The Epigenetics Revolution Nessa Carey

Prev Next

To see a world in a Grain of Sand,

And a Heaven in a Wild Flower,

Hold Infinity in the palm of your hand,

And eternity in an hour.

William Blake

Probably all of us are familiar with the guessing game ‘animal, vegetable or mineral’. The implicit assumption in the name of this game is that plants and animals are completely different from one another. True, they are both living organisms, but that’s where we feel the similarity ends. We may be able to get on board with the idea that somewhere back in the murky evolutionary past, humans and microscopic worms have a shared ancestor. But how often do we ever wonder about the biological heritage we share with plants? When do we ever think of carnations as our cousins?

Yet animals and plants are surprisingly similar in many ways. This is especially the case when we consider the most advanced of our green relatives, the flowering plants. These include the grasses and cereals that we rely on for so much of our basic food intake, and the broad-leaved plants, from cabbages to oak trees and from rhododendrons to cress.

Animals and the flowering plants are each made up of lots of cells; they are multicellular organisms. Many of these cells are specialised for particular functions. In the flowering plants these include cells that transport water or sugars around the plant, the photosynthesising cells of the leaves and the food storing cells of the roots. Like animals, plants have specialised cells which are responsible for sexual reproduction. The sperm nuclei are carried in pollen and fertilise a large egg cell, which ultimately gives rise to a zygote and a new individual plant.

The similarities between plants and animals are more fundamental than these visible features. There are many genes in plants which have equivalents in animals. Crucially, for our topic, plants also have a highly developed epigenetic system. They can modify histone proteins and DNA, just like animal cells can, and in many cases use very similar epigenetic enzymes to those used by animals, including humans.

These genetic and epigenetic similarities all suggest that animals and plants have common ancestors. Because of our common ancestry, we’ve inherited similar genetic and epigenetic tool kits.

Of course, there are also really important differences between plants and animals. Plants can create their own food, but animals can’t do this. Plants take in basic chemicals in the environment, especially water and carbon dioxide. Using energy from sunlight, plants can convert these simple chemicals into complex sugars such as glucose. Nearly all life on planet earth is dependent directly or indirectly on this amazing process of photosynthesis.

There are two other ways in which plants and animals are very different. Most gardeners know that you can take a cutting from a growing plant – maybe just a small shoot – and create an entire new plant from this. There are very few animals where this is possible, and certainly no advanced ones. True, if certain species of lizard lose their tail, the animal can grow a new one. But they can’t do this the other way around. We can’t grow a new lizard from a discarded bit of tail.

This is because in most adult animals the only genuinely pluripotent stem cells are the tightly controlled cells of the germline which give rise to eggs or sperm. But active pluripotent stem cells are a completely normal part of a plant. In plants these pluripotent stem cells are found at the tips of stems and the tips of roots. Under the right conditions, these stem cells can keep dividing to allow the plant to grow. But under other conditions, the stem cells will differentiate into specific cell types, such as flowers. Once such a cell has become committed to becoming part of a petal, for example, it can’t change back into a stem cell. Even plant cells roll down Waddington’s epigenetic landscape eventually.

The other difference between plants and animals is really obvious. Plants can’t move. When environmental conditions change, the plant must adapt or die. They can’t out-run or out-fly unfavourable climates. Plants have to find a way of responding to the environmental triggers all around them. They need to make sure they survive long enough to reproduce at the right time of year, when their offspring will have the greatest chance of making it as new individuals.

Contrast this with a species such as the European swallow (Hirundo rustica) which winters in South Africa. As summer approaches and conditions become unbearable the swallow sets off on an epic migration. It flies up through Africa and Europe, to spend the summer in the UK where it raises its young. Six months later, back it goes to South Africa.

Many of a plant’s responses to the environment are linked to changes in cell fate. These include the change from being a pluripotent stem cell to becoming part of a terminally differentiated flower in order to allow sexual reproduction. Epigenetic processes play important roles in both these events, and interact with other pathways in plant cells to maximise the chance of reproductive success.

