Junk DNA: A Journey Through the Dark Matter of the Genome (13 page)

DNA is found in the nucleus. It’s copied to form RNA, and messenger RNA is transported out of the nucleus to structures where it acts as a template for protein assembly. But analyses showed that Xist RNA never left the nucleus. It doesn’t encode a protein, not even a short one.
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Xist was in fact one of the first examples of an RNA molecule that is functional in its own terms, not as a carrier of information about a protein. It’s a great example of how junk DNA – DNA which doesn’t lead to production of a protein – is anything but junk. It’s extremely important in its own right, because without it X inactivation cannot happen.

An odd feature of Xist is not just that it doesn’t leave the nucleus. It doesn’t even leave the X chromosome that produces it. Instead, it essentially sticks to the inactive X and then spreads along the chromosome. As more and more Xist RNA is produced, it begins to spread out and cover the inactive X chromosome, in a process quaintly referred to as ‘painting’. The fact that this rather descriptive term is used is a quite good indicator that it’s something we don’t particularly understand. No one really knows the physical basis of how the Xist RNA creeps along the chromosome, like the mile-a-minute vine covering a wall. Even after more than twenty years we are still pretty hazy on how this happens. We do know that it’s not based on the sequence of the X chromosome. If the X inactivation centre is transferred on to an autosome in a cell, then the autosome can be inactivated as if it were an X.
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Although Xist is required to initiate the process of X inactivation, it has helpers that strengthen and maintain the process. As Xist paints the X chromosome, it acts as an attachment point for proteins in the nucleus. These bind to the inactivating X, and attract yet more proteins, which shut down expression even more tightly. The only gene that isn’t coated with Xist RNA and these proteins is the Xist gene itself. It remains a little beacon of expression in the chromosomal darkness of the inactive X.
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Left to right, right to left

So we have here a situation where a piece of ‘junk’ DNA – one that doesn’t code for protein – is absolutely essential for the function of half the human race. Scientists have recently discovered that this process of X inactivation requires at least one other piece of junk DNA. Confusingly, this is encoded in exactly the same place on the X chromosome as Xist. DNA, as we know, is composed of two strands (the iconic double helix). The machinery that copies DNA to form RNA always ‘reads’ DNA in one direction, which we could call the beginning and end of a specific sequence. But the two strands of DNA run in opposite directions to each other, a little like one of those funicular railways we find at older seaside and mountain resorts. This means that a particular region of DNA may carry two lots of information in one physical location, running in opposite directions to each other.

A simple example in English is the word DEER, formed by reading from left to right. We could also read the same letters from right to left and in this case we would get the word REED. Same letters, different word, different meaning.

The other key piece of junk DNA involved in X inactivation is called, rather fittingly, Tsix. This is of course Xist spelt backwards, and it is found in the same region as Xist but on the opposite strand. Tsix encodes an RNA of 40,000 bases in length, over twice the size of Xist. Like Xist, Tsix never leaves the nucleus.

Although Tsix and Xist are encoded on the same part of the X chromosome, they are not expressed together. If an X chromosome expresses Tsix, this prevents the same chromosome from expressing Xist. This means that Tsix must be expressed by the active X chromosome, unlike Xist, which is always expressed from the inactive one.

This mutually exclusive expression of Tsix and Xist is of critical importance at a point in early development. The X chromosome in the egg has lost any of the protein marks that show it was inactivated (if it was the inactive version) and the X chromosome in
the sperm had never been inactivated anyway. Following fusion and six or seven rounds of cell division, there will be a hundred or so cells in the embryo. At this stage, each cell in the female embryo switches off one of its two X chromosomes randomly. This requires a fleeting but intense physical relationship between the pair of X chromosomes in a cell. For just a couple of hours the two X chromosomes are physically associated in a brief encounter that ends with one being inactivated. The association is only over a small region of the X chromosome – the X inactivation centre, which codes for both Xist and Tsix RNA.
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A fleeting moment lasts forever

This is the mother of all one-night stands. In those two hours, chromosomal decisions get made which are then maintained for the rest of life. Not just during foetal development, but right up until the woman dies, even if that is more than a hundred years later. And it affects not just the hundred or so cells, but the trillions that come after them, because the same X chromosome is inactivated in all daughter cells.

It’s still not entirely clear what happens during the hours of X chromosome intimacy in early development. The current theory is that there is a reallocation of junk RNA between the two chromosomes, such that one ends up with all the Xist and becomes the inactive X. We don’t know how, but it’s possible that one chromosome expresses slightly more or less of Xist or another key factor. We do know that the process begins just as levels of Tsix start to drop. It may be that once its levels fall below a certain critical threshold, Xist can start getting expressed from one of the X chromosomes.

Gene expression tends to have what’s known as a stochastic component, by which we simply mean there’s a bit of random variability in the levels. If one of the chromosomes is expressing a slightly higher amount of one or more key factors, this may
be sufficient to build a self-amplifying network of proteins and RNA molecules. Because the inequality in expression is essentially stochastic (due to random ‘noise’) the inactivation will also be essentially random across the hundred or so cells.

Here’s a possible way of visualising this. Imagine you get home late one evening and you have a hankering for melted cheese on two slices of toast. Just as you start to make this delicious supper, you realise you don’t have much cheese in the fridge. What do you do? Make two rounds where neither really contains enough cheese to be satisfying? Or concentrate all of it on one slice, so that you get the dairy hit you are craving? Most people probably choose the latter, and in a way this is what the pair of X chromosomes do during the phase when random inactivation is taking place in the embryo. Evolution has favoured a process whereby, rather than each have a sub-critical amount of a key factor, the factor migrates to the chromosome that has slightly more to begin with. The more you have, the more you get.

