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
6.3 The Agouti viable yellow allele in mice
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From the course by The University of Melbourne
Epigenetic Control of Gene Expression
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From the lesson
Week 6 - Mechanisms of Environmental Influence on Epigenetic Control and Transgenerational Epigenetic Inheritance Through the Gametes
A look at the mechanisms underlying some of the observations we discussed in week 5, through the study of model organisms. We’ll learn about metastable epialleles, which have allowed the study of transgenerational epigenetics in mice, and provided some evidence for transgenerational epigenetics in mammals.
Meet the Instructors
Dr. Marnie BlewittHead, Molecular Medicine Laboratory
Walter and Eliza Hall Institute of Medical Research
So, now we are going to go into more detail about trans-generational
epigenetic inheritance via the gametes in terms of mouse models.
So, in this first lecture, we are going to talk about the Agouti viable
yellow allele. So, we will consider both this allele in
terms of the trans-generational effects, but also in the next lecture we will
think about environmental effects on these allele because, it's such a good
model and the one that's best studied. And then in the subsequent lecture, we
will talk about axin fused, another allele which displays transgenerational
epigenetic inheritance in mice. And then towards the end of this week, we
are going to start to think about the mechanism using both of these models, but
mainly Avy as the example. And we will come back to the end thinking
about whats happening in humans. So, in this picture what you can see are
three genetically identical mice sitting on my hand.
So, these mice, despite the fact that one's completely yellow, one's completely
brown, it's called agouti or it basically looks like a normal wild-type mouse.
And then one is mottled, it has patches of brown and patches of yellow.
So, despite the fact I have these three distinctly different coat colours, they
are genetically identical at every locus that you can test for in the mouse.
So, these alleles, these mice are part of a strain called the agouti viable yellow
strain, and they carry the agouti viable yellow allele.
So, how is it they can be so different phenotypically, and yet genetically
identical? It's similar to if you knew monozygotic
twins. Identical twins that had different hair
colour, that were born with different hair colour, or different eye colour.
Something distinctly different about them that wasn't due to one of them dying
their hair, but rather, this is the way they were born.
So, in this case we know how this works. For the agouti viable yellow allele, we
have the agouti gene drawn here with the yellow exons.
But upstream of the agouti gene, about 100 kilobases upstream, there's an
IAP, an intracisternal A-particle that we've spoken about before.
It's a repetitive element that has jumped in upstream.
These IAPs, or intracisternal A-particles, this is actually acting in
this direction. So, it's direction of transcription for
itself is away from the agouti gene. However, it has a very very strong
promoter within its long terminal repeat that also acts in the other direction.
This direction here. So, and this can drive expression of the
agouti gene that's 100 kilobases away. So, the agouti gene normally only comes
on for a very short window during the hair cycle.
And it means that you get a long hair that is mostly brown or black, but has a
small fleck of yellow. Because what the agouti protein does is
that it's a singly molecule that causes a switch of this dark pigment production to
a yellow pigment production. However, if you have this constitutive
promoter that's happening, this very strong strong promoter in the IAP, long
term not repeat. That these driving expression of agouti
all the time constitutive expression of agouti, then you end up with an animal
that has a completely yellow coat. It's because there's, agouti is on all
the time, and therefore you always switch to making a yellow pigment.
It, actually this animal that has a yellow coat also has other issues,
because agouti is not only involved in pigment production, but is also involved
in other aspects of normal biology. And so, if you have agouti on all the
time, this animal ends up also being obese and diabetic.
So, what happens stochastically on occasion, is that this long terminal
repeat of the intracisternal A-particle will be silenced.
And so then, what happens is you get normal expression of the Agouti gene.
So, if we silence it here, and this is associated with DNA methylation, which is
shown here by these nice lollipop structures that I've shown you before.
We now also know that when it's silenced, it's also, this stochastic silencing is
also associated with changes to the chromatin packaging.
So, we have more tightly compacted chromatin, and the appropriate histone
modifications that are made associated with silence chromatin.
And if this happens, it means that the agouti gene can go about its normal
functioning. It's still just turned on for that short
window in the hair cycle, and so, you get an animal that is called pseudoagouti.
It's not like a normal agouti coloured mouse, because it's still has this
possession of these Avy allele, and so, that's why it's pseudoagouti.
It's pretending a normal wild type mouse. So, it will have this normal wild type
coat colour, but it will also be normal weight and will not suffer from type II
diabetes. So, the difference between these two
animals that are shown here is an epigenetic difference, and only an
epigenetic difference. They are genetically identical but
epigenetically different. So, it's these epigenetic differences
that exist at the long terminal repeat of the intercisternal A-particle which is
driving expression of the agouti gene and the agouti viable yellow allele.
