So in this lecture we'll again think about how these imprint control regions bring about the imprinted gene expression. So in the last lecture we spoke about the Kcnq1 cluster and the H19/Igf2 cluster, and how disruptions to the clusters led to Beckwith Wiedemann syndrome. But In this cluster we're going to consider a third cluster, the Snrpn cluster. This Snrpn cluster is also controlled by a long non-coding RNA, however the mechanism of action of this long non-coding RNA is quite different to that of exist, or Kcnq1ot1. When this cluster is disrupted, depending on how it's disrupted, whether it's the maternal chromosome or the paternal chromosome, it results in two different imprinted disorders known as Angelman syndrome and Prader-Willi syndrome. So let's start by thinking about what the cluster looks like. So the notation I'm using here is the same as last time. If a gene is shown in pink then it's expressed from the maternal allele, if a gene is shown in blue it's expressed from the paternal allele. And, of course, they're greyed out if they are not expressed from that chromosome. In this case you can see that we also have a split imprinting center. So, it's called the Angelman Syndrome imprinting centre and the Prader-Willi imprinting center. And I'll explain the function of each of these two. But what I would like to think about from the beginning is that this gene, Ube3a, which is expressed from the maternal allele and happens in this direction, from right to left, so from the negative strand of DNA, is it's expression is only imprinted in the brain. And this really relates to the phenotype of the Angelman Syndrome patients. Okay? So this is an example about where All though it's not imprint, although DNA methylation difference in the Prader-Willi Syndrome imprint clusters shown here is present in all tissues. We don't see imprinted expression of all genes in all tissues, and I'll explain why this occurs. I'll also point out that Atp10a, the imprinted expression of Atp10a is still relatively controversial and we won't talk about this today. So if we think first of all about the Prader Willi syndrome imprint centre this Prader Willi syndrome imprint centre is surrounding the promoter of the start site for the Snurf/Snrpn long noncoding RNA. So if this imprint centre is unmethylated as it is in the paternal allele then it results in expression of this long noncoding RNA. And this long non-coding RNA is extremely long, it's hundreds of kilo bases long. So, it's much longer than Xist which is already very long at 17 kilobases in length. So if this long noncoding RNA is expressed, then it can be processed into the snoRNA's which are small nucleolar RNAs found within it's transcript. But it also can progress through the Ube3a locus in an antisense orientation. And so it's called at this end Ube3a antisense. The reason that Ube3a is only displaying, imprinted expression in the brain, is because, this long noncoding RNA Snurf/Snrpn stops short of the Ube3a region in everywhere except the brain. So this tells you that this, progress of long noncoding RNA through this region is required to set up the imprinted state. In fact, we know it's required for silencing in the, on the paternal allele. So, there have also been mouse experiments that have been done where they deliberately stop the expression of the Snurf/Snrpm long noncoding RNA before it gets to this region. So, what we know here is that this antisense section, this Ube3a antisense section is required for this imprinted expression. So, what's not clear is why it should be that you require the transcription of this long noncoding RNA at this point. Is it because it's antisense and this is why it's setting up silencing, or is the actual active transcription through the locus that sets up the silencing? Now it seems that the active transcription through the locus in other instances as well can set up a silent state. And now this doesn't make perfect sense when you first think about it. Why should the active RNA polymerase which is an active event you're transcribing something lead to silencing? We still don't really know exactly how this occurs, but this it seems to be a common feature of not just the Snurf/Snrpn locus, but also of other imprinted loci and other places throughout the genome. So this action of the long noncoding RNA to produce snoRNAs in this case and also, to have some function in setting up a negative inhibition of gene expression of the Ube3a gene itself which would be going this direction. Is a very different mechanism of action of this long coding RNA. When we compare it to exist or Kcnq1 that we've spoken about previously. So, we've said that this Prader-Willi syndrome imprint cluster is un-methylated in the paternal allele, which allows for expression of the long non-coding RNA, but methylated on the maternal allele, and this is why we don't have expression of the Snurf/Snrpn long non-coding RNA from the maternal allele. But what's the function of the Angelman syndrome imprint cluster. Well, we know the Angelman syndrome imprint cluster is not differentially methylated between the two parental alleles, as you can see here. They're both unmethylated. But the Angelman syndrome imprint cluster acts within the oocyte lineage, so only doing oogenesis in the production of the oocytes. And what its function is, we require this piece of DNA, this Angelman syndrome imprint cluster, to actually set up methylation at the Prada-Willie syndrome imprint cluster. So that's its function, to allow methylation of the neighbouring region. So now let's think about those disorders with all of those, those features that we've mentioned about this particular cluster. So we know disruption to this cluster can result in either Angelman syndrome or Prader-Willi syndrome, but this depends on which parental allele is disrupted, and therefor which genes are or are not expressed So we start with Angelman syndrome. The clinical features are very different to the other imprinting disorder that I mentioned. And that makes sense because it's a different cluster, a different set of genes that are mis-regulated. So in Angelman syndrome we see severe brain growth retardation, and this results in microcephaly, or a small head, and sever mental retardation. There are characteristic puppet-like jerky arm movements and seizures, but actually the patients have an amazingly happy disposition and an actually inappropriately happy disposition, and inappropriate bouts of laughter. Just like Beckwith Wiedemann syndrome that we discussed in the last lecture, this also is a syndrome which is