So now we come to summarise what I've told you so far about X chromosome inactivation and the stages. You remember this slide from before and I've just now added some more details. We start out with the embryonic stem cells or, the cells of the inner cell mass. And watch differentiation as it occurs over the course of about 10 days in vitro. The very early stages are all about controlling which chromosome will express Xist. We know that the pluripotency factors are involved in repressing Xist expression. And that as differentiation is stimulated the pluripotency factors will decrease this will allow the up regulation of RNF 12 and RNF 12 will then be involved in this counting process by it's own expression. We know that X chromosome pairing. This transient chromosome kissing that occurs some how related to the choice of which X chromosome will go on to be inactive. And the SATB1 and SATB2 are acquired at around this time point in order to bring about that silent nuclear compartment. So once Xist begins to be expressed we know that its rapidly brought, we know that RNA Polymerase 2 is rapidly removed. From the region where Xist is found. And we have silencing of repeats and the establishment of a silent nuclear compartment. And we rapidly lose the active histone marks. Then Xist is involved in recruiting PRC1 and PRC2 and possibly other epigenetic modifiers at this time. And we get the accumulation of the repressive histone marks. During this time, we know that there's progressive recruitment into this silent nuclear compartment. And this is why genes are progressively silenced over time. So not all genes are silenced at the same time point. But rather, it depends, when they are recruited into that silent nuclear compartment. In terms of when they will actually be silenced. It's at this stage when we really switch to the maintenance phase of X chromosome inactivation. And one of the things I haven't mentioned to date is that one of the features of the X chromosome is that it undergoes late replication timing. And this is true not only for X inactivation. Or not only for the heterochromatin found on the inactive X, I guess. But also for other heterochromatin. So I'll just spend a moment to explain that. So, within the S phase we know that chromatin can be or the DNA can be replicated at many different stages. It could be replicated early or late during S phase. And so there can be a temporal segregation of the replication of euchromatin. So open chromatin or active chromatin compared with heterochromatin like the inactive X. And so what we know is that this probably relates to the transfer or the transfer of the epigenetic marks from one nucleosome to the daughter nucleosome. So if we want to have mitotic heritability, we need to have that these epigenetic marks can be translated from one nucleosome to the second nucleosome. Just like with DNA methylation. You need to have the second, the daughter strand of DNA being methylated just like the parental strand. So it's only during late S phase when the epigenetic modified complexes that are involved in laying down repressive histone marks are actually located at that replication fork. They need to be there at the replication fork so they can recognise their own epigenetic mark on the parental nucleosome and also on the parental histones. And lay them down on the new keystones being brought in to the the new strand that's being created. So then it makes sense that heterochromatin tends to be replicated very late in S phase whereas euchromatin is replicated early in S phase. So this is true for heterochromatin in general and is true for the inactive X chromosome. It undergoes late replication timing. So this late after the late replication timing happens we know that we also have the association of other chromatin proteins or histone variants on the inactive X chromosome so in fact although you wouldn’t necessarily expect it some of these are also Xist dependent. So we know MacroH2A, this histone variant associated with the inactive X, it's association is dependent on Xist, as is Smchd1's. SAF-A is a protein that I haven't talked about, but it is involved in stabilising Xist on the inactive X chromosome. Very late in X inactivation we know that Atrx, which is a chromatin remodeller, is also associated, which is presumably involved in densely packaging down that inactive X chromosome. And so, almost all of the features, or probably all of the features that I told you about epigenetic control in weeks one and two are demonstrated by this X inactivation process. So DNA methylation, I mentioned, tends to happen very late. It does tend to happen at this very light stage, at around 10 days post differentiation in embryonic stem cells. However, there are some CpG islands, some promoters of gene subject to X inactivation which are methylated relatively early in this process. So, like gene silencing DNA methylation tends to happen over an extended period of time. But it is predominantly happening at this end stage as one of the last events in X inactivation. So, this slide is an extremely busy slide, and so it would seem that we know a huge amount how X inactivation occurs, we know much about the molecular mechanisms. But actually that's not true. While we know many of the marks that are associated with inactive X chromosomes, we don't know exactly how they fit together. So say for example for polycomb repressive complex one and two. These lay down the marks that are associated with inactive chromatin, and they were recruited by Xist. However, we know that even when they are recruited, they're insufficient to actually silence the inactive X chromosome. And so, this is actually, at the moment, is kind of where we're at. We know many of the hallmarks of the inactive X chromosome, but we don't really know how it brings about transcriptional silencing. So I just want to touch on very briefly one of the things that my lab does. So I've mentioned before that one of the things that my lab's interested in doing is using micro RNAs directed against the genes that we think might be involved in epigenetic control. Or the potential epigenetic modifiers. To be able to sort out which epigenetic modifiers are important and when. So in a way these are the potential epigenetic modifiers, this huge pile of puzzle pieces. And each in turn we can reduce the expression of each of these in turn, using microRNAs directed against each one. One of the systems that we used to study actually is X inactivation. So we studied this in the lab hoping to find new epigenetic modifiers that are involved in X inactivation because we believe that perhaps one of the reasons we don't really understand the precise molecular mechanisms yet is because we don't know about all of the puzzle pieces. We don't have all of the players. The way that we do this, we need to be is to use the fluorophore and I will explain why. To be able to form a screen, we need to be able to very rapidly assess X inactivation, and so I will just mention exactly how we do this, and so what we have done is we have made fluorescent proteins that tag the expression from each X chromosome. So, here the paternal X chromosome Xp is green. Whereas Xm, so the maternal X chromosome produces a red florissant protein. So in embryonic stem cells or cells of the inner cell mass or primordial germ cells you'll have two active X chromosomes and the cells will be green and red. However, in differentiated cells, somatic cells of the female, adult cells, then you will find that the cells are either red or green and that's because of random X inactivation. So, what we can do is we can use this system to be able to screen for the factors involved in these many processes, these many stages of X inactivation I have mentioned. So, we can take the embryonic stem cells that are both red and green. We can induce them to differentiate and watch them as they turn either red or green or not both. And as we introduce these micro RNAs to reduce the expression of each of the particular genes we're interested in about a thousand genes. We can study whether the phases of initiation and establishment or of maintenance of X inactivation occur normally or in are in some way disturbed. And this will give us some indication as to whether the gene that we reduced the expression of with microRNAs has any role in this fundamental process of X inactivation. So, the reason we study this is for the same reason that I wanted to tell you all about X inactivation, and that's because everything that we seem to learn about X inactivation is in fact true for epigenetic control for throughout the genome. So, in summary then of what I've told you about X inactivation over the past several lectures. We know that X inactivation is initiated by Xist this long non-coding RNA that is absolutely critical for X inactivation to occur. This long non-coding RNA Xist, coats the inactive chromosome in cis. So from the same chromosome from which it's expressed. We know that the X in activation centres in the surrounding X chromosome pairing regions briefly kiss. They transiently co-localise. And this influences the choice of which X chromosome will actually go on to express, Xist and become inactive. Xist can then create a silent nuclear compartment by excluding RNA polymerase 2 and we get repeats being silenced first followed by genes. And its by the genes being pulled into this silent nuclear compartment that they are actually silenced. And finally we know that Xist can recruit various epigenetic modifiers, histone variants, chromatin proteins like SmchD1 and chromatin remodellers, potentially even chromatin remodellers and this results in a progressive layering of these redundant epigenetic marks that are mitotically heritable. And so therefore we lock in that silence state, and we make sure that it is maintained for the lifetime of that organism. So, in the next lecture, we are going to spend just a very brief amount time thinking about how dosage compensation happens in lower organisms, in worms and flies.