Junk DNA: A Journey Through the Dark Matter of the Genome

6. Two is the Perfect Number

One cell becomes two; two become four; four become eight and, to quote from The King and I, ‘et cetera, et cetera, and so forth’^{84} until there are over 50 trillion cells in a human body. Every time a human cell divides, it has to pass on exactly the same genetic material to both daughter cells as it contains itself. In order to do this, the cell makes a perfect copy of its DNA. This results in a replicate of each chromosome. The two replicates stay attached to each other initially, but then are pulled apart to opposite ends of the cell. A basic schematic for this is shown in Figure 6.1.

Figure 6.1 A normal cell contains two copies of each chromosome, one inherited from each parent. Before a cell divides, each chromosome is copied to create a perfect duplicate. The copies are pulled apart when the cell divides. This creates two daughter cells, containing exactly the same chromosomes as the original cell. For simplicity, this figure shows just one pair of chromosomes, rather than the 23 pairs in a human cell. The different colours indicate different origins of the pair, one from each parent. The diagram only shows division of the nucleus, but this is also accompanied by division of the rest of the cell.

The only exception to this is when the germ cells in the ovaries or testes create eggs or sperm. Eggs or sperm only contain half the number of chromosomes that are found in all the other cells of the body. The result of this is that when an egg and a sperm fuse, the full chromosome number is restored in the single cell (the zygote) which will then divide to become two cells et cetera, et cetera and so forth.

This halving of the chromosome number is possible because all our chromosomes come in pairs. We inherit one of each pair from our mother and one from our father. Figure 6.2 shows how the chromosome number is halved when eggs or sperm are created.

If cell division goes wrong, either when new body cells are created or when the germ cells create eggs or sperm, the effects can be really serious, as we will see later in this chapter. Cell division is an exceptionally complex process, involving hundreds of different proteins working in a highly coordinated fashion. Given how complicated it is, and how vital it is that cell division happens smoothly and successfully, it might seem surprising that quite a lot of it is critically dependent on a long stretch of junk DNA.

This particular stretch of junk DNA is called the centromere, and unlike the telomeres from the last chapter, the centromere is found on the interior of a chromosome. Depending on the chromosome, it may be pretty much in the middle, or it may be near to an end. Its position is consistent in the sense that on human chromosome 1, for example, it’s always near the middle whereas in human chromosome 14 it’s always near the end.

Centromeres are essentially attachment points for a set of proteins that drag the separated chromosomes to opposite ends of the cell. Imagine Spider-Man is standing in a set position and needs to get something. He throws a web at the thing he wants, and then drags it to him. Now imagine that a very tiny Spider-Man is standing at one end of a cell. He throws a web at the chromosome he wants, the web attaches, and he pulls the chromosome to his end of the cell. A tiny Spider-Man clone does the same thing at the opposite end of the cell for the other chromosome in the matching pair.

Figure 6.2 This shows the cell division process that generates gametes (eggs or sperm) each containing just one of every pair of chromosomes. The process initially looks like the standard cell division shown in Figure 6.1. However, this is followed by a second separation of chromosome pairs, to create gametes with only half the normal number of chromosomes. There is also an early event where genetic material is swapped over within chromosome pairs, to create greater genetic diversity in offspring, but this isn’t shown in this figure.

There is a complication for Spider-Man. Most of the surface of the chromosome is coated with web-repellent. There is only one part where his web will stick. This part is the centromere. In the cell the centromere attaches to a long string of proteins which pulls the chromosome away from the centre and to the periphery. This string of proteins is called the spindle apparatus.

Centromeres play a very important and consistent role in all species. They form the essential attachment point for the spindle apparatus. It’s essential that this system works properly, or cell division goes wrong. Given that this is such a vital process, we would expect that the centromere DNA sequence would be highly conserved throughout the evolutionary tree. But weirdly, this isn’t the case at all. Once we move beyond yeast[9] and microscopic worms,[10] the DNA sequence is highly variable when we look at different species.^{85} In fact, the DNA sequence of a centromere may differ between two chromosomes in the same cell. This level of sequence diversity, in the face of functional consistency, is really quite counterintuitive. Happily, we are starting to understand how this vital region of junk DNA manages to pull off this strange evolutionary trick.

In human chromosomes, the centromeres are formed from repeats of a DNA sequence that is 171 base pairs in length.[11] These 171 base pairs are repeated over and over, and may reach lengths of up to 5 million bases in total.^{86} The critical feature of the centromere is that it acts as a location for the binding of the protein called CENP-A (Centromeric Protein-A).^{87} The CENP-A gene is highly conserved between species, in contrast to the centromere DNA.

