Lecture 5 - Genomic imprinting in mammals, epigenetic mechanisms in plants Flashcards
1
Q
Describe genomic imprinting in mammals
A
- 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
Q
What is the pattern of imprinted genes in mammalian development?
A
- imprint established in the germ cells
- And is not removed in the global erasure
3
Q
Describe mouse germ cell development
A
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
Q
What is the experimental evidence for needing a male and female parent when mammals reproduce?
A
- 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
Q
What is the experimental evidence that there are some required factors from the male pronuclei and some required factors from the female pronuclei?
A
- 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
Q
Outline the H19-insulin growth factor 2 (Igf2) as an example of genomic imprinting
A
- 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
Q
How were the identities of the imprinted genes identified?
A
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
Q
Outline two imprinting disorders
A
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
Q
Outline Insulin growth factor 2 receptor (Igf2R) and Air (ncRNA) as an example of genomic imprinting
A
- 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
Q
What organisms imprint, how and why ?
A
- 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
Q
What are the implications for assisted reproductive strategies for epigenetic reprogramming?
A
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
Q
Outline the similarities and differences of epigenetics in plant
A
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
Q
How do epigenetic patterns differ in egg laying mammals?
A
- 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
Q
Describe the parental conflict theory with reference to cats
A
- 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
Q
Outline DNA methylation in plants
A
-
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
Q
What are the similarities in methyltransferases between plants and animals?
A
- 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
Q
How essential is DNA methylation for normal development in plants?
A
- 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
Q
What is the status of the global erasure in plants?
A
- 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
Q
Gernerally - how might stress memories in plant progeny be generated?
A
- Plants take in environmental information
- Change epigenome accordingly
- Stress memory
20
Q
How do the generation of germ cells differ in plants and mammals?
A
- 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
Q
What is the experimental evidence that plants that undergo stress affect epigenetic changes that are passed onto their progeny, giving them a survival advantage?
A
- 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
Q
What is the experimental evidence that ddm1 mutants partially regain DNA methylation levels when WT levels of DDM1 are restored?
A
- 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
Q
What is the experimental evidence that there is a strong correlation between TE methylation and sRNA accumulation?
A
- 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
Q
What is the process of RNA-directed DNA methylation (RdDM) in plants?
A
- sRNAs come from regions of dsRNA
- Dicer like (RNAseIII enzyme) cuts into small interfering (si) RNAs
- siRNA and effctor (Argonaute AGO) proteins form a complex with DRM2 (de novo DNA methyltransferase)
- siRNA-effector complex directs sequence specific DNA methylation with the aid of PolV
- PolIV then transcribes the region
- RNA-dependent RNA polymerase 2 (RDR2) copies the PolIV transcript and produces more double stranded RNA and siRNAs via DCL3 cleavage
- cycle
25
Q
How is the process of RNA-directed DNA methylation (RdDM) affected in ddm1 mutant plants?
A
- 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
Q
What is the primary role of DNA methylation in plants and how does this relate to animal methylation?
A
- the primary role of DNA methylation in plants may be to control transposable elements
- Animals also use small RNAs to epigenetically silence transposons
27
Q
How do plants control gene expression at key developmental stages and what are these stages?
A
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
Q
Why are many plant genomes packed full of transposable elements and how are these recognised and distinguished from host genes?
A
- 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
Q
What are the PCR complexes of plants?
A
- 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
Q
How is epigenetics involved in flowering?
A
- 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
- FLC is a TF that blocks the floral transition
31
Q
What is vernalisation and how is epigenetics involved?
A
- Vernalisation is flowering after a long period of cold
- FLC expression is downregulated by cold
- FLC repression is maintained after plants resume growth in the warm (epigenetic?)
- VRN2 polycomb reperssive complex is involved (H3K27 methylation)
FLC expression is reactivated in the developing embryo
- Vernalisation fits the definition of a true epigenetic process
- FLC expression high at 20degrees, FLC decreases in prolonged cold exposure (trigger), FLC reduction is maintained in the absence of the trigger (20degrees)
32
Q
What is the model for epigenetic silencing of FLC during vernalisation?
A
- Looking at FLC locus
- Before cold (high FLC) VRN is present on the gene but not adding repressive marks
- Vin3 is upregulated by prolonged cold, brings together VEL1 and VRN5 to the VRN complex (PHD-PRC2 complex)
- This complex contains VRN complex to add a methylating mark
- Vin2 leaves and repression spread out and is maintained
33
Q
What is the model of the regulation at VIN3 chromatin by vernalisation?
A
- Vin3 is normally repressed with known repressive marks
- CLF/SWN (polycomb repressive complex)
- lys37 and lys9 methylation
- very repressed state
- Cold trigger
- get slight opening of Vin3
- recruitment of trithorax activity, puts down active marks
- lys4
- allows Vin3 to be activated and transscribed
- even during transcription keeps active and repressive marks (like bivalent domains seen in stem cells - all quick removal of Vin3 when gets hot again)
34
Q
Outline epigenetics in seed development
A
- imprinting occurs primarily in the endosperm of flowering plants
- endosperm is triploid and formed from fusion between sperm cell (haploid) and the central cell (diploid)
- imprinting due to differences in epigenetic marks on the alleles of the central and sperm cells
35
Q
What is the process of seed development~?
A
- producton of maternal and paternal gamete (gametogenesis)
- maternal gametophyte undergoes multiple divisions - some meiotic, some mitiotic. Have a haploid egg cell (contributes to the embryo) and a diploid central cell (contributes to the endosperm)
- In paternal gametophyte (pollen), have 2 sperm cells and a vegetative cell.
- Sperm cells takes place in double fertilisation
- one fertilises the egg cell to generate a diploid embryo which will form the seed
- the diploid central cell gets fertilised by the other sperm cell to generate a triploid endosperm (2 X maternal, 1 X paternal): this balance is essential for normal endosperm development
- Only the embryo contributes to the next generation, endosperm is purely nutritive
36
Q
How is the plant endosperm prevented from proliferating before fertilisation?
A
- The 4 PRC2 FIS complex components
- MEA
- FIS2
- FIE
- MSI1
- are normally expressed in the central cell and repress central cell proliferation until fertilisation
- mutants in these complex components produce endosperm in the absence of fertilisation
- Imprinting: self regulatory system to keep level of endosperm balanced
37
Q
What gene are imprinted in the endosperm?
A
- MEA, FIS, FWA (maternal expression) are imprinted in the endosperm by MET1
- specific de-methylation occurs in the central cell by the demeter (DME) DNA glycosylase
- doesn’t happen in the sperm - proteins stay methylated
- upon fertilisation in the endosperm get balance of the correct level of PcG complex
- genes from the maternal side repress the paternal side and proliferation, and other targets (PHE1 - represses maternal allele but not paternal allele)
- balance ensures optimal growth of the endosperm
- If balance is affected
- e.g. different ploidy
- normally crossing 2 diploid plants
- if cross female 4n with male 2n get a doubling of the female chromosome and too little endosperm
38
Q
Why is studying the epigenetics of the endosperm important?
A
- 70% of worldwide calories come from endosperm
- cereals
- plant breeding efforts to manipulate endosperm
- but this affects the developmetn of the seed
39
Q
What ar teh features of epigenetics in plants?
A
- plants use many of the same epigenetic mechanisms as animals
- DNA methylation is linked to genome defense
- polycomb and trithorax group proteins are involved in kep developmental transitions
- imprinting in closely linked with seed development and is consistent with the parental conflict theory
