While the human genome sequence has transformed our understanding of human biology, it isn’t just the sequence of your DNA that matters, but also how you use it! How are some genes activated and others are silenced? How is this controlled? The answer is epigenetics. Epigenetics has been a hot topic for research over the past decade as it has become clear that aberrant epigenetic control contributes to disease (particularly to cancer). Epigenetic alterations are heritable through cell division, and in some instances are able to behave similarly to mutations in terms of their stability. Importantly, unlike genetic mutations, epigenetic modifications are reversible and therefore have the potential to be manipulated therapeutically. It has also become clear in recent years that epigenetic modifications are sensitive to the environment (for example diet), which has sparked a large amount of public debate and research. This course will give an introduction to the fundamentals of epigenetic control. We will examine epigenetic phenomena that are manifestations of epigenetic control in several organisms, with a focus on mammals. We will examine the interplay between epigenetic control and the environment and finally the role of aberrant epigenetic control in disease. All necessary information will be covered in the lectures, and recommended and required readings will be provided. There are no additional required texts for this course. For those interested, additional information can be obtained in the following textbook. Epigenetics. Allis, Jenuwein, Reinberg and Caparros. Cold Spring Harbour Laboratory Press. ISBN-13: 978-0879697242 | Edition: 1
1.5 DNA methylation at CpG islands
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Do curso por The University of Melbourne
Epigenetic Control of Gene Expression
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Week 1 - Introduction to Epigenetic Control
An introduction to and definition of epigenetic control of gene expression, and its importance in normal development. We will learn what chromatin is, and how its composition and packaging can alter gene expression. We’ll also discuss the best-characterised epigenetic modification, DNA methylation, and how it is not only implicated in regulating gene expression, but also in maintaining genome stability.
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Dr. Marnie BlewittHead, Molecular Medicine Laboratory
Walter and Eliza Hall Institute of Medical Research
So now we are going to go through in the next series of lectures, each of the
specific epigenetic modifications that are called epigenetic marks, and they
have functional consequences for how the genes are expressed and how the chromatin
is packaged. So while we'll go through each of these.
This is the relevance of these, and how they specifically relate to the
epigenetic phenomena, or the particular examples we are going to go through in
the later lectures, we'll deal with that in later
lectures and just have brief highlights of that here.
But we need to go through and learn about how each of these work in order to really
be able to understand at the molecular level how these processes are working in
other instances. So we're going to start with DNA
methylation. So DNA methylation is, as it sounds, the
addition of a methyl group to the DNA, and this happens at cytosines.
So here we're showing a cytosine single ring base but we can add on a 5- methyl
group so its added on to the 5 carbon the fifth carbon here.
And the methyl group is CHC group that's added directly onto that base.
We know that, in mammals, DNA methylation occurs almost exclusively at Cs that are
followed by Gs. So cytosines are followed by guanines.
And this is called a CpG dinucleotide. And that p is just for the phosphate bond
between the two. There's good reason that it's found
almost exclusively at CpG dinucleotides. And that's because these dinucleotides
are symmetrical when you look at the other side, the other strand of DNA, and
this allows them to be maintained through cell division.
And you remember this is one of those hallmark features of epigenetic
modifications. So, how is it then that DNA methylation
can be laid down and how can it be copied mitotically?
So how is it that it can have this
mitotic memory? So he pictured is a very simple
picture of a DNA strand, shown by the black lines, and then in the middle this
CpG dinucleotide. And in each case the cytosine is
methylated. So, this methylation, because it's close
by to one another. The cytosine on the other strand is very
close by, and so it's methylated as well.
What happens when the cell divides?
Sorry, I’ll go back a step and say that the first thing that happens, is that
DNA methyltransferases. So these are enzymes, in mammals
they're know as DNMT3A and DNMT3B, these lay down the methylation.
And they do this in a de novo fashion. In other words, they work on DNA that
begins out being unmethylated. So they lay down these, these methyl-marks
during development. Then how is it that it can be maintained?
Well if we go through cell division, we have the parent strand in each case shown
here in black. And the daughter strand shown in green.
So, when the DNA is replicated, there's only going to be the parent strand that
maintains the methyl group on that side within the CPG dinucleotide.
And then the daughter strand, now, shown in green
we have an unmethylated cytosine in each case.
Now, what happens is we bring in DNMT1, so another DNA methyltransferase enzyme,
and this stain of methyltransferase specifically recognises
hemi-methylated DNA. It has a preference for hemi-methylated
DNA, that is where one strand is methylated and the other is not.
And this hemi-methylated DNA is then bound DNMT1. And DNMT1 the lays down
methylation on the daughter strand and we have the restoration of a fully
methlayted CPG dinucleotide. And this is how we know that DNA methylation can be a
stable epigenetic mark. Because at every cell division, this DNA
methylation will be copied by DNMT1 onto the new daughter strands of DNA.
So we know I said that CpGs are where you, you mostly find methylation,
or almost exclusively find methylation in mammals.
So where are these CpG's found? Well many CpG’s are found in what are CPG
islands. So this is where you find more CG dinucleotides than you would expect by
chance and they tend to be found at the promoters of gene’s. So these, promoter are
just to remind you is the region upstream of the start side of transcription of the
gene and it's where the transcription factors bind.