X inactivation is entirely dependent on ‘junk’ DNA, and really gives the lie to that terminology. The process is absolutely essential in female mammals for normal cell function and a healthy life. It also has consequences in various disease states. Full-blown Fragile X syndrome of mental retardation, which we encountered in Chapter 1, only affects boys. This is because the gene is carried on the X chromosome. Women have two X chromosomes. Even if one of their chromosomes carries the mutation, enough protein is produced from the other (normal) one to avoid the worst of the symptoms. But males only possess one X chromosome and one Y chromosome, which is very small and doesn’t carry many genes apart from the sex determining ones. Consequently, there is no compensatory normal Fragile X gene in males who carry a mutation on their X chromosome. If their sole X chromosome carries the Fragile X expansion, they can’t produce the protein and so they develop symptoms.

This is also true of a whole range of genetic disorders where the mutated gene is carried on the X chromosome. Boys are more likely to have symptoms of an X-linked genetic disorder than girls, because the boys can’t compensate for a faulty gene on their single X chromosome. Relevant medical conditions range from relatively mild issues such as red–green colour blindness to much more severe diseases. These include haemophilia B, the blood clotting disorder. Queen Victoria was a carrier of this condition and one of her sons (Leopold) was a sufferer and died at the age of 31 from a brain haemorrhage. Because at least two of Victoria’s daughters were also carriers, and the royal families of Europe tended to inter-marry, this mutation was passed on to various other dynasties, most famously the Romanov line in Russia.
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Although women carrying the mutation that causes haemophilia only produce 50 per cent of the normal amounts of the clotting factor, this is enough to protect them from symptoms. This is partly because this clotting factor is released from cells and circulates in the bloodstream, where it reaches high enough levels for protection against bleeds, no matter where they happen.

There are, however, circumstances wherein the presence of two X chromosomes in a woman doesn’t guarantee protection from an X-linked disorder. Rett Syndrome is a devastating neurological disease which presents in some ways as a really extreme form of autism. Baby girls appear to be perfectly healthy when born and they reach all the normal developmental milestones for the first six to eighteen months of life. But after that, they begin to regress. They lose any spoken language skills they have developed. They also develop repetitive hand actions, and lose purposeful ones such as pointing. The girls suffer serious learning disability for the rest of their lives.
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Rett Sydrome is caused by mutations in a protein-coding gene
on the X chromosome.
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Affected females have one normal copy of this gene, and one version which is mutated and can’t produce functional protein. Assuming random X inactivation, we expect that on average half of the cells in the brain will express normal amounts of the protein, and there will be no expression from the other ones. It is obvious from the clinical presentation that there are severe problems if half the brain cells can’t express this protein.

Rett Syndome pretty much only affects girls. This is unusual for an X-linked disorder, where girls are usually carriers and boys are affected. This might make us wonder how boys are protected from the effects of a Rett mutation. But the reality is that they are not. The reason we almost never find boys who are affected by Rett Syndrome is because affected male embryos don’t develop properly and the foetuses don’t survive to term.

Never underestimate luck, good or bad

Scientists are trained to think about many things during our education and careers. But something we are rarely asked to ponder is the role played by luck. Even when we do, we usually dress it up with terms like ‘random fluctuations’ or ‘stochastic variation’. And that’s a shame, because sometimes ‘luck’ is probably a better description.

Duchenne muscular dystrophy is a severe muscle wasting disease, which we first met in Chapter 3. Boys with this disorder are fine initially but during childhood their muscles begin to degenerate, in a characteristic pattern. For example, in the legs the thigh muscles begin to waste first. The boys develop very large calves as their bodies try to compensate, but after a while these muscles also
wither. The children are usually wheelchair users by their teens and the average life expectancy is only 27 years of age. The early mortality is caused to a large extent by the eventual destruction of the muscles involved in breathing.
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Duchenne muscular dystrophy is caused by a mutation in a gene on the X chromosome that encodes a large protein called dystrophin.
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This protein seems to act as a sort of shock absorber in muscle cells. Because of the mutation, males can’t produce functional protein and this ultimately leads to destruction of the muscle. Carrier females will usually produce 50 per cent of the normal amounts of functional dystrophin protein. This is generally sufficient, because of an odd anatomical feature. As we develop, individual muscle cells fuse to create a large super-cell with lots of individual nuclei in it. This means each super-cell has access to multiple copies of the necessary genes, in all the different nuclei. So the muscles of carrier females overall contain enough dystrophin protein for normal activity, instead of one cell with enough, and one cell with none.

There was an unusual case of a woman with all the classic symptoms of Duchenne muscular dystrophy. This is very rare but there are ways we could predict this would happen. One possibility would be if her mother was a carrier and her dad was a Duchenne sufferer who survived long enough to father a child. If that was the case she would definitely have inherited a mutated gene from her father (because he would only possess one – affected – X chromosome). There would be a one in two chance that any egg produced by her carrier mother also contained a mutated dystrophin gene. If that scenario had occurred, neither of her X chromosomes would have a normal copy of the gene, and she wouldn’t be able to produce the necessary protein.

But the doctors treating this patient had taken a family history and they knew that her father didn’t have Duchenne muscular dystrophy, so another explanation was necessary. Sometimes
mutations arise quite spontaneously when eggs or sperm are produced. The gene that codes for dystrophin is very large, so just by chance it is at relatively high risk of mutation compared with most other genes in the genome. That’s because mutation is essentially a numbers game. The bigger the gene, the more likely it is that it may mutate. So, one mechanism by which a female could inherit Duchenne muscular dystrophy is if she inherits a mutated chromosome from her carrier mother, and a new mutation in the sperm that fertilised the egg.