Epigenetic reprogramming

Question 1

Problem with Maternal and paternal imprinting

Answer 1

Sometimes (half the time!) males go on to have female offspring
Sometimes (the other half of the time) females go on to have male offspring
This requires resetting of the imprints to the sex of the zygote/embryo

Question 2

How many times does DNA methylation occur?

Answer 2

Twice

Question 3

WHat needs to happen before next generation

Answer 3

Epigenetic signals need resetting.

Question 4

Epigenetic reprogramming occurs twice during development – when?

Answer 4

During gamete formation
Post-fertilisation

Question 5

WHat do imprinted genes always exhibit?

Answer 5

Imprinted genes always exhibit the methylation pattern of the parent in sperm or eggs regardless of whether they came from the maternal or paternal genome.

Question 6

WHen do imprinted genes reset?

Answer 6

Imprinted genes are re-set in the developing gamete and bypass epigenetic reprogramming in the early embryo

Question 7

What is the problem with imprinted genes and what does this mean?

Answer 7

But with imprinted genes, we inherit only one working copy.

Depending on the gene, either the copy from mom or the copy from dad is epigenetically silenced.

Silencing usually happens through the addition of methyl groups during egg or sperm formation.

The epigenetic tags on imprinted genes usually stay put for the life of the organism. But they are reset during egg and sperm formation.

Regardless of whether they came from mom or dad, certain genes are always silenced in the egg, and others are always silenced in the sperm.

Question 8

What happens the epigenetic tags soon after sperm and egg meet?

Answer 8

Soon after egg and sperm meet, most of the epigenetic tags that activate and silence genes are stripped from the DNA.
However, in mammals, imprinted genes keep their epigenetic tags. Imprinted genes begin the process of development with epigenetic tags in place.

Question 9

How might studying imprinting help researchers?

Answer 9

Understand how other genes make it through reprogramming without losing thei epigenetic tags.

Question 10

Mammalian life cycle

Answer 10

Primordial germline cells (PGCs) are specified during early embryonic development
PGCs migrate to the (developing) gonads
Meiosis
Gamete differentiation
Fertilization of oocyte by sperm
Totipotent zygote formation
Primordial germline cells are specified…

Question 11

What is the purpose of genome-wide epigenetic reprogramming

Answer 11

Gamete formation
Pre-implantation embryo

Question 12

Gamete formation

Answer 12

Re-sets imprinted genes for the sex of the embryo
Erases parental of acquired epigenetic memories (e.g. environmental)
Facilitates gametogenesis
Maintain the silencing of transposable elements
Reduces the mutation rate in the germline

Question 13

Pre-implantation embryo

Answer 13

Re-sets zygotic epigenetic genome for naïve pluripotency
Some evidence of maternal vs paternal genome wars

Question 14

What are Primordial germ cells regarded as?

Answer 14

Whats regarded as the first step in acquisition of totipotency and continuation od the life cycle

Question 15

WHat are PGC?

Answer 15

PGCs are the founder cells for the germline
PCGs migrate to the (developing) gonads where they undergo meiosis and differentiate into eggs and sperm

Question 16

What does fertilisation result in?

Answer 16

Fertilisation results in a totipotent zygote; gives rise to all cells, including germ cells again
Figure 1 from Tang et al

Question 17

What do gondadal germ cells undergo?

Answer 17

During foetal development and adulthood gonadal germ cells undergo meiosis and gametogenesis to differentiate into sperm and eggs.

Question 18

What occurs for the generation of a zygote upon fertilisation?

Answer 18

The genome is re-methylated and acquires appropriate epigenetic signatures for the generation of a zygote upon fertilisation

Question 19

What do migratory PGCs undergo?

Answer 19

Migratory PGCs undergo genome-wide epigenetic reprogramming to erase imprints and other somatic epigenetic memories

Question 20

During migration of PGCs, somatic epigenetic memories are erased by?

Answer 20

Global DNA demethylation
Genomic imprint erasure
X-chromosome reactivation
Reorganisation of chromosome

This is the same in male (XY) and female embryos (XX)
End of PGC stage of germline development -
XY germ cells then enter mitotic arrest
XX germ cell then enter meiosis

Question 21

DNA methylation profile in the germline

Answer 21

Migratory primordial germ cells (PGCs) become demethylated in early development.
Remethylation begins in spermatogonial stem cells (SSCs) in males
Remethylation begins after birth in growing oocytes

From 70% to 4% methylation

Question 22

When are Primordial germ cells specificed?

Answer 22

Post-implantation epiblast.

Question 23

Describe genome-wide DNA methylation

Answer 23

DNA is hypermethylated (70%) and the PGCs are primed for differentiation.

At the onset of migration, PMCs undergo genome-wide demethylation (to 4%)

Almost all genomic features (including imprint control elements) become hypomethylated

This is thought to act mostly via a passive mechanism

DNA Methylation is re-established in a sex-specific manner after E13.5 in males

DNA Methylation is re-stablished in a sex-specific manner after birth in females

Question 24

What is DNMT1 loss essential for?

