The Epigenetics Revolution Nessa Carey

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The sound of a kiss is not so loud as that of a cannon, but its echo lasts a great deal longer.

Oliver Wendell Holmes

At a purely biological, and especially an anatomical level, men and women are different. There are ongoing debates about whether or not certain behaviours, ranging from aggression to spatial processing, have a biological gender bias. But there are certain physical characteristics that are linked unequivocally to gender. One of the most fundamental differences is in the reproductive organs. Women have ovaries, men have testicles. Women have a vagina and a uterus, men have a penis.

There is a clear biological basis to this, and perhaps unsurprisingly, it’s all down to genes and chromosomes. Humans have 23 pairs of chromosomes in their cells, and inherited one of each pair from each parent. Twenty-two of these pairs (imaginatively named chromosomes 1 to 22) are called autosomes and each member of a specific pair of autosomes looks very similar. By ‘looks’ we mean exactly that. At a certain stage in cell division the DNA in chromosomes becomes exceptionally tightly coiled up. If we use the right techniques we can actually see chromosomes down a microscope. These chromosomes can be photographed. In pre-digital days, clinical geneticists literally used to cut out the pictures of the individual chromosomes with a pair of scissors and rearrange them in pairs to create a nice orderly picture. These days the image processing can be carried out by a computer, but in either case the result is a picture of all the chromosomes in a cell. This picture is called a karyotype.

Figure 9.1 Karyotype of all the chromosomes in a male (top) and female (bottom) somatic cell. Note that the female cell contains two X chromosomes and no Y chromosome; the male cell contains one X chromosome and one Y chromosome. Note also the substantial difference in size between the X and Y chromosomes. Photos: Wessex Reg Genetics Centre/Wellcome Images.

Karyotype analysis is how scientists originally discovered that there were three copies of chromosome 21 in the cells of people with Down’s syndrome. This is known as trisomy 21.

When we produce a human karyotype from a female, there are 23 pairs of identical chromosomes. But if we create a human karyotype from a male, the picture is different, as we can see in Figure 9.1. There are 22 obvious pairs – the autosomes – but there are two chromosomes left over that don’t look like each other at all. One is very large, one exceptionally small. These are called the sex chromosomes. The large one is called X, and the small one is called Y. The notation to describe the normal chromosome constitution of human males is 46, XY. Females are described as 46, XX because they don’t have a Y chromosome, and instead have two X chromosomes.

The Y chromosome carries very few active genes. There are only between 40 and 50 protein-coding genes on the Y chromosome, of which about half are completely male-specific. The male-specific genes only occur on the Y chromosome, so females have no copies of these. Many of these genes are required for male-specific aspects of reproduction. The most important one in terms of sex determination is a gene called SRY. SRY proteins activate a testis-determining pathway in the embryo. This leads to production of testosterone, the archetypal ‘male’ hormone, which then masculinises the embryo.

Occasionally, individuals who phenotypically appear to be girls have the male 46, XY karyotype. In these cases the SRY gene is often inactive or deleted and consequently the foetus develops down the default female pathway¹. Sometimes, the other scenario arises. Individuals who phenotypically appear to be boys can have the typically female karyotype of 46, XX. In these cases a tiny section of the Y chromosome containing the SRY gene has often transferred onto another chromosome during formation of sperm in the father. This is enough to drive masculinisation of the foetus². The region of the Y chromosome that was transferred was too small to be detected by the karyotyping process.

The X chromosome is very different. The X chromosome is extremely large and carries about 1300 genes. A disproportionate number of these genes are involved in brain function. Many are also required for various stages in formation of the ovaries or the testes, and for other aspects of fertility in both males and females³.

Getting the dose right

So, about 1300 genes on the X chromosome. That creates an interesting problem. Females have two X chromosomes but males only have one. That means that for these 1300 genes on the X, females have two copies of each gene but males only have one. We might speculate from this that female cells would produce twice the amounts of proteins from these genes (referred to as X-linked genes) as males.

