So now we are going to go through in the next series of lectures, each of the specific epigenetic modifications that are called epigenetic marks, and they have functional consequences for how the genes are expressed and how the chromatin is packaged. So while we'll go through each of these. This is the relevance of these, and how they specifically relate to the epigenetic phenomena, or the particular examples we are going to go through in the later lectures, we'll deal with that in later lectures and just have brief highlights of that here. But we need to go through and learn about how each of these work in order to really be able to understand at the molecular level how these processes are working in other instances. So we're going to start with DNA methylation. So DNA methylation is, as it sounds, the addition of a methyl group to the DNA, and this happens at cytosines. So here we're showing a cytosine single ring base but we can add on a 5- methyl group so its added on to the 5 carbon the fifth carbon here. And the methyl group is CHC group that's added directly onto that base. We know that, in mammals, DNA methylation occurs almost exclusively at Cs that are followed by Gs. So cytosines are followed by guanines. And this is called a CpG dinucleotide. And that p is just for the phosphate bond between the two. There's good reason that it's found almost exclusively at CpG dinucleotides. And that's because these dinucleotides are symmetrical when you look at the other side, the other strand of DNA, and this allows them to be maintained through cell division. And you remember this is one of those hallmark features of epigenetic modifications. So, how is it then that DNA methylation can be laid down and how can it be copied mitotically? So how is it that it can have this mitotic memory? So he pictured is a very simple picture of a DNA strand, shown by the black lines, and then in the middle this CpG dinucleotide. And in each case the cytosine is methylated. So, this methylation, because it's close by to one another. The cytosine on the other strand is very close by, and so it's methylated as well. What happens when the cell divides? Sorry, I’ll go back a step and say that the first thing that happens, is that DNA methyltransferases. So these are enzymes, in mammals they're know as DNMT3A and DNMT3B, these lay down the methylation. And they do this in a de novo fashion. In other words, they work on DNA that begins out being unmethylated. So they lay down these, these methyl-marks during development. Then how is it that it can be maintained? Well if we go through cell division, we have the parent strand in each case shown here in black. And the daughter strand shown in green. So, when the DNA is replicated, there's only going to be the parent strand that maintains the methyl group on that side within the CPG dinucleotide. And then the daughter strand, now, shown in green we have an unmethylated cytosine in each case. Now, what happens is we bring in DNMT1, so another DNA methyltransferase enzyme, and this stain of methyltransferase specifically recognises hemi-methylated DNA. It has a preference for hemi-methylated DNA, that is where one strand is methylated and the other is not. And this hemi-methylated DNA is then bound DNMT1. And DNMT1 the lays down methylation on the daughter strand and we have the restoration of a fully methlayted CPG dinucleotide. And this is how we know that DNA methylation can be a stable epigenetic mark. Because at every cell division, this DNA methylation will be copied by DNMT1 onto the new daughter strands of DNA. So we know I said that CpGs are where you, you mostly find methylation, or almost exclusively find methylation in mammals. So where are these CpG's found? Well many CpG’s are found in what are CPG islands. So this is where you find more CG dinucleotides than you would expect by chance and they tend to be found at the promoters of gene’s. So these, promoter are just to remind you is the region upstream of the start side of transcription of the gene and it's where the transcription factors bind. The general rule to remember is that CpG islands although they have many CpG’s there in fact tend to be protected from methylation, so methylation doesn't tend to occur at CpG islands. It tends to be that it occurs at other places in the genome. But if you do find methylation of CpG island. Then, this is almost universally synonymous with silencing of gene expression. So, DNA methylation is an inactive epigenetic mark. So, there are some CpG islands in the genome that are found to be methylated. So, while the general rule is that they are unmethylated There are some that are associated with gene silencing, and these are found at particular times and with dynamic methylation between different cell types. However, it’s mostly being studied, DNA methylation at CpG islands, for the inactive X chromosomes that I have mentioned a few times. So I'd like to go into a little bit more detail here about X inactivation. So, the reason for that is X inactivation really clearly demonstrates the mitotic heritability of DNA methylation. So we know that females have two X chromosomes, where as males