Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
The Epigenetics Revolution (24 page)
This sophisticated epigenetic control in females is a complicated and highly regulated process, and that’s where Mary Lyon’s predictions have provided such a useful conceptual framework. They can be paraphrased as the following four steps:
1. Counting: cells from the normal female would contain only one active X chromosome;
2. Choice: X inactivation would occur early in development;
3. Initiation: the inactive X could be either maternally or paternally derived, and the inactivation would be random in any one cell;
4. Maintenance: X inactivation would be irreversible in a somatic cell and all its descendants.
Unravelling the mechanisms behind these four processes has kept researchers busy for nearly 50 years, and this effort is continuing today. The processes are incredibly complicated and sometimes involve mechanisms that had barely been imagined by any scientists. That’s not really surprising, because Lyonisation is quite extraordinary – X inactivation is a procedure where a cell treats two identical chromosomes in diametrically opposite and mutually exclusive ways.
Experimentally, X inactivation is challenging to investigate. It is a finely balanced system in cells, and slight variations in technique may have a major impact on the outcome of experiments. There’s also considerable debate about the most appropriate species to study. Mouse cells have traditionally been used as the experimental system of choice, but we are now realising that mouse and human cells aren’t identical with respect to X inactivation
6
. However, even allowing for these ambiguities, a fascinating picture is beginning to emerge.
6
. However, even allowing for these ambiguities, a fascinating picture is beginning to emerge.
Counting chromosomes
Mammalian cells must have a mechanism to count how many X chromosomes they contain. This prevents the X chromosome from being switched off in male cells. The importance of this was shown in the 1980s by Davor Solter. He created embryos by transferring male pronuclei into fertilised eggs. Males have an XY karyotype, and when they produce gametes each individual sperm will contain either an X or a Y. By taking pronuclei from different sperm and injecting them into ‘empty’ eggs, it was possible to create XX, XY or YY zygotes. None of these resulted in live births, because a zygote requires both maternal and paternal inputs, as we have already seen. But the results still told us something very interesting, and are summarised in
Figure 9.3
.
Figure 9.3
.
Figure 9.3
Donor egg reconstitution experiments were performed in which the donor egg received a male and female pronucleus or two pronuclei from males. Just as in
Figure 7.2
, the embryos derived from two male pronuclei failed to develop to term. When the nuclei each contained a Y chromosome, and no X chromosome, the embryos failed at a very early stage. Embryos derived from two male pronuclei where at least one contained an X chromosome developed further before they also died.
Donor egg reconstitution experiments were performed in which the donor egg received a male and female pronucleus or two pronuclei from males. Just as in
Figure 7.2
, the embryos derived from two male pronuclei failed to develop to term. When the nuclei each contained a Y chromosome, and no X chromosome, the embryos failed at a very early stage. Embryos derived from two male pronuclei where at least one contained an X chromosome developed further before they also died.
The earliest loss of embryos occurred in those that had been reconstituted from two male pronuclei which each contained a Y chromosome as the sole sex chromosome
7
. In these embryos there was no X chromosome at all, and this was associated with exceptionally early developmental failure. This shows that the X chromosome is clearly essential for viability. This is why male (XY) cells need to be able to count, so that they can recognise that they only contain one X, and thus avoid inactivating it. Turning off the solitary X would be disastrous for the cell.
7
. In these embryos there was no X chromosome at all, and this was associated with exceptionally early developmental failure. This shows that the X chromosome is clearly essential for viability. This is why male (XY) cells need to be able to count, so that they can recognise that they only contain one X, and thus avoid inactivating it. Turning off the solitary X would be disastrous for the cell.
Having counted the number of X chromosomes, there must be a mechanism in female cells by which one X is randomly selected for inactivation. Having selected a chromosome, the cell starts the inactivation procedure.
X inactivation happens early in female embryogenesis, as the cells of the ICM begin to differentiate into the different cell types of the body. Experimentally, it is difficult to work on the small number of cells available from each blastocyst so researchers typically use female ES cells. Both X chromosomes are active in these cells, just like in the undifferentiated ICM. It’s easy to roll ES cells down Waddington’s epigenetic landscape, just by subtly altering the conditions in which the cells are cultured in the lab. Once we change the conditions to encourage the female ES cells to differentiate, they begin to inactivate an X chromosome. Because ES cells can be grown in almost limitless numbers in labs, this provides a convenient model system for studying X inactivation.
Painting an X-rated picture
Initial insights into X inactivation came from studying mice and cell lines with structurally rearranged chromosomes. In some of these studies, various sections of an X chromosome were missing. Depending on which parts were missing, the X chromosome did or did not inactivate normally. In other studies, sections had come off the X chromosome and attached themselves onto an autosome. Depending on which part of the X chromosome had transferred, this could result in switching off the structurally abnormal autosome
8
,
9
.
8
,
9
.
