Okay, in this lecture I'd like to think about the stages of X chromosome inactivation. So we know that X inactivation doesn't happen instantly, but instead happens progressively over a period of developmental time, and during this time we know that there are specific stages that X inactivation needs to go through. So, the first stage of X inactivation is counting. So what I mentioned earlier was that in karyotypically abnormal cells, we know that there's only going to be one X chromosome that remains active per diploid set of autosomes. And so the cell must have, must have some mechanism to count how many X chromosomes there are in relation to how many autosomes there are within the cell. And this is the mechanism of counting. The second stage of X inactivation is choice. So, I mentioned imprinted X inactivation in the last lecture, and clearly here, there is no choice. The choice has already been pre determined that the paternal X chromosome will be silenced. However, in this instance, choice means that in random X inactivation there must be a choice of which of the two X chromosomes in a normal female cell will be the one that's selected to be inactivated. The third stage of X inactivation is the initiation. And really this is all about the expression of that long non-coding RNA that I mentioned in previous lectures called Xist. Xist expression comes from it's particular location on the X chromosome know as the X inactivation centre, which we'll talk about in future lectures. After this initiation of Xist expression the Xist needs to spread to colour the entire X chromosome that will become inactive. And following on from that we need to have establishment of the silencing marks and finally after that we will have the maintenance of this inactivation And maintenance is of course perhaps, the longest lived phase of X inactivation. Because while the first few stages happen just within a few days in the embryo, maintenance must occur for the life of that female organism. And so it can be a go, be occurring for many decades up to perhaps 100 years. So, while we'll go through many of the molecular events that are involved in each of these stages. I think the really key thing to understand and remember. Is that the way X inactivation happens is that there's a progressive layering of an accumulation of epigenetic marks. And it's by this progressive layering of redundant epigenetic marks that you can ensure this mitotic heritability, and the incredible stability of X inactivation. And that's why it can last for the lifetime of an organism. So before going into all of the details for each of these different stages of X inactivation. The most important thing to remember is that it's really all about Xist. So Xist is the critical determinant for X inactivation to occur. This long non-coding RNA is essential in, for X inactivation. And so the first few stages of X inactivation, counting and choice, are really about getting Xist expressed in the first place. And the latter stages of X inactivation are turning these expression of Xist into transcriptional silence. And so, just keep this in mind at all stages. So before, we go any further, let's have a little refresher about what Xist is. So, as I said, Xist is the critical determinate for X inactivation. Without Xist nothing else can happen. So, its a 17 kilo base long, long non-coding RNA. It's spliced, just like a normal RNA would be. It's polyadenylated. But it's constrained to the nucleus. It doesn't, isn't exported from the nucleus to be made into protein. And indeed, there's no open reading frame to be found. So there's no protein product that's produced. We know that Xist is expressed from just one of the two X chromosomes in a normal female cell. And it coats that X chromosome in cis that is it coats the X chromosome from which it is expressed but doesn't act on the other X chromosome. So the first detectable event in X inactivation is indeed the expression of this long non-coding RNA. So here so shown on the right is an image that i've shown you before showing the DNA first of all in the nucleus which is stained in blue and then we also have shown in yellow The Xist DNA itself. Of course, there are two pieces of Xist DNA, one on each of the two X chromosomes. So, in addition to these two Xist DNA foci, which you can see, with the very small yellow dots that are shown indicated with the arrow, each of these arrows. You can also see the Xist RNA, which is stained in red. using instituted hybridisation. But this Xist RNA is not found on both X chromosome but instead just found on the inactive X chromosome where it forms a cloud so its not a punctate RNA like you would expect. what if you just had a small amount of RNA being expressed and then being exported. But rather it's being expressed and retained in the region from which it's expressed and so it forms this cloud, which actually coats the entire X chromosome which will become inactive. So, let's now think about X chromosome counting, this first stage X inactivation. Well there still isn't a lot of knowing about X chromosome counting and over the years there have been many theories about how this might occur. Many of which have been controversial but, I think now the consensus is that they are both X linked factors and autosomal factors and it's the balance between these X linked factors and the autosomal factors which allows the cell to be able to count how many X chromosomes there are compared with how many autosomes. So, so far we know essentially nothing about these autosomal factors. But we do know something about the X-linked factors. It appears that there are many, regions that are quite close to the XIST gene itself, that encode non-coding RNAs. These particular DNA elements, which I'll show you in the next slide, are all regulating Xist expression. And we know if we do something to manipulate these different elements, we get the incorrect number of X chromosomes being inactivated and so they must have something to do with counting. So lets look at those factors now. These DNA elements. So what's pictured here