Okay so in the previous lecture we thought about the DNA elements that actually the long noncoding RNA's that control Xist expression in addition to Rnf12. So the next activation centre is made of the cluster of noncoding RNA's that each control the expression go Xist. And just a little upstream a few hundred kilo bases upstream. Rnf12 also seems to control Xist expression by being an activator. But there are also another set of factors which control Xist expression. And you remember, I said, that really, these first few stages of X inactivation are all about determining whether Xist will be expressed or not and controlling the expression of Xist. So these other factors are known as the pluripotency factors. Pluripotency factors tend to be transcription factors, and what they do is they regulate the expression genes that are involved in maintaining pluripotent cells. So for example embryonic stem cells or primordial germ cells. These pluripotency factors are found to be expressed in these two cell types, and that's what, for example they are opt for Sox2 and Nanog are the most famous three although there are many others and Rex1 is one of those. So these transcription factors activate genes within the pluripotency circuitry, the genes that really need to be there to maintain the pluripotent nature of these, of these cell types. So primordial germ cells and embryonic stem cells are unique because of their pluripotency, but also unique because you remember I mentioned that they express two X chromosomes. They have two active X chromosomes, and this sets them apart from all other cells at any stage of development for a female. And so these two active X chromosomes only ever occur in the context of pluripotency within the normal scenarios. So this suggested to many researchers that perhaps there was a link between the pluripotency factors and expression of Xist. And indeed over the last few years, they've shown that this is the case. So, how would the pluripotency factors be doing this? So, we consider again this picture of the X inactivation centre with Rnf12, a couple of hundred kb outside of the X inactivation center. And now, I'd like to explain how these pluripotency factors are acting within this region, and how it is that they can that also control Xist expression. So, the first thing is that these pluripotency factors that I'm showing here as purple blobs or purple proteins bind in multiple regions within the in multiple places within this region. First of all, they bind within the Xist gene itself. They bind within the first intron of Xist and many controlling elements for the expression of genes are found not only in their promoter but also within the first intron. So by these pluripotency factors binding within the first intron they're actually, directly repress Xist expression. So they're keeping Xist off. If they keep Xist expression off this means X inactivation can't occur. And so, when these pluripotency factors are around in those pluripotent cell types, embryonic stem cells and primordial germ cells, then Xist will not be expressed because of this binding. They also allow for the repression of Xist in two other ways. The second way is that they bind to DXPas34, and also to Xite. Now you might remember I said that these two regions activate Tsix. And by binding here these pluripotency factors activate, Xite in DXPas34 and thereby activate Tsix. And if you have more Tsix, which is a repressor of Xist, you would keep Xist expression off. So, it's somewhat more convoluted to think about it in this case, because there's activating repressor of Xist is what the pluripotency factors are doing. But this is a second mechanism by which they keep Xist expression off. Third mechanism by which they repress Xist is by actually repressing Rnf12. So you'll remember Rnf12 is an activator of Xist. And I'm trying to indicate that to you because all of the green genes activate Xist expression. So if you repress the expression of Rnf12 shown over here, then you are going to repress, the expression of Xist because you are disallowing the expression of an activator. So in conjunction, all of these 3, regions what that they bind together, keep Xist expression off and therefore keep two active X chromosomes. So if we consider that then together. We know that, that these primordial cells that, the primordial germ cells or the X inactivate, the X inactivation that's not occurring in embryonic stem cells, so the two active X chromosomes. We know that X inactivation does occur at later stages. And this happens because the pluripotency factors are decreased in their levels upon differentiation. So, for example, in the embryonic stem cells when you induce differentiation, the levels of the pluripotency factors decline within the next day or two, and they therefore will leave this repression on Xist, and at this stage, we know Xist can be activated. So, for example, if we consider Rnf12, if we remove the repression of Rnf12, Rnf12 would come on early post differentiation. It with then allow activation of Xist and once Xist is activated, we'll have X in activation and Rnf12 will be turned off again because of X inactivation. And so we have this very temporal control of Xist expression and allowing X inactivation to occur only after differentiation ensues. In the next lecture, we'll think about choice of X chromosome inactivation. So which X chromosome will be chosen to inactivate.
