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MBB345 - Human Reproduction and Fertility COPY > Alvey - imprinting > Flashcards

Alvey - imprinting Flashcards

1
Q

Which parent is always wrote 1st in a genetic cross?

A
  • female
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2
Q

In what way are maternal and paternal crosses not equal, example?

A
  • cross dep on which is mother and which is father
  • female tiger x male lion = liger (twice weight of either parent, prob diff in expression of a GF)
  • female lion x male tiger = tigon (size of either parent)
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3
Q

What did imprinting evolve as a mech for, and what have been the results of this?

A
  • to balance parental reproductive resources
  • gen genes from father want offspring to be big and genes from mother want offspring to be equal size and manageable
  • this conflict has resulted in small subsection of genes in mammalian genome being subjected to imprinting
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4
Q

What was the 1st evidence of imprinting?

A
  • 2 studies in mice
  • gynogenetic diploids
  • -> control was haploid nucleus from mother and father implanted into enucleated oocyte, viable embryo when implanted into foster mother
  • -> then injection of 2 female (haploid) pronuclei into enucleated oocyte, embryos arrested early in dev
  • -> conclusion was maternal and paternal genomes not equal in mice
  • androgenetic diploids
  • -> similar study
  • -> injection of 2 male (haploid) pronuclei into enucleated oocyte
  • -> also not viable
  • even though both oocytes contained ‘diploid’ level of DNA, normal embryo did not develop in either case
  • as even XX individuals did not survive, the authors concluded that this could not be due to the sex chromosomes
  • due to genomic imprinting
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5
Q

What did a classical eg. of maternal imprinting of IGF-II in mice involve?

A
  • targeted disruption of insulin like GF II gene results in growth deficient (small) mice
  • IGF-II controlled by paternal side
  • IGF-IIr is receptor for gene and controlled by maternal side
  • WT female x heterozygote KO male
  • -> get some small and some big (WT) mice
  • heterozygotes of both sexes are of normal size
  • can have big and small mice that are male or female
  • -> so not linked to sex, only dep on whether inherited mutant allele from father
  • therefore phenotype of offspring dep only on genotype of paternal allele
  • allele from mother silenced has no function
  • then did reciprocal cross: heterozygote KO mother x WT father
  • -> all offspring are WT
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6
Q

What were the conclusions drawn from the IGF-II imprinting paper?

A
  • transmission of IGF-II mutation through male germline results in growth deficient heterozygous progeny
  • when disrupted gene transmitted maternally, heterozygous offspring are phenotypically normal
  • difference in growth phenotypes dep on the type of gamete contributing the mutated allele
  • homozygous mutants indistinguishable in appearance from growth-deficient heterozygous siblings
  • only paternal allele is expressed in embryos, while maternal allele is silent
  • key finding: in mice the paternal and maternal members of some autosomal gene pairs are functionally non-equivalent
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7
Q

What is maternal imprinting?

A
  • allele of particular gene inherited from mother is transcrip silent (not exp), so direct obs of phenotype governed by paternal allele
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8
Q

What is paternal imprinting?

A
  • allele of particular gene inherited from father is transcrip silent (not exp), so direct obs of phenotype governed by maternal allele
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9
Q

How can imprinting lead to disease?

A
  • usually in diploid species, if defective copy inherited there is a 2nd (functioning) copy from other parent that can compensate for loss
  • not true of imprinted genes
  • even though 2 copies of gene, only 1 copy exp, mutations affected this copy will result in disease
  • situations that result in both copies of gene being silenced will also lead to exp of the disease
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10
Q

How is IGF-II linked to human disease?

A
  • homolog is Ifg2

- imprinted region of human genome that contains Ifg2 causes paternally inherited disease = Beckwith-Wiedemann syndrome

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11
Q

What is Beckwith-Wiedemann syndrome?

A
  • pediatric overgrowth disease, involving predisposition to tumour dev
  • clinical presentation highly variable
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12
Q

What did original mutation causing BWS cause, and how is it inherited?

A
  • mutation in germline resulted in hypermeth of IC1
  • unaffected have active copy of IGFR2 from father, maternal copy silenced
  • affected offspring carry active copy from BOTH parents (maternal copy not silenced due to imprinting defect at IC1), so have BWS
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13
Q

What are the clinical symptoms of PWS?

A
  • obesity
  • behaviour and cognitive problems
  • deficiencies in sexual development
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14
Q

What are the clinical symptoms of AS?

A
  • developmental deficiencies
  • sleep disorders
  • seizures
  • happy disposition
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15
Q

What are the clinical symptoms of Silver-Russel Syndrome?

A
  • pre- and/or postnatal growth restriction
  • small triangular shaped face
  • skeletal asymmetry.
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16
Q

What are the chromosomal and inheritance details of BWS?

