Jeannie Lee (Harvard) 2 – X Chromosome Inactivation: Making the Right Choice

Welcome back, everyone. So, again, I'm Jeanie Lee. I'm a Professor of Genetics at Harvard Medical School, and I'm also a faculty member in the Department of Molecular Biology at Massachusetts General Hospital. Now, in Lecture 1, I gave an overview of X chromosome inactivation. And what we're gonna do now in Lecture 2 is a deeper dive into the initiation phase of inactivation, namely how cells count and then make the correct choice of active and inactive chromosomes. So, here again are the different steps of X inactivation. And by way of review, there's a counting mechanism followed by a choice mechanism, and then the initiation of silencing. So, we're gonna focus first on the counting step. So, again, this takes place in the blastocyst, shortly after the paternal X chromosome is reactivated. And every cell makes its own determination of the X chromosome number. So, it's taking place around the time that the epiblast has 20 cells or so. Alright. And we discussed how it's really the X-to-autosome ratio that cells are sensing, rather than the absolute number of X chromosomes.

And we also mentioned that cells follow the n-1 rule per diploid content, so that males who have an X-to-autosome ratio of 0.5 will not inactivate any chromosomes, regardless of whether it's diploid or tetraploid, with twice the genomic content. Whereas the female, with an X-to-autosome ratio of 1.0, will inactivate one of her two X chromosomes, and if she's tetraploid she'll inactivate two out of those four X chromosomes. And furthermore, if the diploid had three X chromosomes, it will inactivate two out of the three.

And if she has four X chromosomes, she'll inactivate three out four. And so the point is that cells follow this n-1 rule in a diploid content, and every cell makes the determination for itself. And we also mentioned that counting is most likely a titration of X-linked and autosomal factors. We mentioned that the numerators are produced from the X chromosome, in the form of this green blob. And the autosomes also produce their own factors — we call them denominators. And then the red and green factors will titrate each other out, and form a blocking factor that then sits on one X chromosome, at the inactivation center, and prevents that inactivation center from initiating the cascade of inactivation. So, we see that in the male cell. And we also see that in the female cell, where the same factors titrate each other out to form a hypothetical blocking factor, which then sits on the… one X chromosome, preventing that inactivation center from firing, giving rise to this privileged, active X chromosome.

And we mentioned that one hypothesis is that the remaining X chromosomes would then undergo an inactivation by default. So, that's certainly one viable viewpoint. However, we favor the idea that there is a purposeful inactivation — not something that happens by default. Because, in fact, the female produces an extra copy of these green factors, since she has one extra X chromosome. And that green factor is not titrated by the blocking factor, so we propose that that factor goes and forms this additional complex called a competence factor, which has to sit on the remaining X chromosome to purposely induce the initiation of inactivation.

So, that's the two factors hypothesis. Okay. So, then we get down to, what are these molecular factors that make up the X, right? And what are the factors that make up the A of the X-to-autosome ratio? And here we'll start with the numerator, the X. Alright. So, in principle, without knowing all that much about what these factors are, we can say that the factor has to be produced from the X inactivation center — from those transgenesis experiments that I showed you before. And we believe that that factor has to escape X inactivation. So, I haven't mentioned before, but a number of genes on this chromosome are actually immune to the influence of Xist.

They escape silencing. And we believe that numerators have to escape X inactivation in order to serve as a dosage-sensitive readout of that chromosome. And furthermore, that factor has to be diffusible. So, in order to titrate away autosomal factors, it has to be able to move around in the nucleus. And then finally, it has to act at the X inactivation center, which is where the Xist gene ultimately resides. And then, most importantly, the math has to work. And what I mean by that is, if something were truly a numerator, then when we take away one copy of that X-linked numerator, a female's cells should start to behave like male cells and block X inactivation. Right? So, she should think that she's a male cell, because she's missing the extra X-linked factor. And conversely, if we were to give a male cell extra copies of a numerator, that male cell has to start to behave like a female cell and start undergoing X chromosome inactivation. Okay. So, these are the rules. Now, a while back, we started to suspect this non-coding gene, Jpx, which lies just on the other side of Xist.

