L6 DNA Methylation II: Demethylation and Reprogramming Learning Objectives

Distinguish passive from active demethylation, and say which of the two requires DNA replication

• Passive demethylation:

1. Loss of methylation due to lack of maintenance of DNA methylation

2. Two rounds without DNMT1: hemimethylated, then unmethylated

3. Requires DNA replication

• Active demethylation:

1. Removal of existing methyl marks

2. Deamination of 5mC to thymidine by activation-induced deaminase (AID)

3. Creates T:G mismatches – T replaced by C by TDG or MBD4

4. Discovered when 5-hydroxy-methylcytosine (5hmC) was identified

Follow a fully methylated CpG through two rounds of replication without DNMT1, and name the intermediate state

The Starting Point

You start with 1 DNA molecule that is fully methylated (methyl groups on both strands).

Round 1 of Replication (No DNMT1)

• The 2 strands separate to make 2 new DNA molecules.

• Since DNMT1 is missing, the new partner strands are built completely unmethylated.

• Result: 2 DNA molecules that are half-methylated.

• Intermediate State Name: Hemi-methylated DNA (or semimethylated DNA).

Round 2 of Replication (No DNMT1)

The 2 hemi-methylated molecules separate to make 4 total DNA molecules:

• The 2 methylated strands pair with new unmethylated strands —> 2 Hemi-methylated DNA molecules (50%)

• The 2 unmethylated strands pair with new unmethylated strands —> 2 Fully unmethylated DNA molecules (50%)

Summary:

Missing DNMT1 causes the original methyl marks to get diluted by half with every cell division until unmethylated DNA is produced

Trace active demethylation: AID deamination of 5mC giving T:G mismatches repaired by TDG or MBD4, and TET oxidation of 5mC to 5hmC, 5fC and 5caC, returned to C by base excision repair

• Deamination of 5mC to thymidine by activation-induced deaminase (AID)

• Creates T:G mismatches – T replaced by C by TDG or MBD4

• Discovered when 5-hydroxy-methylcytosine (5hmC) was identified

• 5hmC is produced via action of Ten-eleven translocation methylcytosine dioxygenase (TET)

• 5hmC can not act as a substrate for DNMT1 (maintenance DNA methyltransferase) – this leads to passive demethylation

• 5hmC, 5fC (5-formylcytosine), 5caC (5-carboxylcytosine) can be converted to C through base excision repair via glycosylase

Explain why TET feeds both routes — 5hmC is a poor substrate for DNMT1, so oxidation also causes passive loss

• TET feeds both routes: BER removal and dilution

• 5hmC is a poor substrate for DNMT1, so oxidation also causes passive loss

Describe early mammalian development from zygote to blastocyst, and say which lineage makes the embryo proper, and which makes the placenta

• Cells are totipotent between zygote and morula stage. Morula = 16 cells

• Transition from morula to blastocyst produces inner cell mass (ICM)

• ICM produces pluripotent embryonic stem (ES) cells and makes all body organs

• Trophectoderm (TE) cells make placenta

Name the two genome-wide demethylation cycles in the mammalian life cycle, say when each occurs, and give what each accomplishes

• Two distinct cycles of large-scale demethylation and remethylation

• Differentiation of primordial germ cells:

1. Allows sex-specific imprinting

• After fertilization and before implantation:

1. Crucial for establishing pluripotency

Place PGC demethylation on a developmental timeline (specification ~E6.0, migration ~E8.0, complete by ~E11.5) and give the levels reached: under 10%, against over 70% in surrounding somatic cells

• Primordial germ cells (PGC) are precursors of germ cells

• PGC originate from embryonic cells that start to acquire somatic fate

• At their origin, PGC have methylation levels similar to their parental somatic cells

• Chromatin structure and transcription profiles are also similar to parental somatic cells

• As the PGC start migrating to genital ridge, their methylation is mostly removed such that total methylation is <10% compared to >70% methylation in other cells in the embryo

• Parental imprints are removed

• X chromosome in females is reactivated

Distinguish the two waves of PGC demethylation

• There are two waves of PGC demethylation. First is passive and second is active demethylation

• First stage – passive demethylation:

1. De novo DNA methyltransferases DNMT3a/b are suppressed

2. DNMT1 present but its cofactor NP95 is absent

3. Replication dependent demethylation causes higher amount of hemi-methylated DNA

4. X chromosome and imprinted genes retain some methylation during this phase

• Second stage – active demethylation:

1. Involves conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)

2. Involves TET and AID proteins

3. X chromosome and imprinted genes are demethylated

4. Most of DNA methylation is removed in PGC

5. Except some retroelements

Explain sex-specific remethylation of PGCs and say why the male germline accumulates more maintenance errors

• In males, PGC methylation levels are fully established by birth:

1. DNA methylation levels are maintained during life leading to adulthood – several rounds of mitosis

2. Chances of errors in maintaining methylation are higher in males

3. Male germline keeps dividing; female stays in meiotic arrest

• In females, re-methylation occurs after birth during oocyte growth:

1. De novo DNA methylation is required to put sex-specific imprints

2. Loss of de novo DNA methylation causes sperm apoptosis

3. Such loss in oocytes causes death of embryo

Contrast paternal and maternal demethylation after fertilization: Tet3 translocation to the paternal pronucleus against replication-dependent dilution of the maternal genome; say when remethylation begins and give one hypothesis for why the paternal genome is actively demethylated

• Starts after zygote formation – before implantation to uterus

• Imprinted genes are not demethylated

• Maternal and paternal chromosomes show different kinetics of demethylation

• Remethylation begins at the blastocyst, as the ICM methylation goes high while trophectoderm methylation stays low

• Tet3 expressed in oocyte and zygote

• After fertilization, Tet3 protein moves from cytoplasm to male paternal nucleus

• Converts 5mC to 5hmC/5fC/5caC

• Paternal genome undergoes rapid and almost complete demethylation:

1. This is very dramatic as 90% of the sperm genome is methylated!

2. Demethylation of paternal genome is active

3. 5hmC is then diluted as the embryo divides toward the morula

• Maternal genome under passive demethylation:

1. Oocytes have lower (~40%) levels to begin with

2. Maternal genome appears resistant to active demethylation (e.g., TET)

• Why is paternal genome actively demethylated:

1. One hypothesis is that it allows maternal control by removing any distinguishing paternal epigenetic marks

2. Removes any marks that may favor a specific embryo

3. Allows equal distribution of resources to embryos

The two reprogramming cycles (not part of the learning objectives)