, we followed up many years later and asked, what would happen if we removed this critical factor a few days after conception? So, the original experiments were done from the time of conception. We asked, what would happen if we removed it several days later? And again, most of the female embryos perish. Okay? But a few make it to birth. But I think as you can see here, in the red circle, these female animals are almost always runted, growth-retarded, and do not make it past the third week of life. Okay. So, X inactivation is very important during early development. You might also ask the question, well, what if we were to take away X inactivation a little bit later in development, or even in the adult female… adult female cells? Okay? So, this has been a question that’s been under intensive investigation for a number of reasons that will become obvious later. But the answer seems to be that it depends on what cells and when we do it. Okay? So, for example, in the mouse, in highly proliferative cells — so, cells that are destined to divide many, many times, like blood, over the lifetime of a female — there seems to be an increased risk of cancer. But the same is not true in the brain, which… for the most part, once the brain is formed, does not… the cells within the brain do not divide again. Okay. So… and then in humans, there’s also been a link to various cancers, although not currently shown to be… shown to be a direct causality. But what has been observed is that there is an increased risk of certain conditions, like blood cancer, breast cancer, ovarian cancer, and even, in men, testicular germ cell tumors. Okay. So, this increased risk is associated with both men and women. And they seem to be associated with the loss of the Barr body and/or the gain of additional active X chromosomes, suggesting that there’s an increased risk of cancer when the X chromosome dosage exceeds physiological levels in both men and women. Alright. So, here’s an experiment that we performed not so long ago in which we removed this critical factor, Xist, and demonstrated that these female animals — and this was specific to the female animals — developed a fulminant blood cancer with essentially 100% penetrance, meaning that nearly all females succumbed to this disorder. And their blood stem cells become quote “cancerous”. Okay. So, here’s an example of one female that succumbed to the disease. And you can see that her spleen has become extremely large and filled with tumor cells. And just looking at the animal grossly, you can see that there are a number of abdominal masses, or masses that are present elsewhere in the body, indicative of a solid hematopoietic tumor. And if we follow these animals over their lifespan — here I’m showing you a survival curve — you can see that most of the females perish in early to mid-adulthood. Okay. Alright. So, I think I’ve shown you that the X chromosome and its dosage compensation are very important, not only during embryogenesis but also throughout female life. And what I’d now like to turn your attention to is how we think the mechanism of X chromosome inactivation might be working. Okay. So, now, early pioneers in this field knew that there had to be a control center, and that that control center is most likely on the X chromosome, based on a number of genetic studies that we don’t have time to get into right now. But there was a debate in the field about whether there was one inactivation center, located somewhere in the X chromosome, or a series of inactivation centers that would be located up and down the X chromosome, for silencing to spread. So, the experiments that were performed in Hunt Willard’s lab, and also in my lab and the Jaenisch lab, in the 1990s showed that the inactivation center is in fact singular — there’s only one — and that that inactivation center is very small. It’s probably no more than 100-200 kilobases. So, we’re talking about a control center, a brain of the X chromosome, which is just 1/1000th of the size of that sex chromosome. And we were able to show that this inactivation center is indeed extremely powerful, because if we transplanted that inactivation center to a non-sex chromosome, to an autosome, that center will induce the autosome to behave like the sex chromosome — be counted and be chosen for inactivation. In fact, the chromosome will undergo inactivation. And so what these genetic experiments told us is that X inactivation is driven entirely by this master switch that we call the X inactivation center, and that apart from this 200 kilobase sequence there are no… probably no other inactive X-specific elements that drive the process of silencing. Okay. So then, let’s talk about the X inactivation center. So, here it is in all its glory. And you’ll see that it’s populated by an epigenetic… a type of epigenetic factor called a long non-coding RNA. So, I mentioned earlier that this region is probably no more than 200 kilobases in size. It’s very small. And yet it is both necessary and sufficient to drive the different steps of chromosome silencing. Now, in the 1990s, sequencing experiments around this region demonstrated — strangely — that there were few if any protein-coding genes. And that was very surprising for the 1990s because back then we all thought that anything that was important had to be encoded by a protein. So, in other words, we thought that proteins had to be the work… the workhorse for anything related to regulating gene expression. However, many of us felt that that may be an overly simplistic view, because in fact proteins make up only about 2% of our genome. The rest of the genome was termed, decades ago, as “junk DNA”. They account for 98… 98% of the mammalian genome. And what’s become very obvious in the past decade or two is that this so-called junk is extremely active. Nearly every single nucleotide