Okay if we come back to these stages of X chromosome inactivation we've briefly covered counting and choice. And now we need to think about the initiation of X chromosome inactivation. So the initiation of course is all about turning on Xist expression. So, as we've mentioned before that the main thing we've required for X inactivation in fact the critical determinant is Xist. And so initiation requires Xist to be turned on and for it to then coat the inactive X chromosome that will or, the chromosome that will become the inactive X chromosome in cis. In other words, from the very one that it's expressed from. So Xist we know, this Xist expression can be detected in all female somatic cells for the lifetime of the organism. And so, although its the critical determinant for initiating X inactivation, it is there for the life time of the organism. But, there seems to be some sort of switch in the dependence on X inactivation on Xist itself. So, in the very early stages of X inactivation just after Xist is being switched on, there is a critical dependence still on Xist. And during this time, if Xist is switched off then it's reversible and you'll go back to having two active X chromosomes. But later in developmental time, when you're in the later phases of X inactivation, or indeed in the maintenance phase of X inactivation, which of course is predominantly what occurs for the longest period of time, then there's less of a dependence on X inactivation. If you at this stage delete Xist you can destabilise X inactivation but you won't immediately result in upregulation of that inactive X chromosome. So because we know that Xist expression is really critically involved at the early phases but also we know if we turn Xist on in most cell types so we turned on for example, on a trans gene that's located on an autosome, we don't find X inactivation ensuing. So there are very few cell types where X inactivation can actually proceed even with Xist expression, and so this led researchers to postulate that there must be other competence factors, factors that are required to be found within the nucleus or found in the cell in general. Before Xist can actually bring about transcriptional silencing. And so, I'm going to mention some of these other factors as we come through the next few slides. So, after this initiation of Xist expression, the next stage is spreading of Xist and this silencing that ensues. And so over the years, just like with counting and choice, there have been a very number, very large number of theories about how this might, might occur. But I think now the prevailing model, and the one everybody seems to accept, is that there is a progressive recruitment of the X chromosome into a silent Nuclear compartment. So what I mean by silent nuclear compartment is a region where RNA polymerese two is excluded so the RNA polymerase that actually transcribes genes is removed from this region. and the X chromosome is drawn into this region. So what I'm showing you in the picture along the bottom of the slide is first of all a nucleus that's stained with DNA. A DNA stain DAPI so we can see the DNA and you can see two regions that are more densely stained than the others in the top right hand side. These are two inactive X chromosomes. These particular cells are cancer cells that happen to have two inactive X chromosomes. The second oh, sorry, in the third image you can see Xist expression in green, and you can see these two DAPI dense or these heterochromatic regions. These Barr bodies are also where Xist is found, but the, this image in between, which is called h/m Cot-1 is a stain for repetitive RNA, so the expression of repetitive elements, and what you can see indicated by the yellow arrows. Is that these two regions where these Barr bodies are found, where Xist is found, are devoid of this repetitive RNA. So, then when you look at the pixel profile, so this is when a line has been drawn through the image showing the merge, the line is shown in yellow. You can see that throughout this region, you have enrichment of DAPI. So that the DNA stain in blue, and that strain by the mound in the graph. And the mound in the Xist graph, so that the green line. But you can see that the red goes down. So you see much less, repetitive RNA being found in that region. And this is because we know that one of the earliest events after Xist expression is exclusion of RNA polymerase two from this region where, in which the X chromosome is found. And silencing of the repeats, and so we find that this, the repetitive, the repeats that we sometimes be expressed, the ones that are found in this region, so the ones in the X chromosome are not being expressed at this time, and it's a very early event in X inactivation. So this happens, this silent nuclear compartment and the silencing in the repeat actually happens before the genes on the X chromosome are silenced. And we know that the X inactivation spreading, correlates with how the genes are actually drawn into this silent nuclear compartment. And I'll show you this in more detail in a diagrammatic form in the next few slides. But here, I'd like to mention about some of those factors that are differentially expressed or that these competent factors, competence