But our knowledge of disorders like Down’s syndrome makes this seem rather unlikely. Having three copies of chromosome 21 (instead of the normal two) results in Down’s syndrome, which is a major disorder in those individuals who are born with the condition. Trisomies of most other chromosomes are so severe that children are never born with these conditions, because the embryos cannot develop properly. For example, no child has ever been born who has three copies of chromosome 1 in all their cells. If the 50 per cent increase in gene expression from an autosome can cause such problems in trisomic conditions, how do we explain the X chromosome scenario? How is it possible for females to survive when they have twice as many X chromosome genes as males? Or, to put it the other way – why are males viable if they only have half as many X chromosome genes as females?

The answer is that expression of X-linked genes is actually pretty much the same in males and females, despite the different number of chromosomes, a phenomenon called dosage compensation. The XY system of sex determination doesn’t exist in other animal classes so X chromosome dosage compensation is limited to placental mammals.

In the early 1960s a British geneticist called Mary Lyon postulated how dosage compensation would occur at the X chromosome. These were her predictions:

1. Cells from the normal female would contain only one active X chromosome;

2. X inactivation would occur early in development;

3. The inactive X could be either maternally or paternally derived, and the inactivation would be random in any one cell;

4. X inactivation would be irreversible in a somatic cell and all its descendants.

These predictions have proven remarkably prescient⁴^,⁵. So prescient, in fact, that many textbooks refer to X inactivation as Lyonisation. We’ll take the predictions one at a time:

1. Individual cells from a normal female do indeed only express genes from one X chromosome copy – the other copy is, effectively, shut down;

2. X inactivation occurs early in development, at the stage when the pluripotent cells of the embryonic inner cell mass are beginning to differentiate into different lineages (near the top of Waddington’s epigenetic landscape);

3. On average, in 50 per cent of cells in a female the maternally derived X chromosome is shut down. In the other 50 per cent of cells it’s the chromosome inherited from Dad which gets inactivated;

4. 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 100 years of age.

The X chromosome isn’t inactivated by mutation; it keeps its DNA sequence entirely intact. X inactivation is the epigenetic phenomenon par excellence.

X inactivation has proven to be a remarkably fertile research field. Some of the mechanisms involved have turned out to have parallels in a number of other epigenetic and cellular processes. The consequences of X inactivation have important implications for a number of human disorders and for therapeutic cloning. Yet even now, 50 years on from Mary Lyon’s ground-breaking work, there remain a number of mysteries about how X inactivation actually takes place.

The more we ponder X inactivation, the more extraordinary it appears. For a start, the inactivation is only on the X chromosome, not on any of the autosomes, so the cell must have a way of distinguishing X chromosomes and autosomes from one another. Furthermore, the inactivation in the X doesn’t just affect one or a few genes, such as occurs in imprinting. No, in X inactivation, over 1,000 genes are turned off, for decades.

Think of a car manufacturer, with a factory in Japan and another in Germany. Imprinting is the equivalent of a few changes in specification for the different markets. The German factory may switch on the machine that installs the heater on the steering wheel and switch off the robot that inserts the automatic air freshener, whilst the Japanese factory does the opposite. X inactivation is the equivalent of shutting down and mothballing one factory, never to be re-opened unless the company is bought by a new manufacturer.

Random inactivation

The other major difference between X-inactivation and imprinting is that there is no parent-of-origin effect in X imprinting. In somatic cells, it doesn’t matter if an X chromosome was inherited from your mother or your father. Each has a 50 per cent chance of being inactivated. The reason why this is the case makes complete evolutionary sense.

Imprinting is about balancing out the competing demands of the maternal and paternal genomes, especially during development. The imprinting mechanisms that have evolved are specifically targeted at individual genes, or small clusters of genes, that particularly influence foetal growth. There are, after all, only 50–100 imprinted genes in the mammalian genome.

But X inactivation operates on a much greater scale. It’s a mechanism for switching off over 1,000 genes, en masse and permanently. A thousand genes is a lot, about 5 per cent of the total number of protein-coding genes, so there’s always a possibility that any given gene on an X chromosome may have a mutation. Figure 9.2 compares the outcomes of imprinted X inactivation on the left, with random X inactivation on the right. For clarity, the diagram just exemplifies a mutation in a paternally inherited gene, with imprinted inactivation of the maternally derived X chromosome.

By using random X inactivation, cells are able to minimise the effects of mutations in X-linked genes.