Not all plants use exactly the same epigenetic strategies. The best-characterised model system is an insignificant looking little flowering plant called Arabidopsis thaliana. It’s a member of the mustard family and looks like any nondescript weed you can find on any patch of wasteland. Most of the leaves grow close to the ground in a rosette shape. It produces small white flowers on a stem about 20–25 centimetres high. It’s been a useful model system for researchers because its genome is very compact, which makes it easy to sequence in order to identify the genes. There are also well-developed techniques for genetically modifying Arabidopsis thaliana. This makes it relatively straightforward for scientists to introduce mutations into genes to investigate their function.

Arabidopsis thaliana seeds typically germinate in early summer in the wild. The seedlings grow, creating the rosette of leaves. This is called the vegetative phase of plant growth. In order to produce offspring, Arabidopsis thaliana generates flowers. It is structures in the flowers that will generate the new eggs and sperm that will eventually lead to new zygotes, which will be dispersed in seeds.

But here’s the problem for the plant. If it flowers late in the year, the seeds it produces will be wasted. That’s because the weather conditions won’t be right for the new seeds to germinate. Even if the seeds do manage to germinate, the tender little seedlings are likely to be killed off by harsh weather like frost.

The adult Arabidopsis thaliana needs to keep its powder dry. It has a much greater chance of lots of its offspring surviving if it waits until the next spring until it flowers. The adult plant can survive winter weather that would kill off a seedling. This is exactly what Arabidopsis thaliana does. The plant ‘waits’ for spring and only then does it produce flowers.

The rites of spring

The technical term for this is vernalisation. Vernalisation means that a plant has to undergo a prolonged cold period (winter, usually) before it can flower. This is very common in plants with an annual life-cycle, especially in the temperate regions of the earth where the seasons are well-defined. Vernalisation doesn’t just affect broad-leaved plants like Arabidopsis thaliana. Many cereals also show this effect, especially crops like winter barley and winter wheat. In many cases, the prolonged period of cold needs to be followed by an increase in day length if flowering is to take place. The combination of the two stimuli ensures that flowering occurs at the most appropriate time of year.

Vernalisation has some very interesting features. When the plant first begins to sense and respond to cold weather, this may be many weeks or months before it starts to flower. The plant may continue to grow vegetatively through cell division during the cold period. When new seeds are produced, after the vernalisation of the parent plant, the seeds are ‘reset’. The new plants they produce from the seeds will themselves have to go through their own cold season before flowering¹.

These features of vernalisation are all very reminiscent of epigenetic phenomena in animals. Specifically:

1. The plant displays some form of molecular memory, because the stimulus and the final event are separated by weeks or months. We can compare this with abnormal stress responses in adult rodents that were ‘neglected’ as infants.

2. The memory is maintained even after cells divide. We can compare this with animal cells that continue to perform in a certain way after a stimulus to the parent cell, such as in normal development or in cancer progression.

3. The memory is lost in the next generation (the seeds). This is comparable with the way that most changes to the somatic tissues are ‘wiped clean’ in animals so that Lamarckian inheritance is exceptional, rather than common.

So, at a phenomenon level, vernalisation looks very epigenetic. In recent years, a number of labs have confirmed that epigenetic processes underlie this, at the chromatin modification level.

The key gene involved in vernalisation is called FLOWERING LOCUS C or FLC for short. FLC encodes a protein called a transcriptional repressor. It binds to other genes and stops them getting switched on. There are three genes that are particularly important for flowering in Arabidopsis thaliana, called FT, SOC1 and FD. Figure 15.1 shows how FLC interacts with these genes, and the consequences this has for flowering. It also shows how the epigenetic status of FLC changes after a period of prolonged cold.

Figure 15.1 Epigenetic modifications regulate the expression of the FLC gene, which represses the genes which promote flowering. The epigenetic modifications on the FLC gene are controlled by temperature.

