The Epigenetics Revolution (37 page)
Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
The drugs do work
There’s more evidence supporting a significant role for epigenetics in responses to stress. The naturally jumpy B6 mice were the ones with the increased expression of Hdac2 in the nucleus accumbens, and decreased expression from the
Gdnf
gene. We can treat these mice with SAHA, the histone deacetylase inhibitor. SAHA treatment leads to increased acetylation of the
Gdnf
promoter. This is associated with increased expression of the
Gdnf
gene. The crucial finding is that the treated mice stop being jumpy and become chilled instead
14
– changing the histone acetylation levels of the gene changed the mouse’s behaviour. This supports the idea that histone acetylation is really important in modulating the responses of these mice to stress.
Gdnf
gene. We can treat these mice with SAHA, the histone deacetylase inhibitor. SAHA treatment leads to increased acetylation of the
Gdnf
promoter. This is associated with increased expression of the
Gdnf
gene. The crucial finding is that the treated mice stop being jumpy and become chilled instead
14
– changing the histone acetylation levels of the gene changed the mouse’s behaviour. This supports the idea that histone acetylation is really important in modulating the responses of these mice to stress.
One of the tests used to investigate how depressed the mice become in response to stress is called the sucrose-preference test. Normal happy mice love sugared water, but when they are depressed they aren’t so interested in it. This decreased response to a pleasant stimulus is called anhedonia. It seems to be one of the best surrogate markers in animals for human depression
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. Most people who have been severely depressed talk about losing interest in all the things that used to make life joyful before they became ill. When the stressed mice were treated with SSRI anti-depressants, their interest in the sugared water gradually increased. But when they were treated with SAHA, the HDAC inhibitor, they regained their interest in their favourite drink much faster
16
.
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. Most people who have been severely depressed talk about losing interest in all the things that used to make life joyful before they became ill. When the stressed mice were treated with SSRI anti-depressants, their interest in the sugared water gradually increased. But when they were treated with SAHA, the HDAC inhibitor, they regained their interest in their favourite drink much faster
16
.
It’s not just in the jumpy or chilled mice that histone deacetylase inhibitors can change animal behaviour. It’s also relevant to the baby rats who don’t get much maternal licking and grooming. These are the ones that normally grow up to be chronically stressed, with over-activation of the cortisol production pathway. If these ‘unloved’ animals are treated with TSA, the first histone deacetylase inhibitor to be identified, they grow up much less stressed. They react much more like the animals who received lots of maternal care. The levels of DNA methylation at the cortisol receptor gene in the hippocampus go down, increasing expression of the receptor and improving the sensitivity of the all-important negative feedback loop. This is presumed to be because of cross-talk between the histone acetylation and DNA methylation pathways
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.
17
.
In the social defeat model in mice, the susceptible animals were treated with an SSRI anti-depressant drug. After three weeks of treatment, their behaviour was much more like that of the resilient mice. But treatment with this anti-depressant drug didn’t just result in increased levels of serotonin in the brain. The anti-depressant treatment also led to increased DNA methylation at the promoter of the corticotrophin-releasing hormone.
These studies are all very consistent with a model where there is cross-talk between the immediate signals from the neurotransmitters, and the longer-term effects on cell function mediated by epigenetic enzymes. When depressed patients are treated with SSRI drugs, the serotonin levels in the brain begin to rise, and signal more strongly to the neurons. The animal work described in the last paragraph suggests that it takes a few weeks for these signals to trigger all the pathways that ultimately result in the altered pattern of epigenetic modifications in the cells. This stage is essential for restoring normal brain function.
Epigenetics is also a reasonable hypothesis to explain another interesting but distressing feature of severe depression. If you have suffered from depression once, you are at a significantly higher risk than the general population of suffering from it again at some time in the future. It’s likely that some epigenetic modifications are exceptionally difficult to reverse, and leave the neurons primed to be more vulnerable to another bout.
The jury’s out
So far, so good. Everything looks very consistent with our theory about life experiences having sustained and long-lasting effects on behaviour, through epigenetics. And yet, here’s the thing: this whole area, sometimes called neuro-epigenetics, is probably the most scientifically contentious field in the whole of epigenetic research.
