Lecture 5 - Genomic imprinting in mammals, epigenetic mechanisms in plants Flashcards Preview

Epigenetics in Development and Disease > Lecture 5 - Genomic imprinting in mammals, epigenetic mechanisms in plants > Flashcards

Flashcards in Lecture 5 - Genomic imprinting in mammals, epigenetic mechanisms in plants Deck (39)
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1

Describe genomic imprinting in mammals

  • A form of epigenetic regulation that results in mono-allelic expression in a parent-of-origin dependent manner
  • essential for normal development
  • over 90 imprinted genes have been identified
  • often occur in large clusters regulated by an imprinting control region (ICR), non-coding RNAs and differential DNA methylation 

2

What is the pattern of imprinted genes in mammalian development?

  • imprint established in the germ cells
  • And is not removed in the global erasure 

3

Describe mouse germ cell development 

Mouse germ cell development is similar to human development but different timings

  • E3.5: Blastocyst
    • differentiation to epiblast stage
    • extra embryonic tissue and primordial germ cell precursors
    • form a founder population 
    • migrate through the embryo before settling in a final position 
    • Erasure takes place during migration 
  • Depending on whether sperm or eggs are going to be produced the timing of imprinting differs
    • male: quite early before birth, in the development of pre-sperm cells. This is maintained throughout divisions 
    • female: occurs post birth during maturation of the oocyte

4

What is the experimental evidence for needing a male and female parent when mammals reproduce?

  • nuclear transfer
  • took pronuclei from a fertilised egg 
  • generated male/male, female/female and male/female pronuclei in the manipulated fertilised egg
  • only got successful development when have male and female pronuclei

5

What is the experimental evidence that there are some required factors from the male pronuclei and some required factors from the female pronuclei?

  • Looked at whole embro, yolk sack and tropoblast development of male/female pronuclei (PN) control, female/female PN and male/male PN.
  • In F/F: trophoblast fails to develop substantially and the embryo does not develop well
  • In M/M: Placenta gets an overgrowth, the embryo does not develop well
  • only in the control do you get a good balance.
  • Transgenic observations showed that genes were differentially methylated whether mother or father 

6

Outline the H19-insulin growth factor 2 (Igf2) as an example of genomic imprinting

  • H19 encodes a 2.3kb non-coding RNA 
    • active on maternal allele
  • Igf2 encodes a protein that plays a role in promoting embryonic and placental growth and development
    • active on paternal allele
  • Explain placental effects
  • regulated by intra-chromosomal interaction 
    • Igf2 and H19 separaed by an imprinting control region (ICR) and this is differentially regulated in the maternal and paternal chromosomes
    • In females - ICr is not methylated, CTCF (a DNA binding protein that acts as an insulator) binds to the ICR and blocks effect of enhancers which then act on H19
    • In males ICr is methylated, CTCF cannot bind and enhancers act on Igf2

7

How were the identities of the imprinted genes identified?

Myagenesis studies 

  • Female Igf2 mutant crossed to normal male
    • offspring all normal sized
  • Normal female crossed to Igf2 mutant male
    • off spring small 
  • Paternal allele promotes growth, maternal allele supresses growth 

8

Outline two imprinting disorders

Beckwith-Wiedemann (BWS)

  • gain of methylation on the maternal ICR
  • Igf2 is expressed from both alleles
  • overgrowth disease

Silver-Russell syndrome (SRS)

  • Loss of DNA methylation from the paternal ICR
  • No Igf2 expression 
  • growth retardation disease 

Imprinting disorders occur in a pairwise manner 

9

Outline Insulin growth factor 2 receptor (Igf2R) and Air (ncRNA) as an example of genomic imprinting

  • controlled by antisense transcription and differential DNA methylation 
  • Air overlaps Igf2R (growth suppressor) and is transcribed in the antisense direction 
  • Set up by methylation in the maternal region, more likely to ocur in the maternal region 
  • In the maternal chromosome Igf2R is active (when maternal ICR is methylated). 
  • In the paternal chromosome Air (ncRNA) is active (ICR not methylated). IGF2R becomes methylated as a consequence. 

10

What organisms imprint, how and why ?

