Okay so if we come back to these stages of X chromosome inactivation we've been briefly through this counting and what's known about how the chromosomes can be counted. That the X chromosomes relative to the autosomes. Now what I want to think about is how that choice is made, about which chromosome should be the one that's elected to be silenced. So very little is also known about choice. But again a few years ago there was quite a break through because what was found was that the two X chromosomes transiently come together and pair and this transient pairing through the X inactivation centre and some surrounding regions known as the X pairing regions was really involved in upregulating and stabilising Xist expression and therefore determining which chromosome will end up becoming the inactive X chromosome. So why would chromosomes, two X chromosomes that would normally be apart, Why would having them communicate with one another by pairing and co-localising within the nucleus, what would this do? Why would it lead to a choice of which should be inactivated? Essentially we don't really know but we think that there must be some sort of cross talk between the chromosomes and perhaps an exchange of bound factors. So an exchange of the factors that are bound on one and then they'll, well get an unequal, with distribution of this factors between the X-chromosomes. So this transient co-localisation of X chromosomes or X chromosomes pairing is sometimes known a X chromosome kissing and we're going to watch a video of how that occurs now. So you can see the two X chromosomes here shown in orange and this is when they are both active say for example in embryonic stem cell. So, one of those is inherited from the father, the paternal X chromosome and one from the mother. So, shortly after the induction of differentiation, they transiently co-localise, as you can see, the chromosome kissing at this point. There is some exchange of factors, and then one X chromosome upregulates Xist RNA which is shown in blue. It then goes through the next stages of inactivation and localises to the nuclear periphery and goes through all of these phases of X inactivation that we will not talk about. So, we know that this X chromosome pairing is X chromosome kissing is in fact critical for X inactivation choice. The way we know that is that, if you take many copies of the X chromosome pairing region. And so you make a multi-copy array of this autosome. Then this multi-copy array will out-compete the regular X-X pairing that occurs. So this ectopic pairing that occurs at these autosomal integrations of this multi-copy X pairing region. Out competes the normal X, X pairing and therefore, when the X, X pairing doesn't occur we know that you get failure to upregulate Xist expression and therefore, failure to perform X inactivation. And so this experiment really shows that we know that that pairing is critical, and that we need for it to occur. So, choice, although this is essentially all we know about choice. About, they must, they must pair. And, and there must be some decision. And some sort of, decision between which two should happen based on this exchange of factors. We also need choice isn't always necessarily always 50-50 it isn't entirely random instead the choice of the paternal or maternal X chromosome to silence can be skewed and this then results in skewed X inactivation that is that you silence one X chromosome more frequently than the other. So through studies in mice, we know that there are not, there's not only this X chromosome pairing region which is required for the pairing to take place, but there's also another genetic region close by to the X inactivation centre, called the XCE. And this is the choice locus. We still don't know everything about this choice locus we know that between different strains of inbred mice, that there are different choice alleles and some of them are stronger or weaker than others. This means there can be a preference to inactivate one X chromosome compared to the other. Essentially the outcome is that you will end up having some sort of skewing. So if we a population perhaps of say five cells, or six cells, we would expect, by random choice, to have 50% of them silencing the paternal X chromosome and 50% silencing the maternal X chromosome. But in fact, you can have it far more skewed than that. So that you could have this one. You could have five of them choosing to express the maternal X chromosome, for example, and only one expressing the paternal X chromosome, and so this is clearly skewed from the 50/50 proportions you might expect. Now interestingly we know this skewed choice of X inactivation doesn't just happen in mice. But also happens in humans. And in human it has quite extreme consequences for diseases that are caused by mutation on gene's in the X chromosome. I'll go through one example of this and that's for Rett syndrome. Although what I'm going to tell you about Rett syndrome is in fact true for all X linked disorders. And so skewed X inactivation can alter the phenotype in females female patients of any X linked disease. So, Rett syndrome is X linked neurodevelopment disorder. What that means is, it's caused by a mutation in the gene on the X chromosome. And it tends to result in mental retardation. Its caused by mutations in a gene known as MeCP2 or methyl binding domain protein number 2. So, you might remember that I mentioned this gene or the protein, in earlier lectures, because it binds to the methylated cytosine residue. So, what's known is that this mutation, or mutations that occur in MeCP2, are lethal in males before birth. So, males only have one copy of MeCP2 cause they have just a single X chromosome. However, in females that have one copy of a mutant MeCP2 gene and one copy of a wild type MeCP2 gene, these are the patients that have Rett Syndrome, these females that are born. But what's relevant to this case is that we know the phenotype of these female Rett Syndrome patients is variable. So, in humans when you have variable phenotypes of people with a particular mutation with mutations in a particular gene there are many explanations. We know that it could be because of their particular genetic background so they might be predisposed to exhibit more extreme phenotypes or predisposed to be more resistant to these phenotypes. But, in the case of X linked disorders there are there's more at play. And so we also know that sometimes its the severity of the particular mutation itself that can alter the phenotype of the patients. But in the case of X link disorders it can also be the skewing of X inactivation which can alter the patient phenotype, or how well or ill the patient is. So I'll go through this a little bit more slowly in the next slide. So skewing means that, although the random nature of X inactivation should mean we get 50, 50, You can have this skewed all the way to being, say, perhaps, 95 percent of the time, 95 percent of cells, you find one particular X chromosome is inactivated. Or the other way around. So, in 95 percent the other way. So these extreme cases of skewing can alter the outcome for these patients for Rett syndrome or other X link diseases. So if we consider that we call the mutated X chromosome X mute. And the wild type X chromosome, X. So, if you skew, X inactivations, such is, such that the mutated X chromosome, is the one that's chosen to be silenced, 80% of the time, then that means that, 80% of the time, the cell, effectively is wild type. And so therefore there's less severe disease. However, if the reverse is true, the reciprocal, and you actually skew so that the wild-type X chromosome is silenced 80% of the time. Now 80% of the time, it's the mutated MeCP2 which is expressed and therefore, there's more severe disease. And so you can see in this way, the skewing of X inactivation can really dramatically alter the phenotype, because of the proportion of the time or the proportion of cells that express that mutated form. So apart from the skewing, we also know that it really depends on where these clonal patches are found, so for X inactivations. So you know that the calico cat, you can see the nice patchy appearance on their coat. But its not just the coat where we see these clonal patches with a choice of which X chromosome was inactivated can be seen. It can in fact be seen throughout the whole organism throughout the whole adult female in this case and so depending on where those clonal patches are depending on which portions of the brain. have the mutated copy of their X chromosome active compared to the wild type, this may also influence phenotype. So, you'll also remember I mentioned earlier that Stanley Gartler used the Barr body and which Barr body occurred the random nature of X inactivation to look at the clonal nature cancer. And its interesting now to think that this skewing of X and activation in which we can measure in much greater detail than we could then could be done in 1965 is still used to look at the clinality of cancer. In fact its used to look mostly at the clinality of leukemias. In the next lecture, we will move on to thinking about the next stages of X inactivation, the initiation and spreading of X inactivation.
