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(ANDREW) Epigenetics in Development and Disease > Lecture 4 > Flashcards

Lecture 4 Flashcards

1
Q

Which parental genome is more methylated?

A

Paternal

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2
Q

What happens to parental genome after fertilisation?

A

Active demethylation involving TET3 - increasing levels of 5-hmC

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3
Q

Which sequences escape methylation erasure after fertilisation?

A

Imprinted genes and repetitive sequences

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4
Q

How is DNA packaged in sperm?

A

Protamines rather then histones

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5
Q

What is a polar body?

A

Small haploid cell that is formed concomitantly as an egg cell during oogenesis

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6
Q

Why does TET3 only act on the paternal genome?

A

PGC7/Stella protects the maternal genome from TET3

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7
Q

PGC7 is found in both pronuclei, so why is the paternal genome not protected from TET3 action?

A

PGC7/Stella binds with high affinity to H3K9me - a histone mark not present in the paternal genome

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8
Q

How was PGC7’s high affinity binding to H3K9me shown by Nakamura?

A

Two gtreatments PT and TP
PT; first fixation and then triton wash= nothing washed as its fixed
TP; first triton wash weakly bound nucleus and then fixed.
Both genomes have PGC7 in PT treatment.
Paternal genome loses PGC7 in TP treatment, however, maternal does not, because it is bound to the H3K9me.

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9
Q

How does the maternal genome lose its methylation marks?

A

Passive loss as the cells divide due to failure to maintain

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10
Q

Why is the active process required for the paternal?

A

Possibly due the paternal having a higher level to start with

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11
Q

Why demethylate post-fertilisation?

A

To allow expression of some key genes that will be off in the gametes; pluripotency genes and genes required for trophoblast formation (e.g. Elf5)

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12
Q

What does the inner cell mass give rise to?

And the trophoectoderm?

A

The embryo

The placenta and all extra-embryonic tissues

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13
Q

Which gene is first turned off in the ES cells?

A

Elf5, to stop them forming trophoectoderm

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14
Q

When does most remethylation occur?

Why at this stage?

A

Stage of implantation

Up-regulation of DNA methyltransferases; Dnmt3a, Dnmt3b, Dnmt1

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15
Q

What mechanisms have been put forward to explain CpG island hypomethylation?

A

1) Direct inhibition (less evidence)
2) Demethylation (by TET)
3) Steric Hinderance (proteins bound there blocking action)

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16
Q

How are some CpG islands turned off post-implantation?

A

Sequence-specific targeted DNA methylation

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17
Q

What did Feldman show about Oct4 inactivation?

A

Treating embryonic carcinoma cells, Oct4 expressing cells, with retonic acid switches off Oct4. First active histone marks H3K4me3 and H3K9,14A are removed, then repressive histone mark H3K9me3 is added and then the DNA is methylated. DNA methylation changes happen late in repression.

18
Q

Which transferase adds methyl group to H3K9?

A

The G9a methyltransferase

19
Q

What did Feldman show about G9a?

A

Required for maintenance of Oct4 repression but not the initial repression of the histones; G9a mutant still lost Oct4 due to retinoic acid treatment but removing the treatment in these mutants lead to Oct4 expression returning, whereas in wild type the Oct4 stays off.
Also required for DNA methylation of Oct4

20
Q

Outline repression of Oct4

A

Retinoic acid response element (RARE) in the Oct4 promoter binds GCNF transcription factor in presence of retinoic acid . GCNF will recruit G9a and HDACs. G9a methylation of H3K9. HP1 is recruited along with Dnmt3a/b to add 5mC marks and establish irreversible repression. In loss of G9a mutants the HDACs are enough to turn off the Oct4 but will noy keep it off when the retinoic acid is removed as the HP1 and Dnmt’s will not have been recruited

21
Q

How many epigenetic reprogramming events happen during normal mammalian development?

A

2

Once directly post-fertilisation and once during gamete formation

22
Q

How can epigenetic reprogramming events be induced artificially?

A

Somatic cell nuclear transfer

Induced pluripotent stem cells

23
Q

What effect does differentiation have on nuclear transfer?