That is defining the differences in their coat colour in their layered obesity, or
lack of obesity. So, the fact that you've got these two
very different phenotypes, despite genetic identity is termed variable
expressivity. So, this gene, the Agouti gene in the Avy
allele is variably expressed between genetically identical individuals.
Variable expressivity is a term that's been used often in human genetics
to describe this same sort of thing which is a variable outcome.
But in that case in human genetics, we don't necessarily know that the
underlying genetics are identical. It'll be a variable outcome despite
perhaps two people having the same mutation in a disease gene.
but we don't know necessarily know that the rest of their genome is identical.
In the case of mice, we do know the rest of the genome is identical.
So, it's a slight variance in this definition.
So, other than these perfectly yellow and perfectly agouti mice, there are also the
mice that live in the middle. So, they're the ones that have got the
patches of agouti coloured fur, and the patches of yellow fur.
So, these are called mottled mice, and you can really compare them to when we've
looked at the calico cat. We have this variegated expression of the
Avy allele. A variegated constitutive expression of
the agouti gene. So, these mosaics, these that are, these
mottled mice, mean that obviously in some cells you have active expression of
agouti. And that's because you've got an
unmethylated and open common structure at the controlling IAP.
But in contrast, you have other cells, other clones of cells where agouti is
only expressed in the normal hair cycle when it should do.
And this is associated with a silenced IAP because of DNA methylation and other
chromatin marks. So, the reason why you have these clonal
patches, these patches that you can actually see, patches of rather than
just being a mess throughout the whole of the mouse, where you have one cell that's
yellow, neighbouring another that's pseudoagouti.
The reason you have these visible patches, just like in the calico cat, is
because this epigenetic state, this stochastic epigenetic state at the IAP is
established at gastrulation, when there are very small numbers of cells.
This epigenetic state, just like for an X inactivation is then mitotically
heritable. So, you get this spectrum of phenotypes
with the Avy allele mice from purely yellow all the way through to purely
pseudoagouti, and a range in between of mottled animals.
So, this allele shows variegation, because you can get these animals that
display this mottled coat colour, and variable expressivity between these
genetically identical animals. We know that the epigenetic state is
established in early development, and it's heritable for the lifetime of this
organism. So, alleles that display these effects of
variegation and variable expressivity, particularly in the context of genetic
identity, in this case are termed metastable epialleles.
And this is a term which is used to describe them because they are
epialleles. They differ in the state, their
epigenetic state. And they're metastable because, while
they can be switched between generations, which I'll show you in the coming slides,
they are stable for the lifetime of that organism.
So, an animal with a yellow coat will always have a yellow coat.
They're not going to become mottled later in life.
Okay. So, now I want to think about this
switching between generations, and come back to the idea of trans-generational
epigenetic inheritance. So, I'm going to go through a number of
pedigrees so that we can think about how the agouti viable yellow allele phenotype
or their methylations take these inherited.
So, here at the top, we're thinking about a male that carries the agouti viable
yellow allele who has an active allele. He has a yellow coat.
And he is mated with a female that doesn't carry the Avy allele, and she has
a black coat. So, she carries a different allele.
She doesn't carry an agouti allele. She carries a non-agouti allele.
And basically with, from what it means is that you can ignore the contribution of
her alleles. So, while I've drawn her here,
we're not considering the pups that didn't inherit the agouti viable yellow
alleles. So, basically, you can ignore the
contribution from the black animals in these pedigrees.
What we're then looking at is in the offspring looking at their coat colours.
So, a yellow male, if you collect enough offspring, you'll find that 40% of his
offspring will have a yellow coat, 40% of his offspring will be mottled, and 40%
will be pseudo-agouti. So in other, I'm sorry 20% will be
pseudoagouti, I can't add up. So, the 20% that are pseudoagouti will
be methylated at that IAP in the Avy allele.
And the 40% for the yellow will be unmethylated at that IAP.
If you take a male that differs in his coat colour phenotype, here he is
pseudoagouti. So, he has a silent agouti viable yellow
allele, and it's heavily methylated. And you again look at proportions of
these different phenotypes in the offspring, the spectrum of phenotypes.
You find the same thing, 40% yellow, 40% mottled, 20% pseudoagouti.