of this junk DNA is synthesized into what’s known as non-coding RNA, long non-coding RNA. So, the X inactivation center is a really good example of what long non-coding RNAs can do. In fact, it’s enriched for this type of gene. So, I’ll just mention a few here. So, there’s the Xist gene, which was first identified by Willard and Ballabio. And that gene is responsible for spreading across the X chromosome and ushering in this silent state of the chromosome. And then we identified its antisense repressor, Tsix, back in the 1990s. This is an antisense transcript that prevents Xist from doing what it’s supposed to do on the inactive X. And then there’s a mysterious motif here, the repeat A motif, which is important for the transcriptional activation of the Xist gene, and also for recruiting polycomb complexes. And then, on the other side of Xist, we have two additional non-coding RNAs — one called Jpx and the other called Ftx — both of which appear to be associated with the activation of this important Xist gene. Okay. So then, on the other side of Tsix, we have two other non-coding RNAs — a Tsx gene and an enhancer element called Xite, which is responsible for inducing or allowing the expression of the antisense gene to persist. Okay. So, what we have here is a bipartite structure of the X inactivation center, in which on the left side we have all the pro-inactivation genes. These are genes that are necessary to induce the silent state of the X chromosome. And then on the other side, we have genes that are actively trying to prevent that from happening, so we call these the anti-inactivation genes. Okay. So, X inactivation is mechanistically complex, and it takes place in a number of different steps. Okay. So, we’re just gonna go through these steps right now, and then I’ll do a deeper dive in the next lecture. So, in the beginning, there is a counting mechanism, which is determining the number of X chromosomes in the… in the cell, and whether X inactivation should take place. And if the answer is yes, there is an allelic choice mechanism, which will allow the embryo to pick which of the two X chromosomes — the mother’s X chromosome or the father’s X chromosome — for inactivation. And then once the chromosome is chosen, there’s a very intriguing event in which silencing nucleates onto the X inactivation center and then spreads outwardly from that chromosome, to cover the entire 160 megabase or so chromosome. So, all of this is over by embryonic day six and a half, let’s say, which is shortly after implantation in the mouse. Now, thereafter, the chromosome goes into a maintenance phase, a very important maintenance phase, in which the same X chromosome remains the inactive one for the lifetime of that female. So, once it’s chosen, forevermore that chromosome will be inactive. Alright. So, many of us in the field have been trying to understand the mechanisms by which these steps take place. So, I’m gonna just outline some of the conceptual challenges associated with studying these problems. We’ll begin with the counting problem. So, X chromo… X chromosome counting takes place in the blastocyst, shortly after the paternal X chromosome reactivates. Okay, so remember back to the life cycle of the X chromosome. So, the paternal X chromosome reactivates. And then a counting mechanism comes into play, in which every cell in the epiblast makes a determination of the sex chromosome number. So, this takes place around the time that there are about 20 cells in the epiblast, so we say it’s a cell-autonomous decision. So, how does this all work? Now, it turns out that cells aren’t actually counting the absolute number of X chromosomes, because that wouldn’t make any sense. Cells are actually measuring the number of X chromosomes relative to the total genome content, so, the number of autosomes. So, we call that the X-to-autosome ratio. And it’s been empirically observed by pioneers in the field that every cell — if it’s diploid, okay? — is allowed to keep one X chromosome active. So, cells follow this so-called n-1 rule, in which all X chromosomes are inactivated except for that one privileged, active X. Alright. So then, let’s see what happens in males. So, males have an X-to-autosome ratio of 0.5. So, if the male cell is diploid, it keeps its one active X chromosome. If the male is tetraploid — so it has twice the genome content, so we call it 4n, here — then it’s allowed to keep two active X’s. There are no supernumerary X chromosomes, so males, whether it’s diploid or tetraploid, will not undergo X inactivation. So then, contrast that with what happens in females, where the X-to-autosome ratio is 1.0. So, if the female is diploid and following the n-1 rule, it will inactivate one of its two X chromosomes. Now, if she has twice the genomic content, then she will keep two X chromosomes active, and silence the two additional X chromosomes in her cells, Alright. So, now let’s go back to the diploid, and assume for a moment that this female is a mosaic, and she has a mixture of cells that are dip… that are… that have two X chromosomes, and some that have three X chromosomes. Then, by the n-1 rule, she would inactivate two, the two blue chromosomes, out of her three. And suppose, again, that she actually had a mixture of cells that had four X chromosomes. And in this situation, by the n-1 rule, she would inactivate three of her four X chromosomes. So, very complicated. Alright. Now, how do we envision all of this happening at the molecular level. That’s the big question. So, we believe that counting is really a titration of X-linked and autosomal factors. Okay? So, those go to make up the X-to-autosome ratio. And so the question is, what are these numerators? What are these X-linked factors? And what are the denominators, the autosomal factors? So, here’s a model that’s very popular in the field, and