  • placental mammals and flowing plants use imprinting
  • genes expressed from the paternally derived genome are often enhancers of pre- and post-natal growth (Igf2)
  • those expressed from the materally derived genome are often growth supressors (Igf2R)
  • this is the parental conflict theory 

11

What are the implications for assisted reproductive strategies for epigenetic reprogramming?

In vitro fertilisation 

  • children have statistically significanly increased occurance of imprinting disorders 
  • in non IVF, epigenetic changes take place within the mother
  • This doesn't happen in IVF leading to errors of maintaing the imprint, or being removed incorrectly

12

Outline the similarities and differences of epigenetics in plant

Similarities 

  • Use DNA methylation, histone modification and non-coding RNAs
    • histone codes are broadly conserved
  • Undergo dynamic changes in epigenetic modifications during development
  • perform genetic imrinting (in edosperm tissue)

Differences 

  • Cytosine methylation in all sequence contexts in all tissues, predominantly in CG dinucleotides in vertebrates
  • changes in DNA methylation can be transmitted though multiple generations (but not 100% stable) 
  • plant cells can switch fate - they retain potency
  • Do not do a complete erasure and reestablishment as seen in vertebrates 

13

How do epigenetic patterns differ in egg laying mammals?

  • short lived placenta before egg development
  • don't imprint
  • can't directly influence growth of embryo 

placental mammals

  • imprinting common in placental mammals
  • lots of imprinting in brain 
  • evidence that paternally expressed genes affect subtlty of behaviour post birth 

14

Describe the parental conflict theory with reference to cats

  • In a litter of kittens they may not all have the same father as mother has multiple ovulations
  • same mother, different father
  • Mother wants to even out nutrient allocation as equally related to all 
  • Father wants preference for his offspring
  • balance of nutrient allocation due to the parental conflict theory 
  • other theories due to sexual selection 

15

Outline DNA methylation in plants

  • Mosaic pattern over the genome
    • ​Arabidopsis thaliana
    • 'gene body' DNA methylation (occasional)
      • mainly CG configurations
      • significance unknown 
    • Repetitive sequences (ALWAYS methylated)
      • CG, CHG, CHH contexts
      • maintaining heterochromatin and supressing TEs
  • Global DNA methylation 
    • Zea mays 
    • packed full of transposable elements 
    • more extreme version of arabisopsis
    • cereals and grasses 

16

What are the similarities in methyltransferases between plants and animals?

  • plants have different methyltransferases with different substrates like mammals
  • De novo methylation
    • Mammals: Dnmt3a, Dnmt3b
    • Plants: Drm2 (CG, CHG, CHH)
  • Mainenance methylation
    • Mammals: Dnmt1
    • Plants: Met1 (CG), Cmt3 (CHG)
  • Have the same enzymology as the mammal enzymes

17

How essential is DNA methylation for normal development in plants?

  • DNA methylation is essential for normal development in plants as it is in mammals
  • lowered levels of DNA methylation across genome (ddm1 required to maintain methylation) 
  • phenotypesof ddm1/ddm1 lines propagated by self cross
  • seriusly impacts development, very dwarfed
  • the phenotypes of these plants are not the same
    • stochastic effect - action of the transposable elements

18

What is the status of the global erasure in plants?

  • Plants DON'T do a global erasure and resetting of information as mammals do
  • affects the heritability of the epigenetic marks 
  • if methylation pattern changes, this is likely to be maintained in the next generation 

19

Gernerally - how might stress memories in plant progeny be generated?

  1. Plants take in environmental information 
  2. Change epigenome accordingly
  3. Stress memory

20

How do the generation of germ cells differ in plants and mammals?

  • In mammals, germ cells arise very early
  • can still be influenced by the environment of their mother
  • In plants, have a vegetative stage, then flower - can pass epigenetic changes influenced by the environment onto progeny
  • these progeny may have advantages 
    • prepared for stressful conditions
    • useful as plants can't move 

21

What is the experimental evidence that plants that undergo stress affect epigenetic changes that are passed onto their progeny, giving them a survival advantage?