It's an X-linked Xic product. We know that Jpx levels increase about tenfold during the process of X inactivation. And it occurs in the same timeframe that Xist is getting upregulated on that chromosome, and we know that it escapes X inactivation. It's one of the few genes that will escape inactivation. And from the transgenesis experiments… I told you before, when we move this region and put it on an autosome, that autosome behaves like an X chromosome and undergoes inactivation. However, if we were to make a transgene that's missing this Jpx, that transgene could no longer inactivate the autosome. Okay. So, we put this to the test. So, here… this is an RNA fluorescence in situ experiment, in which we're looking at expression of Xist, which is shown here as a pink dot, and it's coating the X chromosome. So, in wildtype cells we see a very robust expression of Xist RNA. Now, if we, in the female cell, deleted just one copy of Jpx…

So, there are two copies normally… we just take away one copy of Jpx… you see that these cells no longer produce that large, robust cloud of RNA that coats the inactive X chromosome. However, if we then take a copy of Jpx and insert it into another chromosome, an autosome, we rescue this expression of Xist in the same female cells. Okay. So, these experiments tell us two very important things. First of all, Jpx is a dosage-sensitive element. So, by removing one copy of Jpx in these female cells, the female cells start to behave like a male cell. And furthermore, Jpx is diffusible, because we put the gene on an autosome.

That autosomally produced Jpx will rescue Xist expression on the X chromosome. And then we did the converse experiment, where, in male cells now, we insert extra copies of Jpx. Male cells normally don't produce Xist at all, so you don't see a big Xist spot in green. But when we insert extra copies of Jpx into these male cells, you start to see Xist expression go up, okay? So, that suggests that Jpx may in fact be a candidate for a numerator factor. And in this experiment, here, we're demonstrating that Jpx is acting as a diffusible RNA, one of these long non-coding RNAs. And it's not simply the genetic element, the DNA, which is responsible for this counting act. And we know that because when we introduce these things called shRNAs…

This is a technology that allows us to degrade the RNA when we introduce the shRNA into cells, without actually touching the underlying gene. Okay, so when we introduce these RNA degradation factors, we see that Xist could no longer be upregulated, like in the wildtype female cell, or the untouched female cell. So, this experiment tells us that Jpx is a diffusible element, acting as a non-coding RNA. Okay. So, the idea then is that Jpx is one of these green factors that's being produced by the X chromosome, and that it is titrating away the autosomal blocking factor. And then the untitrated Jpx factors would be the one that sits on the remaining inactive X to induce the firing of that inactivation center. Okay. So then, we turn our attention to, what are the pink factors? What are these autosomal factors which are getting expressed to titrate Jpx? Now, here, we began to suspect a protein called CTCF.

Now, CTCF is a very famous protein because it does a lot of different things. It has been shown to be a critical chromosome architectural factor. It was first identified by Victor Lobanenkov as an 11-zinc finger transcription factor that can take two distant genetic elements and bring them together to form a loop, as shown here, and can regulate enhancer-promoter interactions. And more recently, CTCF has been shown to reside at the border of these chromosomal topological structures called TADs, and I'll say a lot more about that in lecture number 3. Okay. But importantly, we've known for quite some time that CTCF occupies discrete positions at the X inactivation center and plays an important role in a number of different processes. So, for example, here CTCF binds to the Xist promoter at a number of positions that are shown here in red. And we know that at these sites CTCF is serving as a repressor of the Xist gene. And then, as cells go through X inactivation, these binding sites here — shown, again, in red — pretty much stay the same. They remain bound. Except for one… at one location, the so-called P2 location. Now, at this position, CTCF binding actually goes down during X inactivation.

So, that was really interesting. And we wanted to know, because there are two X chromosomes, from which X chromosome CTCF was getting removed. So, for that, we had to perform an allele-specific analysis. You know… so, that's an analysis that allows us to tell the difference between the future active and the future inactive chromosomes. And the long and the short of this is that it is from the future inactive… the chromosome which will become inactivated… that's where CTCF is losing its binding. Okay. So, during X inactivation, CTCF at P2 is retained only on the active X chromosome. And we know that its role is to block the expression of this critical silencing factor called Xist. So, what I've told you, then, is that CTCF is an autosomal factor. It represses Xist expression.

And at the same time, I've told you that this non-coding RNA that's X-linked induces Xist expression. And so, with one being autosomal, the other one being X-linked, and doing opposite things, we wondered whether these two factors could be functionally interacting with each other, and be part of that titration mechanism that I referred to earlier, part of the X inactiv… the X-to-autosome ratio. So, indeed, we learned that CTCF is an RNA-binding protein. That was not previously known to bind RNA. But in this context, it is a very good RNA-binding protein. In fact, it prefers to bind RNA over DNA.

So, you can see from the same sort of gel shift analysis, here… except that this time we're using Jpx RNA, and you see that very robust shift, indicating a high-affinity binding between the RNA and CTCF protein. And so, in fact, we can biochemically measure the affinity of this complex by measuring the dissociation constant. And that Kd is less than one nanomolar. So, CTCF is a very good RNA binding protein, much better than binding to… its binding to DNA, where the dissociation constant is more than 20 nanomolar. Okay. So, then we have this idea that CTCF may be getting competed away from the promoter by this non-coding RNA, Jpx, and that may underlie this titration mechanism.

