In this lecture we're going to talk about 2 examples of long noncoding RNAs, Xist and HOTAIR. So Xist is named because it's the X inactivation specific transcript. And it was the first long non-coding RNA to be discovered back in 1991. It has a function in X chromosome inactivation, which we've talked about in previous lectures. And this is to remind you that dosage compensation mechanism in female mammals, where one whole X chromosome is densely packaged down in the female nucleus to be able to allow for dosage compensation between the female and the male. So, as you may expect, then, we have two X chromosomes, but only one of them needs to be silenced in the female. And so we need to somehow have allele specificity brought into this process of X inactivation. And this is, you'll remember, one of the features of long noncoding RNA's. Because they're tethered to where they're transcribed from, they can provide this allele specificity. Xist is the critical determinant for X inactivation. So Xist is 17 kilobase long. So 17,000 base pairs, or bases. Long noncoding RNA. It is spliced. It has a poly-A tail, and it's constrained to the nucleus. It does not produce a protein product. So it ticks all of those boxes for being a long non-coding RNA. It's expressed from just 1 of the 2 X chromosomes, so it's expressed in an allele-specific way. And indeed it's the first event that we know happens in X chromosome inactivation. The first detectable event, and without the expression of Xist you cannot have x inactivation proceed. So, it's this expression of Xist that actually determines the chromosome that will become inactive. So this is slightly counterintuitive because you have the transcription meaning that the gene is active, but this will cause the inactivation of the chromosome on which that gene is being transcribed from. So it's a little counterintuitive. But the expression of Xist, which acts in that allele-specific way, in cis on the chromosome from which its expressed. This RNA will then coat the inactive X chromosome. And it does so, only from the one it's expressed from. So the picture here is showing again a nucleus of DNA with a DNA stained by DAPI, a DNA stain that's showing in blue. And there are two very small yellow dots and these yellow dots show each of the X chromosomes here and here. This is marking the DNA of the X chromosome. But in red, what you can see is over the one of these dots is a cloud of Xist RNA. And it's this Xist RNA which allows this chromosome to become inactive, and it's only associated with one of the two X chromosomes. So the way that Xist works, in this case it involves one of the repeats that's found in Xist. So this long 17 kb RNA has at least six repeats that we know of. And one of those repeats, repeat A, actually forms a secondary structure through here. And it's this secondary structure of repeat A, or rep A that causes the binding of Polycomb Repressive complex 2 PRC2 you might remember from the earlier lectures this is one of the examples of histone methyl transferase complexes that I brought up and its the histone methyltransferase that has specificity to H3K27 so lays down methylation of H3K27. So in this way Xist is acting as a guide. It guides PRC2, and then uses the sequence specificity to be attracting to the X chromosome from which it's transcribed. And then PRC2 can lay down this methylation mark. And so the inactive X not only has Xist bound to it, but is also found with H3K 27 trimethylation along that X chromosome. So Xist is an example of a long noncoding RNA that acts in cis in an allele specific way, that also acts in a sequence specific way. It's bound to the X chromosome from which it's transcribed. But finally, it also acts as a guide because it recruits the chromatin modifier complex, PRC2. In the second example, that is HOTAIR, the second long noncoding RNA we'll talk about. It can act both as a guide, similar to PRC2, but also as a scaffold. So HOTAIR is transcribed from one of the HOX cluster of genes. So there are many HOX genes spread throughout the mammalian genome. And there's a cluster of them on chromosome 12 in humans. And a cluster of them on chromosome 2. These are known as the HOXC cluster. And the HOXD cluster. And within the HOXC cluster, there's the HOTAIR gene itself. So this HOTAIR gene is transcribed into the long noncoding RNA. But then it doesn't work on the same chromosome. It doesn't work in cis like Xist did, but rather it acts in trans. And this is extremely controversial. But if we believe that it acts in trans then what it does is it binds to PRC2, similarly to how we found or we discussed for Xist and then lays down it particular. And PRC2 can lay down the methylation mark. In this case the HOX genes are being silenced. And hot air is being, is acting as a guide. And the reason it's acting as a guide is, it's just bringing in one epigenetic modifier complex. This is, this being PRC2. And it's also still being targeted based on the sequence specificity. Because each HOX gene has a high degree of identity to the other genes. The HOXD cluster is quite similar to the HOXC cluster. But we know that HOTAIR can actually have another role. So hot air can actually be misexpressed in cancer. And we'll talk about this a little more in the cancer epigenetics lectures. But here what's relevant, HOTAIR is not only binding to the HOXD cluster. But rather, also at other places throughout the genome. And so, sequence specificity, if it's found at other places, may not necessarily be required. And in this context, it not only binds PRC2, but it also binds a second complex. It binds LSD1. And in this way it seems to be acting as a scaffold because it'll bring together these 2 epigenetic modifier complexes. So PRC2 can lay down H3K27 trimethylation, whereas LSD1 is actually a demethylase. But it removes methylation from H3K4, which you'll remember is an active mark. So HOTAIR tethers together 2 complexes which are both going to end up in transcriptional repression. One because it lays down repressive mark and the other because it removes an active mark. So again if we look at this visually we can see that we have this HOTAIR long non coding RNA and through 2 different regions of the long non-coding RNA it will bind 2 different epigenetic complexes. PRC2 and LSD1 and this is it's function as a scaffold. It can then either act in a sequence specific way or not in a sequence specific way and will be able to the changes in the chromatin will result because of PRC2 and LSD1. And regions where HOTAIR is found because of the association with PRC2 or PRC2 and LSD1 will result in repressive chromatin structure and repressive epigenetic marks.
