The Epigenetics Revolution (28 page)
Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
The
Air
ncRNA gave scientists important insights into how these long ncRNAs repress gene expression. The ncRNA remained localised to a specific region in the cluster of imprinted genes, and acted as a magnet for an epigenetic enzyme called G9a. G9a puts a repressive mark on the histone H3 proteins in the nucleosomes deposited on this region of DNA. This histone modification creates a repressive chromatin environment, which switches off the genes.
Air
ncRNA gave scientists important insights into how these long ncRNAs repress gene expression. The ncRNA remained localised to a specific region in the cluster of imprinted genes, and acted as a magnet for an epigenetic enzyme called G9a. G9a puts a repressive mark on the histone H3 proteins in the nucleosomes deposited on this region of DNA. This histone modification creates a repressive chromatin environment, which switches off the genes.
This finding was particularly important as it provided some of the first insights into a question that had been puzzling epigeneticists. How do histone modifying enzymes, which put on or remove epigenetic marks, get localised to specific regions of the genome? Histone modifying enzymes can’t recognise specific DNA sequences directly, so how do they end up in the right part of the genome?
The patterns of histone modifications are localised to different genes in different cell types, leading to exquisitely well-regulated gene expression. For example, the enzyme known as EZH2 methylates the amino acid called lysine at position 27 on histone H3, but it targets different histone H3 molecules in different cell types. To put it simply, it may methylate histone H3 proteins positioned on gene A in white blood cells but not in neurons. Alternatively, it may methylate histone H3 proteins positioned on gene B in neurons, but not in white blood cells. It’s the same enzyme in both cells, but it’s being targeted differently.
There is increasing evidence that at least some of the targeting of epigenetic modifications can be explained by interactions with long ncRNAs. Jeannie Lee and her colleagues have recently investigated long ncRNAs that bind to a complex of proteins. The complex is called PRC2 and it generates repressive modifications on histones. PRC2 contains a number of proteins, and the one that interacts with the long ncRNAs is probably EZH2. The researchers found that the PRC2 complex bound to literally thousands of different long ncRNA molecules in embryonic stem cells from mice
13
. These long ncRNAs may act as bait. They can stay tethered to the specific region of the genome where they are produced, and then attract repressive enzymes to shut off gene expression. This happens because the repressive enzyme complexes contain proteins like EZH2 that are capable of binding to RNA.
13
. These long ncRNAs may act as bait. They can stay tethered to the specific region of the genome where they are produced, and then attract repressive enzymes to shut off gene expression. This happens because the repressive enzyme complexes contain proteins like EZH2 that are capable of binding to RNA.
Scientists love to build theories, and in some ways a nice one was shaping up around long ncRNAs. It seemed that they bind to the region from which they are transcribed, and repress gene expression on that same chromosome. But if we go back to our analogy from the start of this chapter, we’d have to say that it’s now becoming clear we have built a pretty small shed and already cemented quite a bit of rubble to the roof.
There’s an amazing family of genes, called
HOX
genes. When they’re mutated in fruit flies (
Drosophila melanogaster
) the results are incredible phenotypes, such as legs growing out of the head
14
. There’s a long ncRNA known as
HOTAIR
, which regulates a region of genes called the
HOX-D
cluster. Just like the long ncRNAs investigated by Jeannie Lee,
HOTAIR
binds the PRC2 complex and creates a chromatin region which is marked with repressive histone modifications. But
HOTAIR
is not transcribed from the
HOX-D
position on chromosome 12. Instead it is encoded at a different cluster of genes called
HOX-C
on chromosome 2
15
. No-one knows how or why
HOTAIR
binds at the HOX-D position.
HOX
genes. When they’re mutated in fruit flies (
Drosophila melanogaster
) the results are incredible phenotypes, such as legs growing out of the head
14
. There’s a long ncRNA known as
HOTAIR
, which regulates a region of genes called the
HOX-D
cluster. Just like the long ncRNAs investigated by Jeannie Lee,
HOTAIR
binds the PRC2 complex and creates a chromatin region which is marked with repressive histone modifications. But
HOTAIR
is not transcribed from the
HOX-D
position on chromosome 12. Instead it is encoded at a different cluster of genes called
HOX-C
on chromosome 2
15
. No-one knows how or why
HOTAIR
binds at the HOX-D position.
There’s a related mystery around the best studied of all long ncRNAs,
Xist. Xist
ncRNA spreads out along almost the entire inactive X chromosome but we really don’t know how. Chromosomes don’t normally become smothered with RNA molecules. There’s no obvious reason why
Xist
RNA should be able to bind like this, but we know it’s nothing to do with the sequence of the chromosome. The experiments described in the last chapter, where
Xist
could inactivate an entire autosome as long as it contained an X inactivation centre, showed that
Xist
just keeps on travelling once it’s on a chromosome. Scientists are basically still completely baffled about these fundamental characteristics of this best-studied of all ncRNAs.
