The major repressor works as part of a large complex of proteins[21], and various long non-coding RNAs have been shown to be associated with this complex, suggesting there may be multiple ways of targeting the repressive modifications, depending on the cell type and its behaviour. In Chapter 8 we met a long non-coding RNA whose over-expression drives prostate cancer (see page 108). It has been shown to bind to the major repressor and direct it to certain genes, including ones that normally hold back cell proliferation.{175} This finding reinforces the concept that there is a delicate balance of long non-coding RNAs and epigenetic modifiers and that disturbing the equilibrium may be dangerous for a cell or an individual. So do similar data around binding of the long non-coding RNA that is involved in skeletal deformities and a range of cancers, which we encountered in the same chapter (see pages 106, 108). It binds to the complex containing the major repressor, and simultaneously to another epigenetic enzyme that can deposit an additional repressing modification.{176}
One of the features implicit in the above explanation is that the long non-coding RNA is transcribed at or near the gene whose histones will be targeted by the major repressor or by other epigenetic enzymes. Although it’s difficult to investigate this, the existing data suggest that this is indeed the case. The major repressor can bind to all sorts of long non-coding RNA molecules. The complex containing the major repressor can recognise different types of histone modifications, depending on the components of the complex. These components can vary from cell to cell. As they ‘scan’ the nearby histones, the complexes can recognise various modification patterns and reinforce these by adding the major repressive modifications. Alternatively, if the region is very rich in modifications that lead to gene expression, the complex may be inhibited and the major repressor will leave the histones alone. This is another of those scenarios where it is a disadvantage to think in purely linear terms, of what came first. Instead, patterns are often maintained or created as a consequence of the histone modification combinations that are already present on the genome.{177},{178}
This also seems to hold for the opposite effect, where active regions remain active. Long non-coding RNAs have been reported to be expressed from regions where protein-coding genes are switched on. These long non-coding RNAs stay moored to the genome region where they are produced, possibly by forming a third strand to accompany the double helix of DNA. These long non-coding RNAs bind to the enzymes that place methyl modifications on DNA and stop them doing their job. This keeps the genes in that region in an active state.{179}
Xist, which is critical for switching off expression from one of the X chromosomes in a female cell, was one of the first functional long non-coding RNAs to be identified. Perhaps it’s no surprise that it’s the one whose cross-talk with the epigenetic system has been shown most clearly. As Xist spreads along the X chromosome it attracts other proteins. Many of these are epigenetic enzymes that add chemical modifications to either the DNA or the histone protein. They include the major repressor of histones, and also the enzymes that add methyl groups to DNA.{180} The epigenetic modifications they produce strengthen the shutdown of genes and ultimately lead to hyper-compaction of the inactive X chromosome, and the formation of the Barr Body that we encountered in Chapter 7 (see page 84).
It may seem puzzling that the epigenetic modifications always get re-established on the correct X chromosome after cell division. It may be easier to imagine this using a physical example. You have two wooden baseball bats, and you coat one of them with magnetic paint, which represents Xist. After the paint has dried you drop both bats into a tub containing little iron discs. One side of each disc is coated with hooked Velcro. The discs represent the epigenetic proteins that bind to the Xist-coated chromosome. These discs will stick to the bat that has a magnetic covering, but not to the other one. After that, you drop each bat into a tub containing pretty fabric flowers, each backed with a piece of looped Velcro. These represent the modifications. Clearly, the flowers will only stick to the bat that was originally coated with magnetic paint, even though they aren’t magnetic themselves.
You could even take this slightly bizarre thought experiment further. Even if you take the flowers off the bat, if you drop it into another tub containing Velcroed blooms, it will be covered again. You could even take off the little iron discs, and as long as you put the bat back into the first and second tubs, it will get covered in flowers again.
In fact, the only way in which you can prevent the bat being covered in flowers when you drop it into the two tubs is to remove the magnetic paint. This is essentially what happens when women make eggs. The inactivating marks are all removed from the X chromosomes and all the daughter cells, i.e. all the eggs are ‘fresh’ in the sense that they won’t pass on inactivation to their offspring. The magnetic paint has to be applied anew to one of the two X chromosomes during early development.
Long non-coding RNAs clearly interact with and help regulate the function of epigenetic proteins. But it would be a mistake to think this is the only way in which junk talks to the epigenetic system. Far from it. We saw in Chapter 4 that the human genome has been invaded by vast numbers of repetitive DNA elements and how important it is that these are kept switched off. Some researchers have gone so far as to speculate that epigenetic control of gene expression may originally have evolved to keep certain junk regions under control.{181} It was only later that the epigenetic system struck out into new territory of regulating normal endogenous genes.
A really striking example of the interplay between junk DNA, epigenetics and the final appearance and behaviour of a mammal can be found in a mouse strain called the Agouti viable yellow mouse. All the mice in this strain are genetically identical, but they can look very different. Some are fat and yellow, some are thin and brown, and others are somewhere in between. The differences in their appearance are due to variable epigenetic regulation of a junk DNA region. In these mice, a repetitive DNA element has become inserted into the genome in front of a particular gene. The DNA element can undergo varying and random degrees of methylation. The heavier the methylation, the more the activity of the repetitive DNA element is repressed, and this affects the expression of the nearby gene.{182} It’s the expression levels of the nearby gene that ultimately determine how fat and how yellow the mouse will be. This is summarised in Figure 9.1.
Figure 9.1 In the top panel an insertion drives expression of the Agouti gene, leading to a fat yellow mouse. In the bottom panel the insertion has been modified through DNA methylation. The insertion can no longer drive expression of the Agouti gene, and the mouse is brown and skinny.
21
This complex is known as Polycomb Response Complex 2 or PRC2. The activity of PRC2 is closely coordinated with that of another repressive complex called PRC1. PRC2 usually establishes the first repressive modifications at a genomic region and PRC1 follows on with additional modifications that stabilise the repressive state.