  • the progeny of arabidopsis plants exposed to salt may have a higher tolerance to salt stress than control plants
  • Looked at DNA methylation differences reported between control and stress progeny plants
    • higher in progeny of plants exposed to 25mM or 75mM NaCl
  • Looked at the germination rates of progeny 
    • at higher concentration of NaCl, progeny of parents that had already been exposed had a higher germination level
  • Yes. Methlyation does change as a result of environmental differences in the progeny of stress affected plants 
  • Contraversial: respond to the environment and pass this onto progeny. Led to major plant breeding experiement, does exposing plants to stress make them more tolerant 

22

What is the experimental evidence that ddm1 mutants partially regain DNA methylation levels when WT levels of DDM1 are restored?

  • Molecular analysis of ddm1 mutant and developmental phenotypes
  • Decreased levels of methylation by reducing levels of the ddn1 mutant
  • If ddn1 is put back does this re-establish methylation?
    • wouldn't expect it to as plants don't do erasure and re-establishment
    • area where it fails to respond to WT levels are regions that correspond to gene bodies (GC methylation)
    • when gene body methylation gone, it is not re-established 
    • however do restore methylation levels on transposable elements
    • therefore they must have an ability to recognise and methylate transposable elements 

23

What is the experimental evidence that there is a strong correlation between TE methylation and sRNA accumulation?

  • genome wide analysis of DNA methylation patterns (epigenome single base analysis - where is m, where are the small RNAs that direct m?) 
  • sRNA accumulation analysis
  • strong correlation between TE and sRNA accumulation 
  • compared to gene body methylation, which does not correlated with sRNA accumulation 
  • are sRNAs on TE used to re-establish methylation?

24

What is the process of RNA-directed DNA methylation (RdDM) in plants?

  1. sRNAs come from regions of dsRNA
  2. Dicer like (RNAseIII enzyme) cuts into small interfering (si) RNAs
  3. siRNA and effctor (Argonaute AGO) proteins form a complex with DRM2 (de novo DNA methyltransferase)
  4. siRNA-effector complex directs sequence specific DNA methylation with the aid of PolV
  5. PolIV then transcribes the region 
  6. RNA-dependent RNA polymerase 2 (RDR2) copies the PolIV transcript and produces more double stranded RNA and siRNAs via DCL3 cleavage
  7. cycle

25

How is the process of RNA-directed DNA methylation (RdDM) affected in ddm1 mutant plants?

  • affects the efficiency of the process
  • once ddm1 restored, cycle will again methylate TE regions and genome repeats 
  • RdDM cycle present to keep methyation on in TE and genome repeats 

26

What is the primary role of DNA methylation in plants and how does this relate to animal methylation?

  • the primary role of DNA methylation in plants may be to control transposable elements
  • Animals also use small RNAs to epigenetically silence transposons

27

How do plants control gene expression at key developmental stages and what are these stages?

Plants use Polycomb (PcG) and trithorax (trxG) group proteins to control gene expression at key developmental stages

  • fertilisation 
  • seed development
  • transition to flowering
  • flower organogenesis 

Different to mammals: get post embryonic development and clear developmental transitions

28

Why are many plant genomes packed full of transposable elements and how are these recognised and distinguished from host genes?

  • Over evolutionary time can compare different types of transposable elements/genome rearrangement
  • hybridisation of different species results in genomic shocks
  • lots of methylation changes can occur if bring together two different genomes
  • results in a burst of transposable element activity 
  • Also may be a selective pressure: transposable elements may confer advantages
  • Germ cells TE most highly methylated in all organisms 

29

What are the PCR complexes of plants?

  • mammals have two major PCR complexes
  • plants only have PCR2 but have multiple versions
    • FIS
    • EMF
    • VRN
  • Act at different stages of the arabisopsis life cycle

30

How is epigenetics involved in flowering?

  • plants need to flower in favourable conditions
  • many only flower after an extended period of cold (e.g. arabidopsis)
  • acceleration of flowering by prolonged cold is known as vernalisation 
  • epigenetics is involved
    • FLC is a TF that blocks the floral transition 
      • acts by repressing SOC1 and FT
      • FT is the main flowering promoting gene
    • Arabidopsis must turn off FLC in order to flower
      • mutant that cannot turn of FLC doesn't leave vegetative stage, produces lots of vegetation