Before winter, the FLC gene promoter carries lots of histone modifications that switch on gene expression. Because of this, the FLC gene is highly expressed, and the protein it codes for binds to the target genes and represses them. This keeps the plant in its normal growing vegetative phase. After winter, the histone modifications at the FLC gene promoter change to repressive ones. These switch off the FLC gene. The FLC protein levels drop, which removes the repression on the target genes. The increased periods of sunlight during spring activate expression of the FT gene. It’s essential that FLC levels have gone down by this stage, because if FLC levels are high, the FT gene finds it difficult to react to the stimulus from sunlight².

Experiments with mutated versions of epigenetic enzymes have shown that the changes in histone modifications at the FLC gene are critically important in controlling the flowering response. For example, there is a gene called SDG27 which adds methyl groups to the lysine amino acid at position 4 on histone H3, so it is an epigenetic writer. This methylation is associated with active gene expression. The SDG27 gene can be mutated experimentally, so that it no longer encodes an active protein. Plants with this mutation have less of this active histone modification at the FLC gene promoter. They produce less FLC protein, and so aren’t so good at repressing the genes that trigger flowering. The SDG27 mutants flower earlier than the normal plants³. This demonstrates that the epigenetic modifications at the FLC promoter don’t simply reflect the activity levels of the gene, they actually alter the expression. The modifications do actually cause the change in expression.

Cold weather induces a protein in plant cells called VIN3. This protein can bind to the FLC promoter. VIN3 is a type of protein called a chromatin remodeller. It can change how tightly chromatin is wound up. When VIN3 binds to the FLC promoter, it alters the local structure of the chromatin, making it more accessible to other proteins. Often, opening up chromatin leads to an increase in gene expression. However, in this case, VIN3 attracts yet another enzyme that can add methyl groups to histone proteins. However, this particular enzyme adds methyl groups to the lysine amino acid at position 27 on histone H3. This modification represses gene expression and is one of the most important methods that the plant cell uses to switch off the FLC gene⁴^,⁵.

This still raises the question of how cold weather results in epigenetic changes to the FLC gene specifically. What is the targeting mechanism? We still don’t know all the details, but one of the stages has been elucidated. Following cold weather, the cells in Arabidopsis thaliana produce a long RNA, which doesn’t code for protein. This RNA is called COLDAIR. The COLDAIR non-coding RNA is localised specifically at the FLC gene. When localised, it binds to the enzyme complex that creates the important repressive mark at position 27 on histone H3. COLDAIR therefore acts as a targeting mechanism for the enzyme complex⁶.

When Arabidopsis thaliana produces new seeds, the repressive histone marks at the FLC gene are removed. They are replaced by activating chromatin modifications. This ensures that when the seeds germinate the FLC gene will be switched on, and repress flowering until the new plants have grown through winter.

From these data we can see that flowering plants clearly use some of the same epigenetic machinery as many animal cells. These include modifications of histone proteins, and the use of long non-coding RNAs to target these modifications. True, animal and plant cells use these tools for different end-points – remember the orthopaedic surgeon and the carpenter from the previous chapter – but this is strong evidence for common ancestry and one basic set of tools.

The epigenetic similarities between plants and animals don’t end here either. Just like animals, plants also produce thousands of different small RNA molecules. These don’t code for proteins, instead they silence genes. It was scientists working with plants who first realised that these very small RNA molecules can move from one cell to another, silencing gene expression as they go⁷^,⁸. This spreads the epigenetic response to a stimulus from one initial location to distant parts of the organism.

The kamikaze cereal

Research in Arabidopsis thaliana has shown that plants use epigenetic modifications to regulate thousands of genes⁹. This regulation probably serves the same purposes as in animal cells. It helps cells to maintain appropriate but short-term responses to environmental stimuli, and it also locks differentiated cells in permanent patterns of specific gene expression. Because of epigenetic mechanisms we humans don’t have teeth in our eyeballs, and plants don’t have leaves growing out of their roots.

Flowering plants share a characteristic epigenetic phenomenon with mammals, and with no other members of the animal kingdom. Flowering plants are the only organisms we know of besides placental mammals in which genes are imprinted. Imprinting is the process we examined in Chapter 8, where the expression pattern of a gene is dependent on whether it was inherited from the mother or father.