It’s important to bear in mind that the inactive X really is inactive. Almost all the genes are permanently shut off and this inactivation cannot normally be broken. When we refer to the active X chromosome, we are using slightly ambiguous shorthand. It doesn’t mean that every gene on that X is active all the time in every cell. Rather, the genes have the potential to be active. They are subject to all the normal epigenetic modifications and controls on gene expression, so that selected genes are switched on or off in a controlled manner, in response to developmental cues or environmental signals.

Figure 9.2 Each circle represents a female cell, containing two X chromosomes. The X chromosome inherited from the mother is indicated by the female symbol. The X chromosome inherited from the father is indicated by the male symbol, and contains a mutation, denoted by the white square notch. The left hand side of the diagram demonstrates that imprinted inactivation of the maternally derived X chromosome would result in all cells of the body expressing only the X chromosome carrying the mutation, which was inherited from the father. On the right hand side, the X chromosomes are randomly inactivated, independent of their parent-of-origin. As a result, on average, half of the somatic cells will express the normal version of the X chromosome. This makes random X inactivation a less risky evolutionary strategy than imprinted X inactivation.

Women really are more complicated than men

One interesting consequence of X inactivation is that (epigenetically) females are more complicated than males. Males only have one X chromosome in their cells, so they don’t carry out X inactivation. But females randomly inactivate an X chromosome in all their cells. Consequently, at a very fundamental level, all cells in a female body can be split into two camps depending on which X chromosome they inactivated. The expression for this is that females are epigenetic mosaics.

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 inactivation⁶. 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 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 chromosome⁷. 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 autosome⁸^,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-inactive (X_i) specific transcript¹⁰. 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 gene¹¹ 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 protein¹²^,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-pairs¹⁴). 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.

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 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.

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 desert¹⁵.

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?

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 transgene¹⁶.

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.

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 inactivation¹⁷. 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 expression¹⁸. 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.

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.

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.

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.

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 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 expression¹⁹. 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 reactivated²⁰. 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 …

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.

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.

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.

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 chromosome²¹. 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 stroke²².

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.

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.

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 not²³.

Duchenne muscular dystrophy is a severe muscle wasting disease caused by mutations in the X-linked DYSTROPHIN gene. This is a large gene that encodes a big protein which acts as an essential shock absorber in muscle fibres. Boys carrying certain mutations in DYSTROPHIN suffer major muscle loss that usually results in death in the teenage years. Females with the same mutation are usually symptom-free. The reason for this is that muscle has a very unusual structure. It is called a syncytial tissue, which means that lots of individual cells fuse and operate almost like one giant cell, but with lots of discrete nuclei. This is why most females with a DYSTROPHIN mutation are symptom-free. There is enough normal DYSTROPHIN protein encoded by the nuclei that switched off the mutated DYSTROPHIN gene to keep this syncytial tissue functioning healthily²⁴.

There are occasional cases where this system breaks down. There was a case of female monozygotic twins where one twin was severely affected by Duchenne muscular dystrophy and the other was healthy²⁵. In the affected twin, the X inactivation had become skewed. Early in tissue differentiation the majority of her cells that would give rise to muscle happened, by ill chance, to switch off the X chromosome carrying the normal copy of the DYSTROPHIN gene. Thus, most of the muscle tissue in this woman only expressed the mutated version of DYSTROPHIN, and she developed severe muscle wasting. This could be considered the ultimate demonstration of the power of a random epigenetic event. Two identical individuals, each with two apparently identical X chromosomes, had a completely discordant phenotype, because of a shift in the epigenetic balance of power.

Sometimes, however, it is essential that individual cells express the correct amount of a protein. You may have noticed in Chapter 4 that Rett syndrome only affected girls. One might hypothesise that boys are somehow very resistant to the effects of the MeCP2 mutation, but actually the opposite is true. MeCP2 is carried on the X chromosome so a male foetus that inherits a Rett syndrome mutation in this gene has no means of expressing normal MeCP2 protein. A complete lack of normal MeCP2 expression is generally lethal in early development, and that’s why very few boys are born with Rett syndrome. Girls have two copies of the MeCP2 gene, one on each X chromosome. In any given cell, there is a 50 per cent chance that the cell will inactivate the X that carries the unmutated MeCP2 gene and that the cell will not express normal MeCP2 protein. Although a female foetus can develop, there are ultimately major effects on normal post-natal brain development and function when a substantial number of neurons lack MeCP2 protein.

