Okay. So we've been through counting, choice, initiation, and spreading of X inactivation. And now we're going to consider how silencing is established. Now, there really is quite a fuzzy distinction between the spreading of X inactivation and actually establishment of silencing or the establishment of X inactivation. So keep this in mind. There probably aren't two distinct mechanisms. But some of the earliest events after Xist is expressed and the establishment of a silent nuclear compartment, are the alterations in the epigenetic marks that are found on the inactive X chromosome. One of the very earliest things to happen is that you find a loss of active histone marks. So you lose those histone acetylation marks, histone H3 and histone H4 acetylation and methylation of histone H3 lysine 4. So these three examples of active histone marks are removed from that X chromosome that is now silent or is becoming silent. You secondly get an accumulation of repressive histone marks. So the three that are a hallmark of the inactive X chromosome are histone H3 lysine 27 methylation, histone H3 lysine 9 methylation, and histone H2A lysine 119 ubiquitination. So we know that this accumulation of repressive marks, at least in the case of the H3K27 methylation and the H2AK119Ub occurs because two different Polycomb Repressive Complexes are actually attracted to the inactivate chromosome. These are called Polycomb Repressive Complex 2 and Polycomb Repressive Complex 1. And in each case the Polycomb Repressive Complex is a drawn to the inactive X chromosome by binding to the Xist RNA. So you recall that I've shown you before that Xist has a region within it called Repeat A, and this Repeat A is shown here as a bit of a hairpin structure. So, this hairpin structure combine Polycomb Repressive Complex 2. In Polycomb Repressive Complex 2, then it can then lay down the inactive histone mark, Histone H3 lysine 27 methylation on the inactive X chromosome. And so, it's easy to understand then how you can accumulate these particular epigenetic mark on the X chromosome on the inactive X chromosome. We don't have a clear idea yet of how PRC1 binds to Xist. However, we know the association of PRC1 is Xist dependent. So without Xist, it won't go to the inactive X chromosome. And we know that PRC1 catalyses this particular epigenetic mark ubiquitination of histone H2A lysine 119. At the moment the third epigenetic mark, the histone H3 lysine 9 methylation. And we don't yet know, or it's not yet published which particular enzyme is involved in laying down this mark on the inactive X chromosome. So, let's then think about the stages that we've spoken about so far as a bit of a summary. We know that early on in X inactivation, the the pluripotency factors are still being expressed and they gradually decreased it within the first few days of differentiation. In this case, we're thinking about differentiation of embryonic stem cells whereas, I said, most of the studies have been done. So embryonic stem cells, when induced to differentiate in culture, differentiate over a relatively long period of time, ten or more days. But they mimic exactly what happens in vivo. Just at a slightly slower rate. We know as the pluripotency factors decrease, we know that Rnf12 expression is turned on, but only for a short window in time. And it's at this precise time point in time that activates Xist expression. and then it is itself silenced because of X inactivation proceeding. We know that X pairing, X-X pairing, this chromosome kissing happens at this, around this same time point, and it precedes Xist expression. And we know that Satb and Satb2, these competence factors that are required to form that silent nuclear compartment, need to be expressed at this time, and are indeed expressed for around the first two days of differentiation. So, the slightly later stages once Xist expression is turned on, we know that very rapidly afterwards, you see exclusion of RNA preliminaries 2 from the Xist region. We know that the repeats on the inactive X chromosome or the X chromosome that will become inactive are silenced. We see a rapid loss of active histone marks and an accumulation of repressive histone marks. We know that this repression, the repressive histone marks are laid down because of the recruitment of Polycomb Repressive Complexes 1 and 2, at least for a couple of these epigentic marks. The recruitment of these Polycomb Repressive Complex 1 and 2 is actually dependent on the expression of Xist. So during this time from about two days two and a half days of differentiation onwards, we see progressive silencing of genes on the inactive X chromosome. But exactly when each each gene undergoes silencing and undergoes X inactivation depends on the gene itself. And so, this progressively happens over the course of the next eight days or so. And the reason why they aren't all silenced at once, is because of this drawing of genes into the silent nuclear compartment. So if you remember what we said earlier, the repeats are drawn in first and very early. But then, from then to when we have the final inactive state, there's a progressive recruitment into their silent nuclear compartment. And as a gene is drawn in, it is then silenced, but not beforehand. And so you can imagine that different genes are silenced at different times and it doesn't all happen at once. In the next lecture, we'll think about how this silenced state, which is shown here, can actually be maintained through the lifetime of the organism for decades at a time.