And so to test that, we mixed together purified components of P2 DNA, CTCF bound to the P2 DNA, and increasing concentrations of this Jpx RNA. And what we see here in this gel shift analysis is that CTCF gets pulled away from the DNA by Jpx RNA. Okay. So, we can do the same sort of competition experiment inside of cells. Now, what I've shown you so far occurred within a test tube, right?, but we can do this sort of thing inside a cell as well. So, here, we're overexpressing CTCF — that's the repressor of Xist — and you can see that when we do that the cells no longer upregulate this green cloud of Xist.

So, here's wildtype, as you can see. But in the overexpression system, we no longer see Xist clouds. However, we can overcome these extra quantities, if you will, of CTCF by giving the cells extra Jpx RNA. And so that's what we've done here. And you see that these green spots come back. Okay. So, that supports this idea that CTCF and Jpx RNA are functionally interacting with each other and titrating each other inside of cells. So, what we propose, then, is a functional antagonism between CTCF and Jpx RNA. So, prior to X inactivation, CTCF sits very robustly at the 5' end of Xist, where it blocks the expression of Xist. And then, at the onset of X inactivation, what we have empirically measured is that Jpx RNA increases in expression by tenfold. And when it crosses a certain threshold, as it will do only in female cells — because we have twice the number of Jpx copies as male cells — the Jpx RNA binds to CTCF and titrates it away from one Xist promoter.

And that act enables Xist RNA to be upregulated on that same chromosome. Okay. So, that's how we're presently thinking about this functional antagonism and about the X-to-autosome ratio. What I'd now like to turn your attention to is the second step of X inactivation, which is allelic choice. And I mentioned in the first lecture that this is a conceptually very challenging problem, because, here, choice has to be random. It has to be instantaneous, mutually exclusive, and completely irreversible. Okay. So, how do we make the right choice.

And again, we believe that there is a communication between the two chromosomes, such that when one chromosome is chosen as the inactive one the other one is instantaneously the active chromosome. So, this mutually exclusive choice — which is what we call it — requires two genetic loci at the X inactivation center. So, one is Xist's antisense repressor, called Tsix, shown here in yellow, and the other its enhancer, shown here in brown, called Xite. Okay. So, the region that's responsible for choice is this 15 kilobase domain that encompasses Tsix and Xite.

And what we know — going back to experiments done many, many years ago — is that prior to X inactivation, when the two chromosomes are active, the Tsix antisense RNA is expressed at very high levels, and its expression prevents Xist from turning up. Okay. But then, at the onset of X inactivation, what happens is that the antisense RNA disappears from one X chromosome. And when it disappears, Xist RNA is upregulated from that chromosome, leading to the formation of the inactive X. While on the other X chromosome, the action of the Xite enhancer, right?, allows Tsix to persist on that chromosome, so that the Xist gene continues to be repressed on that chromosome. And that chromosome stays active. So, the action of Tsix is essential for this mutually… for this allelic choice, with its persistence on the active… its persistence determining the active X chromosome, and its loss determining the inactive chromosome. Now, what we also demonstrated in these early studies is that we can genetically manipulate the choice decision by simply removing Tsix from one X chromosome. And when we do that, that chromosome is always the one that becomes inactivated. So, we can influence and manipulate which X chromosome will become the inactive one.

Okay. So, then, what I told you is that normally cells can choose either one or the other X chromosome for inactivation. But very strangely, when we delete both copies of Tsix — not just one, but both copies of Tsix — we see these additional cell types, where both X chromosomes are inactivated or neither X chromosome is inactivated. Right? So, it appeared to us here that the cells are undergoing some kind of a chaotic choice. Or maybe there's no choosing at all. You see all combinations as a result of losing this antisense repressor. So, this is a loss of mutually exclusive choice. And from that, we postulate that maybe there has been a loss of communication between the two X chromosomes, such that now cells… you know, really, the left brain doesn't know what the right brain is doing, going back to the analogy I drew in the first lecture. So, these experiments also tell us that the Tsix repressor is very important for that communication between the two chromosomes. Now, around the same time, we and the Heard lab made an interesting observation, which is that prior to X inactivation the X…

Two X chromosomes behave like they're not even aware of each other. But at the onset of X inactivation, one of the very first things that we see is that the chromosomes come together, and they briefly touch, just at the X inactivation center. And it's very brief. It happens probably in under 15 minutes, but let's say under 30 minutes. And then when they come apart again, one X chromosome is the active one; the other one is expressing Xist, so it's become the inactive one. It's almost as though the cells have flipped on a bistable switch as a result of pairing. And you can see this pairing event, here, by DNA fluorescence in situ hybridization, where you see two dots of the Xic coming close together in a certain timeframe during X inactivation. And so, because of this observation, we propose that the XX pairing process may serve as a bridge by which the two chromosomes can communicate with each other prior to the choice decision.

