0:00
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.
0:50
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.
2:35
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.
5:59
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.
8:41
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.
9:56
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.
Dr. Marnie BlewittHead, Molecular Medicine Laboratory
