So, welcome to week 4. Here we are thinking about epigenetic reprogramming. And particularly about a particular class of genes called the genomically imprinted genes or the imprinted genes, and how they are programmed between generations. So, you will remember that what I have said is that we have generalised reprogramming between generations. And this is to wipe off the marks you find that are being laid down in one life time in the somatic cells. And ensure that in the subsequent generations, that you will have totipotency. So, I've shown this to you before in this manner, so, with this cycle of life, if you like. Where the break in between the generations, shown here, is showing, is trying to depict that we need to undergo reprogramming during this period. And it makes sense that you'd need to do these. So, a sperm has a very specialised function, and it's a specialised function in terms that it has to swim very fast, and so, it needs a particular set of genes that are expressed. And the egg has a very different function. But when these come to fertilisation, we now need to now remove the particular epigenetic marks that made the sperm or the egg have the particular set of genes they need to express. Remove those and allow re-expression of genes required for total totipotency in the zygote and beyond. So, it's not just about sperm and egg gene expression patterns though, but also about particular chromatin types. So, we know that the germ cell chromatin, the sperm chromatin, or the oocyte chromatin, it's highly specialised. This is perhaps easiest to imagine for the sperm chromatin. It has to be very densely packaged down into the head of the sperm. So, the head of the sperm is very small, and so, the DNA needs to be compacted as tightly as possible, so that the sperm can swim as fast as possible. We also know that the sperm chromatin is different to other chromatin, because it's not made up of histones. It's instead made up predominantly of protamines, a different type of molecule. Different type of protein that will package the DNA. So, these differences in germ cell chromatin and the different gene expression signatures required need to be removed before the next generation's can develop. If we think about this in another way, I can show you here that we have two phases of epigenetic reprogramming. The first is during preimplantation development, and the second is during primordial germ cell development. So, preimplantation development as it sounds, is that period before the embryo implants and makes a placenta. So, we know that at this time that when the sperm and the oocyte come together to form the zygote. After this point, we have removal of much of the DNA methylation, and it seems also the histone marks throughout the genome. And this reaches a global low about the blastocyst stage, just before implantation. After this stage, we have resetting of epigenetic marks, but this happens in a cell type specific manner. Now, if you think what I told about last week in terms of X inactivation. During this preimplantation period, we didn't have reprogramming of X inactivation, there's wasn't removal of X inactivation, but rather we had imprinted X inactivation. That is the silencing of the paternal X chromosome was maintained. So, this starts to tell you the different regions of the genome can be treated differently by this reprogramming process. They're not all treated the same. If we then look at the embryo that's developed to mid gestation, we have predominantly somatic cells except for the primordial germ cells. These cells that will go on to become the germ cells, either the egg or the sperm, dependent on the sex of the embryo. This is the second phase of reprogramming. This is because in these cells that will become the germ cells, you need to remove the somatic marks and put, lay down this particular germ cell chromatin that I spoke about. And in particular lay down those particular epigenetic signatures that are required. So, I'm going to show you this in more slowly and in a slightly different way. So, let's first of all think again about this early development, this early development reprogramming. So, I'm showing you here the paternal genome separate to the maternal genome. And I'll explain why. So, if we start out with the gametes after fertilisation, we actually have two pronuclei which is what I'm showing here is these two dots. So, the paternal pronucleus in this case is in blue, and then maternal pronucleus is in pink. So, very early after fertilisation, they still exist as two separate small nuclei or pronuclei before they fuse together. And during this period within the first six hours for post fertilisation. So very rapidly after fertilisation, and in fact, before any cell division or DNA replication has taken place, we know that the paternal genome is actively and rapidly demethylated. And that's what I'm showing here in this graph. On the y axis I'm showing DNA methylation, and you can see the blue line descends very rapidly post fertilisation. So, even before we knew about the identity of the enzymes involved in DNA demethylation, we knew this was an active process which much involve enzymes, because there was no replication at the same time. We weren't just diluting out the DNA methylation that was there by replication. So, we now know that this occurs, this DNA demethylation occurs via hydroxylation. So, demethylation is in fact incredibly difficult to achieve chemically. And that's because you have a very strong carbon-carbon bond. So, there's not a simple snipping off of that methyl group, the CH3 group. Instead what happens is that the bond is destabilised through hydroxylation and then the hydroxyl group is demethylated. So, this is what happens early in development on the paternal side, the paternal genome. In contrast, if you look at the maternal genome now in pink, it's demethylated more slowly so that it reaches a global low of blastocyst. So, it's also low of blastocyst, just like the paternally derived genome. But in this case, it's happening in a very slow pace, and instead is dependent on cell division. So, this passive demethylation we know occurs, not necessarily because of the TET proteins, but in this case because DNA methyltransferase one, that maintains methyltransferase, is excluded from the nucleus. So, it can no longer act on the hemimethylated DNA, and as a consequence the DNA methylation will gradually dilute out in each with each cell division. So, although the paternal and the maternal genome are treated differently in this time, in both cases, we end up with a low at the blastocyst stage. And then after implanting the epigenetic marks are re-established. The epigenetic marks are re-established in a lineage specific fashion. So, each lineage will have its own set of epigenetic marks, and these will be associated with the particular gene expression patterns that are required with each of those lineages. And from this time point on, we are in general maintaining these sematic epigentic marks. Now, this of course is a large generalisation. There are periods of development of each of the particular organs that where you will indeed see some demethylation and remethylation happening for example. Some additional epigentic reprogramming. However, the largest time periods of epigenetic reprogram when you consider the genome in general are during early development that I'm showing you here. And secondly, during primordial germ cell development. So, if we consider these cells that would go on to become the germ cells, they need to be derived from a somatic cell. So, that at mid-gestation they develop in their mid-gestation embryo. And so, we have this second round of reprogramming now. So, both the paternal and maternal genome are cleared at about the same rate. But the resetting of marks in the germ cells happens at a slightly different rate in the male versus the female. You can see they're offset here. So, this seems to be due to the differing dynamics of oogenesis versus spermatogenesis. So, while we know that say a female is born with all of the oocytes she'll ever have in her life, they haven't undergone complete maturation. And they actually completely mature a little later than sperm do, in terms of life span. So, you can see this is why there's this offset here. So, if we put together this early embryonic reprogramming and then the primordial germ cell reprogramming, we can see that the paternal and maternal genome are treated slightly differently at each phase. But that in general reprogram the genome both times, both in early development and then again in primordial germ cell development. But if you look into the mid-gestation embryo and beyond, in general, if you're not thinking about those germ cells, we're in a period of somatic maintenance where these epigenetic marks that were established in a lineage specific way post implantation are just being maintained. So, in the next lecture, we'll think not about how the genomes in general, the paternal or maternal genome is treated, but rather how specific classes of genes are treated.