The Epigenetics Revolution (25 page)
Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
We know a bit more now about the mechanism of chromosome counting. Cells don’t normally count their autosomes. Both copies of chromosome 1, for example, operate independently. But we know that the two copies of the X chromosome in a female ES cell somehow communicate with each other. When X inactivation is getting going, the two X chromosomes in a cell do something very weird.
They kiss.
That’s a very anthropomorphic way of describing the event, but it’s a pretty good description. The ‘kiss’ only lasts a couple of hours or so, and it’s startling to think this sets a pattern that can persist in cells for the next hundred years, if a woman lives that long. This chromosomal smooch was first shown in 1996 by Jeannie Lee, who started out as a post-doctoral researcher in Rudi Jaenisch’s lab, but who is now a professor in her own right at Harvard Medical School, where she was one of the youngest professors ever appointed. She showed that essentially the two copies of the X find each other and make physical contact. This physical contact is only over a really small fraction of the whole chromosome, but it’s essential for triggering inactivation
17
. If it doesn’t happen, then the X chromosome assumes it is all alone in the cell,
Xist
never gets switched on, and there is no X inactivation. This is a key stage in chromosome counting.
17
. If it doesn’t happen, then the X chromosome assumes it is all alone in the cell,
Xist
never gets switched on, and there is no X inactivation. This is a key stage in chromosome counting.
It was Jeannie Lee’s lab that also identified one of the critical genes that controls
Xist
expression
18
. DNA is double-stranded, with the bases in the middle holding the strands together. Although we often envisage it as looking like a railway track, it might be better to think of it as two cable cars, running in opposite directions. If we use this metaphor, then the X Inactivation Centre looks a bit like
Figure 9.4
.
Xist
expression
18
. DNA is double-stranded, with the bases in the middle holding the strands together. Although we often envisage it as looking like a railway track, it might be better to think of it as two cable cars, running in opposite directions. If we use this metaphor, then the X Inactivation Centre looks a bit like
Figure 9.4
.
There is another non-coding RNA, about 40kb in length, in the same stretch of DNA as
Xist
. It overlaps with
Xist
but is on the opposite strand of the DNA molecule. It is transcribed into RNA in the opposite direction to
Xist
and is referred to as an antisense transcript. Its name is
Tsix
. The eagle-eyed reader will notice that
Tsix
is
Xist
backwards, which has an unexpectedly elegant logic to it.
Xist
. It overlaps with
Xist
but is on the opposite strand of the DNA molecule. It is transcribed into RNA in the opposite direction to
Xist
and is referred to as an antisense transcript. Its name is
Tsix
. The eagle-eyed reader will notice that
Tsix
is
Xist
backwards, which has an unexpectedly elegant logic to it.
Figure 9.4
The two strands of DNA at a specific location on the X chromosome can each be copied to create mRNA molecules. The two backbones are copied in opposite directions to each other, allowing the same region of the X chromosome to produce
Xist
RNA or
Tsix
RNA.
The two strands of DNA at a specific location on the X chromosome can each be copied to create mRNA molecules. The two backbones are copied in opposite directions to each other, allowing the same region of the X chromosome to produce
Xist
RNA or
Tsix
RNA.
This overlap in location between
Tsix
and
Xist
is really significant in terms of how they interact, but it makes it exceedingly tricky to perform conclusive experiments. That’s because it’s very difficult to mutate one of the genes without mutating its partner on the opposite strand, a sort of collateral damage. Despite this, considerable strides have been made in understanding how
Tsix
influences
Xist
.
Tsix
and
Xist
is really significant in terms of how they interact, but it makes it exceedingly tricky to perform conclusive experiments. That’s because it’s very difficult to mutate one of the genes without mutating its partner on the opposite strand, a sort of collateral damage. Despite this, considerable strides have been made in understanding how
Tsix
influences
Xist
.
