So, welcome to week three in this Coursera course on Epigentic control. You'll remember, in the first two weeks, we've run through chromatin modifications and their various epigentic marks. How chromatin is packaged, and how this all relates to transcriptional controls. So whether the gene is likely to be silenced or expressed. And now in the third week what we want to consider is dosage compensation. We've spoken a little bit about dosage compensation in mammals. About X in activation, on a few occasions as a means to explain some of the things that we've been speaking about so far. But now we're going to delve into more detail. The reason we're doing this is because not only is it a fascinating process where a whole X chromosome, about a thousand genes, goes unused and densely packaged in the nucleus. Also, what we learn about dosage compensation tends to be true for epigenetic control throughout the rest of the genome, and so it makes a really good model system to think about when we want to try and put all of these epigenetic marks together that we've spoken about in the previous 2 weeks, to try and understand at a molecular level how epigenetic control actually functions. So we'll start out with a bit of a historical view of X inactivation. We'll go through the various stages that exist in X inactivation in mammals, and the molecular mechanisms about how these work. At least, what's known about them so far. We're then going to take a very brief foray into thinking about how dosage compensation exists in lower organisms in flies and worms and finally we'll spend a little bit more time thinking about a few other things that we've learned about from the fly. And in fact much of what we know about epigenetics control in mammals has come from work that's been done in flies. And then being translated into the mammalian system. So let's think about X inactivation in mammals then. Well it was way back in 1949 when Barr and Bertram discovered that when they're looking at the nuclei of, neurons from cats. That in the male nuclei compared with the female nuclei, they didn't observe this dense heterochromatic body. In the female nuclei, what they saw was a region of densely packed DNA, of heterochromatin, that was found at the nuclear periphery. And so, you can see in the image that's shown here on the right of the slide, that here indicated by the arrow, that there is a region that is stained more densely, this heterochromatic region. and this is now known as the Barr body. So it was then in 1959 that Setsuko Ohno discovered that this wasn't just any old region of heterochromatin. But in fact this region which was exclusively found in female cells was indeed an X chromosome. So moving on from that in 1959 this discovery in 1959, in 1961 Mary Lyon proposed. the that this X chromosome was inactivated, in order to perform dosage compensation for male cells which had only one X chromosome in comparison with female cells that had two. And really one of the hallmarks of Mary Lyon's hypothesis was that this choice of which X chromosome would be inactivated was random. So you remember that we've spoken about on other occasions, that this, that there is random X inactivation, and either the paternally inherited or the maternally inherited X chromosome could be chosen to be inactivated, and that this choice is then heritable for the life of that cell and all of its progeny. So we'll think about this again in a couple of slide's time and go through each of these features that Mary Lyon proposed, but in it's nice to note that 50 years after Mary Lyon first made her her hypothesis said Lyon's hypothesis was changed to Lyon's Law. So now this is taken as law, that is is indeed what happens in a mammalian cell. So you can also see another image, which ill show you, which is a further image related to this first one. That on the lower panel, we're staining just DNA, and you can see a denser region of DNA in the nuclear periphery indicated with the arrow, the Barr body. But in the upper panel, what we're looking at now is X chromosome DNA. And this is painted with a chromosome paint. And, so the one that's densely stain are in 2 regions. One that is densely staining and one that is less densely staining and we actually don't see any dense staining here at all, where there is this second X chromosome. And so these denote that inactive X chromosome which is densely staining and that the nuclear periphery, this Barr body while the open chromatin so, therefore, this not particularly well stained region is the active X chromosome. Another way to see the Barr body is shown in this slide where you can see And on the left we're staining the DNA in blue or with a DNA integrating dye and again you can see indicated with the arrow, the Barr body this region that's more densely staining at the nuclear periphery which is only found in the female cells. However you can see density in the right panel we're staining for a histone variant that I mentioned in previous lectures, called macroH2A. and macro H2A is known to be accumulated on the in active X X chromosome. And so there is a co-localisation of this densely Duffy staining or density DNA density in either heterochromatin, this Barr body and macroH2A as you can see as indicated by the arrow in this inflorescence image. So it's nice to think about how it was that Mary Lyon and also Setsuko Ohno were able to come up with their theory, Mary Lyon's theory of X inactivation and Setsuko Ohno, really amazing discoveries about the X chromosome. So one of the ways they were able to propose this, was because they looked in karyotypically abnormal cells. So cells that didn't contain normal numbers of chromosomes. So lets think about the two normal cases first. We know that in female mammals, you have two X chromosomes and 44 autosomes. This is in a human. This is a normal diploid female. In normal males, you have one X chromosome, one Y chromosome, and still 44 autosomes. But then, there are different cases where this is, not the case. We have an abnormal number of sex chromosomes, or X or Y chromosomes. So the first case is, trisomy X and then, as it sounds like, you have three X chromosomes. These patients have three X chromosomes, but still the normal number of autosomes. Klinefelter patients who are, who are males, they have an X and a Y like your average male would, but they have an additional X chromosome. So two X chromosomes and a Y chromosome. Plus their normal 44 autosomes. And finally, an additional type that was studied were tetraploid female cells. These aren't from patients. But what they found was that