The general rule to remember is that CpG islands although they have many CpG’s
there in fact tend to be protected from methylation, so methylation doesn't tend
to occur at CpG islands. It tends to be that it occurs at other
places in the genome. But if you do find methylation of CpG island.
Then, this is almost universally synonymous with silencing of gene
expression. So, DNA methylation is an inactive
epigenetic mark. So, there are some CpG islands in the
genome that are found to be methylated.
So, while the general rule is that they are unmethylated There are some that are
associated with gene silencing, and these are found at particular times and with
dynamic methylation between different cell types.
However, it’s mostly being studied, DNA methylation at CpG islands, for the
inactive X chromosomes that I have mentioned a few times.
So I'd like to go into a little bit more detail here about X inactivation.
So, the reason for that is X inactivation really clearly demonstrates
the mitotic heritability of DNA methylation.
So we know that females have two X chromosomes, where as males have
one X and one Y. So if we just consider the nucleus of
these female and male cells, then the female cell will have twice the dose of
all the genes that reside on the X chromosome, of which there are over 1000
genes. This in fact is not what ends up
happening. What happens is that one of those two X
chromosomes, either the one you inherited from your mom or the one you inherited
from your dad, is chosen and its densely packed down in a cell as shown here.
And this is called the inactive X chromosome.
So while females have two X chromosomes they only ever use one.
The second goes by unused. And is put in indeed is even put to the
side in the nucleus literally put to the side.
So what's interesting is this - when this X inactivation first occurs it occurs
when there are only a few hundred cells in the embryo at gastrulation.
And we know that when this choice is made it's a random choice.
So each cell at that couple-of-hundred-cell stage can make the
choice individually as to which X chromosome to inactivate - the one from
your mom or the one from your dad. That choice is then mitotically heritable
to all of the daughter cells. So, this X inactivation involves DNA
methylation of the CpG islands. All thousand of them or so, or almost all
thousand of them on the inactive X chromosome. And it's this mitotic
heritability after the choice is made is partly ensured by the DNA methylation
of those CpG islands. So, this is actually able to be observed
in a visible way in the coat colour of cats.
So, I'm going to show you a small movie here which is about Calico cats.
What we can see, is that Calico cats, that are shown in this picture here, have
genes that encode the coat colour genes are on the X chromosome.
So, they can either have the ginger version or the black version.
And then the choice is made early in development to have the ginger one being
active or the black one being active. And the other one is silenced.
The other chromosome is silenced. And so you end up with these cats that
have a mottled appearance based on when that choice of which X to be inactivated
was made early in development. So this could, is also the case not
only in cats, of course, but is also true in humans.
So in humans if we had a coat colour marker like this, which of course we
don't, but if we had a coat colour marker like this, you would actually see these
patches of green skin and patches of pink skin, based on when that choice was made
early in development. Just as in female, humans just as you see
in female cats they have this Calico appearance if they happen to have the
right genetics. It's also interesting to note then, that
you actually can't get male Calico cats. Male Calico cats, if they exist, have had
a mutation where they actually have two copies an X chromosome but still have a Y
chromosome. Otherwise they could not have this Calico
appearance, traditional mottled appearance.
So, how is then that DNA methylation which we've just discussed is heritable
for many cell divisions and if you think about it in human mammals
can can be heritable for maybe a 100 years.
How is it that the DNA methylation actually is associated with gene
silencing? There are probably at least a couple of
mechanisms by which this can happen. So perhaps the primary mechanism is
because the CpG, these methylated CPGs. Are associated with a condensation of the
chromatin, and the way that this happens in the primary case is because the
methylated CpG is bound by methylated CpG binding proteins, which are otherwise
known as MeCP1 and MeCP2. So these MeCP1 and MeCP2.proteins bind to
the methylated CpG dinucleotide and they themselves can alter transcription
because they possess a transcriptional repression domain.
Alternatively the MeCP2 or CeCP1 protein can itself bring in it's own protein
partners and they can condense the chromatin. But for this primary mechanism
it seems to be the binding of the methylated CPG by the methylated binding
protein domain family. Probably the secondary mechanism, which
doesn't seem to be is important, but can occasionally occur is that the methylated
CpG will stop a transcription factor binding.
So, transcription factors have particular binding sites.
Say for example C,G,A,T. So, this binding site might be bound by a
protein, a transcription factor, and it will then enable the transcription of a
neighbouring gene, or the nearby gene. However, if this particular site now has
the same sequence C,G,A,T, but its a methylated C,G,A,T,
this will then block the binding of that
transcription factor. So just that small addition of the
methyl group will not allow the transcription factor to bind and
therefore we don't have transcription in ensuing.
So we don't think, although there are some specific examples of this occurring, for
example for the transcription factor SP1 we don't think it's a generalisable
mechanism and instead it seems to be just true For promoters that don't have so
many CpGs. And so therefore, even single CpGs will have a large consequence.
Rather we think that primary mechanism where the methylated CpG binding proteins
bind to the methylated CpG is likely to be the most dominant mechanism within the nucleus.
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