Of course, the beauty of working with mice, and especially with highly inbred strains, is that it’s relatively easy to perform detailed genetic and epigenetic studies, especially when we already have a reasonable idea of where to look. In this case, the region to examine was the agouti gene.
Mouse geneticists knew how the yellow phenotype was caused in Avy yellow mice. A piece of DNA had been inserted in the mouse chromosome just before the agouti gene. This piece of DNA is called a retrotransposon, and it’s one of those DNA sequences that doesn’t code for a protein. Instead, it codes for an abnormal piece of RNA. Expression of this RNA messes up the usual control of the downstream agouti gene and keeps the gene switched on continuously. This is why the hairs on the Avy mice are yellow rather than banded.
That still doesn’t answer the question of why genetically identical Avy/a mice had variable coat colour. The answer to this has been shown to be due to epigenetics. In some Avy/a mice the CpG sequences in the retrotransposon DNA have become very heavily methylated. As we saw in the previous chapter, DNA methylation of this kind switches off gene expression. The retrotransposon no longer expressed the abnormal RNA that messed up transcription from the agouti gene. These mice were the ones with fairly normal banded mouse coat colour. On other genetically identical Avy mice, the retrotransposon was unmethylated. It produced its troublesome RNA which messed up the transcription from the agouti gene so that it was switched on continuously and the mice were yellow. Mice with in-between levels of retrotransposon methylation had in-between levels of yellow fur. This model is shown in Figure 5.4.
Figure 5.4 Variations in DNA methylation (represented by black circles) influence expression of a retrotransposon. The variation in expression of the retrotransposon in turn affects expression of the agouti gene, leading to coat colour variability between genetically identical animals.
Here, DNA methylation is effectively working like a dimmer switch. When the retrotransposon is unmethylated, it shines to its fullest extent, producing lots of the abnormal RNA. The more the retrotranposon is methylated, the more its expression gets turned down.
The agouti mouse has provided a quite clear-cut example of how epigenetic modification, in this case DNA methylation, can make genetically identical individuals look phenotypically different. However, there is always the fear that agouti is a special case, and maybe this is a very uncommon mechanism. This is particularly of concern because it’s proved very difficult to find an agouti gene in humans – it seems to be in that 1 per cent of genes we don’t share with our mouse neighbours.
There is another interesting condition found in mice, in which the tail is kinked. This is called Axin-fused and it also demonstrates extreme variability between genetically identical individuals. This has been shown to be another example where the variability is caused by differing levels of DNA methylation in a retrotransposon in different animals, just like the agouti mouse.
This is encouraging as it suggests this mechanism isn’t a one off, but kinked tails still don’t really represent a phenotype that is of much concern to the average human. But there’s something we can all get on board with: body weight. Genetically identical mice don’t all have the same body weight.
No matter how tightly scientists control the environment for the mice, and especially their access to food, identical mice from inbred mouse strains don’t all have exactly the same body weight. Experiments carried out over many years have shown that only about 20–30 per cent of the variation in body weights can be attributed to the post-natal environment. This leaves the question of what causes the other 70–80 per cent of variation in body weight[35]. Since it isn’t being caused by genetics (all the mice are identical) or by the environment, there has to be another source for the variation.
In 2010, Professor Emma Whitelaw, the terrifically enthusiastic and intensely rigorous mouse geneticist working at the Queensland Institute of Medical Research, published a fascinating paper. She used an inbred strain of mice and then used genetic engineering to create subsets of animals which were genetically identical to the starting stock, except that they only expressed half of the normal levels of a particular epigenetic protein. She performed the genetic engineering independently in a number of mice, so that she could create separate groups of animals, each of which was mutated in a different gene coding for epigenetic proteins.
When Professor Whitelaw analysed the body weights of large numbers of the normal or mutated mice, an interesting effect appeared. In a group of normal inbred mice, most of the animals had relatively similar body weights, within the ranges found in many other studies. In the mice with low levels of a certain epigenetic protein, there was a lot more variability in the body weights within the group. Further experiments published in the same paper assessed the effects of the decreased expression of these epigenetic proteins. Their decreased expression was linked to changes in expression levels of selected genes involved in metabolism[36], and increased variability in that expression. In other words, the epigenetic proteins were exerting some control over the expression of other genes, just as we might expect.
Emma Whitelaw tested a number of epigenetic proteins in her system, and found that only a few of them caused the increased variation in body weight. One of the proteins that had this effect was Dnmt3a. This is one of the enzymes that transfers methyl groups to DNA, to switch genes off. The other epigenetic protein that caused increased variability in body weight was called Trim28. Trim28 forms a complex with a number of other epigenetic proteins which together add specific modifications to histones. These modifications down-regulate expression of genes near the modified histones and are known as repressive histone modifications or marks. Regions of the genome that have lots of repressive marks on their histones tend to become methylated on their DNA, so the Trim28 may be important for creating the right environment for DNA methylation.
These experiments suggested that certain epigenetic proteins act as a kind of dampening field. ‘Naked’ DNA is rather prone to being switched on somewhat randomly, and the overall effect is like having a lot of background chatter in our cells. This is called transcriptional noise. The epigenetic proteins act to turn down the volume of this random chat. They do this by covering the histones with modifications that reduce the genes’ expression. It’s likely that different epigenetic proteins are important for suppressing different genes in some tissues rather than in others.
It’s clear that this suppression isn’t total. If it were, then all inbred mice would be identical in every aspect of their phenotype and we know this isn’t the case. There is variation in body weight even in the inbred strains, it’s just that there’s even more variation in the mice with the depressed levels of the epigenetic proteins.
This sophisticated balancing act, in which epigenetic proteins dampen down transcriptional noise but don’t entirely repress gene expression, is a cellular compromise. It leaves cells with enough flexibility of gene expression to be able to respond to new signals – be these hormones or nutrients, pollutants or sunlight – but without the genes being constantly ready to fire up just for the heck of it. Epigenetics allows cells to perform the difficult compromise between becoming (and remaining) different cell types with a variety of functions, and not being so locked into a single pattern of gene expression that they become incapable of responding to changes in their environment.