have one X and one Y. So if we just consider the nucleus of these female and male cells, then the female cell will have twice the dose of all the genes that reside on the X chromosome, of which there are over 1000 genes. This in fact is not what ends up happening. What happens is that one of those two X chromosomes, either the one you inherited from your mom or the one you inherited from your dad, is chosen and its densely packed down in a cell as shown here. And this is called the inactive X chromosome. So while females have two X chromosomes they only ever use one. The second goes by unused. And is put in indeed is even put to the side in the nucleus literally put to the side. So what's interesting is this - when this X inactivation first occurs it occurs when there are only a few hundred cells in the embryo at gastrulation. And we know that when this choice is made it's a random choice. So each cell at that couple-of-hundred-cell stage can make the choice individually as to which X chromosome to inactivate - the one from your mom or the one from your dad. That choice is then mitotically heritable to all of the daughter cells. So, this X inactivation involves DNA methylation of the CpG islands. All thousand of them or so, or almost all thousand of them on the inactive X chromosome. And it's this mitotic heritability after the choice is made is partly ensured by the DNA methylation of those CpG islands. So, this is actually able to be observed in a visible way in the coat colour of cats. So, I'm going to show you a small movie here which is about Calico cats. What we can see, is that Calico cats, that are shown in this picture here, have genes that encode the coat colour genes are on the X chromosome. So, they can either have the ginger version or the black version. And then the choice is made early in development to have the ginger one being active or the black one being active. And the other one is silenced. The other chromosome is silenced. And so you end up with these cats that have a mottled appearance based on when that choice of which X to be inactivated was made early in development. So this could, is also the case not only in cats, of course, but is also true in humans. So in humans if we had a coat colour marker like this, which of course we don't, but if we had a coat colour marker like this, you would actually see these patches of green skin and patches of pink skin, based on when that choice was made early in development. Just as in female, humans just as you see in female cats they have this Calico appearance if they happen to have the right genetics. It's also interesting to note then, that you actually can't get male Calico cats. Male Calico cats, if they exist, have had a mutation where they actually have two copies an X chromosome but still have a Y chromosome. Otherwise they could not have this Calico appearance, traditional mottled appearance. So, how is then that DNA methylation which we've just discussed is heritable for many cell divisions and if you think about it in human mammals can can be heritable for maybe a 100 years. How is it that the DNA methylation actually is associated with gene silencing? There are probably at least a couple of mechanisms by which this can happen. So perhaps the primary mechanism is because the CpG, these methylated CPGs. Are associated with a condensation of the chromatin, and the way that this happens in the primary case is because the methylated CpG is bound by methylated CpG binding proteins, which are otherwise known as MeCP1 and MeCP2. So these MeCP1 and MeCP2.proteins bind to the methylated CpG dinucleotide and they themselves can alter transcription because they possess a transcriptional repression domain. Alternatively the MeCP2 or CeCP1 protein can itself bring in it's own protein partners and they can condense the chromatin. But for this primary mechanism it seems to be the binding of the methylated CPG by the methylated binding protein domain family. Probably the secondary mechanism, which doesn't seem to be is important, but can occasionally occur is that the methylated CpG will stop a transcription factor binding. So, transcription factors have particular binding sites. Say for example C,G,A,T. So, this binding site might be bound by a protein, a transcription factor, and it will then enable the transcription of a neighbouring gene, or the nearby gene. However, if this particular site now has the same sequence C,G,A,T, but its a methylated C,G,A,T, this will then block the binding of that transcription factor. So just that small addition of the methyl group will not allow the transcription factor to bind and therefore we don't have transcription in ensuing. So we don't think, although there are some specific examples of this occurring, for example for the transcription factor SP1 we don't think it's a generalisable mechanism and instead it seems to be just true For promoters that don't have so many CpGs. And so therefore, even single CpGs will have a large consequence. Rather we think that primary mechanism where the methylated CpG binding proteins bind to the methylated CpG is likely to be the most dominant mechanism within the nucleus.