If an X chromosome expresses
Tsix
, this prevents
Xist
expression from the same chromosome. Oddly enough, it may be the simple action of transcribing
Tsix
that prevents the
Xist
expression, rather than the
Tsix
ncRNA itself. This is analogous to a mortice lock. If I lock a mortice from the inside of my house and leave the key in the lock, my partner can’t unlock the door from the outside of the house. I don’t need to keep locking the door, just having the key in there is enough to stop the action of someone on the other side. So, when
Tsix
is switched on,
Xist
is switched off and the X chromosome is active.
Tsix
, this prevents
Xist
expression from the same chromosome. Oddly enough, it may be the simple action of transcribing
Tsix
that prevents the
Xist
expression, rather than the
Tsix
ncRNA itself. This is analogous to a mortice lock. If I lock a mortice from the inside of my house and leave the key in the lock, my partner can’t unlock the door from the outside of the house. I don’t need to keep locking the door, just having the key in there is enough to stop the action of someone on the other side. So, when
Tsix
is switched on,
Xist
is switched off and the X chromosome is active.
This is the situation in ES cells, where both X chromosomes are active. Once the ES cells begin to differentiate, one of the pair stops expressing
Tsix
. This allows expression of
Xist
from that X chromosome, which drives X inactivation.
Tsix
. This allows expression of
Xist
from that X chromosome, which drives X inactivation.
Tsix
alone is probably not enough to keep
Xist
repressed. In ES cells, the proteins Oct4, Sox2 and Nanog bind to the first intron of
Xist
and suppress its expression
19
. Oct4 and Sox2 were two of the four factors used by Shinya Yamanaka when he reprogrammed somatic cells to the pluripotent iPS cell type. Subsequent experiments showed that Nanog (named after the mythical Celtic land of everlasting youth) can also work as a reprogramming factor. Oct4, Sox2 and Nanog are highly expressed in undifferentiated cells like ES cells, but their levels fall as cells start to differentiate. When this happens in differentiating female ES cells, Oct4, Sox2 and Nanog stop binding to the
Xist
intron. This removes some of the barriers to
Xist
expression. Conversely, when female somatic cells are reprogrammed using the Yamanaka approach, the inactive X chromosome is reactivated
20
. The only other time the inactive X is reactivated is during the formation of primordial germ cells in development, which is why the zygote starts out with two active X chromosomes.
alone is probably not enough to keep
Xist
repressed. In ES cells, the proteins Oct4, Sox2 and Nanog bind to the first intron of
Xist
and suppress its expression
19
. Oct4 and Sox2 were two of the four factors used by Shinya Yamanaka when he reprogrammed somatic cells to the pluripotent iPS cell type. Subsequent experiments showed that Nanog (named after the mythical Celtic land of everlasting youth) can also work as a reprogramming factor. Oct4, Sox2 and Nanog are highly expressed in undifferentiated cells like ES cells, but their levels fall as cells start to differentiate. When this happens in differentiating female ES cells, Oct4, Sox2 and Nanog stop binding to the
Xist
intron. This removes some of the barriers to
Xist
expression. Conversely, when female somatic cells are reprogrammed using the Yamanaka approach, the inactive X chromosome is reactivated
20
. The only other time the inactive X is reactivated is during the formation of primordial germ cells in development, which is why the zygote starts out with two active X chromosomes.