the idea is as follows, where in male cells, the X chromosome will make a limited set… or it will make a set of these so-called numerators, which are in green, in limited quantities. Okay? And likewise, the autosomes, shown in pink here, will make these denominators. They’re also produced in limited quantities. And so the numerators and the denominators — so, the green and red factors — will titrate each other out to form what the field calls a blocking factor, which will then go and bind to one — the one and only — inactivation center in the cell, and prevent that inactivation center from firing to induce X chromosome inactivation. So then, the male is spared of X chromosome silencing. So, then a similar thing would happen in the female. So, she also makes red and green factors, and the red and green factors titrate each other to form this blocking factor. And then the blocking factor goes and sits on one inactivation center, prevents a firing in that center, and that chromosome remains active. Now, again, the early pioneers in the field felt that the blocking factor was sufficient to explain X chromosome counting, because then all of the remaining X chromosomes that weren’t lucky enough to get the blocking factor would undergo silencing by default. Okay. So, now, that model certainly works very well, and it could in fact be what’s happening in cells. But we’d like to think that biology is purposeful, right?, so that… nothing happens in life sort of randomly, or by default. And that there should be a purposeful inactivation of the remaining X chromosomes. And so, in fact, there is something different here about the female. So, has one extra X chromosome, which means that she’s producing extra green factors. Now, these green factors go untitrated by the blocking factor. And so we suggest that these additional green factors would go and make their own complex — a complex that we call the competence factor. And that factor would then go and bind to the remaining X chromosome, and purposefully induce that inactivation center to fire and initiate the cascade of silencing. Okay. So, we call that the two factors hypothesis. And I’m gonna have a lot more to say about that in the second lecture. But for now, I’d like to move on to the next conceptual challenge, which is allelic choice. And this is one of my favorite problems. Because what this allelic choice really means is that that decision has to take place instantaneously. In order for it to be a robust mechanism, it has to be mutually exclusive, and irreversible. Right? Because we know that once a decision is made, well, you don’t go back on that decision. And the same X chromosome remains inactive for the lifetime of that female. Okay, so here’s the big problem. How do we make the right choice? And how do we make that choice in a mutually exclusive fashion? So, we postulate that there has to be a mechanism of communication between the two X chromosomes — so, trans, we say… trans communication — such that when one chromosome is chosen as the inactive one, this other X chromosome has to instantaneously become the active X chromosome. Okay. So, the idea is that one hand has to know what the other hand is doing at all times. And in fact, we have that problem with our brain. Because, you know, we have two halves to our brain, and one… the right brain controls the left hand, and the left brain controls the right hand. And so how do we communicate between the two halves of the brain? And so the brain does it very well, through a bridge called the corpus callosum. And so we postulate that there has to be a bridge between the two X chromosomes as well, that allows the left hand to know what the right hand is doing. Okay? So, in the next lecture, I’m gonna be talking a lot about this hypothesis that that communication bridge is built during a very transient pairing event between the two X chromosomes. And so, briefly, the idea here is that prior to X inactivation we observe empirically that the two X chromosomes sort of do their own dance. Then, at the onset of… just before… actually, just before the initiation of X inactivation, we see that the two X chromosomes come together in three-dimensional space, and they briefly touch each other at the X inactivation center. And then, when they come apart again, one X chromosome is active and the other one has become inactive. Okay, so we have suggested that this mechanism of pairing may underlie the ability of cells to determine choice instantaneously, in a mutually exclusive fashion, and in an irreversible fashion. So, more about that in Lecture 2. Alright. So then, the final conceptual challenge is how the chromosome initiates and spreads silencing. So, as we briefly touched upon already… so, inactivation starts within the XIC, the X inactivation center, and then it spreads throughout the rest of that chromosome without touching the other X chromosome, or for that matter any other chromosome in the cell. Now, that’s a big conceptual problem, because we know that proteins, and just about all biological factors that we know, can diffuse in some way. And so… and the two X chromosomes and all the other chromosomes lie in the same nucleoplasm. And so, how is it that we render all other chromosomes immune to the inactivation process except for this one? Alright. So, that’s the conceptual challenge. And so this gene, Xist, discovered many, many years ago now, is a 17-20 kilobase long non-coding RNA. And it has a distinction of being the only gene which is transcribed from the inactive X chromosome. Now, all other genes are transcribed from the active X chromosome, by definition. This gene is made only from the inactive one. And from experiments done many, many years ago, we know that Xist is absolutely essential for X chromosome inactivation. And this image, here, is an RNA fluorescence in situ hybridization