So in this final week final lecture for Week 3, I'd like to think about other things that we've learned about epigenetic control from the fly. So in last lecture we thought about fly dosage compensation. And actually this alerted us to the idea that maybe there could be up regulation of that active x chromosome in mammals. But, we've actually learned a huge amount of [INAUDIBLE] about epigenetic control from studies that have been done in the fly. And much of this has come from studies of position effect variegation, which I'll explain in the coming slides. And screens, mutagenesis screens that have been done in the fly to find modifiers of position effect variegation. And really, this is a way to find new epigenetic regulators. So what's position effect variegation? Well, by position effect, what we mean is that the position of the gene relative to heterochromatin can help influence its expression. So if a gene is neighboring heterochromatin, it's more likely to be silenced than if a gene is distal to heterochromatin. I've explained variegation before but let me just recap on it. A variegated expression, like you might see in variegated ivy or in variegated coat colors in dogs, like beagles, for example. We know that you get the mosaic expression of a gene in cells of the same tissue. So this means that a particular gene can either be on and active and expressed, or silent. But within the very same cell type. So say for example in a leaf of variegated ivy, you can have the green pigment being, green pigment being expressed and on in some cells and [INAUDIBLE] those parts of the leaf are green or in other parts of the leaf that green is turned off, and you have white sections of the leaf. So putting the two together then, position effect variegation is saying that some genes when located or genes, when located need a heterochromatin, can display variegation. So they can be sometimes on and sometimes off. And this is determined by the fact that they are nearby to heterochromatin. So in the fly, position effect variegation is observed in the eye color phenotype. And this is when position effect variegation was first described. So if you think about a gene called the white gene, what's confusing is the white gene actually codes for red pigment. This is because historically genes were named not by their function but by the phenotype that resulted when the gene was mutated. So, if you have a mutation in the white gene, that means you can't produce red pigment, and you have a white eye, and hence why it's called the white gene. We know that in the DNA, the white gene is normally located in euchromatins, and this orange euchromatin, distal to the centromere and distal to the heterochromatin that surrounds the centromere, this pericentromeric heterochromatin. And, in this case, the white gene is always active and, therefore, you have a completely red eye in the fly. But sometimes what happens is that this white gene is translocated due to a DNA rearrangement, and it's then located close the heterochromatin, which is found at the centromere. When the white genes translocated in this way, the heterochromatin found at the centromere can spread out to sometimes encapsulate and cover that white gene. So if the heterochromatin spreads through the white gene, it's then silenced it's turned off, and you have white pigment being produced rather than red pigment. And so, in this case what we know is that this spreading is not completely efficient. It doesn't happen in every cell in the same way, and so what ends up happening in the fly eye is a variegated expression. And so, in my naive drawing of this here, is that you have little dots of red within an otherwise white eye. And so this is the variegated eye phenotype which is [INAUDIBLE] is the the result of position effect variegation in the fly. Interestingly, this spreading of heterochromatin is not restricted to occuring in the fly. We now know that it occurs in essentially all organisms and is a common feature of heterochromatin. We also know that it's not restricted to spreading from the pericentromeric heterochromatin, this heterochromatin need a centromere, but can also occur from other regions of heterochromatin, such as the telomere or repetitive elements that were, that are densely heterochromatinized. And if you think back to what I said about X inactivation, we know that the heterochromatin there also spreads throughout the whole X chromosome. So if left unchecked, you can imagine that heterochromatin spreading would mean that the whole genome would end up being densely packaged down and heterochromatinized and clearly that's not what happens. And so heterochromatin spreading is limited by DNA elements, known as boundary elements. And these boundary elements insulate the surrounding region from this spreading. So, now to look at, at position effect variegation and how they've used position effect variegation, or PEV, to find genes involved in epigenetic control. So, down the bottom, here, we've got three pictures of fly eyes. These are the real eyes, now, rather than my naive drawings. And you can see this one has a lot of white and only a little bit of red. And then we've got two here that have