So, despite the fact that these two males had, themselves had a different
expression of the Avy allele, their offspring show the same spectrum of
phenotypes. So, this suggests that there's clearing
and resetting between generations, because there's no memory of the
phenotype of the father. We don't have a skewing based on the DNA
methylation state and the expression state seen in the dad in this case.
But something quite different occurs when you look following maternal inheritance
of the Avy allele. So, now, on this right hand panel, we are
looking at a female that carries the Avy allele and it has a yellow coat which has
unmethylated allele mated with a black male.
Again, he's not contributing the coat colour alleles we are interested in.
so, we don't show the pups that inherit only his, that don't inherit the Avy
allele. Here, what we see is when the mother has
an active Avy allele which is hypomethylated.
And you find she has 50% of her pups have a yellow coat, 50% are mottled, and she
doesn't have any that are pseudoagouti. There are none that inherit the silent
allele. Whereas, if we look at a female that is
Pseudoagouti, so, has a silent methylated allele, she'll have 40% yellow, 40%
Mottled, and 20% Pseudoagouti. So, the difference between these two
spectrums coat colours in the offspring, there's a difference
because of the epigenetic state in the mother.
So, this is what would appear to be some sort of memory of the phenotype of the
mother. Not the genotype, these two animals are
genetically identical, the yellow mother, and the pseudoagouti mother.
But they are epigenetically different. So, this memory of the phenotype of
mother raises the possibility that this trans-generaltional epigenetic
inheritance through the gametes. But we need to do further experiments to
show this is true, and I'll explain what's being done in this case.
But this says that potentially, there's some of the epigenetic marks which are
found on one of these alleles which are passed through to the next generation.
Because of the difference in the phenotypes in the off spring.
So, what are the possibilities here if we think about those controls and the
questions that you could say. In the mouse, because we know and we can
prove that these animals are genetically identical, we actually don't need to
worry so much about genetic alterations. However, what we do know is that this
female will go on to be an obese, diabetic adult.
And when she's conceived and had babies, she's certainly at least overweight, if
not already obese. And so, could it be that the development
in the environment of an obese mother is what changes this spectrum of phenotypes
in the babies. So, could it be that it's the
intra-uterine environment that was experienced?
So, we need to think about this, and we also need to think about when is the
effect established? So, when is this coat colour effect
established? And is there any different behaviour of a
mother, this mother compared with that mother.
Okay. So, first of all we'll think about the
intra-uterine environment. The experiments that were done is they
took this same sort of breeding. They had a yellow female breeding with a
black male. But instead of allowing that mother to go
on and litter, they took the zygotes so, the fertilised eggs and they transferred
them into a mother that is not obese. She in fact is just a normal mouse in
that strain that doesn't carry the Avy allele, the black female.
So, she's normal weight, and she's not, she's not obese.
And if you take these eggs and transfer them over just after fertilisation, you
still get 40%, sorry 50% yellow, 50% mottled, the same as if you let the the
breeding happen naturally. So, we don't all of a sudden by changing
around the intrauterine environment in this case, cause some sort of shift in
the spectrum of phenotypes, or shift in the silencing of the Agouti viable yellow allele.
And this tells you that this effect is via the gametes.
We took the gametes that were from here, the fertilised egg, transfer them over,
and already, this was already predetermined back at those, in those
gametes, in the fertilised egg at least.
And so, this rules out the contribution of the intrauterine environment in this
case. What it doesn't rule out is that
oogenesis happens differently in this obese female or overweight female.
This was ruled out by separate experiments that were done through
breeding that I won't go through in detail in these experiments, but you'll
just have to believe me that they ruled out these differences as well.
So, in the case of the agouti viable yellow allele, we know that this is set
up in utero, and it's set up shortly after gestation.
We know this through detail of molecular analysis, but also because of those
patchy the size of the patches on that mottled mouse, just like with the calico cat.
So, we can now say that this is the
example, this is an example, at least in mice, where we do indeed see
trans-generational epigenetic inheritance through the gametes.
All of the controls have been performed. And it's because of this difference in
the phenotypes on this spectrum of phenotypes following female transmission
that we can see where we can see there's a memory of the phenotype of the mother.
A memory of the epigenotype of the mother.
So, in the next lecture what we're going to do is consider how the
environment can alter the epigenetic state of the agouti viable yellow allele.
And we'll think about this being a very epigenetically sensitive allele and
really a model to work with in terms of this, because first of all, we know a lot
about it. But secondly, coat colour’s very easy to
examine and so, it really gives us a tractable system to look at these sorts of questions.
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