So the next thing we're going to talk about are histone variants. So histone variants are different forms of the canonical histones that normally make up the nucleosome, so rather than being one of the regular histones, they have some difference about them, which allows a different function. So there are known to be different variants for histone H2A, H3, and H1. And today I'm going to take you through three examples, and these are going to be only of histone H2A and H3. So there are very many variants that can be found in for histones. And each of these have different properties, which will allow them to change something about their nucleosome For example, they might allow increased stability of the nucleosome or decreased stability. They might have particular amino acids that can be modified in a different way that the normal core histones don't have. Or they may have some other functionality that we don't yet understand. So, you'll remember in the last lecture that I mentioned that histone variants need to be deposited and this involves the chromatin remodellers. The ATP-dependent chromatin remodelling proteins. So importantly, the histone variant deposition can happen at the time of replication, so it can be dependent on replication, or it can be replication independent. In other words, the cell wouldn't have to divide for this happen for a nucleosome to be changed and the histone variant to be included. And this is important depending on the type of nucleosome or the type of histone variant and its function. We wouldn't always want to have to wait until the cell divides to be able to incorporate these variants. So we'll now go through those three examples I said we'd go through. The first of which is a variant in histone H3, and this called CENP-A. This centromeric histone variant, and you find some form of CENP-A in basically all organisms, because all organisms with chromosomes need to have a centromere. The function of the centromere as it's shown here down in this metaphase chromosome that's pictured is it's the central constriction of the chromosome. And it is where these chromosomes during division attach to the mitotic spindle. And it's the mitotic spindle, that, when a nucleus is actually dividing the chromosomes. That, when the chromosomes are attached, it pulls them to the opposite poles of the cell. So it's very important to have a centromere to ensure that when replication, happens, and division happens, you get one copy of each chromosome to each daughter cell. So it's very important for genomic stability in that way. What's interesting about CENP-A is that, it binds through this region here at the centromere. And this is the same region where we have a lot of repetitive DNA. And it's been thought for a long time that it's the repetitive DNA which defines the centromere. However it's relatively recently been shown that this isn't the case. You can form a centromere somewhere else. If for example, you have a truncation which gets rid of the portion of the chromosome that would normally contain the centromere, the remaining part sometimes will be able to form a neo- centromere, and this neo-centromere does not form necessarily where there's repetitive DNA but rather is simply defined by the CENP-A protein. This particular histone variant which changes the nucleosomes in that region. In this way, CENP-A really has some kind of a structural role. It does change the packaging of the DNA in the region, but it's having a structural role in maintaining genomic stability. The second Histone variant we want to think about is called H2A.X, so its a variant of Histone H2A. And this variant is involved in DNA repair. So it's a universal histone variant, somewhat like CENP-A. It's found in all organisms, and it's very highly conserved. And in this case, it's the C terminal motif that differs from the normal histone H3 that's found in regular nucleosomes. And this particular C terminal motif contains a serine at residue 139. And serine is an amino acid that can be phosphorylated. And it's the phosphorylated form of H2A.X, which we, we call gamma H2A.X, which is found in double strand breaks. So, double strand breaks can happen through a variety of insults to the cell. And, but of course, these double strand breaks need to be repaired. And so by having this gamma-H2A.X that marks those double-strand breaks, this enables repair to happen at the right places. So, the serine 139 is phosphorylated by kinases, and this makes the gamma-H2A.X, and this happens at the double-strand breaks. And then it is this gamma H2A.X which is then able to recruit DNA repair proteins. So it's not only DNA repair proteins that are recruited by gamma H2AX, but also epigenetic factors. And this is quite interesting, because, to be able to repair the DNA, just like to be able to transcribe it or to replicate it, you need to have accessibility to each individual base within the region. And so, the chromatin needs to be unwound in the region that needs to be repaired. It will be repaired and then wound back up again. So DNA repair is an intimate association between the repair machinery and the epigenetic machinery. Once this repair is complete, then we have a phosphotase that comes along and cleaves the phosphate group. So if you have, this image here is a nucleus which, where the DNA is stained by DAPI, and this is a blue dye. But the gamma H2A.X is stained with green. And so you can see these densely green phosi spread throughout the nucleus and these are all where the double strand breaks are clustering. This a nucleus that's been exposed through a radiation and this is why we can say gamma H2A.X at all within the nucleus. You remember that I mentioned that these, the histone variants can be incorporated into nucleosomes, either in a replication dependent manner or replication independent manner. And this one of those cases where it's really important that it is a replication-independent manner. We certainly wouldn't want the cell to have to divide, a cell that has DNA damage to divide, in order to incorporate gamma H2A.X, in order to be able to then repair the DNA. You certainly, that's exactly when you want to stop, when a cell has DNA damage. So this is an instance where gamma H2A.X can be incorporated before DNA replication takes place. The 3rd example is another variant of histone H2A called macroH2A. So, as you might expect this, histone variant has a macro domain. And the macro domain is just inferring that it has a very large, domain. In this case, 200 amino acids at its C terminus. And we don't yet understand the function of this macro domain. What we do know is that, unlike the previous two histone variants I mentioned, this macroH2A is only found in vertebrates. And that's particularly interesting because what we know it's involved in is X inactivation. It seems to be associated quite densely with the inactive X chromosome. X inactivation only happens in precisely the same way in mammals. So perhaps, then it wouldn't be surprising that macroH2A would only be can only be found in higher organisms, only found in vertebrates. So, you'll also remember that the inactive X chromosome, it's made up of facultative heterochromatin. That is, that it's not the same in every cell type. It can vary, because it could be either the paternal or the maternal X chromosome that is silenced. And that can vary by cells, within each cell. So it's possible that macroH2A is found in other forms of facultative heterochromatin as well. So to summarise what we've said about histone variants, we know there are many different variants, and we've just covered 3 in very brief detail. These variants, the cool thing about these variances that have particular domains or particular residues or some difference to the core histones, the canonical histones, and it's these differences that allow particular functionalities. So we know that histone variants can be involved in many different things. They can be involved in structural aspects. In other words like CENP-A, which is involved which is involved in making centromeres. They can be involved in DNA repair, such as H2A.X, which can be phosphorylated and called gamma-H2A.X. Or they can be involved in transcriptional silencing like macroH2A. There are also other histone variants that can be involved in transcriptional activation. And, so, in these ways histone variants contribute to epigenetic control.