Our Spider-Man analogy might be useful again here in terms of understanding the apparent evolutionary conundrum we laid out earlier. Spidey’s web can bind to CENP-A protein. It doesn’t matter if the CENP-A protein is bound to meat, bricks, potatoes or lightbulbs. So long as the CENP-A protein is bound to something, Spider-Man’s web will stick to it, and pull the CENP-A and the something towards our superhero.

So, the DNA sequence at the centromere can vary enormously between species, ranging from meat to lightbulbs. What matters is that the CENP-A protein remains the same, so that the highly conserved spindle apparatus can stick to it and pull the chromosomes apart to opposite poles of the dividing cell.

CENP-A isn’t the only protein that is found at the centromere; many others are also present. It’s possible to knock out the expression of CENP-A in cells in the laboratory. When this happens, the other proteins that should bind to the centromere stop doing so.^{88},{89} However, when the experiment is performed the other way around — knocking out expression of one of the other proteins — CENP-A continues to bind at the centromere.^{90} This demonstrates that CENP-A acts as a foundation stone.

When researchers over-expressed CENP-A in cells from fruit flies, they found that the chromosomes began to create centromeres in unusual positions.^{91} But the situation in human cells seems to be more complicated, because over-expression of CENP-A doesn’t result in new, abnormally located centromeres.^{92} It seems that in humans, CENP-A is necessary for centromere formation, but it’s not sufficient.

The CENP-A acts as the essential cornerstone for the recruitment of all the other proteins that are also required for the spindle apparatus to do its job. When a cell is actively dividing, over 40 different proteins build up from the CENP-A. They do so in a step-wise fashion, like adding on LEGO bricks in a particular order. Immediately after the duplicated chromosomes have been pulled to the opposite ends of the cell, this big complex falls apart again. This whole process can take less than an hour. We don’t know what controls all of this, but some of it is down to a simple physical feature. Normally, the nucleus has a membrane around it, and large protein molecules find it really difficult to get through this. When the cell is ready to separate its replicated chromosomes, this barrier breaks down temporarily and the proteins can join on to the complex at the centromere.^{93} It’s like having a removal company outside your house. They are ready to shift your furniture but can’t get on with the job unless you open the door and let them in.

We are still left with a difficult conceptual problem. If the DNA sequence at the centromere isn’t very conserved, and the critical factor is the placement of the CENP-A protein, how does the cell ‘know’ where the centromere should be on each chromosome? Why is it always near the middle of chromosome 1, but near the end of chromosome 14?

To understand this, we have to develop a more sophisticated image of the DNA in our cells. The DNA double helix is an iconic image, probably the defining image in biology. But it doesn’t really represent what DNA is like. DNA is a very long spindly molecule. If you stretched out the DNA from one human cell it would reach for two metres, assuming you joined up the material from all the chromosomes. But this DNA has to fit into the nucleus of a cell, and the nucleus has a diameter of just one hundredth of a millimetre.

This is like trying to fit something that is the vertical height of Mount Everest into a capsule the size of a golf ball. If you are trying to fit a climbing rope the height of Mount Everest into a golf ball, that clearly won’t work. On the other hand, if you replace the climbing rope with a filament thinner than a human hair, you’ll probably be OK.

Although human DNA is long, it’s very thin, so it is possible to fit it into the nucleus. But there is, as always, a complication. It’s not enough just to jam the DNA into a small space. The easiest way to visualise why not is to think of strings of Christmas tree lights. If at the end of the festive season you take the lights off the tree and shove them into a box, they will take up a lot of space. You will also almost certainly find that when you come to use them again the following year they are all tangled up. It will take you ages to unravel them and there is a fair chance you will break some of them. In their tangled state, you would also really struggle to get to just one particular bulb.

But, if you are a freakishly organised person, you will wrap each string of lights around a piece of cardboard before storing them away. And your organisational acumen will be rewarded next Christmas when you take the lights out of the surprisingly small box you were able to use for storage. Not only did you save on loft space, you also will find that it’s very easy to unwind the lights, none of the strands get tangled around each other or snapped, and you can access your one favourite bulb very easily.

The same process happens in our cells. DNA is not stored as a random bundle of scrunched-up genetic material. Instead it is wrapped around certain proteins. This stops the DNA getting tangled and broken, allows it to be squeezed in an orderly fashion into a small space, and also keeps it structured so that the cell can access different regions as necessary, in order to switch individual genes on or off.