maternally transmitted. So this tells you that it's going to be the maternal allele which is disrupted in this case. So Angelman syndrome is caused by a disruption to Ube3aA. We don't think there's actually any role for Atp10a, and so this is part of the reason we're not sure about it's role and whether it's imprinted. So, commonly, what can happen is you can have deletion or inappropriate silencing of the maternal copy, such that Ube3a is not expressed. And then we know Ube3a is not expressed from the paternal allele, and this means that no Ube3a is made, at least in the brain. So, remember, Ube3a's expression is only imprinted in the brain. So if you think back to those phenotypes of the Angelman Syndrome patients, They are all related to brain phenotypes. So this is because of the lack of Ube3a expression in the brain, but the way that this can, come about can be through multiple different mechanisms. So you could have a deletion of the Angelman syndrome imprint centre. Without this Angelman syndrome imprint centre, then during the development of the eggs, you cannot lay down the DNA methylation at the Prader-Willi Syndrome imprint center. And therefore the allele looks like the paternal allele where both are unmethylated. You'll have expression of that long non coding RNA Snurf/Snrpn. And you'll end up with two alleles that behave like the expression from the paternal allele. This of course means that you'll get no expression of Ube3a. And as in the brain and as a consequence, Angelman syndrome results. So it can occur, Angelman syndrome similar to Beckwith-Wiedemann syndrome can result from uniparental disomy. In this case you would have to have two copies of the paternal allele and no copies of the maternal allele. And this would be resulting in what’s called paternal unit parental disomy. It could also result from actually very specific mutations just in Ube3a because Angelman syndrome is all about the expression of Ube3a or lack of expression of Ube3a in the brain. But finally it can result from a similar sort of mechanism as Beckwith-Wiedemann. It can be an epigenetic disruption with no underlying genetic cause. And so this can be a failure to methylate, this Prader-Willi Syndrome imprint cluster. So, again, just like with Beckwith-Wiedemann Syndrome, the cases that are resulting form disruption to epigenetic disruption but no underlying genetic disruption are extremely rare in comparison to the genetic cases of Angelman Syndrome. If we now think about the partner disorder, Prader-Willi syndrome, we know it's also resulting from disruption to this Snurf/Snurpn but it's on the opposite allele. And so, Prader-Willi syndrome is paternally transmitted. So the clinical features for Prader-Willi syndrome are a low muscle tone and failure to thrive. So in general fairly small children, hypogonadism and severe mental retardation. But then later in life, around teenage years, there's an obsessive compulsive behaviour that results. And perhaps the behaviour that has the largest effect on the phenotype of these patients is over-eating, obsessive over-eating to the point of morbid obesity. So these two things, the failure to thrive as children, and then the consequent and then following on from that, this obsessive behaviour when you end up with severe overeating, can actually now be treated. So, they can be treated with growth hormone as kids to make sure that they're slightly larger and they grow so they're not so small. And, then caloric restriction, which can be through various means. And this can get over at least some of the phenotypes so low it doesn't overcome completely the mental retardation for these patients. So, if we think about what the molecular events are that lead to this disorder. We again think back to this Snruf/Snrpn cluster. So here it leads, it's because of inappropriate silencing of the paternal allele, so you have two alleles that look like the maternal allele instead. So there are very many genes that are expressed from this paternal chromosome. So we have Frat 3, Mkrn3, Magel2 and Ndn. We also have this long non coding RNA, and within this long non coding RNA are the snoRNAs. We know if you fail to express the snoRNAs, these are the ones that seem to have a consequence for the for the phenotype, in the brain, at least. So how can this happen? Well, we can have deletion of the Prader-Willi Syndrome imprint cluster entirely. So if you delete this imprint cluster, it actually behaves like the maternal allele. That's basically because you've removed the start site of this long non-coding RNA, Snurf/Snrpn, and in the absence of this long non-coding RNA, then it behaves like the maternal allele when you wouldn't normally express this long non-coding RNA. So then, let's think about a summary of what we've learned in these last two lectures on how the imprint control regions work, and then about the imprinted disorders. We know that each cluster has at least one imprint control region, and it displays differential methylation between the two parental alleles, which is established during primordial germ-cell development. The mechanism of action differs for each of these three clusters. We know that DNA methylation can be used to silence a long noncoding RNA and this is what happens to the Kcnq1 cluster and also for the the Snrpn cluster. If it's the Kcnq1 long noncoding RNA we are thinking about well, it can recruit epigenetic modifiers in cis just like Xist. A second cluster, the H19/Igf2 cluster that we spoke about, instead has a different mechanism action, which is insulator blocking. So you have an insulator which binds to the imprint control region, dependent on the methylation of this cluster. And finally, we can have what happened with the Snrpn cluster, DNA methylation will be there to silence a long non-coding RNA. But this long non-coding RNA works in a very distinct fashion, where it seems to be either the act of transcription itself or the antisense transcription, which is required to establish silencing. So these are these three different mechanisms by which these imprint control regions can work. In terms of the imprinted disorders. We know that they're all associated with misexpression of genes within these particular imprinted clusters. All of them show parent of origin dependent inheritance. And we went through these for Beckwith Wiedemann Syndrome. And they can result because of genetic disruptions. Either a deletion, or mutation, uniparental disomy, or from an epigenetic basis which means that you get disruption of the imprint itself, the DNA methylation imprint within the cluster.
