The Epigenetics Revolution (29 page)
Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
Room for manoeuvre
The fact that 98 per cent of the human genome does not code for protein suggests that there has been a huge evolutionary investment in the development of complicated ncRNA-mediated regulatory processes. Some authors have even gone so far as to speculate that ncRNAs are the genetic features that have underpinned the development of
Homo sapiens
’ greatest distinguishing feature – our higher thought processes
20
.
Homo sapiens
’ greatest distinguishing feature – our higher thought processes
20
.
The chimpanzee is our closest relative and its genome was published in 2005
21
. There isn’t one simple, meaningful average figure that we can give to express how similar the human and chimp genomes are. The statistics are actually very complicated, because you have to take into account that different genomic regions (for example repetitive sections versus single copy protein-coding gene regions) affect the statistics differently. However, there are two things we can say quite firmly. One is that human and chimp proteins are incredibly similar. About a third of all proteins are exactly the same between us and our knuckle-dragging cousins, and the rest differ only by one or two amino acids. Another thing we have in common is that over 98 per cent of our genomes don’t code for protein. This suggests that both species use ncRNAs to create complex regulatory networks which govern gene and protein expression. But there is a particular difference which may be very important between chimps and humans. This lies in how ncRNA is treated in the cells of the two species.
21
. There isn’t one simple, meaningful average figure that we can give to express how similar the human and chimp genomes are. The statistics are actually very complicated, because you have to take into account that different genomic regions (for example repetitive sections versus single copy protein-coding gene regions) affect the statistics differently. However, there are two things we can say quite firmly. One is that human and chimp proteins are incredibly similar. About a third of all proteins are exactly the same between us and our knuckle-dragging cousins, and the rest differ only by one or two amino acids. Another thing we have in common is that over 98 per cent of our genomes don’t code for protein. This suggests that both species use ncRNAs to create complex regulatory networks which govern gene and protein expression. But there is a particular difference which may be very important between chimps and humans. This lies in how ncRNA is treated in the cells of the two species.
It’s all to do with a process called editing. It seems that human cells just can’t leave well-enough alone, particularly when it comes to ncRNA
22
. Once an ncRNA has been produced, human cells use various mechanisms to modify it yet further. In particular, they will often change the base A to one called I (inosine). Base A can bind to T in DNA, or U in RNA. But base I can pair with A, C or G. This alters the sequences to which an ncRNA can bind and hence regulate.
22
. Once an ncRNA has been produced, human cells use various mechanisms to modify it yet further. In particular, they will often change the base A to one called I (inosine). Base A can bind to T in DNA, or U in RNA. But base I can pair with A, C or G. This alters the sequences to which an ncRNA can bind and hence regulate.
We humans, more than any other species, edit our ncRNA molecules to a remarkable degree. Not even other primates carry out this reaction as well as we do
23
. We also edit particularly extensively in the brain. This makes editing of ncRNA an attractive candidate process to explain why we are mentally so much more sophisticated than our primate relatives, even though we share so much of our DNA template in common.
23
. We also edit particularly extensively in the brain. This makes editing of ncRNA an attractive candidate process to explain why we are mentally so much more sophisticated than our primate relatives, even though we share so much of our DNA template in common.
In some ways, this is the beauty of ncRNAs. They create a relatively safe method for organisms to use to alter various aspects of cellular regulation. Evolution has probably favoured this mechanism because it is simply too risky to try to improve function by changing proteins. Proteins, you see, are the Mary Poppins of the cell. They are ‘practically perfect in every way’.
Hammers always look pretty similar. Some may be big, some may be small, but in terms of basic design, there’s not much you can change that would make a hammer much better. It’s the same with proteins. The proteins in our bodies have evolved over billions of years. Let’s take just one example. Haemoglobin is the pigment that transports oxygen around our bodies, in the red blood cells. It’s beautifully adept at picking up oxygen in the lungs and releasing it where it’s needed in the tissues. Nobody working in a lab has been able to create an altered version of haemoglobin that does a better job than the natural protein.
Creating a haemoglobin molecule that’s worse than normal is surprisingly easy to do, unfortunately. In fact, that’s what happens in disorders like sickle cell disease, where mutations create poor haemoglobin proteins. A similar situation is true for most proteins. So, unless environmental conditions change dramatically, most alterations to a protein turn out to be a bad thing. Most proteins are as good as they’re going to get.