One, two, many

There are other issues that can develop around the X chromosome. One of the questions we need to answer about X inactivation, is how good mammalian cells are at counting. In 2004 Peter Gordon of Columbia University in New York reported on his studies on the Piraha tribe in an isolated region of Brazil. This tribe had numbers for one and two. Everything beyond two was described by a word roughly equating to ‘many’²⁶. Are our cells the same, or can they count above two? If a nucleus contains more than two X chromosomes, can the X inactivation machinery recognise this, and deal with the consequences? Various studies have shown that it can. Essentially, no matter how many X chromosomes (or more strictly speaking X Inactivation Centres) are present in a nucleus, the cell can count them and then inactivate multiple X chromosomes until there is only one remaining active.

This is the reason why abnormal numbers of X chromosomes are relatively frequent in humans, in contrast to abnormalities in the number of autosomes. The commonest examples are shown in Table 9.1.

Table 9.1 Summary of the major characteristics of the commonest abnormalities in sex chromosome number in humans.

The infertility that is a feature of all these disorders is in part due to problems when creating eggs or sperm, where it’s important that chromosomes line up in their pairs. If there is an uneven total number of sex chromosomes this stage goes wrong and formation of gametes is severely compromised.

Leaving aside the infertility, there are two obvious conclusions we can draw from this table. The first is that the phenotypes are all relatively mild compared with, for example, trisomy of chromosome 21 (Down’s syndrome). This suggests that cells can tolerate having too many or too few copies of the X chromosome much better than having extra copies of an autosome. But the other obvious conclusion is that an abnormal number of X chromosomes does indeed have some effects on phenotype.

Why should this be? After all, X inactivation ensures that no matter how many X chromosomes are present, all bar one get inactivated early in development. But if this was the end of the story there would be no difference in phenotype between 45, X females compared with 47, XXX females or with the normal 46, XX female constitution. Similarly, males with the normal 46, XY karyotype should be phenotypically identical to males with the 47, XXY karyotype. In all of these cases there should be only one active X chromosome in the cells.

One thought as to why people with these karyotypes were clinically different was that maybe X inactivation is a bit inefficient in some cells, but this doesn’t seem to be the case. X inactivation is established very early in development and is the most stable of all epigenetic processes. An alternative explanation was required.

The answer has its origin about 150 million years ago, when the XY system of sex determination in placental mammals first developed. The X and Y chromosomes are probably descendants of autosomes. The Y chromosome has changed dramatically, the X chromosome much less so²⁷. However, both retain shadows of their autosomal past. There are regions on both the X and the Y called pseudoautosomal regions. The genes in these regions are found on both the X and the Y chromosome, just in the same way as pairs of autosomes have the same genes in the same positions, one inherited from each parent.

When an X chromosome inactivates, these pseudoautosomal regions are spared. This means that, unlike most X-linked genes, those in the pseudoautosomal regions don’t get switched off. Consequently, normal cells potentially express two copies of these genes in all cells. The two copies are expressed either from the two X chromosomes in a normal female or from the X and the Y in a normal male.

But in Turner’s syndrome, the affected female only has one X chromosome, so she expresses only one copy of the genes in the pseudoautosomal region, half as much as normal. In Trisomy X, on the other hand, there are three copies of the genes in the pseudoautosomal regions. As a result, the cells in an affected region will produce proteins from these genes at 50 per cent above the normal level.

One of the genes in the X chromosome pseudoautosomal regions is called SHOX. Patients with mutations in this gene have short stature. It is likely that this is also why patients with Turner’s syndrome tend to be short – they don’t produce enough SHOX protein in their cells. By contrast, patients with Trisomy X are likely to produce 50 per cent more SHOX protein than normal, which is probably why they tend to be tall²⁸.

It’s not just humans who have trisomies of the sex chromosomes. One day you may be happily amazing your friends with your confident statement that their tortoiseshell cat is female when they deflate you by telling you that their pet has been sexed by the vet and is actually a Tom. At this point, smile smugly and then say ‘Oh, in that case he’s karyotypically abnormal. He has an XXY karyotype, rather than XY’. And if you’re feeling particularly mean, you can tell them that Tom is infertile. That should shut them up.

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