Answer 24

Maintenance of DNA methylation

Question 25

What does TET1 replace>

Answer 25

Meth cyt with unmethylation cytosine. (liten to lecture slide)

Question 26

However, why only 4% of genomic loci remain methylated in PGC

Answer 26

Vast majority of ‘escapees’ are associated with retrotransposable elements
The mechanism is not known
The current assumption is that it is due to incomplete repression of DNA methylation pathway – but that’s a best guess

Question 27

WHat is DNA methylation essential for?

Answer 27

Imprint erasure.

Question 28

WHat is erased in PGCs?

Answer 28

Allele-specific methylation at imprinting control regions (ICRs)

Question 29

When is imprinting reestablished?

Answer 29

Re-established in a sex-specific manner later in gamete development.

Question 30

Maternal locus is methylated and silenced?

Question 31

What’s happening in male gametes to the (imprinted) IGFR2 locus?

Answer 31

Methylation status of the IGFR2 gene is examined by Southern blot analysis.

DNA digestion with the methylation-sensitive restriction enzyme. (MluI digestion is blocked at CpG methylated sites)

Distinguishes between methylated and unmethylated alleles

In male ES cells both copies of Igf2r are unmethylated (lane 1)
Geijson 2004

Question 32

Demethylation folloing fertilisation (Epigenetic resetting in the pre-implantation embryo)

Answer 32

Immediately following fertilization the paternal genome is demethylated by an active mechanism

The female genome is demethylated by a passive mechanism that requires DNA replication

Question 33

DNA methylation patterns in pre-implantation embryos.

Answer 33

At fertilisation and implantation both genomes are methylated

The paternal genome is demethylated by an active mechanism immediately after fertilisation

The maternal genome is demethylated by a passive mechanism

Methylated imprinted genes do not become demethylated

Unmethylated imprinted genes do not become re-methylated

Question 34

WHat is essential for normal development and establishing totipotency?

Answer 34

These waves of demethylation and re-methylation in the early embryo are essential for normal development and establishing totipotency.

Question 35

Demethylation and erasure of imprinting marks during gametogenesis

Answer 35

Maternal germline requires all oocytes to carry the maternal epigenetic mark
Paternal genome must carry paternal marks.

Question 36

Post-fertilisation, how are paternal and maternal imprints maintained in the developing zygote?

Answer 36

When the male pronucleus enters the oocyte (nearly) all methylation is actively removed from the paternal genome.

The mechanism by which some genes bypass the reprogramming is not known, but long non-coding RNAs are a candidate mechanism

Whatever the mechanism, parent-of-origin specific DNA methylation is transmitted at imprinted loci. Imprints must be maintained during global DNA methylation in the pre-implantation embryo and persist in somatic cells

Question 37

Establishment, maintenance and erasure of genomic imprints in mouse development 1
- What do Primordial germ cells undergo?

Answer 37

In the germline, primordial germ cells (PGCs) undergo multiple changes in chromatin structure and DNA demethylation during migration into the genital ridge (gonad).

Question 38

Establishment, maintenance and erasure of genomic imprints in mouse development 2
- How are imprints acquired?

Answer 38

Imprints are then acquired in a sex-specific manner in the germline (green shading).

Question 39

Establishment, maintenance and erasure of genomic imprints in mouse development 3
- How is DNA methylation targeted?

Answer 39

DNA methylation is targeted specifically to paternally and maternally DNA-methylated ICEs—prenatally in prospermatogonia and postnatally during oocyte maturation.

Question 40

Establishment, maintenance, and erasure of genomic imprints in mouse development 4 - How are these imprints maintained?

Answer 40

These imprints are maintained despite global changes in DNA methylation after fertilization (orange shading): active demethylation of the paternal genome in the zygote and passive maternal demethylation in the preimplantation embryo.

Question 41

Establishment, maintenance, and erasure of genomic imprints in mouse development 5 - WHat protects methylation regions?

Answer 41

Candidates for protection of
methylation regions include ZFP57 and PGC7/STELLA.

Question 42

Establishment, maintenance, and erasure of genomic imprints in mouse development 6 - What does De novo DNA methylation of the genome begin?

Answer 42

De novo DNA methylation of the genome begins at the morula stage, during which time unmethylated alleles of imprinted genes must be protected.

Question 43

Establishment, maintenance, and erasure of genomic imprints in mouse development 7 - What the difference in the maintenance of imprints in somatic cells and extraembryonic tissue

Answer 43

These imprints are maintained in somatic cells throughout the lifetime of the organism, whereas imprinting in extraembryonic tissues is thought to be less dependent on maintenance of DNA methylation.

Question 44

Establishment, maintenance, and erasure of genomic imprints in mouse development 8 - In the germline what happens to imprints?

Answer 44

In the germline, imprints are erased and reset
for the next generation (red shading).
PTM, post-translational modification;
MAT, maternal genome;
PAT, paternal
genome.

Question 45

Self-assessment

Answer 45

When does genome-wide demethylation occur during mammalian development? (Hint – it is more than once!)

What is the purpose of epigenome resetting in the germline?

What would happen to an imprinted gene (e.g. UBE3A (causes Angelman Syndrome)) in the germline?

Describe a mechanism that has been suggested to protect imprinted genes from the wave of genomic reprogramming in the developing embryo? (Advanced question - requires reading!)

Name an enzyme (or two!) responsible for establishing imprinting marks (Requires reading!)