This seems full of internal contradictions, but it becomes a little easier to understand if we once again visit the world of the musical. Not Oscar Hammerstein this time but Hal David, who was the lyricist who worked for a long time with Burt Bacharach. They wrote the songs for the 1973 flop film musical Lost Horizon. One of the songs from this became famous and contains a quite useful concept for us: ‘The world is a circle without a beginning and nobody knows where it really ends.’ Developmental processes become much easier to visualise if we think of them as never-ending circles rather than in straight lines. The ‘put it on — take it off — put it on’ cycle in the generation of the imprinted ICE is shown in Figure 10.2. This shows how eggs always pass on a maternal pattern of ICE methylation. A similar process allows sperm always to pass on the reciprocal paternal pattern.
Of course, one of the questions this schema raises is how the developing eggs and sperm identify ICE regions and how they ‘know’ which should be methylated and which unmethylated. This is an area of very active research and it may be different for each ICE, and between male and female germ cells. Some of it is frankly still a mystery but there are certain features that have been elucidated. We know that in the maternal germline, i.e. the cells that give rise to eggs, the process is critically dependent on the enzymes that can add DNA methylation to previously unmethylated CpG motifs.[22],{194} After that, the pattern is actively sustained by an enzyme whose job it is to maintain existing methylation patterns.[23],{195} Other proteins are also likely to be involved in establishing the correct methylation patterns, some of which are likely to be selectively expressed in the developing germ cells.
Figure 10.2 Cycles of methylation and demethylation ensure that chromosomes are passed on to children with the correct modifications to indicate parent of origin.
How do the enzymes in the germ cells recognise the ICE regions among all the other genomic DNA? Again there are gaps in our knowledge, although it has been suggested that certain repeated sequences in these special junk DNA regions may play a role.{196} These are quite poorly conserved at the sequence level between species, but may look more similar when we consider their three-dimensional structures. The cell may have a way of recognising them through their shape, rather than their sequence.{197} This is similar to the findings for long non-coding RNAs we saw in Chapter 8.
Although there are obviously plenty of questions that remain about imprinting, we are confident that this is absolutely the reason why both sexes have to contribute to the offspring. In a complex set of breeding experiments using genetically modified mice, researchers showed in 2007 that they could generate viable mice by inserting two egg nuclei into one fertilised egg. The reason they were able to do this was that they artificially altered the pattern of imprinting at two regions in the mouse genome. In one of the egg nuclei, they had created a methylation pattern that looked like the normal paternal pattern, not the maternal one. This fooled the developmental pathways into believing that the genetic material was from a male rather than a female. This demonstrated a particularly strong role for these two imprinted regions in controlling development. It also showed that the only real block to bi-maternal reproduction is the DNA methylation pattern at key genes. It disproved a previous hypothesis that sperm were required because the sperm themselves carried certain necessary accessory factors such as particular proteins or RNA molecules required to kick-start development properly.{198}
Going back to Figure 10.2 we can see that imprinting patterns may change during development. Imprinted control of gene expression seems to be particularly important during development. In mice, for example, most of the 140 or so imprinted genes are only imprinted in the placenta. In adult tissues both or neither copy of the genes may be expressed. This confirms that control of growth during early development was probably the major reason why imprinting evolved. There seems to be almost a geographical reason for this. In the imprinting clusters, the genes nearest the ICE may remain imprinted in all tissues but the ones further from the control centre may only be imprinted in the placenta. Selected cell types in the brain seem to be particularly likely to retain imprinting, although there is no clear consensus on why this would be favoured evolutionarily in most cases. There have been suggestions that the long non-coding RNA produced from the ICE attracts DNA methylation to the nearest genes but attracts histone modifications to the more distant genes in the cluster.{199} Because histone modifications can be more easily altered than DNA methylation, this may provide a mechanism for releasing more distant genes from imprinting as tissues mature.
So, imprinting occurs, and we have insights into at least some of the mechanisms by which this happens. In light of the theory that imprinting has evolved to balance out the competing evolutionary drives of the mother and foetus (and thus indirectly the father), it’s not surprising that the majority of protein-coding genes controlled by imprinting are ones involved in foetal growth and infant suckling, along with metabolism.{200} It’s also not surprising that when imprinting goes wrong, defects in growth are the commonest symptoms.
Studies of imprinting disorders really took off in the 1980s, when it was first becoming possible to identify genes associated with inherited diseases. The techniques involved finding families with more than one individual affected by a condition, and then analysing these families to narrow down the region on a chromosome that caused the disease. We can do this pretty easily now because we have the sequence of the normal human genome and access to very cheap sequencing technologies. But back in the 1980s, finding a mutation which caused a disease was akin to being asked to find a specific broken light bulb when all you knew was that it was in a house in America. It took years of work by large teams of scientists to identify the mutations underlying a condition.
A number of groups were looking into a disease called Prader-Willi syndrome. Babies born with Prader-Willi syndrome have a low birth weight and poor suckling responses. Their muscle tone doesn’t develop properly until after weaning, so the babies are quite floppy. As the children get older, their appetite becomes completely insatiable and as a consequence they develop early and extreme obesity. The children also suffer from mild mental disability.{201}
A completely different set of researchers was working on a condition with very different symptoms. This is called Angelman syndrome. Children suffering from this condition have small, under-developed heads, severe learning disabilities and are very late at moving on to solid food. The children are prone to outbursts of laughter for no reason, but thankfully the previous appallingly insensitive description of these patients as ‘happy puppets’ is falling into disuse.{202}
Imagine building a railway across a continent, where one set of workers starts in the east and builds westward, and the other starts in the west and builds eastwards. At first the workers are in completely different territories, but as time goes on they begin to get closer and closer to each other, and eventually there is a point (assuming all has gone well) where they meet, drive in the last spike, shake hands and have a drink. This is pretty much what happened to the researchers investigating Prader-Willi syndrome and Angelman syndrome. The difference, of course, compared with our railway analogy is that the scientists never expected to meet. They thought they were building independent railways, to completely different cities, and yet they each ended up in exactly the same spot as the other.