The DNA in our cells is wrapped around particular proteins, called histones. The basic structure is shown in Figure 6.3. Eight histone proteins — two each of four different types — form an octamer. DNA wraps around this octamer, like a skipping rope around eight tennis balls. There are huge numbers of these octamers all along our genome.

CENP-A is a close cousin of one of these histone proteins, sharing much of the same amino acid sequence, but with some important differences. At the centromere, both copies of one of the standard histone proteins are missing,[12] and CENP-A is present in the octamer instead, as shown in Figure 6.4.^{94} There are thousands of these octamers containing CENP-A at the centromere of each chromosome.

Figure 6.3 DNA, represented by the solid black band, is wrapped around packages of eight histone proteins (two each of four different types).

Figure 6.4 The octamer of histone proteins on the left represents the standard arrangement found throughout most of the genome. The octamer on the right represents the specialised octamers found at the centromeres. One of the standard pairs of histone proteins has been replaced by a pair of specialised centromere histone proteins, called CENP-A. These are represented by the striped globes.

The CENP-A in these thousands of octamers at the centromeres gives the spindle apparatus something to hang on to, when it’s trying to pull the chromosomes apart. One of the effects of inserting CENP-A into the octamers is that it makes the centromere regions more rigid.^{95} If we think about trying to pull a blob of jelly, compared with a boiled sweet, it’s obvious that the increased rigidity will be an advantage for the actions of the spindle apparatus.

But we still keep coming back to the same problem. Why is CENP-A inserted into the octamers at the centromere, but not at other regions? This isn’t driven by the DNA sequence. Other regions of our genome also contain junk DNA with similar sequences to those found at the centromeres, but CENP-A doesn’t accumulate at these.^{96} CENP-A is only found at centromeres, but in some ways it’s the presence of CENP-A that actually defines what a centromere is. How have human cells evolved in such a way that an inherently unstable situation has led to complete genetic stability in terms of cell division?

The answer lies in a self-seeding paradigm, whereby once CENP-A is deposited it continues to direct the maintenance of its own position, and to ensure that this is passed on to all daughter cells.^{97} This is independent of DNA sequence. Instead it seems to depend on small chemical modifications to the histone octamers.

Histone proteins in the octamers can be modified in a huge number of different ways. Proteins are made up of combinations of 20 different amino acids, many of which can be modified. And there are lots of different modifications that can be made to a protein. This is just as true of histones as of any other proteins.

In human centromeres, the octamers that contain CENP-A don’t have a complete monopoly. Instead, blocks of these octamers alternate with ones containing the standard histone protein, as shown in Figure 6.5. The standard octamers carry a very characteristic combination of chemical modifications. These in turn attract other proteins that bind to these modifications, part of whose function is to make sure these modifications are maintained.^{98} This all acts to keep the octamers that contain CENP-A localised to the same region of the genome, and means that they only form at one position on the chromosome. This is probably why the junk DNA sequence at centromeres is so variable between species, even though it provides the geographical scaffold for one of the most fundamental processes in any cell.

Figure 6.5 The alternating pattern of standard and CENP-A histone octamers at the centromeres. For clarity, only small numbers of octamers are shown, whereas there are thousands present in the cell. Each circle represents an entire octamer.

The chemical modifications at the centromere also have the effect of keeping that region of the genome silent. Although there are recent data suggesting that there may be low-level expression of RNA from some centromeric regions, it’s very unclear if this has any functional significance. Essentially, the DNA at the centromeres has no real function except to be junk. It just acts as the regions where CENP-A and all its associated proteins can bind. That’s the only thing the cell needs from it. It’s better that it doesn’t have any other purpose, because that might be disrupted when the octamers containing CENP-A bind. That’s why this region of DNA has been able to change so much during evolution, because the sequence really doesn’t matter.

It might seem that there is still a missing stage in this. How does the CENP-A ‘know’ to bind to the right region of junk DNA in the first place? Because that tends to be how we all think, wanting to know what starts something off. But if we examine that assumption, we realise it leads us into a dead-end. Once again in this chapter we can invoke the lyricist Oscar Hammerstein, although this time in Austria rather than Siam/Thailand.

In The Sound of Music, Captain von Trapp and Maria sing that ‘Nothing comes from nothing. Nothing ever could’.^{99}

How right they were.