that shows that Xist, which is here in red, “coats”, in quotes, the inactive X chromosome without diffusing to any of the other chromosomes in the same cell, including the other active… the other X chromosome. So, we say that the RNA localizes strictly “in cis”. Okay. Now, we know that this property originates from the X inactivation center. So, we did these transgenesis experiments I showed you a few minutes ago, in which we move the Xist gene onto an autosome, and now that autosome gets coated by Xist RNA. And again, none of the other chromosomes get coated by Xist RNA. So, the property is derived from the X inactivation center, and there do not appear to be other X-specific elements responsible for this behavior. Alright. So, Xist starts… here at the top, Xist is synthesized. And it spreads in three dimensions to the rest of the chromosome. And then it does at least three things that we’re aware of. So, firstly, it has to push away all of the existing activating factors. You know, you’re trying to convert an active chromosome to an inactive chromosome, so one of the first things that Xist will do is actually push away the activating factors. And then it recruits silencing factors to that chromosome, to begin the process. And at the same time, it is changing the topology of that chromosome, changing the three-dimensional architecture of that chromosome. And the net result of all of this is a highly stable, a very robust, gene silencing mechanism. So, we’re gonna say a lot more about that in Lecture 3. Now, before I conclude this first lecture, I would just like to say a few words about some efforts that are underway at the present time to leverage our understanding of how X inactivation works to treat human disease. So, this is a very active area of research because, as I mentioned before, there are more than 200 disease genes on the X chromosome. And here, though, through the lifetime of the female, the inactive X chromosome lies completely dormant. So, the idea is… well, here’s a silent chromosome, but it’s a silent chromosome that carries 1000 very good genes, the genes that could function. So, it’s a reservoir of genes that could potentially be reawakened for therapeutic purposes, especially when there is a mutation expressed on the active X chromosome. So, what we’d like to know is whether we could unlock the inactive X chromosome to treat X-linked disease, using all of the knowledge that we have gathered over the past half-century. Okay. And the poster child for this X-reactivation platform is Rett syndrome. So, Rett syndrome is a devastating disorder that’s caused by a mutation in the protein called MECP2, which is carried on the X chromosome. And this disorder arises in girls who unfortunately inherit one mutant copy of MECP2. So, she’s a mosaic. She’s… 50% of her cells, approximately, will express normal MECP2, and the other half of cells will not have a functioning MECP2. So, the girls are born normal, but then during the first year of life, they begin to manifest very severe symptoms. They lose their learned abilities. They develop severe intractable seizures, get repeated lung infections, and they have autism and repetitive behaviors. And many of the girls never learn to walk or talk. So, it is a very devastating, life-long disorder. And there are currently no disease-specific treatments. Okay. But there was a very inspiring study from Adrian Bird’s lab about ten years ago, in which they showed that, at least in a mouse model, they could reverse neurological defects of Rett syndrome if they could resupply, or give back to the brain, normal quantities of MECP2, even after the onset of disease symptoms. And so that’s led many of us to ask this question — can we use our knowledge of X-inactivation to reactivate the inactive X chromosome, and thereby restore expression of MECP2 protein? And maybe partially treat Rett syndrome? So, we’ve demonstrated that 5-10% of normal MECP2 expression in the brain can have significant phenotypic impact. So, for example, here in this mouse model, a mouse that has no MECP2 function at all will live about 76 days. But if we give it even as little as 1% MECP2, those mice will live a month longer. Now, if we were to give them 5% MECP2 expression in the brain, those mice will have their lives extended by 2-3-fold. And furthermore, if we went up to 10-20% MECP… MECP2 expression in the brain, their life span is extended up to 5-fold, with a proportionate improvement to their neuromotor function. And so, the point is that a small amount of MECP2 protein can go a long ways towards good brain function. And so with that in mind, what many of us are trying to do in the field, now, is to partially reactivate the X chromosome, and thereby boost expression of MECP2, specifically in the brain. What we have identified in… within the last year or so is a specific therapeutic cocktail that would boost MECP2 significantly inside cells. And so this is a combination of an anti-Xist molecule and an anti-DNA methylation molecule. And what you can see in these graphs, here, is that the anti-Xist molecule by itself does essentially nothing. And the anti-methylation molecule by itself does ever so little. But if we combine the two of them, we get a huge boost, a 30,000-fold boost, in MECP2 expression. And so we’re presently testing this drug candidate in preclinical models in hopes of identifying, eventually, a clinical candidate that could be translated to the clinic. Okay. So, that then brings us to the end of Lecture 1. And what I’ve told you in this lecture is that X inactivation is an essential developmental process. It in many ways exemplifies the challenges that we as scientists have in trying to understand epigenetic regulation. And I mentioned also that our knowledge of X inactivation may eventually be leveraged to treat certain X-linked diseases.