Lecture 4 - Epigenetics in Animal development

Question 1

What is the pattern of DNA methylation dynamics in mammalian developement?

Answer 1

Paternal/maternal gene methylation patterns vary

Paternal genes demethylated rapidly in the embryo, maternal slower. Imprinted genes maintain DNA methylation

Question 2

What are the dynamics of mammalian DNA methylation?

Answer 2

DNA methyltransferases

Dnmt3

• methylates unmethylated templates
• to restablish patterns

Maintanance methylases (Dnmt1)

• closely assocaited with replication fork and keep pattern going during replication

Different ways of losing

• Active demethylation
• Passive demethylation

Question 3

What proteins are involved in the pathways for DNA demethylation in mammals?

Answer 3

• TET proteins convert 5mc to 5hmc
• Can be removed in many ways - active removal

Question 4

Describe DNA methylation dynamics in mammalian development

Answer 4

• large scale erasure of DNA methylation post-fertilisation
• some sequences (e.g. imporinted genes and some repetitive elements) escape this
• paternal demethylation is active and involves TET3
• DNA demethylation starts at the point of fertilisation
• Paternal genome is more heavily methylated than the maternal genome, rapidly demethylated and accumulates 5hmc

Question 5

What is the methylation status of the sperm and the eggs?

Answer 5

• sperm and eggs are differentially methylated
• also are differently packaged
• paternal: few histones, extra compact chromatin
• paternal genome undergoes a rapid demethylation in the zygote prior to the first mitosis
• looked at pronucleus levels of 5hmc in male which rise as 5mc fall
• Antibody staining of pronuclei (5hmc/5mc) in cell

Question 6

What is a polar body?

Answer 6

Defunct products of meiosis not passed on

Question 7

How is TET3 involved in paternal/maternal demethylation?

Answer 7

• Tet3 is highly expressed in the oocyte and acts on the paternal genome
• PGC7/Stella (expressed in oocytes) is required for the protection of the maternal genome
• In PGC7/Stella mutant staining 5MeC and DAPI, maternal genome is also demethylated

Question 8

Where does PGC7/Stella localise to?

Answer 8

• PGC7/Stella localises to both pronuclei following fertilisation
• Fertilised egg stained by antibodies for PGC7/Stella and DAPI
• but only protects the maternal DNA from active demethylation

Question 9

What is the experimental evidence that PGC7 is less strongly bound in the paternal pronucleus?

Answer 9

• PGC7 in the paternal pronucleus can be removed with treatment with triton
• H3K9me2 - found in the maternal but not paternal pronucleus
• PGC7 protects the maternal genome as it binds with higher affinity to the presence of H3K9me2 (histone methylation mark)
• Preventing the action of TET3

1. 2 treatments
   
   1. PT condition - treated with PFA fixation (paraformaldehye - fixes proteins in place) then triton (removes molecules that are not fixed in place) treatment then immunostaining of PGC7
   2. TP treament - treated with triton, then PFA fixation, then immunostaining of PGC7
2. PT results: PGC7 in both pronuclei, H3K9me2 just in maternal pronuclei (also stained with DAPI)
3. TP results: PGC7/Stella see loss in the paternal nuclei - less tightly bound

Explains quick loss of parental methylation whereas maternal is diluted by failure to maintain during replciation

Question 10

Why demethylate post fertilisation?

Answer 10

• To allow expression of some key genes e.g.
  
  • pluripotency genes e.g. Oct4 - methylated in gametes, essential to demethylate to activate
  • genes required for trophoblast formation e.g. the transcription factor Elf5

Question 11

When does transcription start to occur in the embryo?

Answer 11

Transcription starts to occur at the 2 cell embryo stage

Question 12

What happens at the Morula stage?

Answer 12

• dividing cells give rise to the embryo/extra embryonic tissue
• EIf5 methylated in gametes, needs to be unmethylated to form the trophectoderm by remethylated very quickly in the inner cell mass
• ESC is not fully pluripotent, ICM won’t give rise to trophectoderm - occurs v early in blastula stage

Question 13

What occurs at the start of implantation in terms of methylation?

Answer 13

• most remethylation occurs around the stage of implantation
• up regulation of DNA methyltransferases, Dnmt3a, Dnmt3b, Dnmt1
• Global DNA remethylation apart from at CpG islands

Question 14

What are the mechanisms for CpG island hypomethylation?

Answer 14

1. Direct inhibition: dense clustering of CpGs not a good target for methyltransferases (DNMT)
2. Demethylation: Undergoes active demethylation by action of TET enzymes
3. Steric hindrance/binding inhibition: Factors that block the activity of enzymes. RNApol may be present but not actively transcribing - blocking methylation by DNMT3, DNMT3L. Active H3K4me3 marks also help to inhibit methylases.

Question 15

What is the pattern of DNA methylation in DNA replication, cell division and post implantation ?