These experiments showed that there was a region on the X chromosome that was vitally important for X inactivation. This region was dubbed the X Inactivation Centre. In 1991 a group from Hunt Willard’s lab at Stanford University in California showed that the X Inactivation Centre contained a gene that they called
Xist
, after
X
-
i
nactive (X
i
)
s
pecific
t
ranscript
10
. This gene was only expressed from the
inactive
X chromosome, not from the active one. Because the gene was only expressed from one of the two X chromosomes, this made it an attractive candidate as the controller of X inactivation, where two identical chromosomes behave non-identically.
Xist
, after
X
-
i
nactive (X
i
)
s
pecific
t
ranscript
10
. This gene was only expressed from the
inactive
X chromosome, not from the active one. Because the gene was only expressed from one of the two X chromosomes, this made it an attractive candidate as the controller of X inactivation, where two identical chromosomes behave non-identically.
Attempts were made to identify the protein encoded by the
Xist
gene
11
but by 1992 it was clear that there was something very strange going on. The
Xist
gene was transcribed to form RNA copies. The RNA was processed just like any other RNA. It was spliced, and various structures were added to each end of the transcript to improve its stability. So far, so normal. But before RNA molecules can code for protein, they have to move out of the nucleus and into the cytoplasm of the cell. This is because the ribosomes – the intracellular factories that join amino acids into long protein chains – are only found in the cytoplasm. But the
Xist
RNA never moved out of the nucleus, which meant it could never generate a protein
12
,
13
.
Xist
gene
11
but by 1992 it was clear that there was something very strange going on. The
Xist
gene was transcribed to form RNA copies. The RNA was processed just like any other RNA. It was spliced, and various structures were added to each end of the transcript to improve its stability. So far, so normal. But before RNA molecules can code for protein, they have to move out of the nucleus and into the cytoplasm of the cell. This is because the ribosomes – the intracellular factories that join amino acids into long protein chains – are only found in the cytoplasm. But the
Xist
RNA never moved out of the nucleus, which meant it could never generate a protein
12
,
13
.
This at least cleared up one thing that had puzzled the scientific community when the
Xist
gene was first identified. Mature
Xist
RNA is a long molecule, of about 17,000 base-pairs (17kb). One amino acid is coded for by a three base-pair codon, as described in
Chapter 3
. Therefore, in theory, the 17,000 base-pairs of
Xist
should be able to code for a protein of about 5,700 amino acids. But when researchers analysed the
Xist
sequence with protein prediction programs, they simply couldn’t see how it could encode anything this long. There were stop codons (which signal the end of a protein) all through the
Xist
sequence and the longest predicted run without stop codons was only enough to code for 298 amino acids (894 base-pairs
14
). Why would a gene have evolved which created a 17kb transcript, but only used about 5 per cent of this to encode protein? That would be a very inefficient use of energy and resources in a cell.
Xist
gene was first identified. Mature
Xist
RNA is a long molecule, of about 17,000 base-pairs (17kb). One amino acid is coded for by a three base-pair codon, as described in
Chapter 3
. Therefore, in theory, the 17,000 base-pairs of
Xist
should be able to code for a protein of about 5,700 amino acids. But when researchers analysed the
Xist
sequence with protein prediction programs, they simply couldn’t see how it could encode anything this long. There were stop codons (which signal the end of a protein) all through the
Xist
sequence and the longest predicted run without stop codons was only enough to code for 298 amino acids (894 base-pairs
14
). Why would a gene have evolved which created a 17kb transcript, but only used about 5 per cent of this to encode protein? That would be a very inefficient use of energy and resources in a cell.
But since
Xist
never actually leaves the nucleus, its lack of potential protein coding is irrelevant.
Xist
doesn’t act as a messenger RNA (mRNA) that transmits the code for a protein. It is a class of molecule called a non-coding RNA (ncRNA).
Xist
may not code for protein, but this doesn’t mean it has no activity. Instead, the
Xist
ncRNA itself acts as a functional molecule, and it is critical for X inactivation.
Xist
never actually leaves the nucleus, its lack of potential protein coding is irrelevant.
Xist
doesn’t act as a messenger RNA (mRNA) that transmits the code for a protein. It is a class of molecule called a non-coding RNA (ncRNA).
Xist
may not code for protein, but this doesn’t mean it has no activity. Instead, the
Xist
ncRNA itself acts as a functional molecule, and it is critical for X inactivation.
Back in 1992 ncRNAs were a real novelty, and only one other was known at the time. Even now, there is something very unusual about
Xist
. It’s not just that it doesn’t leave the nucleus.
Xist
doesn’t even leave the chromosome that produces it. When ES cells begin to differentiate, only one of the chromosomes produces
Xist
RNA. This is the chromosome that will be the inactive one.
Xist
doesn’t move away from the chromosome that produced it. Instead, it binds to the chromosome and starts to spread out along it.
Xist
. It’s not just that it doesn’t leave the nucleus.
Xist
doesn’t even leave the chromosome that produces it. When ES cells begin to differentiate, only one of the chromosomes produces
Xist
RNA. This is the chromosome that will be the inactive one.
Xist
doesn’t move away from the chromosome that produced it. Instead, it binds to the chromosome and starts to spread out along it.