So how has evolution solved the problem of creating ever more complex and sophisticated organisms? Basically, by altering the
regulation
of proteins, rather than altering the proteins themselves. This is what can be achieved using complicated networks of ncRNA molecules to influence how, when and to what degree specific proteins are expressed – and there is evidence to show this actually happens.
regulation
of proteins, rather than altering the proteins themselves. This is what can be achieved using complicated networks of ncRNA molecules to influence how, when and to what degree specific proteins are expressed – and there is evidence to show this actually happens.
miRNAs play major roles in control of pluripotency and control of cellular differentiation. ES cells can be encouraged to differentiate into other cell types by changing the culture conditions in which they’re grown. When they begin to differentiate, it’s essential that ES cells switch off the gene expression pathways that normally allow them to keep producing additional ES cells (self-renewal). There is a miRNA family called
let-7
which is essential for this switch-off process
24
.
let-7
which is essential for this switch-off process
24
.
One of the mechanisms the
let-7
family uses is the down-regulation of a protein called Lin28. This implies that Lin28 is a pro-pluripotency protein. It’s therefore not that surprising to discover that Lin28 can act as a Yamanaka factor. Over-expression of Lin28 protein in somatic cells increases the chances of reprogramming them to iPS cells
25
.
let-7
family uses is the down-regulation of a protein called Lin28. This implies that Lin28 is a pro-pluripotency protein. It’s therefore not that surprising to discover that Lin28 can act as a Yamanaka factor. Over-expression of Lin28 protein in somatic cells increases the chances of reprogramming them to iPS cells
25
.
Conversely, there are other miRNA families that help ES cells to stay pluripotent and self-renewing. Unlike
let-7
, these miRNAs promote the pluripotent state. In ES cells, the key pluripotency factors such as Oct4 and Sox2 are bound to the promoters of these miRNAs, activating their expression. As the ES cells start to differentiate, these factors fall off the miRNA promoters, and stop driving their expression
26
. Just like the Lin28 protein, these miR-NAs also improve reprogramming of somatic cells into iPS cells
27
.
let-7
, these miRNAs promote the pluripotent state. In ES cells, the key pluripotency factors such as Oct4 and Sox2 are bound to the promoters of these miRNAs, activating their expression. As the ES cells start to differentiate, these factors fall off the miRNA promoters, and stop driving their expression
26
. Just like the Lin28 protein, these miR-NAs also improve reprogramming of somatic cells into iPS cells
27
.
When we compare stem cells with their differentiated descendants, we find that they express very different populations of mRNA molecules. This seems reasonable, as the stem and differentiated cells express different proteins. But some mRNAs can take a long time to break down in a cell. This means that when a stem cell starts to differentiate, there will be a period when it still contains many of the stem cell mRNAs. Happily, when the stem cell starts differentiating, it switches on a new set of miRNAs. These target the residual stem cell mRNAs and accelerate their destruction. This rapid degradation of the pre-existing mRNAs ensures that the cell moves into a differentiated state as quickly and irreversibly as possible
28
.
28
.
This is an important safety feature. It’s not good for cells to retain inappropriate stem cell characteristics – it increases the chance they will move down a cancer cell pathway. This mechanism is used even more dramatically in species where embryonic development is very rapid, such as fruit flies or zebrafish. In these species this process ensures that maternally-inherited mRNA transcripts supplied by the egg are rapidly degraded as the fertilised egg turns into a pluripotent zygote
29
.
29
.
miRNAs are also vital for that all-important phase in imprinting control, the formation of primordial germ cells. A key stage in creation of primordial germ cells is the activation of the Blimp1 protein that we met in
Chapter 8
. Blimp1 expression is controlled by a complex interplay between Lin28 and let-7 activity
30
. Blimp1 also regulates an enzyme that methylates histones, and a class of proteins known as PIWI proteins. PIWI proteins in turn bind to another type of small ncRNAs known as PIWI RNAs
31
. PIWI ncRNAs and proteins don’t seem to play much of a role in the somatic cells but are required for generation of the male germline
32
. PIWI actually stands for
P
element-
i
nduced
wi
mpy testis. If the PIWI ncRNAs and PIWI proteins don’t interact properly, the testes in a male foetus don’t form normally.
Chapter 8
. Blimp1 expression is controlled by a complex interplay between Lin28 and let-7 activity
30
. Blimp1 also regulates an enzyme that methylates histones, and a class of proteins known as PIWI proteins. PIWI proteins in turn bind to another type of small ncRNAs known as PIWI RNAs
31
. PIWI ncRNAs and proteins don’t seem to play much of a role in the somatic cells but are required for generation of the male germline
32
. PIWI actually stands for
P
element-
i
nduced
wi
mpy testis. If the PIWI ncRNAs and PIWI proteins don’t interact properly, the testes in a male foetus don’t form normally.