Answer 15

• global pattern of CpG DNA methylation is established quickly and maintained through DNA replication and cell division
• followed by sequence specific targeted DNA methylation of some genes post implantation
• CpG islands stay unmethylated throughout the life of the organism

Question 16

What is the experimental evidene for the dynamics of repression of pluripotency genes?

Answer 16

Oct4

• embryonic carcinoma cells express high levels of Oct4
• Oct4 is rapidly switched off when cells are treated with retinoic acid
• DNA methylation is a late event in the inactivation of Oct4
• on in embryonic stem cells
• off in differentiated cells

Question 17

What is the experimental evidence that G9a H3K9 methyltransferase is required for the maintenance of Oct4 repression ?

Answer 17

• Use cell culture model for looking at Oct4 repression
  
  • is this appropritate for looking at the actual developing embryo?
• Treat with retinoic acid (RA) see that Oct4 is rapidly turned off
• Looked at different markers
  
  • oct4
  • methylation
  • histone marks
• Found
  
  • oct4 repressed quickly
  • Me-H3 (acitve histone mark) and Ac-H3 lost quickly
  • K9 repressive mark increases, followed by DNA methylation

Question 18

What is the experimental evidence that G9a H3K9 methyltransferase is required for DNA methylation of Oct4 (and other genes) during ES cell differentiation?

Answer 18

• Used cells mutant G9a compared to WT
• RA triggers loss of stem-ness and start of differentiation
• In WT (with RA) have methylation of Oct4 and other genes including Nanog
• In G9a -/- (with RA) no methylation of Oct4 and other genes
• In WT oct4 stays off when RA is removed (shows process is an epigenetic mechanism)

G9a not involved in initial repression but involved in maintenance of repression

Question 19

What is the model for the repression of Oct4?

Answer 19

• Oct4 has a retionic acid response element (RARE) in its promoter
• GCNF transcription factor binds to the RARE sequecce in the presence of RA
• This recruits G9a and HDACs (remove active marks)
• G9a will methylate H3K9me - preventing
• HP1 is recruited along with DNMT3a/b to add 5mC marks and establish irreversible repression
• can’t perpetuate mark is not got modified histones

Question 20

When do epigenetic reprogramming events occur?

Answer 20

• occur twice during normal mammalian development
• can be induced artificially
  
  • somatic cell nuclearr transfer (SCNT)
  • creation of stem cells from somatic cells (induced pluripotent stem cells iPS cells)

Question 21

What did Yamanaka and Gurdon win the Nobel Price in Medicene for?

Answer 21

Teh discovery that mature cells can be reprogrammed to become pluripotent

Question 22

Can re-programming be induced artifically?

Answer 22

• If take donor nuclei from different embryonic stages there is a bigg difference in the nuclear transfer success rate
• Nuclear transfer success rate decreases as donor cells differentiate
• Due to leveepigenetic marks at different stages
• For genetic engineering - take from fetal fibroblasts

Question 23

What defects do cloned animals derived via SCNT show?

Answer 23

• majority fail to develop after implantation
• newborn clones often show Large Offspring Syndrome
• Aging cloned mice often become obese, develop immune problems or die prematurely
• failure to reset epigenetic marks is likely to be a contributing factor
• Effects from the HongerWinter - some genes escape reprogramming

Question 24

How did Takahashi et al produce pluripotent stem cells from adult human fibroblasts?

Answer 24

• intoduction of Oct3/4, Sox2, Klf4 and c-Myc into human dermal fibroblasts using retroviral vectors
• induced pluripotent stem cells were obstained with similar morphology, proliferation, gene expression and epigenetic status to normal ES cells
• therefore showed that histone marks are dynamic
• proteins bound to DNA are not bound permenently, can reverse state by overexpressing SC master regulators
• but experiments were very ineffecicent

Question 25

How is DNA methylation status analysed?

Answer 25

DNA methylation staus is analysed by bisulfite sequencing * can look at the methylated cysteine marks in the promoter regions of genes * looked at three iPS lines and the methylation status of Oct3/4, Rex1 and Nanog compared to the methylation status of these genes in human dermal fibroblasts (much more methylated) * needed actual stem cell as control (does it look like iPS?)

Question 26

* What is the process of bisulfide sequencing\>

Answer 26

1. cytosine converted to uracil (read as T in DNA) by sodium bisulfide 2. methylated-c does not get converted by NaBS 3. DNA extracted, treated with NaBS, PCR used to amplify 4. PCR generated DNA strands 5. These are sequenced 6. Results in a mixed profile whether c or methylated c 7. Take PCR product (lots of different strands) and clone in plasmid vector 8. each plasmid will have an independent PCR product, reflecting one of the original strands of DNA that was treated Each row is a single clone If write as % lose information about how strands differ

Question 27

What is dosage compensation and X inactivation?