Naked human DNA is a completely non-functional molecule. It does nothing at all, and certainly can’t direct the production of a new human being. It needs all the accessory information, such as the histones and their modifications, and it needs to be in a functioning cell. When the replicated chromosomes are separated and pulled to opposite ends of the cell, they each carry off some histone octamers in the correct positions, and with appropriate modifications. There are enough of these that they can act as the seed region to recreate the full picture of histones and modifications in the daughter cells. This is true not just of standard histone octamers, but also of the ones that contain CENP-A and thus show where the centromeres are formed. For these non-standard octamers, the regions of the CENP-A protein that contain different amino acids from the standard histones are important for attracting the appropriate proteins.^{100}

This information — the chemical modifications — is even retained when eggs and sperm are produced.^{101} The octamers that contain CENP-A stay in place when the egg and sperm fuse to form the one cell that will ultimately give rise to all the trillions of others in the human body. Our centromeres have been passed down through all of human evolution, and long before that in our distant ancestors, based on the position of the proteins, and not the DNA sequence to which they bind.

There are drugs that interfere with the way in which the spindle apparatus pulls the replicated chromosomes to opposite ends of the cells. The spindle apparatus is formed by the coming together of a large number of proteins, and these only combine at the time when a cell is ready to pull the chromosomes apart. A drug called paclitaxel works by making the spindle apparatus too stable, so that the complex of proteins can’t disaggregate.^{102}

We can visualise why this is a bad thing for a cell by comparing the scenario with one of those fire engines that carries an extending ladder. It’s great that the ladder can be extended to rescue people from upper storeys of a burning building. But if the fire crew can’t get the ladder folded back down again after the emergency and have to drive around with it fully extended, it won’t be long before they have a pretty serious accident. The same happens in the cells treated with paclitaxel. Systems in the cell recognise that the spindle apparatus hasn’t been deactivated properly, and this triggers destruction of the cell. In the UK, paclitaxel is licensed for use in a number of cancers including non-small cell lung cancer, breast cancer and ovarian cancer.^{103}

Paclitaxel is probably effective because cancer cells divide rapidly. By using a drug that targets cell division, it’s possible to kill the cancer cells at a higher rate than the normal body cells, which are not proliferating so quickly. But we also know that abnormal separation of chromosomes is itself a hallmark of many cancers.

If the separation of chromosomes goes wrong, one daughter cell may inherit both the ‘original’ chromosome and its replicate. The other daughter cell won’t inherit either. The first daughter cell will have one chromosome too many, the other daughter cell will have one too few. This situation, where the number of chromosomes is wrong, is known as aneuploidy. The word is derived from Greek. In this case, an means ‘not’, eu means ‘good’ and ploos means ‘-fold’ (as in ‘twofold’, ‘threefold’, etc.). In other words, it represents an unbalanced genomic state.

Astonishingly, about 90 per cent of solid tumours contain cells that are aneuploid, i.e. contain the wrong number of chromosomes.^{104} The pattern of aneuploidy can be really complicated, as there is probably a strong degree of randomness to how the chromosomes are mis-segregated if the process is going wrong. In a single cancer cell there may be four copies of one chromosome, two copies of another and one copy of a third, or some other combination. Because of this variability, it’s very difficult to determine if the aneuploidy itself drives the cancer process, or if it’s just an innocent marker of the cancer status of the cells. The likelihood, because of the essentially random patterns of abnormal chromosome numbers, is that there’s probably a spectrum. Some cancer cells may develop combinations of chromosomes that drive cell proliferation faster. Other cells may have combinations with the opposite effect, and which may even trigger the cancer cell’s suicide system. And in some cells the combination may be ultimately neutral.^{105}

Remarkably, aneuploidy also seems to occur in certain normal cells. It’s been reported that perhaps as many as 10 per cent of cells in the brains of mice and humans are aneuploid.^{106} During development, the proportion is even higher, at around 30 per cent, but many of these are eliminated.^{107} As far as we can tell, the remaining aneuploid cells in the brain are functionally active.^{108} There is no clear understanding of why we have these brain cells with abnormal numbers of chromosomes, or the significance of similar findings of aneuploidy reported in the liver.^{109}

In the situations outlined above, the aneuploidy has developed after the main bulk of the cells of the body have been produced. It occurred during cell divisions that were creating new body cells, albeit in some cases cancerous ones. The effects of these failures in chromosome segregation seem relatively mild, if any. That’s probably because there are plenty of normal cells to compensate.

But the situation is very different if the aneuploidy occurs during the formation of the eggs or sperm (gametes). If a pair of chromosomes fails to separate properly, then one of the resulting gametes will have an extra copy of the chromosome, and the other will be lacking that chromosome. Let’s say that happens in the formation of the egg, and chromosome 21 is abnormally segregated when the eggs are created. One of the eggs will have two copies of chromosome 21, the other will have none.