We are still a bit vague as to why X inactivation is so mutually exclusive between the pair of chromosomes. One theory is that it’s all down to what happens when the X chromosomes kiss. This happens at a developmental point where
Tsix
levels are starting to fall, and the levels of the Yamanaka factors are also declining. The theory is that the pair of chromosomes reaches some sort of compromise. Rather than each ending up with a sub-optimal amount of non-coding RNAs and other factors, the binding molecules all get shunted together onto one of the pair. There’s not a great deal of clarity on how this happens. It could be that one of the pair of chromosomes just by chance carries slightly more of a key factor than the other. This makes it slightly more attractive to certain proteins. Complexes may build up in a self-sustaining way, so that the more of a complex one chromosome starts with, the more it can drag off its partner. The rich get richer, the poor get poorer …
Tsix
levels are starting to fall, and the levels of the Yamanaka factors are also declining. The theory is that the pair of chromosomes reaches some sort of compromise. Rather than each ending up with a sub-optimal amount of non-coding RNAs and other factors, the binding molecules all get shunted together onto one of the pair. There’s not a great deal of clarity on how this happens. It could be that one of the pair of chromosomes just by chance carries slightly more of a key factor than the other. This makes it slightly more attractive to certain proteins. Complexes may build up in a self-sustaining way, so that the more of a complex one chromosome starts with, the more it can drag off its partner. The rich get richer, the poor get poorer …
It’s quite remarkable how many gaps remain in our understanding of X inactivation, 50 years after Mary Lyon’s formative work. We don’t even really understand how the
Xist
RNA ends up coating the chromosome from which it is expressed, or how it recruits all those negative repressive epigenetic enzymes and modifications. So perhaps it’s timely to move off the shifting sands and step back onto more solid ground.
Xist
RNA ends up coating the chromosome from which it is expressed, or how it recruits all those negative repressive epigenetic enzymes and modifications. So perhaps it’s timely to move off the shifting sands and step back onto more solid ground.
Let’s return to this statement from earlier in the chapter: ‘Once a cell has switched off one of a pair of X chromosomes, that particular copy of the X stays switched off in all the daughter cells for the rest of that woman’s life, even if she lives to over a hundred years of age.’ How do we know that? How can we be so certain that X inactivation is stable in somatic cells? It is now possible to perform genetic manipulation to show this in species like mice. But long before that became feasible scientists were already pretty certain this was the case. For this piece of information we thank not mice, but cats.
Learning from the epigenetic cat
Not just any old cats, but specifically tortoiseshell ones. You probably know how to recognise a classic tortoiseshell cat. It’s the one that’s a mixture of black and ginger splodges, sometimes on a white background. The colour of each hair in a cat’s coat is caused by cells called melanocytes that produce pigment. Melanocytes are found in the skin, and develop from special stem cells. When melanocyte stem cells divide, the daughter cells stay close to each other, forming a little patch of clonal cells from the same parent stem cell.
Now, here’s an amazing thing: if a cat’s colour is tortoiseshell, it’s a female.
There is a gene for coat colour that encodes either black pigment or orange pigment. This gene is carried on the X chromosome. A cat may receive the black version of the gene on the X chromosome inherited from her mother and the orange version on the X chromosome inherited from her father (or
vice versa
).
Figure 9.5
shows what happens next.
vice versa
).
Figure 9.5
shows what happens next.
So the tortoiseshell cat ends up with patches of orange and patches of black, depending on the X chromosome that was randomly inactivated in the melanocyte stem cell. The pattern won’t change as the cat gets older, it stays the same throughout its life. That tells us that the X inactivation stays the same in the cells that create this coat pattern.
We know that tortoiseshell cats are always female because the gene for the coat colour is only on the X chromosome, not the Y. A male cat only has one X chromosome, so it could have black fur or ginger fur, but never both.
Figure 9.5
In female tortoiseshell cats, the genes for orange and black fur are carried on the X chromosome. Depending on the pattern of X chromosome inactivation in the skin, clonal patches of cells will give rise to discrete patterns of orange and black fur.
In female tortoiseshell cats, the genes for orange and black fur are carried on the X chromosome. Depending on the pattern of X chromosome inactivation in the skin, clonal patches of cells will give rise to discrete patterns of orange and black fur.