To get a sense of just how controversial, consider this. We’ve met Professor Adrian Bird in this book before. He is acknowledged as the father of the DNA methylation field. Another scientist with a very strong reputation in the science behind DNA methylation is Professor Tim Bestor from Columbia University Medical Center in New York. Adrian and Tim are about the same age, of similar physical type, and both are thoughtful and low key in conversation. And they seem to disagree on almost every issue in DNA methylation. Go to any conference where they are both scheduled in the same session and you are guaranteed to witness inspiring and impassioned debate between the two men. Yet the one thing they both seem to agree on publicly is their scepticism about some of the reports in the neuro-epigenetics field
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.
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There are three reasons why they, and many of their colleagues, are so sceptical. The first is that many of the epigenetic changes that have been observed are relatively small. The sceptics are unconvinced that such small molecular changes could lead to such pronounced phenotypes. They argue that just because the changes are present, it doesn’t mean they’re necessarily having a functional effect. They worry that the alterations in epigenetic modifications are simply correlative, not causative.
The scientists who have been investigating the behavioural responses in the different rodent systems counter this by arguing that molecular biologists are too used to quite artificial experimental models, where they can study extensive molecular changes with very on-or-off read-outs. The behaviourists suspect that this has left molecular biologists relatively inexperienced at interpreting real-world experiments, where the read-outs tend to be more ‘fuzzy’ and prone to greater experimental variation.
The second reason for scepticism lies in the very localised nature of the epigenetic changes. Infant stress affects specific regions of the brain, such as the nucleus accumbens, and not other areas. Epigenetic marks are only altered at some genes and not others. This seems less of a reason for scepticism. Although we refer to ‘the brain’, there are lots of highly specialised centres and regions within this organ, the product of hundreds of millions of years of evolution. Somehow, all these separate regions are generated and maintained during development and beyond, and thus are clearly able to respond differently to stimuli. This is also the case for all our genes, in all our tissues. It’s true that we don’t really know how epigenetic modifications can be targeted so precisely, or how the signalling from chemicals like neurotransmitters leads to this targeting. But we know that similarly specific events occur during normal development – so why not during abnormal periods of stress or other environmental disturbances? Just because we don’t know the mechanism for something, it doesn’t mean it doesn’t happen. After all, John Gurdon didn’t know how adult nuclei were reprogrammed by the cytoplasm of eggs, but that didn’t mean his experimental findings were invalid.
The third reason for scepticism is possibly the most important and it relates to DNA methylation itself. DNA methylation at the target genes in the brain is established very early, possibly pre-natally but certainly within one day of birth, in rodents. What this means is that the baby mice or baby rats in the experiments all started life with a certain baseline pattern of DNA methylation at their cortisol receptor gene in the hippocampus. The DNA methylation levels at this promoter alter in the first week of life, depending on the amount of licking and grooming the rats receive. As we saw, the DNA methylation levels are higher in the neglected mice than in the loved ones. But that’s not because the DNA methylation has gone up in the neglected mice. It’s because DNA methylation has gone
down
in the ones that were licked and groomed the most. The same is also true at the arginine vasopressin gene in the baby mice removed from their mothers. It’s also true for the corticotrophin-releasing hormone gene in the adult mice that were susceptible to social defeat.
down
in the ones that were licked and groomed the most. The same is also true at the arginine vasopressin gene in the baby mice removed from their mothers. It’s also true for the corticotrophin-releasing hormone gene in the adult mice that were susceptible to social defeat.
So, in every case, what the scientists observed was decreased DNA methylation in response to a stimulus. And that’s where, molecularly, the problem lies, because no-one knows how this happens. In
Chapter 4
we saw how copying of methylated DNA results in one strand that contains methyl groups and one that doesn’t. The DNMT1 enzyme moves along the newly synthesised strand and adds methyl groups to restore the methylation pattern, using the original strand as a template. We could speculate that in our experimental animals, there was less DNMT1 enzyme present and so the methylation levels at the gene dropped. This is referred to as passive DNA demethylation.
