The reason this experiment is so important is that it offers hope that we may be able to find new treatments for really complex neurological conditions. Prior to the publication of this Science paper, there had been an assumption that once a complex neurological condition has developed, it is impossible to reverse it. This was especially presumed to be the case for any condition that arises developmentally, i.e. in the womb or in early infancy. This is a critical period when the mammalian brain is making so many of the connections and structures that are used throughout the rest of life. The results from the Mecp2 mutant mice suggest that in Rett syndrome, maybe all the bits of cellular machinery that are required for normal neurological function are still there in the brain – they just need to be activated properly. If this holds true for humans (and at a brain level we aren’t really that different from mice) this offers hope that maybe we can start to develop therapies to reverse conditions as complex as mental retardation. We can’t do this the way it was done in the mouse, as that was a genetic approach that can only be used in experimental animals and not in humans, but it suggests that it is worth trying to develop suitable drugs that have a similar effect.
DNA methylation is clearly really important. Defects in reading DNA methylation can lead to a complex and devastating neurological disorder that leaves children with Rett syndrome severely disabled throughout their lives. DNA methylation is also essential for maintaining the correct patterns of gene expression in different cell types, either for several decades in the case of our long-lived neurons, or in all daughters of a stem cell in a constantly-replaced tissue such as skin.
But we still have a conceptual problem. Neurons are very different from skin cells. If both cells types use DNA methylation to switch off certain genes, and to keep them switched off, they must be using the methylation at different sets of genes. Otherwise they would all be expressing the same genes, to the same extent, and they would inevitably then be the same types of cells instead of being neurons and skin cells.
The solution to how two cell types can use the same mechanism to create such different outcomes lies in how DNA methylation gets targeted to different regions of the genome in different cell types. This takes us into the second great area of molecular epigenetics. Proteins.
DNA is often described as if it’s a naked molecule, i.e. DNA and nothing else. If we visualise it at all in our minds, a DNA double helix probably looks like a very long twisty railway track. This is pretty much how we described it in the previous chapter. But in reality it’s actually nothing like that, and many of the great breakthroughs in epigenetics came about when scientists began to appreciate this fully.
DNA is intimately associated with proteins, and in particular with proteins called histones. At the moment most attention in epigenetics and gene regulation is focused on four particular histone proteins called H2A, H2B, H3 and H4. These histones have a structure known as ‘globular’, as they are folded into compact ball-like shapes. However, each also has a loose floppy chain of amino acids that sticks out of the ball, which is called the histone tail. Two copies of each of these four histone proteins come together to form a tight structure called the histone octamer (so called because it’s formed of eight individual histones).
It might be easiest to think of this octamer as eight ping-pong balls stacked on top of each other in two layers. DNA coils tightly around this protein stack like a long liquorice whip around marshmallows, to form a structure called the nucleosome. One hundred and forty seven base-pairs of DNA coil around each nucleosome. Figure 4.3 is a very simplified representation of the structure of a nucleosome, where the white strand is DNA and the grey wiggles are the histone tails.
Figure 4.3 The histone octamer (2 molecules each of histones H2A, H2B, H3 and H4) stacked tightly together, and with DNA wrapped around it, forms the basic unit of chromatin called the nucleosome.
If we had read anything about histones even just fifteen years ago, they would probably have been described as ‘packaging proteins’, and left at that. It’s certainly true that DNA has to be packaged. The nucleus of a cell is usually only about 10 microns in diameter – that’s 1/100th of a millimetre – and if the DNA in a cell was just left all floppy and loose it could stretch for 2 metres. The DNA is curled tightly around the histone octamers and these are all stacked closely on top of each other.
Certain regions of our chromosomes have an extreme form of that sort of structure almost all the time. These tend to be regions that don’t really code for any genes. Instead, they are structural regions such as the very ends of chromosomes, or areas that are important for separating chromosomes after DNA has been duplicated for cell division.
The regions of DNA that are really heavily methylated also have this hyper-condensed structure and the methylation is very important in establishing this configuration. It’s one of the mechanisms used to keep certain genes switched off for decades in long-lived cell types such as neurons.
But what about those regions that aren’t screwed down tight, where there are genes that are switched on or have the potential to be switched on? This is where the histones really come into play. There is so much more to histones than just acting as a molecular reel for wrapping DNA around. If DNA methylation represents the semi-permanent additional notes on our script of Romeo and Juliet, histone modifications are the more tentative additions. They may be like pencil marks, that survive a few rounds of photocopying but eventually fade out. They may be even more transient, like Post-It notes, used very temporarily.
A substantial number of the breakthroughs in this field have come from the lab of Professor David Allis at Rockefeller University in New York. He’s a trim, neat, clean-shaven American who looks much younger than his 60 years and is exceptionally popular amongst his peers. Like many epigeneticists, he began his career in the field of developmental biology. Just like Adrian Bird, and John Gurdon before him, David Allis wears his stellar reputation in epigenetics very lightly. In a remarkable flurry of papers in 1996, he and his colleagues showed that histone proteins were chemically modified in cells, and that this modification increased expression of genes near a specific modified nucleosome[26].
The histone modification that David Allis identified was called acetylation. This is the addition of a chemical group called an acetyl, in this case to a specific amino acid named lysine on the floppy tail of one of the histones. Figure 4.4 shows the structures of lysine and acetyl-lysine, and we can again see that the modification is relatively small. Like DNA methylation, lysine acetylation is an epigenetic mechanism for altering gene expression which doesn’t change the underlying gene sequence.
Figure 4.4 The chemical structures of the amino acid lysine and its epigenetically modified form, acetyl-lysine. C: carbon; H: hydrogen; N: nitrogen; O: oxygen. For simplicity, some carbon atoms have not been explicitly shown, but are present where there is a junction of two lines.
So back in 1996 there was a nice simple story. DNA methylation turned genes off and histone acetylation turned genes on. But gene expression is much more subtle than genes being either on or off. Gene expression is rarely an on-off toggle switch; it’s much more like the volume dial on a traditional radio. So perhaps it was unsurprising that there turned out to be more than one histone modification. In fact, more than 50 different epigenetic modifications to histone proteins have been identified since David Allis’s initial work, both by him and by a large number of other laboratories[27]. These modifications all alter gene expression but not always in the same way. Some histone modifications push gene expression up, others drive it down. The pattern of modifications is referred to as a histone code[28]. The problem that epigeneticists face is that this is a code that is extraordinarily difficult to read.
26
The most important papers from the Allis lab in 1996 were: Brownell et al. (1996),
27
A useful review by one of the leading researchers in the field is Kouzarides, T. (2007)