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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The University of Melbourne

Epigenetic Control of Gene Expression

391 ratings

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

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.

6.1 Mouse and rat studies on paternal effects of chemical exposure, effects of maternal behaviour on epigenetic makeup14:52

6.2 Transgenerational epigenetic inheritance via the gametes10:13

6.3 The Agouti viable yellow allele in mice15:53

6.4 Environmental effects on the Agouti viable yellow allele11:41

6.5 The Axin fused allele in mice and metastable epialleles14:05

6.6 Potential mechanisms of transgenerational epigenetic inheritance: incomplete epigenetic reprogramming9:18

6.7 Paramutation in plants and paramutation-like effects in mice13:14

6.8 Constitutional epimutation in humans - not transgenerational epigenetic inheritance12:30

Meet the Instructors

Dr. Marnie Blewitt
Head, 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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