Lecture 1 - DNA methylation as an epigenetic mark

Question 1

Where are the origins of epigenetics?

Answer 1

The origins of epigenetics are in developmental biology

Related to the maintenance of patterns of gene expression

Question 2

How did Waddington describe epigenetics?

Answer 2

• How genotypes give rise to phenotypes during development.
• Beings with a pluripotent cell becoming commited to a particular cell lineage to result in cells with the same genotype but different phenotypes.
• An increasing loss of potency.

Question 3

Post-waddington, What is epigenetics?

Answer 3

• A collection of biological/genetic phenomena that were hard to explain using standard medelian genetics.
• Not reversible
  
  • during normal development cells don’t de-differentiate or switch fate once differentiated
• The same genotype establishing different phenotypes

Question 4

How are patterns of gene expression initially establish and maintained?

Answer 4

Patterns of gene expression are initially established by regulatory proteins (e.g. TFs) and epigenetic mechanisms are involved in maintaining these patterns of gene expression

Question 5

Explain the observations of the position effect variation as described by Muller, 1930

Answer 5

• X ray mutagenesis of drosophila
  
  • results in large chromosome abnormalities
• identification of white eye colour mutation
  
  • due to chromosome inversion
• Unusual patchy phenotype
  
  • fly able to make red phenotype just not all the time

Question 6

Describe how the position effect variation is produced as described by Muller, 1930

Answer 6

1. The centromeric region (centromere) is made up of heterochromatin (low gene expression)
2. Following inversion, the white+ allele is placed next to the heterochromatin
3. If the heterochromatin spreads (normally would have insulator sequences to prevent spreading but this is no longer present in the right place) into the region containing the white+ allele, expression is prevented leading to the white facet
4. The spreading of the heterochromatin causes the white eye phenotype - the DNA sequence has shifted. Exmaple of a packaging effect on expression.

Question 7

Where is Paramutation seen?

Answer 7

In Maize and mice

Question 8

Describe paramutation in Maize (Brink)

Answer 8

• Involves the pigmentation of maize
• R gene in maize has two alleles (Rst (stippled phenotype) and Rr) which have distinct phenotypes as homozygotes
• Rst and Rr alleles have a diffferent DNA sequence
• RrRr is the stippled phenotype
• Rst is able to induce a change in Rr gene expression that is stable
• Rst is normally methylated, and has the ability to transmit its methylation status to the Rr allele
• Rst = paramutagenic and Rr = paramutable
  
  • due to a covelent modification involving noncoding RNA

Question 9

Give an example of an Epi-mutant

Answer 9

The peloria mutant of linaria vulgaris (Linnaeus)

• flower phenotype differed in radial symtery - normal bilateral flower symmetry lost

Question 10

How did Cubas attribute epigenetics to natural varaition in floral symmetry? (peloria/linaria and cycloidea/wt antirrhinum majus)

Answer 10

Cohen noted that the peloria mutant looked like a cycloidia mutant in Antirrhinum majus (radial symmetry mutant)

• sequnced the cycloidia gene and found no difference in the DNA sequence
• Looked at the DNA methylation profile and found that the cycloidia gene in peloria (Linaria) is heavily methylated
• Resulting in gene reprezssion appearing as a loss of function mutant in a cycloidia gene

Question 11

Outline an example of the transgenerational stress response

Answer 11

• exhibited during th edutch famine (Hongerwinter) of 1944
  
  • one generational period of starvation
  • generational effects could be observed as subsequent generations had normal eating habits
  • Individuals prenatally exposed to famine during this period were shown 6 decardes later to have less DNA methylation in key metabolism genes (Heijmans et al 2008) like IGF, compared to unexposed same sex siblings
  • offspring had lower birth weight
  • higher propensity for sterility and disease
  • pattern also seen in the F2 generation who didnt experience famine

Question 12

What is the best agreed upon definition of epigenetics?

Answer 12

• The study of mitotically and/or meitically heritable changes in gene expression that cannot be explained by changes in DNA seuqence
• relates to the concept of a cellular memory

Question 13

What mechanisms are required for epigenetics?

Answer 13

• mechanisms to create specific ‘expression states’ that result in differential gene expression
• mechanisms that allow these expression states to be maintained during cell dividion and development

Question 14

How does eipgenetics differ to transient changes in gene expression?

Answer 14

• Transient gene expression: gene expression state changes upon a signal, reverts to original state once the signal is removed
• epi-genetic gene expresion: gene expression state changes upon a signal, new expression state maintained with the removal of the signal

Epigenetics is ‘self-maintaining’

Question 15

What are the factors involved in epigenetics?

Answer 15

• DNA methylation
• Histone modifications
• Non-coding RNAs (involved in recruiting other mechanisms or directing DNA methylation/histone modifications to specific genomic regions)

Huge interplay between the three.

Question 16

What needs to be considered when looking at epigenetics?

Answer 16

• how the epigenetic mechanisms influence gene expression
• whether the epigenetic marks are heritable (mitotic and/or meiotic stability)
• how the epigenetic marks are directed to specific regions of the genome
• biological importance of epigenetic mechanism
• the consequences when epigenetic mechanisms fail

Question 17

Outline DNA as associated with epigenetics

Answer 17

• associated with many epigenetic phenomena (but some eukaryotes don’t use it)
• occurs on cytosine residues in many eucaryotes
• generally assocaited with transcriptional inhibition (when located at promoter sequences)
• well studied in vertebrates and plants where it is essential for normal development
  
  • loss in vertebrates = deadly
  • loss in plant = developmentally compromised
• Not all invertebrates and fungi use DNA methylation

Question 18

Outline the evolution of eukaryotic DNA methylation patterns

Answer 18

Protists

use neither

Plants

• gene body methylation
• targeted transposon methylation

Fungi

• Targeted transposon (not in all fungi)

Vertebrates

• Gene body methylation
• Targeted transposon methylation

Invertebrates

• Gene-body methylation

Question 19

What are the two main funcitons of DNA methylation?

