This human condition and the experiments in mice are impossible to reconcile with a model based only on the DNA code, where DNA is a naked molecule, which carries information only in its sequence of A, C, G and T base-pairs. DNA alone isn’t carrying all the necessary information for the creation of new life. Something else must be required in addition to the genetic information. Something epigenetic.
Eggs and sperm are highly specialised cells – they are at the bottom of one of Waddington’s troughs. The egg and the sperm will never be anything other than an egg and a sperm. Unless they fuse. Once they fuse, these two highly specialised cells form one cell which is so unspecialised it is totipotent and gives rise to every cell in the human body, and the placenta. This is the zygote, at the very top of Waddington’s epigenetic landscape. As this zygote divides, the cells become more and more specialised, forming all the tissues of our bodies. Some of these tissues ultimately give rise to eggs or sperm (depending on our sex, obviously) and the whole cycle is ready to start again. There’s effectively a never-ending circle in developmental biology.
The chromosomes in the pro-nuclei of sperm and eggs carry large numbers of epigenetic modifications. This is part of what keeps these gametes behaving as gametes, and not turning into other cell types. But these gametes can’t be passing on their epigenetic patterns, because if they did the fertilised zygote would be some sort of half-egg, half-sperm hybrid when it clearly isn’t this at all. It’s a completely different totipotent cell that will give rise to an entirely new individual. Somehow the modifications on eggs and sperm get changed to a different set of modifications, to drive the fertilised egg into a different cell state, at a different position in Waddington’s epigenetic landscape. This is part of normal development.
Almost immediately after the sperm has penetrated the egg, something very dramatic happens to it. Pretty much all the methylation on the male pronucleus DNA (i.e. from the sperm) gets stripped off, incredibly quickly. The same thing happens to the DNA on the female pronucleus, albeit a lot more slowly. This means that a lot of the epigenetic memory gets wiped off the genome. This is vital for putting the zygote at the top of Waddington’s epigenetic landscape. The zygote starts dividing and soon creates the blastocyst – the golf ball inside the tennis ball from Chapter 2. The cells in the golf ball – the inner cell mass, or ICM – are the pluripotent cells, the ones that give rise to embryonic stem cells in the laboratory.
The cells of the ICM soon differentiate and start giving rise to the different cell types of our bodies. This happens through very tightly regulated expression of a few key genes. One specific protein, for example OCT4, switches on another set of genes, which results in a further cascade of gene expression, and so on. We have met OCT4 before – it is the most critical of all the genes that Professor Yamanaka used to reprogram somatic cells. These cascades of gene expression are associated with epigenetic modification of the genome, changing the DNA and histone marks so that certain genes stay switched on or get switched off appropriately. Here’s the sequence of epigenetic events in very early development:
The male and female pronuclei (from the sperm and the egg respectively) are carrying epigenetic modifications;
The epigenetic modifications get taken off (in the immediate post-fertilisation zygote);
New epigenetic modifications get put on (as the cells begin to specialise).
This is a bit of a simplification. It’s certainly true that researchers can detect huge swathes of DNA demethylation during stage 2 from this list. However, it’s actually more complicated than this, particularly in respect of histone modifications. Whilst some histone modifications are being removed, others are becoming established. At the same time as the repressive DNA methylation is removed, certain histone marks which repress gene expression are also erased. Other histone modifications which increase gene expression may take their place. It’s therefore too naïve to refer to the epigenetic changes as just being about putting on or taking off epigenetic modifications. In reality, the epigenome is being reprogrammed.
Reprogramming is what John Gurdon demonstrated in his ground-breaking work when he transferred the nuclei from adult toads into toad eggs. It’s what happened when Keith Campbell and Ian Wilmut cloned Dolly the Sheep by putting the nucleus from a mammary gland cell into an egg. It’s what Yamanaka achieved when he treated somatic cells with four key genes, all of which code for proteins highly expressed naturally during this reprogramming phase.
The egg is a wonderful thing, honed through hundreds of millions of years of evolution to be extraordinarily effective at generating vast quantities of epigenetic change, across billions of base-pairs. None of the artificial means of reprogramming cells comes close to the natural process in terms of speed or efficiency. But the egg probably doesn’t quite do everything unaided. At the very least, the pattern of epigenetic modifications in sperm is one that allows the male pronucleus to be reprogrammed relatively easily. The sperm epigenome is primed to be reprogrammed[58].
Unfortunately, these priming chromatin modifications (and many other features of the sperm nucleus), are missing if an adult nucleus is reprogrammed by transferring it into a fertilised egg. That’s also true when an adult nucleus is reprogrammed by treating it with the four Yamanaka factors to create iPS cells. In both these circumstances, it’s a real challenge to completely reset the epigenome of the adult nucleus. It’s just too big a task.
This is probably why so many cloned animals have abnormalities and shortened lifespans. The defects that are seen in these cloned animals are another demonstration that if early epigenetic modifications go wrong, they may stay wrong for life. The abnormal epigenetic modification patterns result in permanently inappropriate gene expression, and long-term ill-health.
All this reprogramming of the genome in normal early development changes the epigenome of the gametes and creates the new epigenome of the zygote. This ensures that the gene expression patterns of eggs and sperm are replaced by the gene expression patterns of the zygote and the subsequent developmental stages. But this reprogramming also has another effect. Cells can accumulate inappropriate or abnormal epigenetic modifications at various genes. These disrupt normal gene expression and can even contribute to disease, as we shall see later in this book. The reprogramming of the egg and the sperm prevent them from passing on from parent to offspring any inappropriate epigenetic modifications they have accumulated. Not so much wiping the slate clean, more like re-installing the operating system.
But this creates a paradox. Azim Surani’s experiments showed that the male and female pro-nuclei aren’t functionally equivalent; we need one of each to create a new mammal. This is known as a parent-of-origin effect, because it essentially shows that there are ways for a zygote and its daughter cells to distinguish between chromosomes from the mother and father. This isn’t a genetic effect, it is an epigenetic one, and so there must be some epigenetic modifications that do get transmitted from one generation to the next.
In 1987 the Surani lab published one of the first papers to give an insight into this mechanism. They hypothesised that parent-of-origin effects could be caused by DNA methylation. At that time, this was really the only chromatin modification that had been identified, so it was an excellent place to start. The researchers created genetically modified mice. These mice contained an extra piece of DNA that could get inserted randomly anywhere in the genome. The DNA sequence of this extra bit wasn’t particularly important to the experimenters. What was important was that they could easily measure how much DNA methylation was present on this sequence, and whether the amount of methylation was transmitted faithfully from parent to offspring.