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So now we come to summarise what I've told you so far about X chromosome
inactivation and the stages. You remember this slide from before and
I've just now added some more details. We start out with the embryonic stem
cells or, the cells of the inner cell mass.
And watch differentiation as it occurs over the course of about 10 days in
vitro. The very early stages are all about
controlling which chromosome will express Xist.
We know that the pluripotency factors are involved in repressing Xist expression.
And that as differentiation is stimulated the pluripotency factors will decrease this
will allow the up regulation of RNF 12 and RNF 12 will then be involved in this
counting process by it's own expression. We know that X chromosome pairing.
This transient chromosome kissing that occurs some how related to the choice of
which X chromosome will go on to be inactive.
And the SATB1 and SATB2 are acquired at around this time point in order to bring
about that silent nuclear compartment. So once Xist begins to be expressed we
know that its rapidly brought, we know that RNA Polymerase 2 is rapidly removed.
From the region where Xist is found. And we have silencing of repeats and the
establishment of a silent nuclear compartment.
And we rapidly lose the active histone marks.
Then Xist is involved in recruiting PRC1 and PRC2 and possibly other epigenetic
modifiers at this time. And we get the accumulation of the
repressive histone marks. During this time, we know that there's
progressive recruitment into this silent nuclear compartment.
And this is why genes are progressively silenced over time.
So not all genes are silenced at the same time point.
But rather, it depends, when they are recruited into that silent nuclear
compartment. In terms of when they will actually be
silenced. It's at this stage when we really switch
to the maintenance phase of X chromosome inactivation.
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And one of the things I haven't mentioned to date
is that one of the features of the X chromosome is that it undergoes late
replication timing. And this is true not only for X
inactivation. Or not only for the heterochromatin found
on the inactive X, I guess. But also for other heterochromatin.
So I'll just spend a moment to explain that.
So, within the S phase we know that chromatin can be or the DNA can be
replicated at many different stages. It could be replicated early or late
during S phase. And so there can be a temporal
segregation of the replication of euchromatin. So open chromatin or active
chromatin compared with heterochromatin like the inactive X.
And so what we know is that this probably relates to the transfer or
the transfer of the epigenetic marks from one nucleosome to the daughter
nucleosome. So if we want to have mitotic
heritability, we need to have that these epigenetic marks can be translated from
one nucleosome to the second nucleosome. Just like with DNA methylation.
You need to have the second, the daughter strand of DNA being methylated
just like the parental strand. So it's only during late S phase when
the epigenetic modified complexes that are involved in laying down repressive
histone marks are actually located at that replication fork.
They need to be there at the replication fork so they can recognise their own
epigenetic mark on the parental nucleosome and also on the parental histones.
And lay them down on the new keystones being brought in to the the new strand
that's being created. So then it makes sense that
heterochromatin tends to be replicated very late in S phase whereas euchromatin
is replicated early in S phase. So this is true for heterochromatin in
general and is true for the inactive X chromosome.
It undergoes late replication timing. So this late after the late replication
timing happens we know that we also have the association of other chromatin
proteins or histone variants on the inactive X chromosome so in fact although
you wouldn’t necessarily expect it some of these are also Xist dependent.
So we know MacroH2A, this histone variant associated with the inactive X, it's
association is dependent on Xist, as is Smchd1's.
SAF-A is a protein that I haven't talked about, but it is involved in stabilising
Xist on the inactive X chromosome. Very late in X inactivation we know that
Atrx, which is a chromatin remodeller, is also associated, which is presumably
involved in densely packaging down that inactive X chromosome.
And so, almost all of the features, or probably all of the features that I told
you about epigenetic control in weeks one and two are demonstrated by this X
inactivation process. So DNA methylation, I mentioned, tends to
happen very late. It does tend to happen at this very
light stage, at around 10 days post differentiation in embryonic stem cells.
However, there are some CpG islands, some promoters of gene subject to X
inactivation which are methylated relatively early in this process.
So, like gene silencing DNA methylation tends to happen over an extended period
of time. But it is predominantly happening at this
end stage as one of the last events in X inactivation.
So, this slide is an extremely busy slide, and so it would seem that we know
a huge amount how X inactivation occurs, we know much about the molecular
mechanisms. But actually that's not true.
While we know many of the marks that are associated with inactive X chromosomes,
we don't know exactly how they fit together.
So say for example for polycomb repressive complex one and two.
These lay down the marks that are associated with inactive chromatin, and
they were recruited by Xist. However, we know that even when they are
recruited, they're insufficient to actually silence the inactive
X chromosome. And so, this is actually, at the moment,
is kind of where we're at. We know many of the hallmarks of the
inactive X chromosome, but we don't really know how it brings about
transcriptional silencing. So I just want to touch on very briefly
one of the things that my lab does. So I've mentioned before that one of the
things that my lab's interested in doing is using micro RNAs directed against the
genes that we think might be involved in epigenetic control.
Or the potential epigenetic modifiers. To be able to sort out which epigenetic
modifiers are important and when. So in a way these are the potential
epigenetic modifiers, this huge pile of puzzle pieces.
And each in turn we can reduce the expression of each of these in turn,
using microRNAs directed against each one.
One of the systems that we used to study actually is X inactivation.
So we studied this in the lab hoping to find new epigenetic modifiers that are
involved in X inactivation because we believe that perhaps one of the reasons
we don't really understand the precise molecular mechanisms yet is because we
don't know about all of the puzzle pieces.
We don't have all of the players. The way that we do this, we need to be is
to use the fluorophore and I will explain why.
To be able to form a screen, we need to be able to very rapidly assess X
inactivation, and so I will just mention exactly how we do this, and so what we
have done is we have made fluorescent proteins that tag the expression from
each X chromosome. So, here the paternal X chromosome Xp is
green. Whereas Xm, so the maternal X chromosome
produces a red florissant protein. So in embryonic stem cells or cells of
the inner cell mass or primordial germ cells you'll have two active X
chromosomes and the cells will be green and red.
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However, in differentiated cells, somatic cells of the female, adult cells, then
you will find that the cells are either red or green and that's because of random
X inactivation. So, what we can do is we can use this
system to be able to screen for the factors involved in these many processes,
these many stages of X inactivation I have mentioned.
So, we can take the embryonic stem cells that are both red and green.
We can induce them to differentiate and watch them as they turn either red or
green or not both. And as we introduce these micro RNAs to
reduce the expression of each of the particular genes we're interested in
about a thousand genes. We can study whether the phases of
initiation and establishment or of maintenance of X inactivation occur
normally or in are in some way disturbed. And this will give us some indication as
to whether the gene that we reduced the expression of with microRNAs has any role
in this fundamental process of X inactivation.
So, the reason we study this is for the same reason that I wanted to tell you all
about X inactivation, and that's because everything that we seem to learn about X
inactivation is in fact true for epigenetic control for throughout the
genome. So, in summary then of what I've told
you about X inactivation over the past several lectures.
We know that X inactivation is initiated by Xist this long non-coding RNA that is
absolutely critical for X inactivation to occur.
This long non-coding RNA Xist, coats the inactive chromosome in cis.
So from the same chromosome from which it's expressed.
Dr. Marnie BlewittHead, Molecular Medicine Laboratory