A
  • 11p15.5 (paternal) –> contains Ifg2

- hypometh ICR

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17
Q

What are the chromosomal and inheritance details of PWS?

A
  • 15q11-q13 (paternal)
  • PW region deletion (70% cases)
  • or maternal uniparental disomy (25% cases)
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18
Q

What are the chromosomal and inheritance details of AS?

A
  • 15q11-q13 (maternal)
  • deletion of PW region (70% cases)
  • mutation of UBE3a (10%)
  • paternal uniparental disomy (3%)
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19
Q

What are the chromosomal and inheritance details of Silver-Russell Syndrome?

A
  • 11p15.5 (paternal)
  • hypermeth (35-65%)
  • maternal UPD (10%)
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20
Q

What genetic mechs can cause PWS?

A
  • deletion of paternal PW region –> so don’t get any functioning gene (70%)
  • maternal UPD disomy 15 –> so both copies meth and silenced (25%)
  • mutations in imprinting control region (ICR)
  • translocation that sep ICR from PW region –> ie. if move imprinting centre away from genes to be imprinted then can’t be imprinted
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21
Q

How do chromatin states control gene activity to the active state?

A
  • HAT (histone acetyltransferase) targets H3 (histone) tail
  • once acetylated it is docking site for Bromo-dom prots
  • stims nucleosome accessibility, by making whole thing more open, so more transcrip –> more euchromatin
  • this is reversible by HDAC
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22
Q

How do chromatin states control gene activity to the repressed state?

A
  • histone lysine methyltransferase (KMT) methylated H3 tail
  • methylated histone is docking site for heterochromatin prot 1 (HP1)
  • promotes heterochromatin formation, impairs nucleosome accessibility
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23
Q

What is the mol basis of epigenetic silencing, how does it occur?

A
  • DNA meth
  • demeth of CpG islands at promoter sequences is assoc w/ active genes (transcrip)
  • methylation at CpG islands assoc w/ silencing of that locus
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24
Q

At maternally imprinted locus would the promoter be meth or demeth on the maternal allele?

A
  • promoter would be meth, as gene silenced
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25
Q

What is the overall pattern of meth and demeth?

A
  • DIAG*

- in paternal/maternal imprinted gene opp of this diag will happen

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26
Q

When does epigenetic reprogramming occur?

A
  • during gamete formation

- post fertilisation

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27
Q

What meth pattern do imprinted genes exhibit?

A
  • always meth pattern of the parent in sperm or eggs, regardless of whether they came from maternal or paternal genome
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28
Q

How do imprinted genes differ in epigenetic reprogramming?

A
  • reset in dev gamete and bypass epigenetic reprogramming in early embryo
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29
Q

What can PGCs be regarded as?

A
  • 1st step in acquisition of totipotency and the continuation of the life cycle
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30
Q

What occurs during mammalian life cycle of PCGs?

A
  • PGCs are specified during early embryonic dev
  • PGCs migrate to the (dev) gonads
  • meiosis
  • gamete differentiation
  • fertilisation of oocyte by sperm
  • totipotent zygote formation
  • PGCs are specified…..
31
Q

What is the purpose genome wide epigenetic reprogramming in gamete formation (** also true of imprinted genes)?

A
  • resets imprinted genes for sex of embryo
  • erases parental of acquired epigenetic memories (eg. env)
  • facilitates gametogenesis
  • maintain silencing of transposable elements (might be via chromatin formation and/or long non coding RNAs
  • reduces mutation rate in the germline
32
Q

What is the purpose genome wide epigenetic reprogramming in pre-implantation embryo (** not true of imprinted genes)?

A
  • re-sets zygotic epigenetic genome of naive pluripotency

- some evidence of maternal vs paternal wars

33
Q

What are the maternal vs paternal wars?

A
  • maternal genomes tend to want manageable and equal size, whereas paternal wants as big as poss
34
Q

How does epigenetic resetting occur in the germline?

A
  • migratory PGCs undergo genome wide epigenetic reprogramming to erase imprints and other somatic epigenetic memories
  • during fetal dev and adulthood gonadal germ cells undergo meiosis and gametogenesis to differentiate into sperm and eggs
  • genome remeth and acquires approp epigenetic signatures for gen of a zygote upon fertilisation –> so able to form healthy gametes
35
Q

How are somatic epigenetic memories erased during migration of PGCs

A
  • global DNA demeth
  • genomic imprint erasure
  • X chrom inactivation
  • reorg of chrom
36
Q

How do males and females differ in epigenetic memory erasure in germline?

A
  • same in male and female embryos
  • but XY germ cells then enter mitotic arrest
  • XX germ cells enter meiosis
  • this is the end of the PGC stage of germline dev
37
Q

What is the DNA meth profile in germline?