factors which need to be present in the nucleus for Xist to be able to bring about silencing. So these factors, some of these factors are SATB1 and SATB2. So, SATB1 and SATB2 were found because they were expressed in both embryonic stem cells, early post differentiation but also in the limited number of other cell types. that are actually competent to perform this initiation of X inactivation. And SATB1 and SATB2 are factors that are involved in nuclear reorganisation. So in other words, they move the chromosomes around. They help to move the chromosomes around. So we know that these factors have to be there for Xist to be able to bring about that silent nucleic compartment. So I'm going to go through all these things I've just said about the spreading of Xist and silencing in a diagrammatic form. So pictured here is an X chromosome as it might exist in an embryonic stem cell. Fairly open because it's being expressed. And so shown on there in yellow are the repetitive elements so, that would be for example the intracisternal a particles or the Line1 elements. These repeats that are found spread throughout the genome whereas in green are the genes that are currently being expressed because it's an active X chromosome at this point. Shortly after these embryonic stem cells or the inner cell mass is induced to differentiate, then you get a change in how the X chromosome looks and how much space it takes up. We know that Xist expression comes on that I am showing here as a blue squiggly line, and we know that SATB1 and SATB2 are involved. So, what happens is within this X chromosome, it's recruited into the Xist domain. But, to begin with it's only really those repetitive elements, which is shown in yellow here that have been recruited into this region that contains Xist. So it's only, the repeats that are silenced. This recruitment into that Xist region contains these purple stars, these SATB1 and SATB2 that I mentioned, these competence factors that are acquired. But at this early stage, while the repeats have been silenced, because they are within this silent nuclear compartment, this Xist domain. We know that the genes remain on the outside and so the genes remain active and I'm showing that here is being green. Then in the final stages when you reach the somatic cell we know that now more of those genes have been pulled into that region, pulled into the silent nuclear department by SATB1 and SATB2 or other competence factors. And now, many of the genes are indeed silenced. And I'm showing that here being a black gene, with, a red rim. So, once the genes are within the silent nuclear compartment and pulled into the Xist domain, then they too have been silenced and X activation has occurred. What I haven't mentioned up to now in the lectures, is that, in fact there are a small handful of genes. That don't undergo X inactivation on the X chromosome. So these genes that escape X inactivation, remain outside this silent nuclear compartment in somatic cells. So they're never drawn into the region which isn't transcribed because it's void of RNA polymerase two. What we know about these escape genes is that they differ slightly between mouse and human. So here shown on the left-hand side is an image where you can see that all of the X chromosome is laid out on the left in the human and on the right in the mouse. And what the centromeres are shown in purple. So, we know that a human centromere is quite central where as a mouse centromere is at the very end right at acrocentric at one end. And the genes that escape are showing in orange. So most of the escaped genes are in the regions that are showing as PAR, the pseudoautosomal regions. So in humans there are two of these regions, PAR1 and PAR2, and in mouse there's just one of these regions. And these pseudoautosomal regions are the parts that are required for pairing of the X and Y chromosome in cell division. And so these genes that are found in the pseudoautosomal region tend to have a homologue on the Y chromosome. And so this means that they have one copy that is expressed from the X and one copy from the Y in the male cell but in a female cell you'll have both copies being expressed from each X chromosome even if that X chromosome is actually the inactive X chromosome. But they are also a smattering of other gene's you can see orange coloured gene's spread along of these 2 X chromosomes and there are more in humans than they're are in mice and so these orange escape genes that aren't in the pseudo autosomal regions they don't have a wide linked homolog. In which case they're not silenced. You'll have twice the dose of them in females than you would in males. And so, perhaps these escapee genes that don't have a Y homologue might have something to do with, with the differences between the sexes. What’s important to note at this stage, is that all of these genes, whether they're in the pseudoautosomal region or not, all of these escape genes reside outside of that silent nuclear compartment. So, the reason that they escape silencing is that they are never drawn into that region that contains the Xist cloud, and that is devoid of RNA polymerase two. So in the next lecture, we'll think about how X inactivation is established and how it spreads.