much more red in them. And tell you this variable phenotype can occur. In this case it's because there have been mutations that have occurred in other genes that are important in this position effect variegation. So in epigenetic modifiers, in fact. So what do software genetics, geneticists did 30 years ago, they started about 20 or 30 years ago was they took this mosaic eye pigment phenotype, this position effect variegation. And they knew there seemed to be an epigenetic effect. They couldn't, it seemed to be that this, this phenotype could be determined by the expression of the gene and it was epigenetic. So what they did was they performed a mutagenesis screen. I won't go through exactly the details of what a mutagenesis screen is, but sufficed to say they take a parental fly with a given level of variegation, and they expose it to mutagens and this introduces mutations spread throughout the genome. They, then, breed and look at the phenotype of the eye in the next generation. And so, they're looking for where the inheritable DNA mutations, just regular mutations, which alter the expression of that variegated eye phenotype, alter the position effect variegation. But, then, go back and find where those mutations occurred, and the genes in which they've occurred are likely to be important for epigenetic control. So, in fact what we know is that they were able to identify they identified hundreds of different strains and these worked out to mutations in, in close to 200 different genes. And they classified these, each of these strains as being suppressors of variegation. And a suppressor of variegation really means that you get less variegation, and so the eye is more red, like what's shown in these two images here. There is more red in each of these two eyes compared with the original eye. So these are suppressors of variegation and so you would expect because you get more activation that the mutations occur in a repressive protein. And second they, they identified enhancers of variegation or Evars. And in this case, they increase the variegation, so they result in more white in the eye. And so you expect to have mutated an activator protein. So when they've mapped each of these gene's, they've then identified new genes in epigenetic control. We now know these genes are some of the the very most fundamental epigenetic regulators, some of which I've mentioned to you. So three examples of the Suvars are an H3K9 methyltransferases. Histone deacetylases and the chromobox protein known as HP1, which itself binds to methylated H3K9. But, indeed, many of the proteins that we know are important in epigenetic control in mammals, were first discovered in these PEV screens in fly. And then, we looked for a similar gene in mammals. So really much of what we know about epigenetic control in mammals comes from these fundamental studies which were performed in a fly. But people and mice or other mammals really aren't a fly, so we're not, there are many things we do differently to flies. And x inactivation is just one example of that. And so, what's been happening more recently is over the last ten years or so screen, similar sort of screens in mice. So, in this model system we would hope we could find things about mammalian epigenetic control. In this case, they also used a variegated expression. In this case, it's a transgene, a GFP transgene, this green fluorescent protein transgene is directed to express in red blood cells. So, here is shown a smear of red blood cells, and you can see that not all of those red blood cells express the green fluorescent protein. So, in the same way as was performed in the fly this these animals were exposed to mutagen, and then, they were bred. And it was, and this variegated phenotype in the red blood cells, that expression of GFP was tested in the offspring. And they, then, looked for those who had a different expression. For example, here you could see a greatly increased proportion of cells, that red blood cells expressed GFP. And then, the mutations were mapped to find out which genes were they in, so that we could find new epigenetic modifiers. Now, as you'd expect, many of the homologs of the fly proteins important in epigenetic control were found, and that's reassuring because it meant that screen was working in the same sort of way. But importantly, this screen has also identified novel proteins and in particular, it identified Smchd1, which I mentioned earlier. And these novel proteins are unique to higher organisms. And they're, in particular, involved in epigenetic processes, which only happen in higher organisms. And in particular, as you remember, I mentioned Smchd1 is important in x inactivation. And so, through these screening approaches using variegated expression, we can find novel genes that are important in epigenetic control, and therefore deepen our understanding of how this exciting process occurs. So next week we are going to think about epigenetic reprogramming, that I have briefly touched on because we get clearing of x inactivation. We're going to go into more detail about epigenetic reprogramming and also genomic imprinting. But I've hoped, I hope you've enjoyed dosage compensation for this week.