Okay. So, in support of that idea, here… which we've shown… that the center responsible for pairing is the same region that's responsible for allelic choice. It's the same white bar that you show… that you saw a few slides earlier. So, this is a 15 kilobase region. And intriguingly, if we were to take this white line — take this genetic region — and insert that into an autosome, far away, that autosome is now induced to pair with the X chromosome. So, this region, this very, very small region, is both necessary and sufficient to direct pairing.

Alright. So, here are some real-life experiments. When we delete both copies of Tsix, the chromosomes no longer pair. And as I mentioned, there's a loss of mutually exclusive choice. On the other hand, if we insert extra copies of the pairing region into an autosome, which is shown here in blue, that autosome does something very strange, which is that it attracts one of the X chromosomes… one of the X chromosomes to come and pair with it, and in doing so it prevents the two X chromosomes from interacting with each other, and X inactivation is arrested. And so what these experiments tell us is that XX pairing is very important to somehow properly initiate X chromosome choice. So, we propose that pairing is a mechanism by which the two X chromosomes can break their epigenetic symmetry. So, prior to the onset of X inactivation, Tsix is expressed from both X chromosomes.

And then the process of pairing results in the loss of antisense expression from one X chromosome, and it is from that chromosome that Xist becomes upregulated. And on the opposite X chromosome, Tsix persists, and that blocks the upregulation of Xist, allowing this chromosome to remain active in the female cell. So, we propose, then, that XX pairing is a mechanism of crosstalking which allows the two chromosomes to adopt mutually exclusive fates, of active and inactive X chromosome. So, we've also observed that CTCF, this very versatile zinc finger transcription factor, is essential for X chromosome pairing, by serving as an inter-chromosomal glue. So, in these complicated experiments, what you can see is that CTCF binds to Tsix and Xite RNA, and CTCF also binds to the DNA — that white line, that 15 kb region I demonstrated before — to specific regions of the pairing and choice center. So, this binding to the RNA is essential for CTCF to be recruited as an inter-chromosomal glue. So, before concluding this lecture, I would like to demonstrate what we think is taking place during that process of allelic choice.

So, prior to the onset of X inactivation, this pluripotency factor, OCT4, binds to both Tsix and Xite, and transactivates the expression of Tsix and Tsix. So, I didn't mention that in my lecture, but this is the case. And then the expression of Tsix and Xite recruits CTCF to this pairing region. At the onset of X inactivation, what we see is that the two chromosomes come together and pair exclusively through this 15 kilobase region. And we believe that this pairing event serves as a platform on which the two X chromosomes can communicate with each other, and make the determination of who will be the active versus the inactive x chromosome. Now, exactly what they're saying to each other and how this is done is something that's under very active investigation. We do not presently know. However, we suspect that what happens is that when the two chromosomes come apart again, these transcription factors — like CTCF, and very likely many other factors — will repartition onto one X chromosome. And so CTCF is serving as a transcriptional repressor.

Its binding to this chromosome will downregulate expression of Tsix as well as Xite. And their downregulation is what allows Xist to be upregulated from that chromosome. And that chromosome becomes the inactive X. But on the other hand, on the future active chromosome, Tsix and Xite persist, and their persistence prevents Xist from becoming upregulated, and that chromosome remains an active chromosome. Alright. So, before concluding Lecture 2, then, I just want to mention one last thing, which is that the ends of the sex chromosomes — the telomeres — play a very important role during XX pairing.

Now, XX pairing is not taking place in a random place in the nucleus. Instead, it's taking place within what we call a tetrad, okay? So, what we've now shown is that the ends of both sex chromosomes — the X and Y — produce a non-coding RNA called PAR-TERRA. And PAR-TERRA agglomerates… this RNA brings the two telomeres together… both X chromosomes, and even the X and Y chromosomes. It brings the two sex chromosomes together at the nuclear envelope. And then that RNA serves as a tether, and reels in the X inactivation center so that pairing takes place within this tetrad of two telomeres and two inactivation centers. And you can see real-life examples, here, by DNA FISH, where a pair of the telomeres, shown in red, and a pair of inactivation centers, shown in green, have agglomerated at the nuclear envelope to enable XX pairing. So, why would they even bother to do this? Well, because the nucleus is a vast space. And it would take time for the two inactivation centers…

a lot of time for the two inactivation centers to come together. And so this tethering mechanism facilitates this homology searching process through this process that we called a constrained diffusion in 3-dimensional space. So, that then concludes the second lecture. And we will talk about the initiation and spreading of X inactivation in Lecture 3..

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