Xist
is often described as ‘painting’ the inactive X and it’s a very good description. Let’s revert yet again to our analogy of the DNA code as a script. This time we’ll imagine that the script is written on a wall, maybe it’s an inspiring poem or speech in a classroom. At the end of the summer term the school building closes down and is sold for conversion to apartments. The decorators arrive and paint over the script. Now there’s nothing to tell the new residents to ‘play up and play the game’, or exactly how they should ‘meet with Triumph and Disaster’. But the instructions are actually still there, they’re just hidden from view.
is often described as ‘painting’ the inactive X and it’s a very good description. Let’s revert yet again to our analogy of the DNA code as a script. This time we’ll imagine that the script is written on a wall, maybe it’s an inspiring poem or speech in a classroom. At the end of the summer term the school building closes down and is sold for conversion to apartments. The decorators arrive and paint over the script. Now there’s nothing to tell the new residents to ‘play up and play the game’, or exactly how they should ‘meet with Triumph and Disaster’. But the instructions are actually still there, they’re just hidden from view.
When
Xist
binds over the X chromosome that produced it, it induces a kind of creeping epigenetic paralysis. It covers more and more genes, switching them off. It first seems to do this by acting as a barrier between the genes and the enzymes that normally copy them into mRNA. But as the X inactivation gets better established, it changes the epigenetic modifications on the chromosome. The histone modifications that normally turn genes on are removed. They are replaced by repressive histone modifications that turn genes off.
Xist
binds over the X chromosome that produced it, it induces a kind of creeping epigenetic paralysis. It covers more and more genes, switching them off. It first seems to do this by acting as a barrier between the genes and the enzymes that normally copy them into mRNA. But as the X inactivation gets better established, it changes the epigenetic modifications on the chromosome. The histone modifications that normally turn genes on are removed. They are replaced by repressive histone modifications that turn genes off.
Some of the normal histones are removed altogether. Histone H2A is replaced by a related but subtly different molecule called macroH2A, strongly associated with gene repression. The promoters of genes undergo DNA methylation, an even more stringent way of turning the genes off. All these changes lead to binding of more and more repressor molecules, coating the DNA on the inactive X and making it less and less accessible to the enzymes that transcribe genes. Eventually, the DNA on the X chromosome gets incredibly tightly wound up, like a giant wet towel being turned at each end, and the whole chromosome moves to the edge of the nucleus. By this stage most of the X chromosome is completely inactive, except for the
Xist
gene, which is a little pool of activity in the midst of a transcriptional desert
15
.
Xist
gene, which is a little pool of activity in the midst of a transcriptional desert
15
.
Whenever a cell divides, the modifications to the inactive X are copied over from mother cell to daughter cell, and so the same X remains inactivated in all subsequent generations of that starter cell.
While the effects of
Xist
are amazing, the description above still leaves a lot of questions unanswered. How is
Xist
expression controlled? Why does it switch on when ES cells start to differentiate? Is
Xist
only functional when it’s in female cells, or could it act in males cells too?
Xist
are amazing, the description above still leaves a lot of questions unanswered. How is
Xist
expression controlled? Why does it switch on when ES cells start to differentiate? Is
Xist
only functional when it’s in female cells, or could it act in males cells too?
The power of a kiss
The last question was first addressed in the lab of Rudi Jaenisch, whom we met in the context of iPS cells and Shinya Yamanaka’s work in
Chapter 2
. In 1996, Professor Jaenisch and his colleagues created mice carrying a genetically engineered version of the X Inactivation Centre (an X Inactivation Centre transgene). This was 450kb in size, and included the
Xist
gene plus other sequences on either side. They inserted this into an autosome (non-sex chromosome), created male mice carrying this transgene, and studied ES cells from these mice. The male mice only contained one normal X chromosome, because they have the XY karyotype. However, they had two X Inactivation Centres. One was on the normal X chromosome, and one was on the transgene on the autosome. When the researchers differentiated the ES cells from these mice, they found that
Xist
could be expressed from either of the X Inactivation Centres. When
Xist
was expressed, it inactivated the chromosome from which it was expressed, even if this was the autosome carrying the transgene
16
.
Chapter 2
. In 1996, Professor Jaenisch and his colleagues created mice carrying a genetically engineered version of the X Inactivation Centre (an X Inactivation Centre transgene). This was 450kb in size, and included the
Xist
gene plus other sequences on either side. They inserted this into an autosome (non-sex chromosome), created male mice carrying this transgene, and studied ES cells from these mice. The male mice only contained one normal X chromosome, because they have the XY karyotype. However, they had two X Inactivation Centres. One was on the normal X chromosome, and one was on the transgene on the autosome. When the researchers differentiated the ES cells from these mice, they found that
Xist
could be expressed from either of the X Inactivation Centres. When
Xist
was expressed, it inactivated the chromosome from which it was expressed, even if this was the autosome carrying the transgene
16
.
These experiments showed that even cells that are normally male (XY) can count their X chromosomes. Actually, to be more specific, it showed they could count their X Inactivation Centres. The data also demonstrated that the critical features for counting, choosing and initiation were all present in the 450kb of the X Inactivation Centre around the
Xist
gene.
Xist
gene.
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