is the X inactivation center. it's around 100 kilo bases in size although this picture isn't to scale. An added sensor is the Xist noncoding RNA gene here shown in blue. In the darker blue small box I'm indicating the repeat A, RepA and this is the region the region of Xist which is very important for silencing because it binds to polychrome repressive complex 2 which I've mentioned before and I will come back to again. But all of these other elements that are shown here, all of these other gene also contribute to the expression of Xist. They all control Xist expression in some way shape or form. So in general the ones that are shown in the yellow or red tones that are on this end of the X inactivation center are involved in repressing Xist expression. Where as the ones shown here in green, Ftx and Jpx are involved in activating Xist expression. Now what we know is that this X inactivation centre, if you take this region from, in its entirety, is necessary and sufficient to X inactivation to occur. So what I mean by that is if you take. If you delete this region, then X inactivation won't occur. Therefore, it's necessary. However, if you take this X inactivation center, this whole region and place it onto an autosome, then you'll get inactivation of at least part of that autosome, and so it's sufficient to get this inactivation to take place. We know that we need more than just the Xist gene itself. Instead we need this whole region with all of these other elements. This can also be seen because in some patients there are trans-locations between the X inactivation centre and autosome. So, it's not just through experimental methods that we've, we've placed the X inactivation centre on another autosome. So what are all of these different factors doing and how are they controlling Xist expression. Well in all cases they are long non-coding RNAs. So the centre is made up of a bunch of non-coding RNAs which control another non-coding RNA. I'm not going to through the specialised features of each one of these but perhaps the most important one to remember is Tsix or Tsix this long non-coding RNA is transcribed antisense to Xist and it represses Xist expression. Whereas DXPas34 and Xite themselves activate Tsix and there by indirectly, impress Xist expression because they do that also by activating Tsix. So again these precise details aren't important except to remember that we have a predominance of factors which repress Xist expression. With just a couple that activate Xist expression within this X inactivation center. All of which are long noncoding RNAs. So if we look upstream of this X inactivation centre, a couple of hundred kb upstream we find another gene called Rnf12. And this gene Rnf12 Rather than being a non-coding RNA, actually does code for a protein. The protein is called Rnf12 or Rlim. It has two names that are interchangeably used. So this gene, Rnf12, being that it's on an X chromosome, is subject to X inactivation. So, what's very interesting was that in 2009 a real breakthrough came in our understanding of X chromosome counting. And this was a discovery for this gene of a role for this gene Rnf12 which sits a few hundred kilo-bases outside of the X inactivation center. So what was proposed and shown was that you needed a threshold level of RNF 12 for X inactivation to begin to occur so I'm going to explain this in a series of diagrams so to start out with if you can see that you have cells with 2 active X chromosomes then the female nucleus as two copies of RNF 12. And the male has one, and therefore with two active X chromosomes, you'll have twice the number of molecules of RNF12 here, showing as a red blob. So this is Rnf12. You know, you'll have twice the numbers of molecules in a female nucleus that has two active X chromosomes, compared with a male nucleus, which has only one. However then once X inactivation proceeds you will inactivate one of those copies of Rnf12 and then have only the same amount of Rnf12 as in a male cell and so this is why it proposed that there must be threshold so if you had what was shown here as 8 copies 8 molecules of Rnf12 protein. That would be above the threshold an X inactivation would be initiated whereas once you have 4 copies as shown now this would be insufficient to initiate X inactivation. However, although this was really a ground breaking discovery it still isn't enough to explain the mechanism X chromosome counting completely. And that's for two reasons. First of all we know if you completely delete Rnf12 in a female cell X inactivation will eventually occur. It will occur much later than it should have done and it occurs very inefficiently but it can occur in some instances. And so this suggest that there are indeed other factors that are still important in counting, and that these can in some way compensate for a loss of Rnf12. So, the question is then how does Rnf12 actually work? So how is it really going to bring about, this initiation of X inactivation. So what we think is that Rnf12 activates Xist so just like those other two green elements those long noncoding RNAs. Rnf12 also activates Xist expression but this time as a protein activator of Xist expression. We think that male cells have insufficient Rnf12 to activate Xist and after X inactivation proceeds with a female cell we believe that we've reduced the level Rnf12 to such a level that the second enact X chromosome will not be inactivated because we are below the threshold level of Rnf12. So, clearly Rnf12 must somehow be involved in this counting process, this first stage X inactivation, but we still don't know and this by the levels of its own expression, this threshold, but we really still don't know anything about the autosomal factors involved. And we don't know anything, and it's clear from the Rnf12 results that there are other factors that are also involved in counting. But they're yet to be identified. In the next lecture, we'll think about X inactivation and pluripotency, which I've mentioned in earlier slides. where there are two active X chromosomes in the pluripotent cell types.