Xist. Xist
ncRNA spreads out along almost the entire inactive X chromosome but we really don’t know how. Chromosomes don’t normally become smothered with RNA molecules. There’s no obvious reason why
Xist
RNA should be able to bind like this, but we know it’s nothing to do with the sequence of the chromosome. The experiments described in the last chapter, where
Xist
could inactivate an entire autosome as long as it contained an X inactivation centre, showed that
Xist
just keeps on travelling once it’s on a chromosome. Scientists are basically still completely baffled about these fundamental characteristics of this best-studied of all ncRNAs.
Here’s another surprising thing. Until very recently, all long ncRNAs were thought to repress gene expression. In 2010, Professor Ramin Shiekhattar at the Wistar Institute in Philadelphia identified over 3,000 long ncRNAs in a number of human cell types. These long ncRNAs showed different expression patterns in different human cell types, suggesting they had specific roles. Professor Shiekhattar and his colleagues tested a small number of the long ncRNAs to try to determine their functions. They used well-established experimental methods to knock down expression of their test ncRNAs and then analysed expression of their neighbouring genes. The predicted outcome, and the actual results, are shown in
Figure 10.2
.
Figure 10.2
.
Figure 10.2
ncRNAs were thought to repress expression of target genes. If this hypothesis were correct, then decreasing the expression of a specific ncRNA should result in more expression of the target gene, as the repression diminishes. This is shown in the middle panel. However, it is now becoming clear that a large number of ncRNAs actually drive
up
expression of their target genes. This has been shown by cases in which experimentally decreasing the expression of an ncRNA has the effect shown in the right hand side of this figure.
ncRNAs were thought to repress expression of target genes. If this hypothesis were correct, then decreasing the expression of a specific ncRNA should result in more expression of the target gene, as the repression diminishes. This is shown in the middle panel. However, it is now becoming clear that a large number of ncRNAs actually drive
up
expression of their target genes. This has been shown by cases in which experimentally decreasing the expression of an ncRNA has the effect shown in the right hand side of this figure.
Twelve ncRNAs were tested, and in seven cases the scientists found the result shown in the right-hand panel of
Figure 10.2
. This was contrary to expectations, because it suggests that about 50 per cent of long ncRNAs may actually increase expression of neighbouring genes, not decrease it
16
.
Figure 10.2
. This was contrary to expectations, because it suggests that about 50 per cent of long ncRNAs may actually increase expression of neighbouring genes, not decrease it
16
.
Rather pithily, the authors of the paper stated, ‘The precise mechanism by which our ncRNAs function to enhance gene expression is not known.’ It’s a statement that is very hard to argue with. It has considerable merit as it makes clear that we currently have no idea how this is happening. Ramin Shiekhattar’s work does demonstrate rather convincingly that there is a lot we don’t understand about long ncRNAs, and that we should be wary of creating new dogma too quickly.
Small is beautiful
We should also be wary of assuming that size is everything and that big is best. The long ncRNAs clearly have major importance in cell function, but there is another equally importance class of ncRNAs that also has a significant impact in the cell. The ncRNAs in this class are short (usually 20–24 bases in length), and they target mRNA molecules, not DNA. This was first shown in our favourite worm,
C. elegans
.
C. elegans
.
As we have already discussed,
C. elegans
is a very useful model system because we know exactly how every cell should normally develop. The timing and sequence of the different stages is very tightly regulated. One of the key regulators is a protein called LIN-14. The
LIN-14
gene is highly expressed (a lot of LIN-14 protein is produced) during the very early embryo stages, but is down-regulated as the worms move from larval stage 1 to larval stage 2. If the
LIN-14
gene is mutated the worm gets the timing of the different stages wrong. If LIN-14 protein stays on for too long the worm starts to repeat early developmental stages. If LIN-14 protein is lost too early the worm moves into later larval stages prematurely. Either way, the worm gets very messed up, and normal adult structures don’t develop.
C. elegans
is a very useful model system because we know exactly how every cell should normally develop. The timing and sequence of the different stages is very tightly regulated. One of the key regulators is a protein called LIN-14. The
LIN-14
gene is highly expressed (a lot of LIN-14 protein is produced) during the very early embryo stages, but is down-regulated as the worms move from larval stage 1 to larval stage 2. If the
LIN-14
gene is mutated the worm gets the timing of the different stages wrong. If LIN-14 protein stays on for too long the worm starts to repeat early developmental stages. If LIN-14 protein is lost too early the worm moves into later larval stages prematurely. Either way, the worm gets very messed up, and normal adult structures don’t develop.
In 1993 two labs working independently showed how
LIN-14
expression was controlled
17
,
18
. Unexpectedly, the key event was binding of a small ncRNA to the
LIN-14
mRNA molecule. This is shown in
Figure 10.3
. It is an example of post-transcriptional gene silencing, where an mRNA is produced but is prevented from generating a protein. This is a very different way of controlling gene expression from that used by the long ncRNAs.