At first glance, this similarity between flowering plants and mammals seems positively bizarre. But there’s an interesting parallel between us and our floral relations. In all higher mammals, the fertilised zygote is the source of both the embryo and the placenta. The placenta nourishes the developing embryo, but doesn’t ultimately form part of the new individual. Something rather similar happens when fertilisation occurs in flowering plants. The process is slightly more complicated, but the final fertilised seed contains the embryo and an accessory tissue called the endosperm, shown in Figure 15.2.

Just like the placenta in mammalian development, the endosperm nourishes the embryo. It promotes development and germination but it doesn’t contribute genetically to the next generation. The presence of any accessory tissues during development, be this a placenta or an endosperm, seems to favour the generation of imprinted control of the expression of a select group of genes.

Figure 15.2 The major anatomical components of a seed. The relatively small embryo that will give rise to the new plant is nourished by the endosperm, in a manner somewhat analogous to the nourishment of mammalian embryos by the placenta.

In fact, something very sophisticated happens in the endosperm of seeds. Just like most animal genomes, the genomes of flowering plants contain retrotransposons. These are usually referred to as TEs – transposable elements. These are the repetitive elements that don’t encode proteins, but can cause havoc if they are activated. This is especially because they can move around in the genome and disrupt gene expression.

Normally such TEs are tightly repressed, but in the endosperm these sequences are switched on. The cells of the endosperm create small RNA molecules from these TEs. These small RNAs travel out from the endosperm into the embryo. They find the TEs in the embryo’s genome that have the same sequence as themselves. These TE small RNA molecules then seem to recruit the machinery that permanently inactivates these potentially dangerous genomic elements. The risk to the endosperm genome through reactivation of the TEs is high. But because the endosperm doesn’t contribute to the next generation genetically, it can undertake this suicide mission, for the greater good¹⁰^,¹¹^,¹²^,¹³.

Although mammals and flowering plants both carry out imprinting, they seem to use slightly different mechanisms. Mammals inactivate the appropriate copy of the imprinted gene by using DNA methylation. In plants, the paternally-derived copy of the gene is always the one that carries the DNA methylation. However, it’s not always this methylated copy of the gene that is inactivated¹⁴. In plant imprinting, therefore, DNA methylation tells the cell how a gene was inherited, not how the gene should be expressed.

There are some fundamental aspects of DNA methylation that are quite similar between plants and animals. Plant genomes encode active DNA methyltransferase enzymes, and also proteins that can ‘read’ methylated DNA. Just like primordial germ cells in mammals, certain plant cells can actively remove methylation from DNA. In plants, we even know which enzymes carry out this reaction¹⁵. One is called DEMETER, after the mother of Persephone in Greek myths. Demeter was the goddess of the harvest and it was because of the deal that she struck with Hades, the god of the Underworld, that we have seasons.

But DNA methylation is also an aspect of epigenetics where there are clear differences in the way plants and higher animals use the same basic system. One of the most obvious differences is that plants don’t just methylate at CpG motifs (cytosine followed by a guanine). Although this is the most common sequence targeted by their DNA methyltransferases, plants will also methylate a cytosine followed by almost any other base¹⁶.

A lot of DNA methylation in plants is focused around non-expressed repetitive elements, just like in mammals. But a big difference becomes apparent when we examine the pattern of DNA methylation in expressed genes. About 5 per cent of expressed plant genes have detectable DNA methylation at their promoters, but over 30 per cent are methylated in the regions that encode amino acids, in the so-called body of the genes. Genes with methylation in the body regions tend to be expressed in a wide range of tissues, and are expressed at moderate to high levels in these tissues¹⁷.

The high levels of DNA methylation at repetitive elements in plants are very similar to the pattern at repetitive elements in the chromatin of higher animals such as mammals. By contrast, the methylation in the bodies of widely expressed genes is much more like that seen in honeybees (which don’t methylate their repetitive elements). This doesn’t mean that plants are some strange epigenetic hybrid of insects and mammals. Instead, it suggests that evolution has a limited set of raw materials, but isn’t too obsessive about how it uses them.

Prev Next