These modifications to DNA don’t change the essential nature of the A, C, G and T alphabet of our genetic script, our blueprint. When a gene is switched on and copied to make mRNA, that mRNA has exactly the same sequence, controlled by the base-pairing rules, irrespective of whether or not the gene is carrying an epigenetic addition. Similarly, when the DNA is copied to form new chromosomes for cell division, the same A, C, G and T sequences are copied.
Since epigenetic modifications don’t change what a gene codes for, what do they do? Basically, they can dramatically change how well a gene is expressed, or if it is expressed at all. Epigenetic modifications can also be passed on when a cell divides, so this provides a mechanism for how control of gene expression stays consistent from mother cell to daughter cell. That’s why skin stem cells only give rise to more skin cells, not to any other cell type.
The first epigenetic modification to be identified was DNA methylation. Methylation means the addition of a methyl group to another chemical, in this case DNA. A methyl group is very small. It’s just one carbon atom linked to three hydrogen atoms. Chemists describe atoms and molecules by their ‘molecular weight’, where the atom of each element has a different weight. The average molecular weight of a base-pair is around 600 Da (the Da stands for Daltons, the unit that is used for molecular weight). A methyl group only weighs 15 Da. By adding a methyl group the weight of the base-pair is only increased by 2.5 per cent. A bit like sticking a grape on a tennis ball.
Figure 4.1 shows what DNA methylation looks like chemically.
Figure 4.1 The chemical structures of the DNA base cytosine and its epigenetically modified form, 5-methylcytosine. 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.
The base shown is C – cytosine. It’s the only one of the four DNA bases that gets methylated, to form 5-methylcytosine. The ‘5’ refers to the position on the ring where the methyl is added, not to the number of methyl groups; there’s always only one of these. This methylation reaction is carried out in our cells, and those of most other organisms, by one of three enzymes called DNMT1, DNMT3A or DNMT3B. DNMT stands for DNA methyltransferase. The DNMTs are examples of epigenetic ‘writers’ – enzymes that create the epigenetic code. Most of the time these enzymes will only add a methyl group to a C that is followed by a G. C followed by G is known as CpG.
This CpG methylation is an epigenetic modification, which is also known as an epigenetic mark. The chemical group is ‘stuck onto’ DNA but doesn’t actually alter the underlying genetic sequence. The C has been decorated rather than changed. Given that the modification is so small, it’s perhaps surprising that it will come up over and over again in this book, and in any discussion of epigenetics. This is because methylation of DNA has profound effects on how genes are expressed, and ultimately on cellular, tissue and whole-body functions.
In the early 1980s it was shown that if you injected DNA into mammalian cells, the amount of methylation on the injected DNA affected how well it was transcribed into RNA. The more methylated the injected DNA was, the less transcription that occurred[19]. In other words, high levels of DNA methylation were associated with genes that were switched off. However, it wasn’t clear how significant this was for the genes normally found in the nuclei of cells, rather than ones that were injected into cells.
The key work in establishing the importance of methylation in mammalian cells came out of the laboratory of Adrian Bird, who has spent most of his scientific career in Edinburgh, Conrad Waddington’s old stomping ground. Professor Bird is a Fellow of the Royal Society and a former Governor of the Wellcome Trust, the enormously influential independent funding agency in UK science. He is one of those traditional British scientific types – understated, soft-spoken, non-flashy and drily funny. His lack of self-promotion is in contrast to his stellar international reputation, where he is widely acknowledged as the godfather of DNA methylation and its role in controlling gene expression.
In 1985 Adrian Bird published a key paper in Cell showing that most CpG motifs were not randomly distributed throughout the genome. Instead the majority of CpG pairs were concentrated just upstream of certain genes, in the promoter region[20]. Promoters are the stretches of the genome where the DNA transcription complexes bind and start copying DNA to form RNA. Regions where there is a high concentration of CpG motifs are called CpG islands.
In about 60 per cent of the genes that code for proteins, the promoters lie within CpG islands. When these genes are active, the levels of methylation in the CpG island are low. The CpG islands tend to be highly methylated only when the genes are switched off. Different cell types express different genes, so unsurprisingly the patterns of CpG island methylation are also different across different cell types.
For quite some time there was considerable debate about what this association meant. It was the old cause or effect debate. One interpretation was that DNA methylation was essentially a historical modification – genes were repressed by some unknown mechanism and then the DNA became methylated. In this model, DNA methylation was just a downstream consequence of gene repression. The other interpretation was that the CpG island became methylated, and it was this methylation that switched the gene off. In this model the epigenetic modification actually causes the change in gene expression. Although there is still the occasional argument about this between competing labs, the vast majority of scientists in this field now believe that the data generated in the quarter of a century since Adrian Bird’s paper are consistent with the second, causal model. Under most circumstances, methylation of the CpG island at the start of a gene turns that gene off.
Adrian Bird went on to investigate how DNA methylation switches genes off. He showed that when DNA is methylated, it binds a protein called MeCP2 (Methyl CpG binding protein 2)[21]. However, this protein won’t bind to unmethylated CpG motifs, which is pretty amazing when we look back at Figure 4.1 and think how similar the methylated and unmethylated forms of cytosine really are. The enzymes that add the methyl group to DNA have been described as writers of the epigenetic code. MeCP2 doesn’t add any modifications to DNA. Its role is to enable the cell to interpret the modifications on a DNA region. MeCP2 is an example of a ‘reader’ of the epigenetic code.
Once MeCP2 binds to 5-methylcytosine in a gene promoter it seems to do a number of things. It attracts other proteins that also help to switch the gene off[22]. It may also stop the DNA transcription machinery from binding to the gene promoter, and this prevents mRNA messenger molecule from being produced[23]. Where genes and their promoters are very heavily methylated, binding of MeCP2 seems to be part of a process where that region of a chromosome gets shut down almost permanently. The DNA becomes incredibly tightly coiled up and the gene transcription machinery can’t get access to the base-pairs to make mRNA copies.
23
For a recent review of the actions of MeCP2, see Adkins and Georgel (2011),