<https://zh.coursera.org/lecture/epigenetics/3-6-stages-of-x-inactivation-initiation-and-spreading-of-silencing-NNgjH#lecture>
<http://schema.org/transcript>
"Okay if we come back to these stages of X  chromosome inactivation we've briefly  covered counting and choice.  And now we need to think about the  initiation of X chromosome  inactivation.  So the initiation of course is all about  turning on Xist expression.  So, as we've mentioned before that the  main thing we've required for X  inactivation in fact the critical  determinant is Xist.  And so initiation requires Xist to be  turned on and for it to then coat the  inactive X chromosome that will or, the  chromosome that will become the inactive  X chromosome in cis.  In other words, from the very one that  it's expressed from.  So Xist we know, this Xist expression can  be detected in all female somatic cells  for the lifetime of the organism.  And so, although its the critical  determinant for initiating X inactivation,  it is there for the life time of the  organism.  But, there seems to be some sort of  switch in the dependence on X  inactivation on Xist itself.  So, in the very early stages of X  inactivation just after Xist is being  switched on, there is a critical  dependence still on Xist.  And during this time, if Xist is switched  off then it's reversible and you'll go  back to having two active X chromosomes.  But later in developmental time, when  you're in the later phases of X  inactivation, or indeed in the  maintenance phase of X inactivation,  which of course is predominantly what  occurs for the longest period of time,  then there's less of a dependence on X  inactivation.  If you at this stage delete Xist you can  destabilise X inactivation but you won't  immediately result in upregulation of  that inactive X chromosome.  So because we know that Xist expression  is really critically involved at the  early phases but also we know if we turn  Xist on in most cell types so we turned  on for example, on a trans gene that's  located on an autosome, we don't find X inactivation ensuing.  So there are very few cell types where X  inactivation can actually proceed even  with Xist expression, and so this led  researchers to postulate that there must  be other competence factors, factors that  are required to be found within the  nucleus or found in the cell in general.  Before Xist can actually bring about  transcriptional silencing.  And so, I'm going to mention some of  these other factors as we come through  the next few slides.  So, after this initiation of Xist  expression, the next stage is spreading  of Xist and this silencing that ensues.  And so over the years, just like with  counting and choice, there have been a  very number, very large number of  theories about how this might, might  occur.  But I think now the prevailing model, and  the one everybody seems to accept, is  that there is a progressive recruitment  of the X chromosome into a silent Nuclear  compartment.  So what I mean by silent nuclear  compartment is a region where RNA  polymerese two is excluded so the RNA  polymerase that actually transcribes  genes is removed from this region.  and the X chromosome is drawn into this  region.  So what I'm showing you in the picture  along the bottom of the slide is first of  all a nucleus that's stained with DNA.  A DNA stain DAPI so we can see the DNA  and you can see two regions that are more  densely stained than the others in the  top right hand side.  These are two inactive X chromosomes.  These particular cells are cancer cells  that happen to have two inactive X  chromosomes.  The second oh, sorry, in the third image  you can see Xist expression in green, and  you can see these two DAPI dense or these  heterochromatic regions.  These Barr bodies are also where  Xist is found, but the, this image in  between, which is called h/m Cot-1 is a  stain for repetitive RNA, so the  expression of repetitive elements, and  what you can see indicated by the yellow  arrows. Is that these two regions where these  Barr bodies are found, where Xist is  found, are devoid of this repetitive RNA.  So, then when you look at the pixel  profile, so this is when a line has been  drawn through the image showing the  merge, the line is shown in yellow.  You can see that throughout this region,  you have enrichment of DAPI.  So that the DNA stain in blue, and that  strain by the mound in the graph.  And the mound in the Xist graph, so that  the green line.  But you can see that the red goes down.  So you see much less, repetitive RNA  being found in that region.  And this is because we know that one of  the earliest events after Xist expression  is exclusion of RNA polymerase two from  this region where, in which the X  chromosome is found.  And silencing of the repeats, and so we  find that this, the repetitive, the  repeats that we sometimes be expressed,  the ones that are found in this region,  so the ones in the X chromosome are not  being expressed at this time, and it's a  very early event in X inactivation.  So this happens, this silent nuclear  compartment and the silencing in the  repeat actually happens before the genes  on the X chromosome are silenced.  And we know that the X inactivation  spreading, correlates with  how the genes are actually drawn into  this silent nuclear compartment.  And I'll show you this in more detail in  a diagrammatic form in the next few  slides.  But here, I'd like to mention about some  of those factors that are differentially  expressed or that these competent  factors, competence factors which need to  be present in the nucleus  for Xist to be able to bring  about silencing.  