In the same year, a group led by Gene Robinson in Illinois showed that the predicted DNA methyltransferase proteins encoded in the honeybee genome were active. The proteins were able to add methyl groups to the cytosine residue in a CpG motif in DNA[265]. Honeybees also expressed proteins that were able to bind to methylated DNA. Together, these data showed that honeybee cells could both ‘write’ and ‘read’ an epigenetic code.
Until these data were published, nobody had really wanted to take a guess as to whether or not honeybees would possess a DNA methylation system. This was because the most widely used experimental system in insects, the fruit fly Drosophila melanogaster, whom we met earlier in this book, doesn’t methylate its DNA.
It’s interesting to discover that honeybees have an intact DNA methylation system. But this doesn’t prove that DNA methylation is involved in the responses to royal jelly, or the persistent effects of this foodstuff on the physical form and behaviour of mature bees. This issue was addressed by some elegant work from the laboratory of Dr Ryszard Maleszka at the Australian National University in Canberra.
Dr Maleszka and his colleagues knocked down the expression of one of the DNA methyltransferases in honeybee larvae, by switching off the Dnmt3 gene. Dnmt3 is responsible for adding methyl groups to regions of DNA that haven’t been methylated before. The results of this experiment are shown in Figure 14.1.
Figure 14.1 When royal jelly is fed to honeybee larvae for extended periods, the larvae develop into queens. The same effect is seen in the absence of prolonged feeding with royal jelly if the expression of the Dnmt3 gene is decreased experimentally in the larvae. Dnmt3 protein adds methyl groups to DNA.
When the scientists decreased the expression of Dnmt3 in the honeybee larvae, the results were the same as if they had fed them royal jelly. Most of the larvae matured as queens, rather than as workers. Because knocking down Dnmt3 had the same effects as feeding royal jelly, this suggested that one of the major effects of royal jelly is connected with alterations of the DNA methylation patterns on important genes[266].
To back up this hypothesis, the researchers also examined the actual DNA methylation and gene expression patterns in the different experimental groups of bees. They showed that the brains of queens and worker bees have a different DNA methylation pattern. The DNA methylation patterns in the bees where Dnmt3 had been knocked down were like those of the normal royal jelly-induced queens. This is what we would expect given the similar phenotypes in the two groups. The gene expression patterns in the normal queens and the Dnmt3-knockdown queens were also very similar. The authors concluded that the nutritional effects of continual feeding on royal jelly occurred via DNA methylation.
There are still a lot of gaps in our understanding of how nutrition in the honeybee larva results in altered patterns of DNA methylation. One hypothesis, based on the experiments above, is that royal jelly inhibits the DNA methyltransferase enzyme. But so far nobody has been able to demonstrate this experimentally. It’s therefore possible that the effect of royal jelly on DNA methylation is indirect.
What we do know is that royal jelly influences hormonal signalling in honeybees, and that this changes gene expression patterns. Changes in the levels of expression of a gene often influence the epigenetic modifications at that gene. The more highly a gene is switched on, the more its histones become modified in ways which promote gene expression. Something similar may happen in honeybees.
We also know that the DNA methylation systems and histone modification systems often work together. This has created interest in the role of histone-modifying enzymes in the control of honeybee development and activity. When the honeybee genome was sequenced, four histone deacetylase enzymes were identified. This was intriguing because it has been known for some time that royal jelly contains a compound called phenyl butyrate[267]. This very small molecule can inhibit histone deacetylases but it does so rather weakly. In 2011, a group led by Dr Mark Bedford from the MD Anderson Cancer Center in Houston published an intriguing study on another component of royal jelly. One of the other senior authors on this paper was Professor Jean-Pierre Issa, who has been so influential in promoting use of epigenetic drugs for the treatment of cancer.
The researchers analysed a compound found in royal jelly called (E)-10-hydroxy-2-decenoic acid, or 10HDA for short. The structure of this compound is shown in Figure 14.2, along with SAHA, the histone deacetylase inhibitor we saw in Chapter 11 that is licensed for the treatment of cancer.
Figure 14.2 The chemical structure of the histone deacetylase inhibitor SAHA and 10HDA, a compound found in royal jelly. 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 two structures aren’t identical by any means, but they do share some similarities. Each has a long chain of carbon atoms (the bit that looks like a crocodile’s back in profile), and the right hand side of each compound also looks fairly similar. Mark Bedford and his colleagues hypothesised that 10HDA might be an inhibitor of histone deacetylases. They performed a number of test tube and cell experiments and showed that this was indeed the case. This means that we now know that one of the major compounds found in royal jelly inhibits a key class of epigenetic enzymes[268].
Epigenetics influences more than whether bees develop into workers or queens. Ryszard Maleszka has also shown that DNA methylation is involved in how honeybees process memories. When honeybees find a good source of pollen or nectar, they fly back to the hive and tell the other members of the colony where to head to find this great food supply. This tells us something really important about honeybees; they can remember information. They must be able to, or they wouldn’t be able to tell the other bees where to go for food. Of course, it’s equally important that the bees can forget information and replace it with new data. There’s no point sending your co-workers to the great patch of thistles that were in flower last week, but that have now been eaten by a grazing donkey. The bee needs to forget last week’s thistles and remember the location of this week’s lavender.
It’s actually possible to train honeybees to respond to certain stimuli associated with food. Dr Maleszka and his colleagues showed that when the bees undergo this training, the levels of Dnmt3 protein go up in the parts of the honeybee brains which are important in learning. If the bees are treated with a drug that inhibits the Dnmt3 protein, the compound alters the way the bees retain memories, and also the speed with which memories are lost[269].
Although we know that DNA methylation is important in honeybee memory, we don’t know exactly how this works. This is because it’s not clear yet which genes become methylated when honeybees learn and acquire new memories.