Something rather similar happens in a rare human disorder called X-linked hypohidrotic ectodermal dysplasia. This condition is caused by mutations in a gene called
ECTODYSPLASIN-A
, carried on the X chromosome
21
. A male with a mutation in his sole copy of
ECTODYSPLASIN-A
on his single X chromosome has a variety of symptoms, including a total lack of sweat glands. This might sound socially advantageous, but is actually incredibly dangerous. Sweating is one of the major routes by which we lose excess heat, and men with this condition are at serious risk of tissue damage or even death as a result of heat stroke
22
.
ECTODYSPLASIN-A
, carried on the X chromosome
21
. A male with a mutation in his sole copy of
ECTODYSPLASIN-A
on his single X chromosome has a variety of symptoms, including a total lack of sweat glands. This might sound socially advantageous, but is actually incredibly dangerous. Sweating is one of the major routes by which we lose excess heat, and men with this condition are at serious risk of tissue damage or even death as a result of heat stroke
22
.
Females have two copies of the
ECTODYSPLASIN-A
gene, one on each of their X chromosomes. In female carriers of X-linked hypohidrotic ectodermal dysplasia, one X carries a normal copy of the gene, and one a mutated version. There will be random inactivation of one X chromosome in different cells. This means some cells will express a normal copy of
ECTODYSPLASIN-A
. Other cells will randomly shut down the X carrying the normal copy of the gene, and won’t be able to express the ECTODYSPLASIN-A protein. Because of the clonal way in which areas of skin develop, just like in the tortoiseshell cat, these women have some patches of skin that express ECTODYSPLASIN-A and some that don’t. Where there’s no ECTODYSPLASIN-A, the skin can’t form sweat glands. As a consequence, these women have patches of skin that can sweat and cool down, and others that can’t.
ECTODYSPLASIN-A
gene, one on each of their X chromosomes. In female carriers of X-linked hypohidrotic ectodermal dysplasia, one X carries a normal copy of the gene, and one a mutated version. There will be random inactivation of one X chromosome in different cells. This means some cells will express a normal copy of
ECTODYSPLASIN-A
. Other cells will randomly shut down the X carrying the normal copy of the gene, and won’t be able to express the ECTODYSPLASIN-A protein. Because of the clonal way in which areas of skin develop, just like in the tortoiseshell cat, these women have some patches of skin that express ECTODYSPLASIN-A and some that don’t. Where there’s no ECTODYSPLASIN-A, the skin can’t form sweat glands. As a consequence, these women have patches of skin that can sweat and cool down, and others that can’t.
Random X inactivation can significantly influence how females are affected by mutations in genes on the X chromosome. This depends not just on the type of gene that is mutated but also on the tissues that express and require the protein encoded by that gene. The disease called mucopolysaccharidosis II (MPSII) is caused by mutations in the
LYSOSOMAL IDURONATE-2-SULFATASE
gene, on the X chromosome. Boys with this mutation on their single X chromosome are unable to break down certain large molecules and these build up to toxic levels in cells. The main symptoms include airway infections, short stature and enlargement of the spleen and liver. Severely affected boys also suffer mental retardation, and may die in their teenage years.
LYSOSOMAL IDURONATE-2-SULFATASE
gene, on the X chromosome. Boys with this mutation on their single X chromosome are unable to break down certain large molecules and these build up to toxic levels in cells. The main symptoms include airway infections, short stature and enlargement of the spleen and liver. Severely affected boys also suffer mental retardation, and may die in their teenage years.
Females with a mutation in the same gene are usually perfectly healthy. LYSOSOMAL IDURONATE-2-SULFATASE protein is usually secreted out of the cell that makes it and taken up by neighbouring cells. In this situation it doesn’t matter too much which X chromosome has been mutated in a specific cell. For every cell that has inactivated the X carrying the normal version of the gene, there is likely to be another cell nearby which inactivated the other X chromosome and is secreting the protein. This way, all cells end up with sufficient LYSOSOMAL IDURONATE-2-SULFATASE protein, whether they produce it themselves or not
23
.
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.
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