So now let's consider the role of Smchd1 or smooch d1 in the maintenance of X chromosome in activation. I haven't mentioned Smchd1 before it's an Smc containing, hinge domain containing protein and that's how it gets its name Smchd1 but for simplicity I'm going to call it smooch d1 because it's m easier to say. So the background information is that we know Smchd1 is bound to the inactive X chromosome. We can find it co-localising with the inactive X chromosome right out at embryonic day 13.5 in a mouse. So, since initiation of X inactivation happened at E5.5 And we know that you're into the maintenance phase of X inactivation by E8 or E9.5 of gestation, and by time you're out of E13.5 you're well into the just the maintenance of X inactivation and so, Smchd1's localisation during this period suggests that it may have some role in this maintenance of X inactivation. We also know in embryos that are null for Smchd1. The females die at around embryonic day ten or eleven. Again, this is in the maintenance phase of X inactivation. Not the very initiation or early stages of X inactivation. Again, suggesting they make it through those initial stages but fail at the last stage. So we know that in these cases, that the summary of what know is that both imprinted and random X inactivation are abnormal. And this differs from what I told you about Dnmt1 and this accompany the imprinted defect is a imprinted X inactivation defect is accompanied by a placental abnormality. So when you look in these embryos you can see that Xist expression is normal and that Xist can recruit polycomplex 2 normally as well. But there is very little DNA methylation that ever ends up on the inactive X chromosome. But since DNA methylation doesn't appear to be involved in the placental form of all the imprinted form of X inactivation presumable Smchd1 is doing something else other then some how directing DNA methylation. And finally what ends up resulting is that you'll get a failure of the transcriptional silencing on the chromosome that should be the inactive X. So I'm going to go through the same sort of embryology, the same processes than looking at the expression of the next linked trans gene. For Smchd1 as I did for Dmnt1. In this case though it wasn't a LacZ transgene rather its a green fluorescent protein transgene or a GFP transgene. So this GFP transgene is on the X chromosome and it too is subject to X inactivation like the LacZ gene. And so we can use the expression of green fluorescent protein which is of course very easy to detect under a fluorescent light the activity of the X chromosome on which it resides. So there's an additional complicating factor in the genetics of these embryos. So on the paternal X chromosome we have the GFP trans gene just like the Dmnt1 there was a LacZ trans gene. But now in addition to that on the maternal X chromosome Xm we have an Xist knockout. So since Xist is the key determiner of X inactivation. If a chromosome doesn’t possess Xist because Xist has been knocked out, then that chromosome cannot be inactivated. We know Xist has to have, has to work in cis. So this maternal X chromosome, we know has to be active. And this means that the paternal X chromosome, the one with the GFP should be silent. So, this is forced non-random, primary non-random X inactivation, and means that we expect the embryo with this genetics up here, will not be green and its placenta will not be green. So then, we looked at this, these embryos that we're either Smchd1 null, or wild types. And the Smchd1 wild type's exactly as you expect. You find that the embryo is not green, and the placenta is not green. However, when you look in the embryos that are Smchd1 null You now find an entire green embryo and an entirely green placenta. And this indicates that there's been a failure of X inactivation. And so X inactivation, at some point, has meant that you aren't able to silence this GFP on the paternal X chromosome. So this suggests that X Smchd1 is required for the maintenance. Of both imprinted and random X inactivation however its not formally proven because I haven't shown you that there was the detection of a previously active X chromosome, a previously inactive X chromosome. And I'll show you this is instead shown in a slightly different set of experiments. So in this case this reactivation of a previously silent inactive X chromosome can be displayed in cells that are grown in culture rather than in embryos. And this has indeed been done not only Smchd1, but also for Dnmt1 and MacroH2A. That histone variant that is enriched on the inactive X chromosome. Among, among other genes, as well. So here fibroblasts are made. mouse embryonic fibroblasts, which are commonly called MEFs. These are made from Mid-gestation embryos. E13.5 embryos. And remember, at embryonic day 13.5 in the mouse. You're well and truly just into this maintenance phase of X inactivation. X inactivations have been fully established several days earlier in development. So if these fibroblasts are grown from in a mid-gestation embryo, and we have the same sort of genetics as we had in the previous slide, so we have already determined that the X-linked GFP transgene is on the inactive X chromosome. That is, that it should be always silenced. Now you can test whether or not you can promote the expression of GFP, so you can destabilise X inactivation. So what's found is if you have a control and you add in with that a drug called 5-azacytidine. This drug inhibits DNA methyl transferase 1 and so it inhibits this laying down of this final locking in inactive state and if you do that you really don't get any reactivation even though you have destabilised some Dnmt1. But if you do this in conjunction with a test. So if you do this in conjunction with either reducing the expression of Smchd1, reducing the expression of Dnmt1, or reducing the expression of MacroH2A, now in conjunction by having the drug. And having the knock down reducing expression through use of micro RNA which I've mentioned in previous lectures, then, what you can do is you can do id reactivate GFG in a small proportion of the cells. It still doesn't happen very often because I've said maintenance of X inactivation is incredibly stable but it does happen in a small proportion of cells because in combination if you deplete. DNA methylase transfer through a drug and either additionally deplete Dnmt1 with a knock down with a microRNA against Dnmt1 to reduce its expression. Or remove the expression of some of Smchd1 or MacroH2A then in each case you can get up-regulation or reactivation of that previously silenced X chromosome. And so this really serves to remind you that there are several layers that are locking in the inactive state on that inactive X chromosome once we reach the maintenance phase. And so really you need to destabilise them. And you need to destabilise not just Smchd1, but Smchd1 in addition to DNA methylation to be able to get this reactivation to occur. And that's because the reason that it's so stable, the reason it's actually hard to get it occur at all, is because you have these redundant layers of care of epigentic marks that are mitotically heritable and this is really the key feature of the maintenance phase of X inactivation. In the next lecture, I'll go through a summary of X inactivation and all that I've told you so far this week.