A
  • migratory PGCs become demeth early in dev
  • remeth begins in spermatogonial stem cells (SSCs) in males
  • remeth begins after birth in growing oocytes
38
Q

How does genome wide DNA demeth of PGCs occur?

A
  • mouse PGCs (mPGCs) are specified at the post implantation epiblast
  • DNA hypermeth (70%) and PGCs primed for differentiation
  • at onset of migration, PGCs undergo genome wide demeth (to 4%)
  • almost all genomic features (inc imprint control elements) become hypometh
39
Q

Is genome wide DNA demeth of PGCs active or passive?

A
  • thought to act mostly via passive mech –> ie. not actively removed just not replaced
40
Q

When is DNA meth 1st re-estab?

A
  • in sex specific manner after E13.5 in males, and after birth in females
41
Q

What prop genomic loci escape global DNA demeth?

A
  • approx 4% remain meth in PGCs
42
Q

How do some genomic loci escape global DNA demeth?

A
  • vast majority ‘escapees’ assoc w/ retrotransposable elements
  • mech not known
  • current assumption is that it’s due to incomplete repression of DNA meth pathway
43
Q

What is 1st DNA demeth essential for?

A
  • imprint erasure

- allele specific meth at ICRs erased in PGCs

44
Q

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

A
  • meth status of IGFR2 gene examined by SB
  • DNA digestion w/ meth sensitive restriction enz –> digestion blocked at CpG meth sites (so can differentiate between meth and unmeth alleles) –> meth create bigger product as not chopped up, demeth smaller
  • in male ES cells both copies of IGF2R are unmeth
45
Q

What occurs during DNA methylation in pre-implantation embryos?

A
  • at fertilisation and implantation both genomes are meth
  • paternal genome demeth by an active mech immed after fertilisation
  • maternal genome is demeth by passive mech
  • methylated imprinted genes do not become demeth
  • unmeth imprinted genes do not become remeth
46
Q

What are waves of demeth and remeth in early embryo essential for?

A
  • normal dev and estab totipotency
47
Q

Why does demeth and erasure of imprinting marks makes sense during gametogenesis?

A
  • maternal germline req all oocytes to carry the maternal epigenetic mark
  • paternal genome must carry paternal marks
48
Q

Post fertilisation how are paternal and maternal imprints maintained in dev zygote?

A
  • when male pronucleus enters the oocyte (nearly) all meth actively removed from paternal genome
  • mech by which some genes bypass reprogramming not known, but long non-coding RNAs are candidate mech
  • whatever the mech, parent of origin specific DNA meth is transmitted at imprinted loci, imprints must be maintained during global DNA meth in the preimplantation embryo and persist in somatic cells
49
Q

How does when imprints are placed de novo differ paternally and maternally?

A
  • paternal = prenatally

- maternal = postnatally

50
Q

Where does erasure of imprints occur, what happens elsewhere?

A
  • only in PGCs

- imprints have to stay correct in rest of dev embryo, ie. all somatic tissues and placenta

51
Q

How might it be poss to treat imprinting disorders?

A
  • imprinted genes silenced using normal epigenetic machinery of the cell
  • this machinery restricted to just 1 allele in dev gamete
  • could reactivate the exp of silent parental alleles to reduce symptoms
52
Q

What are imprinted loci a barrier to?

A
  • cloning mammals
  • engineering reproductive cells
  • prod of bimaternal and bipaternal mammals
  • the gen of artificial gametes from somatic tissue
53
Q

How was Dolly cloned?

A
  • SCNT
  • implantation of an adult somatic nucleus into an enucleated oocyte
  • blastocyst was implanted into surrogate ewe
54
Q

Was Dolly a success?

A
  • yes, she was fertile

- but died young (lung cancer)

55
Q

What was the implication of putting somatic nucleus into enucleated oocyte, ie. Dolly?

A
  • no re-setting of epigenetic marks
  • got mature diploid oocyte w/ all epigenetic marks of differentiated cell
  • problem w/ having epigenetic marks of undifferentiated cell in egg cell is 2 fold:
    1) cell not pluripotent and assumed that when implanted, demeth happened in delayed and nonspecific way, so marks wrong for dev embryo
    2) other problem was that epigenetic marks in this cell had been copied many times, copy of epigenetic marks v error prone, so cell used to make Dolly would have already accum lots of epigenetic mutations already
56
Q

Ideally how would artificial gametes be used as a fertility treatment, which of these steps take place in IVF?

A
  • take somatic cell (ideally easily accessible, eg. skin)
  • perform SCNT and induce embryo development in vitro (allows to have lots of undifferentiated cells but w/ desired gDNA)
  • extract embryonic stem cells from blastocyst
  • induce gametogenesis in vitro
  • use functional gamete to fertilise partner’s egg or sperm
  • dev into embryo
  • implant into mother
    • fertilisation to implantation takes place in normal IVF
57
Q

What were man made mouse eggs made from?