If the one that lacks a chromosome 21 is fertilised, the resulting embryo only has one copy of chromosome 21 and very quickly dies. But if the egg that contains two copies of chromosome 21 is fertilised, it will have three copies of this chromosome. And although such embryos are at higher than normal risk of spontaneous abortion, many do develop fully and the child is born.

Most of us have met or at least seen people with three copies of chromosome 21 (having three copies is known as a trisomy, so this condition is known as trisomy 21): this failure of chromosome segregation is the cause of Down’s Syndrome.^{110} It can also occur because of a sperm with two copies of the chromosome, or through failure of chromosome separation in the first few divisions after fertilisation, but the maternal route is the most common.

Down’s Syndrome affects about one in 700 live births, and is a complex and variable disorder commonly associated with heart defects, a characteristic physical and facial appearance and a greater or lesser degree of learning disability. People with Down’s Syndrome are much more likely to reach adulthood than in the past, thanks to better medical and surgical interventions, but are at high risk of a relatively early onset of Alzheimer’s disease.^{111}

The complex nature of the characteristics of Down’s Syndrome demonstrates very clearly that it’s really important that our cells contain the correct number of chromosomes. Patients with Down’s Syndrome have three copies of chromosome 21 instead of two. But this 50 per cent increase in the chromosome number, and therefore of the genes on the chromosomes, has dramatic effects on the cell and on the individual. Our cells are simply unable to deal with this excess, showing that control of gene expression must normally be tightly regulated and is so finely balanced that we are only able to compensate for changes within relatively narrow parameters.

Two other trisomies have been found in humans, both associated with much more severe conditions than Down’s Syndrome. Edward’s Syndrome is caused by trisomy of chromosome 18, and affects one in 3,000 live births. Approximately three-quarters of foetuses with trisomy 18 die in utero. Of the babies who survive to term, about 90 per cent die in the first year of birth due to cardiovascular defects. The babies grow very slowly in the womb, their birth weight is low and they have a small head, jaw and mouth plus a range of other multisystem problems including severe learning disabilities.^{112}

The rarest of all these conditions is Patau’s Syndrome, trisomy 13, which affects one in 7,000 live births. The babies who survive to full term have severe developmental abnormalities and rarely survive their first year. A wide range of organ systems is involved, including the heart and kidneys. Severe malformations of the skull are common and the learning disability is extremely severe.^{113}

It’s notable that having an extra chromosome from conception onwards results in obvious developmental problems. In each of these trisomies, it is very clear that the baby has a major problem from the moment they are born. Indeed, with access to prenatal scanning, most of the affected foetuses are detected during pregnancy. This tells us that having the right dose of chromosomes is vitally important for the highly coordinated process of development.

It’s tempting to wonder if there is something unusual about chromosomes 13, 18 and 21. Is there, perhaps, something different about their centromeres that makes them more susceptible to unequal segregation of the chromosomes during the formation of the egg and the sperm? Or could it be that trisomies of the other chromosomes do occur, but there’s no clinical effect so we don’t think to look for them?

This is falling into the surprisingly common trap of focusing on what we see, rather than what we don’t see. The reason that we see babies born with trisomies of chromosomes 13, 18 and 21 is because these are relatively benign, unlikely though that sounds. These are three of the smallest chromosomes and they each contain relatively few genes. Generally, the larger the chromosome, the greater the number of genes it contains. So the reason we never see trisomy of chromosome 1, for example, is because of its size. Chromosome 1 is very large and contains a lot of genes. If an egg and sperm fuse and create a zygote with three copies of this chromosome, there will be overexpression of such a large number of genes that the cell function will be disrupted catastrophically, leading to extremely early destruction of the embryo. This probably occurs before the woman is even aware she is pregnant.

For women aged between 25 and 40, the success rates for in vitro fertilisation using donated eggs are not affected by age.^{114} But the likelihood of a woman becoming pregnant naturally does decline after her mid-20s. The difference between these two situations suggests that the mother’s age critically affects her eggs, rather than her uterus. We already know from Down’s Syndrome that maternal age influences the success of chromosome segregation into the eggs. So it’s not too big a leap to hypothesise that the decline in pregnancy rates after the mid-20s may be in part due to very early failures of embryo development, as a result of malfunctioning centromere activity and the creation of eggs with disastrous misallocation of large chromosomes.