We are finding more and more instances of cross-talk and interactions between ncRNAs and epigenetic events. Remember that the genetic interlopers, the retrotransposons, are normally methylated in the germline, to prevent their activation. The PIWI pathway is involved in targeting this DNA methylation
33
,
34
. A substantial number of epigenetic proteins are able to interact with RNA. Binding of non-coding RNAs to the genome may act as the general mechanism by which epigenetic modifications are targeted to the correct chromatin region in a specific cell type
35
.
33
,
34
. A substantial number of epigenetic proteins are able to interact with RNA. Binding of non-coding RNAs to the genome may act as the general mechanism by which epigenetic modifications are targeted to the correct chromatin region in a specific cell type
35
.
ncRNAs have recently been implicated in Lamarckian transmission of inherited characteristics. In one example, fertilised mouse eggs were injected with a miRNA which targeted a key gene involved in growth of heart tissue. The mice which developed from these eggs had enlarged hearts (cardiac hypertrophy) suggesting that the early injection of the miRNA disturbed the normal developmental processes. Remarkably, the offspring of these mice also had a high frequency of cardiac hypertrophy. This was apparently because the abnormal expression of the miRNA was recreated during generation of sperm in these mice. There was no change in the DNA code of the mice, so this was a clear case of a miRNA driving epigenetic inheritance
36
.
36
.
Murphy’s Law (if something can go wrong, it usually will)
But if ncRNAs are so important for cellular function, surely we would expect to find that sometimes diseases are caused by problems with them. Shouldn’t there be lots of examples where defects in production or expression of ncRNAs lead to clinical disorders, aside from the imprinting or X inactivation conditions? Well, yes and no. Because these ncRNAs are predominantly regulatory molecules, acting in networks that are rich in compensatory mechanisms, defects may only have relatively subtle impacts. The problem this creates experimentally is that most genetic screens are good at detecting the major phenotypes caused by mutations in proteins, but may not be so useful for more subtle effects.
There is a small ncRNA called BC1 which is expressed in specific neurons in mice. When researchers at the University of Munster in Germany deleted this ncRNA, the mice seemed fine. But then the scientists moved the mutant animals from the very controlled laboratory setting into a more natural environment. Under these conditions, it became clear that the mutants were not the same as normal mice. They were reluctant to explore their surroundings and were anxious
37
. If they had simply been left in their cages, we would never have appreciated that loss of the BC1 ncRNA actually had a quite pronounced effect on behaviour. A clear case of what we see being dependent on how we look.
37
. If they had simply been left in their cages, we would never have appreciated that loss of the BC1 ncRNA actually had a quite pronounced effect on behaviour. A clear case of what we see being dependent on how we look.
The impact of ncRNAs in clinical conditions is starting to come into focus, at least for a few examples. There is a breed of sheep called a Texel, and the kindest description would be that it’s chunky. The Texel is well known for having a lot of muscle, which is a good thing in an animal that’s being bred to be eaten. The muscularity of the breed has been shown to be at least partially due to a change in a miRNA binding site in the 3´ UTR of a specific gene. The protein coded for by this gene is called myostatin, and it normally slows down muscle growth
38
. The impact of the single base change is summarised in
Figure 10.4
. The final size of the Texel sheep has been exaggerated for clarity.
38
. The impact of the single base change is summarised in
Figure 10.4
. The final size of the Texel sheep has been exaggerated for clarity.
Tourette’s syndrome is a neurodevelopmental disorder where the patient frequently suffers from involuntary convulsive movements (tics) which in some cases are associated with involuntary swearing. Two unrelated individuals with this disorder were shown to have the same single base change in the 3´ UTR of a gene called
SLITRK1
39
.
SLITRK1
appears to be required for neuronal development. The base change in the Tourette’s patients introduced a binding site for a short ncRNA called miR-189. This suggests that
SLITRK1
expression may be abnormally down-regulated via such binding, at critical points in development. This alteration is only present in a few cases of Tourette’s but raises the tantalising suggestion that mis-regulation of miRNA binding sites in other neuronal genes may be involved in other patients.
SLITRK1
39
.
SLITRK1
appears to be required for neuronal development. The base change in the Tourette’s patients introduced a binding site for a short ncRNA called miR-189. This suggests that
SLITRK1
expression may be abnormally down-regulated via such binding, at critical points in development. This alteration is only present in a few cases of Tourette’s but raises the tantalising suggestion that mis-regulation of miRNA binding sites in other neuronal genes may be involved in other patients.
Earlier in this chapter we encountered the theory that ncRNAs may have been vitally important for the development of increased brain complexity and sophistication in humans. If that is the case, we might predict that the brain would be particularly susceptible to defects in ncRNA activity and function. Indeed, the Tourette’s cases in the previous paragraph give an intriguing glimpse of such a scenario.
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