Lecture 4 Flashcards
Which parental genome is more methylated?
Paternal
What happens to parental genome after fertilisation?
Active demethylation involving TET3 - increasing levels of 5-hmC
Which sequences escape methylation erasure after fertilisation?
Imprinted genes and repetitive sequences
How is DNA packaged in sperm?
Protamines rather then histones
What is a polar body?
Small haploid cell that is formed concomitantly as an egg cell during oogenesis
Why does TET3 only act on the paternal genome?
PGC7/Stella protects the maternal genome from TET3
PGC7 is found in both pronuclei, so why is the paternal genome not protected from TET3 action?
PGC7/Stella binds with high affinity to H3K9me - a histone mark not present in the paternal genome
How was PGC7’s high affinity binding to H3K9me shown by Nakamura?
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.
How does the maternal genome lose its methylation marks?
Passive loss as the cells divide due to failure to maintain
Why is the active process required for the paternal?
Possibly due the paternal having a higher level to start with
Why demethylate post-fertilisation?
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)
What does the inner cell mass give rise to?
And the trophoectoderm?
The embryo
The placenta and all extra-embryonic tissues
Which gene is first turned off in the ES cells?
Elf5, to stop them forming trophoectoderm
When does most remethylation occur?
Why at this stage?
Stage of implantation
Up-regulation of DNA methyltransferases; Dnmt3a, Dnmt3b, Dnmt1
What mechanisms have been put forward to explain CpG island hypomethylation?
1) Direct inhibition (less evidence)
2) Demethylation (by TET)
3) Steric Hinderance (proteins bound there blocking action)
How are some CpG islands turned off post-implantation?
Sequence-specific targeted DNA methylation
What did Feldman show about Oct4 inactivation?
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.
Which transferase adds methyl group to H3K9?
The G9a methyltransferase
What did Feldman show about G9a?
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
Outline repression of Oct4
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
How many epigenetic reprogramming events happen during normal mammalian development?
2
Once directly post-fertilisation and once during gamete formation
How can epigenetic reprogramming events be induced artificially?
Somatic cell nuclear transfer
Induced pluripotent stem cells
What effect does differentiation have on nuclear transfer?
Rapid loss in successful nuclear transfer events as cells differentiate
What defects are common from SCNT?
- Majority fail to develop after implantation
- Large offspring syndrome
- Obesity, immune problems and premature death
- Failure to re-set epigenetic marks
How did Taskashi induce pluripotent stem cells?
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
Outline bisulfite sequencing
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
What does bisulfite sequencing show about iPS cells?
Less methylation than present in the dermal fibroblasts
What is dosage compensation?
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
What three methods are used to cause transcriptional silencing in X-inactivation?
1) Histone Modifications
2) DNA methylation of CpG islands
3) Establishment of heterochromatin
What are the features of X inactivation?
Happens early in development, is clonal and regulated by the x-inactivation centre (Xic)
Where is the inactive X-chromosome found in the nucleus?
In the barr body - highly condensed and associated with the nuclear membrane
What two types of X-inactivation can be seen in female mammals?
1) Metatherians inactivate the paternal X chromosome (imprinted inactivation)
2) Eutherians (placental) use imprinted followed by random
Outline X inactivation in eutherians embryos
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
Why is this type of X inactivation good?
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
What are the three steps to X-inactivation?
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
What region of the X chromosome is important for X inactivation?
A 1Mb region called the X inactivation centre (Xic)
What element of Xic drives the inactivation?
X (inactive)-specific transcript (Xist)
17kb non-coding RNA, coats the inactivated X and recruits chromatin remodelling proteins (polycomb)
How is Xist expression regulated?
Tsix (anti-sense to Xist)
Outline the molecular events of X-inactivation
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
Where is DNA methylation focused on Xi?
and Xa
On Xi it is in the CpG islands
On Xa it is within the gene (at a higher level overall)
Why do you see transient pairing of X chromosomes?
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