in the dish if you had, cells that instead of having a diploid content, but instead had doubled all of that. So now they had 4 X chromosomes and 88 autosomes. By looking at each of these and counting the number of Barr bodies, they were able to learn something about X inactivation and to make for their proposals. So what they found was, of course, in females you have 1 inactive X chromosome and in males you have none. However, in triploid X there are two inactive X chromosomes and klinefelter male patients have 1 inactive X chromosome and Kleinfelter male patients although they are Kleinfelter’s patients, have one inactive X chromosome this really tells you that the presence of a Barr body isn’t strictly to do with the gender of the individual or the sex of the individual. Finally, in the tetraploid female cells, there are two inactive X chromosomes. And so, part of Mary Lyon's hypothesis was then that only one X chromosome remains active per diploid set of autosomes, and all others are silenced. So in the case of Trisomy X, you can see that two need to be inactivated so that you only have one remaining active for those diploid set of autosomes. Where as in the tetraploid female cells, because you have not just a diploid set of autosomes, but in fact twice as many, now you need 2 active X’s for that and therefore you have only, then you have two that are being inactivated. And as I mentioned in Klinefelter’s, you have one active X chromosome and one inactive X chromosome. So it’s not dependent on whether or not a Y chromosome is present in the cell, or whether the patient is male or female. But rather on the how many X chromosomes should remain active within each nucleus. What's quite interesting is that Mary Lyon made the hypothesis in 1961 but relatively quickly within five years, there’d already being a change, at least in terms of how you would test for sex within the Olympics. So, in the 1966 Olympics they actually used some tests to look at whether or not athletes had a Barr body to be able to determine whether or not they were truly female. Now, this is rather interesting, because of course given what I've just told you the Klinefelter's male patients were, were there any to be in the the Olympics would also come up as female, but this is not the way around that we're concerned about. They were really concerned that males are of course competing as females. But it's interesting it really changed how Olympic testing happened within five years. So, other than how many X chromosomes will be inactivated the second part of Mary Lyons hypothesis. Is the random nature of X inactivation. So we've discussed this before, thinking about a calico cat. And I'd like to come back to it again because it's really a nice and important finding that she had. So, she wasn't actually studying calico cats. She was studying something very similar, the coat colours in mice. An experimental model system. And what she found, was that if you looked, at variegated phenotypes, and this, what this means is phenotypes of the coat colour that have multiple different or at least 2 different coat colour phenotypes. Or in other cases, other phenotypes. So you can variegated ivy or variegated other plants. And they have two different options of colours within their leaf. But cells within the same tissue can have mosaic expressions. So they can have different outcomes. Different colours for variegated ivy. Different colours for mice or cats for example. And she noticed that if you had, animals that females that were obligate heterozygotes. You had to know that they were a heterozygote for a particular coat colour allele. Then they could have this mosaic, this variegated coat colour. But males, never, never showed this variegated coat colour. And of course, the same effect is seen in humans. If you have, if you're able to see this within the scheme colours. And the same is seen in cats and I'd like go through that cat example, the calico cat example in more detail. So this is really about the visual appearance of random X inactivation. So if you consider that there are two alleles of the ginger gene, you have the ginger itself which is given as a big G. And then you have the black version of this. It's a null allele and it' will be little g Well, normally ginger would be dominant over the black allele and therefore, the cat would end up being ginger. However, if now we think that there's, we know there's X inactivation we know that some half of the time then the ginger allele will be on the active X chromosome as shown on the left hand side here and so that cell will end up producing ginger pigment. However half of the time for the rest of the time you'll have the ginger allele rather than the black allele being on the inactive chromosome. And this will result in black pigment being produced. So this choice, when it's set up, happens at around gastrulation. And is then mitotically heritable, for the rest of the lifetime of that female cat. And so you end up getting these clonal patches of coat colour as shown in the picture of the cats on the right. And the same is true if you look at a similar sort of a phenotype in mice, and this is what Mary Lyon was looking at. So, as I've mentioned before, we know that we really don't see male cats that display this calico appearance, and if they are, the ones that are found, in fact karyotypically XXY. And so, from what I've just told you, there Klinefelter's cats, if you like, and they have one X chromosome that is inactivated, and that's why they display this similar phenotype to the females. So, it was studies like this that led Mary Lyon to make her hypothesis in 1961. So, the key point here is that random X inactivation occurs very early in embyogenisis, around gastrulation and once that choice is made, it's mitotically heritable. It's also interesting to think, remember I mentioned the 1966 Olympics, they were already testing for the Barr body to tell whether or not it was a female or a male athlete. But in 1965, Stanley Gartler also used this presence of the Barr body, or random X inactivation. To prove that cancer was clonal in origin so he could find that in fact if you looked at which X chromosome was inactivated then all of the cancer had the same X chromosome inactivated in females. And this therefore suggested that, that they all were derived from the same single cell. And so you can see within a very short space of time of X inactivation being proposed it already infiltrated other fields within biology. So in the next lecture we'll think about the different types of X inactivations that occur in mammals.