Chapter 4
we saw how copying of methylated DNA results in one strand that contains methyl groups and one that doesn’t. The DNMT1 enzyme moves along the newly synthesised strand and adds methyl groups to restore the methylation pattern, using the original strand as a template. We could speculate that in our experimental animals, there was less DNMT1 enzyme present and so the methylation levels at the gene dropped. This is referred to as passive DNA demethylation.
The problem is that this can’t work in neurons. Neurons are terminally differentiated – they are right at the bottom of Waddington’s landscape, and cannot divide. Because they don’t divide, neurons don’t copy their DNA. There’s no reason for them to do so. As a result, they can’t lose their DNA methylation by the method described in
Chapter 4
.
Chapter 4
.
One possibility is that maybe neurons simply remove the methyl group from DNA. After all, histone deacetylases remove acetyl groups from histones. But the methyl group on DNA is different. In chemical terms, histone acetylation is a bit like adding a small Lego brick onto a larger Lego brick. It’s pretty easy to take the two bricks apart again. DNA methylation isn’t like that. It’s more like having two Lego bricks and using superglue to stick them together.
The chemical bond between a methyl group and the cytosine in DNA is so strong that for many years it was considered completely irreversible. In 2000, a group from the Max Planck Institute in Berlin demonstrated that this couldn’t be the case. They showed that in mammals the paternal genome undergoes extensive DNA demethylation, during very early development. We came across this in
Chapters 7
and
8
. What we glossed over at the time was that this demethylation happens before the zygote starts to divide. In other words, the DNA methylation was removed without any DNA replication
19
. This is referred to as active DNA demethylation.
Chapters 7
and
8
. What we glossed over at the time was that this demethylation happens before the zygote starts to divide. In other words, the DNA methylation was removed without any DNA replication
19
. This is referred to as active DNA demethylation.
This means there is a precedent for removing DNA methylation in non-dividing cells. Perhaps there’s a similar mechanism in neurons. There’s still a lot of debate about how DNA methylation is actively removed, even in the well-established events in early development. There’s even less consensus about how it takes place in neurons. One of the reasons this has been so hard to investigate is that active DNA demethylation may involve a lot of different proteins, carrying out a number of steps one after another. This makes it very difficult to recreate the process in a lab, which is the gold standard for these kinds of investigations.
Silencing the silencer
As we’ve seen repeatedly, scientific research often throws up some very unexpected findings and so it happened here. While many people in epigenetics were looking for an enzyme that removed DNA methylation, one group discovered enzymes that added something extra to methylated DNA. This is shown in
Figure 12.3
. Very surprisingly, this has turned out to have many of the same consequences as demethylating the nucleic acid.
Figure 12.3
. Very surprisingly, this has turned out to have many of the same consequences as demethylating the nucleic acid.
A small molecule called hydroxyl, consisting of one oxygen atom and one hydrogen atom, is added to the methyl group, to create 5-hydroxymethylcytosine. This reaction is carried out by enzymes called TET1, TET2 or TET3
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.
20
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Figure 12.3
Conversion of 5-methylcytosine to 5-hydroxymethylcytosine. 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.
Conversion of 5-methylcytosine to 5-hydroxymethylcytosine. 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.
This is highly relevant to the question of DNA demethylation, because it’s the effects of DNA methylation that make this change important. Methylation of cytosine affects gene expression because methylated cytosine binds certain proteins, such as MeCP2. MeCP2 acts with other proteins to repress gene expression and to recruit other repressive modifications like histone deacetylation. When an enzyme such as TET1 adds the hydroxyl group to the methylcytosine to form the 5-hydroxymethylcytosine molecule, it changes the shape of the epigenetic modification. If a methylated cytosine is like a grape on a tennis ball, the 5-hydroxymethylcytosine is like a bean stuck to a grape stuck to a tennis ball. Because of this change in shape, the MeCP2 protein can’t bind to the modified DNA any more. The cell therefore ‘reads’ 5-hydroxymethylcytosine in the same way as it reads unmethylated DNA.
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