A

Rapid loss in successful nuclear transfer events as cells differentiate

24
Q

What defects are common from SCNT?

A
  • Majority fail to develop after implantation
  • Large offspring syndrome
  • Obesity, immune problems and premature death
  • Failure to re-set epigenetic marks
25
Q

How did Taskashi induce pluripotent stem cells?

A

Introduction of Oct3/4. Sox2, Klf4 and c-Myc into adult human dermal fibroblasts using retroviral factors. The iPS cells have similar morphology, proliferation, gene expression and epigenetic status to normal ES cells

26
Q

Outline bisulfite sequencing

A

Treating cytosine and methylcytosine with bisulfite forms thymine and cytosine receptively. PCR the products and add into plasmid and sequence the clones, can see if methylated or un-methylated

27
Q

What does bisulfite sequencing show about iPS cells?

A

Less methylation than present in the dermal fibroblasts

28
Q

What is dosage compensation?

A

Ensures equal levels of X-chromosome gene products in males in females, in mammals= x-inactivation of female, in drosophila= x-activation in males and in c. elegans= half expression of X chromosomes in females

29
Q

What three methods are used to cause transcriptional silencing in X-inactivation?

A

1) Histone Modifications
2) DNA methylation of CpG islands
3) Establishment of heterochromatin

30
Q

What are the features of X inactivation?

A

Happens early in development, is clonal and regulated by the x-inactivation centre (Xic)

31
Q

Where is the inactive X-chromosome found in the nucleus?

A

In the barr body - highly condensed and associated with the nuclear membrane

32
Q

What two types of X-inactivation can be seen in female mammals?

A

1) Metatherians inactivate the paternal X chromosome (imprinted inactivation)
2) Eutherians (placental) use imprinted followed by random

33
Q

Outline X inactivation in eutherians embryos

A

Imprinted X inactivation between 4 cell and morula stage. In blastocyst stage - the trophoectoderm cells keep imprinted. In embryo cells both turned on and then the inactivation is random, and a patches arise within the developing foetus of which is on/off

34
Q

Why is this type of X inactivation good?

A

Paternal is easier to turn off because of its chromosome structure.
Turning of the paternal X in extra-embryonic tissue limits the chance off immune response?
Random later on means that in X-linked diseases not always the disease allele is expressed; sometimes appears selective as defective allele commonly selected against

35
Q

What are the three steps to X-inactivation?

A

1) Counting; ensures one X is inactivated per normal female diploid
2) Choosing; one X is chosen in a mutually exclusive manner
3) Silencing; be able to turn off an entire chromosome

36
Q

What region of the X chromosome is important for X inactivation?

A

A 1Mb region called the X inactivation centre (Xic)

37
Q

What element of Xic drives the inactivation?

A

X (inactive)-specific transcript (Xist)

17kb non-coding RNA, coats the inactivated X and recruits chromatin remodelling proteins (polycomb)

38
Q

How is Xist expression regulated?

A

Tsix (anti-sense to Xist)

39
Q

Outline the molecular events of X-inactivation

A

Tsix expressed form both X’s
Short RepA transcript expressed from both X’s, and interact with PRC2
Tsix expression switched off on future inactive X (Xi) and stays on on future active X (Xa)
Xist can now be expressed by Xi, which is further upregulated by PRC2-RepA
Xist co-transcriptionally recruits PRC2. This complex is recruited to the nucleation centre on Xic by YY1.
Xist-PRC2 complex spreads in cis across the Xi, possibly binding to LINE elements.
Blocking RNA POLII, adding repressive marks to histones and methylating the DNA

40
Q

Where is DNA methylation focused on Xi?

and Xa

A

On Xi it is in the CpG islands

On Xa it is within the gene (at a higher level overall)

41
Q

Why do you see transient pairing of X chromosomes?

A

Thought that the X chromosome come together to share transcription factors the future Xi losses all transcription factor for Tsix -allowing Xist expression and X-inactivation
Also important for counting how many X chromosomes they have

(ANDREW) Epigenetics in Development and Disease (8 decks)

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