Answer 27

* Dosage compensation ensures equal levels of X chromosome gene products in males and females * X-inactivation in female mammals * X-activation in male drosophila * havling the rate of transcription from each X chromosome in C elegans * Dosage compensation mutants are lethal * Occurs early in development, maintained throughout the biology of the organism

Question 28

What are the features of X-inactivation in mammals?

Answer 28

* Occurs at a large scale * Clonal * Transcriptional silencing involving histone modifications, DNA methylation of CpG islands and establishment of heterochromatin * established early in development * regulated by the X-inactivation centre (Xic)

Question 29

What are the two different X-inactivation modes of regulation identified in female mammals?

Answer 29

* Metatherians (e.g. kangeroo) inactivate the paternal X (imprinted inactivation) * Eutherians (placental) use imprinted X inactivation followed by random reversal process

Question 30

Give a visual exaple of X inactivation in mammals

Answer 30

Demonstrated by tortoishell cat * patches of colour * X chromosomes have 2 alleles for coat colour, which is turned off in different patches * this occurs early in individual cells, resulting in patches *

Question 31

What was the earliest indication of X inactivation involvement in disease?

Answer 31

Microscopy identified regions of Barr bodies with inactivated X chromosomes associated with the nuclear membrane

Question 32

What are the developmental dynamics of X-inacitvation in placental mammals?

Answer 32

1. between 4 cell stage and morula stage, Xp is inactivated 2. At the blastocyst stage: Trophectoderm keeps paternal X inactivated, but in the innercell mass get paternal X reactivation. ES cells are XaXa 3. At the epiblast stage, get random x inactivation resulting in clonal patches of paternal/maternal X inactivation 4. Germline cells are identified in the epiblast - X inactivation reverses again - both p/m off

Question 33

What are the 3 steps to X inactivation?

Answer 33

1. Counting: Ensures one X chromosome is inactivated per normal female diploid nucleus 2. Choosing: One X is vhosen in a mutually exclusive way to be the future inactive X (Xi) 3. silencing: initiated by coating the future Xi by the non-coding Xist RNA, recruitment of silencing factors and condensation of Xi chromatin

Question 34

What are the features of the X inactivation centre (Xic)?

Answer 34

* 1 Mb region on all X chromosomes * several elements that have a role in X inactivation * X(inactive)-specific transcipt (XIST) * 17kb non coding RNA * coats the inactived X * recruits chromatin modifying proteins (polycomb) * Tsix (anti-sense to Xist) regulates Xist expression

Question 35

How can the coating of Xi by Xist be viewed?

Answer 35

By in situ hybridisation * take cell that has undergone X inactivation * Yellow- x chromosome * Red - inactive X chromosome

Question 36

What are the molecular events during the onset of X inactivation?

Answer 36

1. **Before inactivation:** 1. Tsix expressed from both X's 2. Short RepA transcript expressed from both X's 3. RepA transcript thought to interact with PRC2 2. **Onset of X-inactivation:** 1. ​Tsix expression switched off from the future inactive X (Xi) 2. PRC2-RepA recruited to XIC locus of Xi 3. Xist transcript is upregulated from Xi 4. Continued expression of Tsix on the future active X (Xa) prevents recruitment of PRC2-RepA 3. 1. Xist co-transcriptionally recruits PRC2 (Xa) 2. A transcription factor (YY1) binds the nucleation centre on the Xi 3. continued expression of Tsix from Xa 4. Xist-PCR2 cotranscriptionally loads onto the YY1 nucleation centre and spreads in cis across the Xi by binding repetitive LINE elements

Question 37

What X chromosome changes are triggered by Xist?

Answer 37

* Xist RNA produced from future Xi * Coats the future Xi * Recruits polycomb complexes PCR2 and PCR1 * H3K27me, H4K20me, H2AK119-ubiquitination * DNA methylation changes occur late, where it becomes self-maintaining

Question 38

How are CpG islands involved in X inactivation?

Answer 38

* X inactivation involves methylation of CpG islands on the inactive X * Overall Xi has less methylation than Xa but at these specific regions

Question 39

What is seen if the X-inactivation process is followed by in situ hybridisation?

Answer 39

* DNA FISH (fluoresence in situ hybridisation) * Transient homolgous chromosome pairing marks the onset of X-inactivation in mouse embryonic fibroblasts * Fluoresent probes to chromosomes * See chromosomes come together (differentiation) and apart again. Coming together may be required for counting and/or chosing the future Xi. * E.g.exchanging TFs to ensure the mono-alleleic expression of Tsix and Xist 1. Prior to X inactivation, TF keep Tsix on (and other pluripotency factors) 2. When x's come together TFs shuffled over to one X chromosome which gains all the regulatory information, the other Tsix is switched off * Mutually exclusive process

Question 40

Outline some X chromosome disorders

Answer 40

**klinefelter syndrome** * XXY: acts like a female, with one X inactivated **XXX syndrome** * turn off two Xs, keep one on, removes extras Very few multichromosomal disorders as most are intolerable but X multichromal disorders are relatively unsevere due to X inactivation