A
  • embryonic stem cells

- induced stem cells –> from embryonic fibroblasts or adult tail tip fibroblasts

58
Q

Was experimentally gen man made mouse eggs successful?

A
  • yes, 26 fertile pups born
59
Q

When man made mouse eggs were made, why was it good that mature oocytes could be used to gen more ESCs?

A
  • so could start cycle again, important if want to use as source of material for experiments
60
Q

Were the man made mouse eggs in vitro?

A
  • yes, full cycle in vitro
61
Q

How were man made mouse eggs made?

A
  • took pluripotent SCs and cultured w/ specific hormones and culture medium to prod primordial germ cell like cells (PGCLCs)
  • then induce differentiation into oocyte by co culture w/ reconstituted ovary
  • selected 2° follicles and grew them
  • then induced maturation to make metaphase II oocyte
62
Q

What was the weakness of the methodology of making a man made mouse egg and what is the next aim?

A
  • req mouse ovaries from other fetus, so fetus lost in this process
  • next aim is to make these synthetically too, so have complete synthetic system
63
Q

Was the method of making a man made egg cell novel?

A
  • part from differentiation to maturation was
64
Q

In prod of man made mouse eggs, where was demeth and remeth occuring?

A
  • demeth happens in germ line cells

- remeth happens somewhere between differentiation and maturation (but unsure where exactly)

65
Q

Why is the method of making man made mouse eggs not yet quite ready for application in humans?

A
  • only 3.5% oocytes went on to make pups (comp w/ 60% when cells implanted into surrogate mice to develop mature oocytes)
66
Q

Did making man made egg cells lead to problems w/ imprinted loci?

A
  • checked for maternal pattern of DNA meth in DMR –> was almost completely the same at loci tested (but not perfect)
  • imprints sufficiently maintained to gen fertile mice –> both male and female
  • no evidence of premature death
67
Q

How were bimaternal mice prod?

A
  • start w/ activated oocyte and isolate haploid ESCs
  • then cultured cells in medium resulting in demeth (to mimic 1st wave of demeth)
  • counted no. passages needed to remove meth to 4% –> for females was 20-24
  • loss of meth non specific, so instead of targeted demeth at specific loci, gradually everything getting less meth as enzs not there to maintain it
  • then CRISPR to delete imprinted regions
  • -> started w/ 1 imprinted region (over 12kb), chosen as had biggest diff between WT and cultured situ
  • -> when deleted problems w/ pups and died soon after birth
  • -> so deleted 2nd region and survived longer but still not healthy
  • -> then deleted 3rd region and injected this haploid ESC into mature oocytes and got almost normal mouse
  • -> therefore it is these 3 imprinted loci that are the barrier to bimaternal prod in mammals, so if remove these can create healthy offspring
  • only done in 1 of parents –> 1 haploid nuclei is totally normal, and other has these deletions
68
Q

What is passage no.?

A
  • times transfer cells and move to new medium to grow
69
Q

How was the prod of bipaternal mice attempted?

A
  • inserted sperm into enucleated oocyte by injection
  • from dev blastocyst isolated androgenetic haploid ESCs
  • cultured in media that caused nonspecific demeth –> needed 40 passages
  • systematically deleted maternally imprinted regions, started w/ 6 diff regions deleted
  • -> pups had problems and died shortly after birth
  • -> looked for another differentially demeth region in the demeth culture (diff to WT situation), found 1 and deleted this 7th region w/ CRISPR
  • -> took this haploid paternal genome w/ these 7 deletions and injected w/ sperm into enucleated oocyte
  • -> these pups survived a bit longer, but not healthy and not fertile
  • so still some way to go before viable option for prod mammals
70
Q

Is there hope for prod of bipaternal mice?

A
  • thought there are around 100 imprinted loci across mouse genome and in total have KO only 7 –> so are many others that could be candidates
71
Q

What was the impact of work on bipaternal/bimaternal mice on imprinting disorders?

A
  • could be used to model reg of imprinting genes
  • technique for accurate, reversible epigenetic switch between maternal and paternal imprinting
  • offspring from same-sex parents
  • altering reg of imprinted genes might offer approach to treat imprinting disorders
72
Q

How would meth patterns vary in PGCs made in vivo or by in vitro methods?

A
  • if PGCs made in vivo, cells would prob have correct DNA meth pattern
  • but if demeth wave in the PGCs happened in vitro, then may be non-specific and full of errors
73
Q

How would you check imprinting status of key imprinted genes in resulting gametes?

A
  • bisulphate sequncing

MBB345 - Human Reproduction and Fertility COPY (3 decks)

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