So these factors, some of these factors  are SATB1 and SATB2.  So, SATB1 and SATB2 were found because  they were expressed in both embryonic  stem cells, early post differentiation  but also in the limited number of other  cell types.  that are actually competent to perform  this initiation of X inactivation.  And SATB1 and SATB2 are factors that are  involved in nuclear reorganisation.  So in other words, they move the  chromosomes around.  They help to move the chromosomes around.  So we know that these factors have to be  there for Xist to be able to bring about  that silent nucleic compartment.  So I'm going to go through all these  things I've just said about the spreading  of Xist and silencing in a diagrammatic  form.  So pictured here is an X chromosome as  it might exist in an embryonic stem cell.  Fairly open because it's being expressed.  And so shown on there in yellow are the  repetitive elements so, that would be for  example the intracisternal a particles or  the Line1 elements.  These repeats that are found spread  throughout the genome whereas in green  are the genes that are currently being  expressed because it's an active X  chromosome at this point.  Shortly after these embryonic stem cells  or the inner cell mass is induced to  differentiate, then you get a change in  how the X chromosome looks and how much  space it takes up.  We know that Xist expression comes on  that I am showing here as a blue squiggly  line, and we know that SATB1 and SATB2  are involved.  So, what happens is within this X  chromosome, it's recruited into  the Xist domain.  But, to begin with it's only really those  repetitive elements, which is shown in  yellow here that have been recruited into  this region that contains Xist.  So it's only, the repeats that are  silenced.  This recruitment into that Xist region  contains these purple stars, these SATB1  and SATB2 that I mentioned, these  competence factors that are acquired.  But at this early stage, while the  repeats have been silenced, because they  are within this silent nuclear  compartment, this Xist domain.  We know that the genes remain on the  outside and so the genes remain active  and I'm showing that here is being green.  Then in the final stages when you reach  the somatic cell we know that now more of  those genes have been pulled into that  region, pulled into the silent nuclear  department by SATB1 and SATB2 or other  competence factors.  And now, many of the genes are  indeed silenced.  And I'm showing that here being a black  gene, with, a red rim.  So, once the genes are within the silent  nuclear compartment and pulled into the  Xist domain, then they too have been  silenced and X activation has occurred.  What I haven't mentioned up to now in the  lectures, is that, in fact there are a  small handful of genes.  That don't undergo X inactivation on the  X chromosome.  So these genes that escape X  inactivation, remain outside this silent  nuclear compartment in somatic cells.  So they're never drawn into the region  which isn't transcribed because it's void  of RNA polymerase two.  What we know about these escape genes is  that they differ slightly between  mouse and human.  So here shown on the left-hand side is an  image where you can see that all of the X  chromosome is laid out on the left in the  human and on the right in the mouse.  And what the centromeres are shown in  purple.  So, we know that a human centromere is  quite central where as a mouse centromere  is at the very end right at acrocentric  at one end.  And the genes that escape are showing in  orange.  So most of the escaped genes are in the  regions that are showing as PAR, the  pseudoautosomal regions.  So in humans there are two of these  regions, PAR1 and PAR2, and in mouse  there's just one of these regions.  And these pseudoautosomal regions are the  parts that are required for pairing  of the X and Y chromosome in cell division. And so these genes that are found in the  pseudoautosomal region tend to have a  homologue on the Y chromosome.  And so this means that they have one copy  that is expressed from the X and one copy  from the Y in the male cell but in a  female cell you'll have both copies being  expressed from each X chromosome even if  that X chromosome is actually the  inactive X chromosome.  But they are also a smattering of other  gene's you can see orange coloured gene's  spread along of these 2 X chromosomes and  there are more in humans than they're are  in mice and so these orange escape genes  that aren't in the pseudo autosomal  regions they don't have a wide linked  homolog.  In which case they're not silenced.  You'll have twice the dose of them in  females than you would in males.  And so, perhaps these escapee  genes that don't have a Y homologue might  have something to do with, with the  differences between the sexes.  What’s important to note at this stage, is  that all of these genes, whether they're  in the pseudoautosomal region or not, all  of these escape genes reside outside of  that silent nuclear compartment.  So, the reason that they escape silencing  is that they are never drawn into  that region that contains the Xist  cloud, and that is devoid of RNA  polymerase two.  So in the next lecture, we'll think about  how X inactivation is established and how it spreads. "
<https://zh.coursera.org/lecture/epigenetics/3-6-stages-of-x-inactivation-initiation-and-spreading-of-silencing-NNgjH>