LIN-14
expression was controlled
17
,
18
. Unexpectedly, the key event was binding of a small ncRNA to the
LIN-14
mRNA molecule. This is shown in
Figure 10.3
. It is an example of post-transcriptional gene silencing, where an mRNA is produced but is prevented from generating a protein. This is a very different way of controlling gene expression from that used by the long ncRNAs.
The importance of this work is that it laid the foundation for a whole new model for the regulation of gene expression. Small ncRNAs are now known to be a mechanism used by organisms throughout the plant and animal kingdoms to control gene expression. There are various different types of small ncRNAs, but we’ll concentrate mainly on the microRNAs (miRNAs).
Figure 10.3
Schematic to demonstrate how expression of microRNAs at specific developmental stages can radically alter expression of a target gene.
Schematic to demonstrate how expression of microRNAs at specific developmental stages can radically alter expression of a target gene.
At least 1,000 different miRNAs have been identified in mammalian cells. miRNAs are about 21 nucleotides (bases) in length (sometimes slightly smaller or longer) and most of them seem to act as post-transcriptional regulators of gene expression. They don’t stop production of an mRNA, instead they regulate how that mRNA behaves. Typically, they do this by binding to the 3´ untranslated region (3´ UTR) of an mRNA molecule. This region is shown in
Figure 10.3
. It’s present in the mature mRNA, but it doesn’t code for any amino acids.
Figure 10.3
. It’s present in the mature mRNA, but it doesn’t code for any amino acids.
When genomic DNA is copied to make mRNA, the original transcript tends to be very long because it contains both exons (which code for amino acids) and introns (which do not). As we saw in
Chapter 3
, introns are removed during splicing to create an mRNA which codes for protein. But the
Chapter 3
description passed over something. There are stretches of RNA at the beginning (known as 5´ UTR) and the end (3´ UTR) which don’t code for amino acids, but don’t get spliced out like introns either. Instead, these non-coding regions are retained on the mature mRNA and act as regulatory sequences. One of the functions of the 3´ UTR in particular is to bind regulatory molecules, including miRNAs.
Chapter 3
, introns are removed during splicing to create an mRNA which codes for protein. But the
Chapter 3
description passed over something. There are stretches of RNA at the beginning (known as 5´ UTR) and the end (3´ UTR) which don’t code for amino acids, but don’t get spliced out like introns either. Instead, these non-coding regions are retained on the mature mRNA and act as regulatory sequences. One of the functions of the 3´ UTR in particular is to bind regulatory molecules, including miRNAs.
How does a miRNA bind to an mRNA and what happens when it does? The miRNA and the 3´ UTR of the mRNA only interact if they recognise each other. This uses base-pairing, quite similar to that in double stranded DNA. G can bind C, A can bind U (in RNA, T is replaced by U). Although miRNAs are usually 21 bases in length, they don’t have to match the mRNA over the entire 21 nucleotides. The key region is positions 2 to 8 on the miRNA.
Sometimes the match from 2 to 8 is not perfect, but it’s still close enough for the two molecules to pair up. In these cases, binding of the miRNA prevents translation of the mRNA into protein (this is what happened in the case shown in
Figure 10.3
). If, however, the match is perfect, the binding of miRNA to mRNA triggers destruction of the mRNA, by enzymes that attach to the miRNA
19
. It’s not yet clear if positions 9 to 21 on the miRNAs also influence in a less direct way how these small molecules are targeted, or what the consequences of their targeting are. One thing we do know, however, is that a single miRNA can regulate more than one mRNA molecule. We saw in
Chapter 3
how one gene could encode lots of different protein molecules, by altering the way in which messenger RNA is spliced. A single miRNA can influence many of these differently spliced versions simultaneously. Alternatively, a single miRNA can also influence quite unrelated proteins that are encoded by different genes but have similar 3´ UTR sequences.
Figure 10.3
). If, however, the match is perfect, the binding of miRNA to mRNA triggers destruction of the mRNA, by enzymes that attach to the miRNA
19
. It’s not yet clear if positions 9 to 21 on the miRNAs also influence in a less direct way how these small molecules are targeted, or what the consequences of their targeting are. One thing we do know, however, is that a single miRNA can regulate more than one mRNA molecule. We saw in
Chapter 3
how one gene could encode lots of different protein molecules, by altering the way in which messenger RNA is spliced. A single miRNA can influence many of these differently spliced versions simultaneously. Alternatively, a single miRNA can also influence quite unrelated proteins that are encoded by different genes but have similar 3´ UTR sequences.
This can make it very difficult to unravel exactly what a miRNA is doing in a cell, as the effects will vary depending on the cell type and the other genes (protein-coding and non-protein-coding) that the cell is expressing at any one time. That can be important experimentally, but also has significant consequences for normal health and disease. In conditions where there are an abnormal number of chromosomes, for example, it won’t just be protein-coding genes that change in number. There will also be abnormal production of ncRNAs (large and small). Because miRNAs in particular can regulate lots of other genes, the effects of disrupting miRNA copy numbers may be very extensive.
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