Answer 19

1. control of gene expression
2. genome defence

Question 20

What is the process of DNA methylation?

Answer 20

• cytosine is converted to 5-methyl-cytosine by a DNA methyltransferase with S-adenosyl methionine (SAM) acting as a methyl donor

Question 21

What is the involvement of S-adenosyl-methionine in DNA methylation?

Answer 21

• acts as a methyl donor in many cellular reactions (methyltransferase reaction including the addion of a methyl group to cytosine)
• has a complex metabolism
• levels can be influenced by diet
• ‘one carbon metaboolism’
  
  • increased by folic acid
  • decreased by alcohol

Question 22

How does DNA methylation affect gene expression?

Answer 22

Two broad ways

• Directly: locking the ability of the TF to bind to DNA (minority)
• Indirectly: the region of methylated DNA recruits other proteins which cause a change in chromatin strcuture and interfere with transcription

Question 23

What is the experimental evidence for the repression of transcription initiation by indirect DNA methylation?

Answer 23

Kass et al (1997)

DNA methylation directs a time dependent repression of transcription initiation

• used xenopus oocytes
  
  • advantages: big, good to inject and get expression

1. Introduced constructs into oocytes which were then trancribed
   
   1. Used HSV tk promoter to drive the expression of a chloroamphenicol acetyltransferase (CAT reporter gene)
2. Methylated the promoter - tk promoter methylation reduces transcription but not immediately
3. Looked at a time course after injection of CH3 compared to the control (to show levels of injection), and CMV (control unmethylated DNA) to HSVtk
   
   1. measured RNA at 30 mins, 1h, 4h and 12h after injection
   2. at 30 mins see methylation and non methylated expression
   3. methylated expression lost from 12 hours onwards
   4. rules out direct action, if it blocked the TF, expression should be effected from the start

Question 24

What is the experimental evidence that the repressive effect of the tk promoter DNA methylation can be removed by adding methylated competitor DNA?

Answer 24

Used a competition experiement to show the repressive effect of the tk promoter DNA methylation can be removed by adding methylated competitor DNA

• Injected methylated or nonmethylated ptk::CAT construct with competitor DNA either methylated or unmethylated
• Measured DNA from constructs under increasing concentrations of competitor DNA (CMV as control - should be equal in all samples)
• when the tk construct is methylated = no expression
• when the competitor construct is unmethylated = no expression
• when competitor construct methylated = expresssion returns
• (some non-specific effects seen at higher concentrations)
• regain of expression as competitive construct stealing away transacting factors (which are at a finite level and therefore can’t be used by the ptk construct) leading to a dilution of the repressive effect

Question 25

What is the experimental evidence to show that methylated DNA forms a nucleosome structure that is relatively resistant to nuclease treatment? Kass et al 1997

Answer 25

Used micrococcul nuclease

• non-specific nuclease, acts on DNA if it is accessible, cannot act on DNA if it is tightly packaged
• observed: at an early time point, (1hr) see no MN activity when probed with the promoter
• at a late time point (16hr) see micrococcul activity
  
  • see a ladder - DNA wrapped around nucleosomes (tightly packaged)
• methylated DNA forms a nucelosome strcuture that is relative resistant to nuclease treatment (compact)

Question 26

What binds methylated DNA?

Answer 26

Mammalian proteins that bind methy-cytosines are:

• MeCP2
• MBD1
• MBD2
• MBD3
• MBD4
• Kaiso

Share methylcytosine binding domains (mostly) and transcriptional repressor domains

Directly recognise methylated cytosines

Question 27

What is the experimental evidence that the proteins involved in binding methylated cytosines work together?

Answer 27

Jones: MeCP”, Sin3 and histone deacetylase activity co-purify

• looked at candidates though to be involved in binding
• used xenopus oocytes
• factionaction of xenopus oocytes by ion exchange chromatography
• sin3 was already known as being involved in the repression of gene expression as well as histone deacetlyase activity
• Do all of these interact in vivo

1. Used antibodies against Sin3/MeCP2 in an immunoprecipitation reaction to see which are assocaited with histone deacetylase activity
2. MeCP2, Sin3 and histone deacetylase actvity co-immuno precipitate (however this may not be a direct link, may be other protins involved)

Question 28

What is the evidence that the interaction of MeCP2, Sin3 and histone deacelyase activity in vivo represses gene expression?

Answer 28

• Looked at the histone deacetylase activity with trichostatin A (relieves transcriptional repression)
• Measured hsp70 gene expression under increasing concentration os TAS (unmethylated DNA and CMV gene (consitiutive) as control)
• identified a positive correlation with increasing expression with an increasing concentration of TAS
• this is likely due to relaxed histone modification

Question 29

How do MeCP2, Sin3a and a histone deacetlylase inhibit transcription by DNA methylation?

Answer 29

Transciption is inhibited by DNA modification

• MeCP2
  
  • complex protein
  • interacts with a diverse number of repressor complexes
  • can also act as an activator at some targets
  • highly abundant in neurons (at levels approaching the level of histones)
  1. binds to region of methylated DNA
  2. recruits the sin3a co-repressor and a histone deacetlyase (HDAC)
  3. also recruits a histone lysine methyltranferase (HKMT)
  4. results in a change in chromatin structure

Question 30

What is Rett syndrome?

Answer 30

• severe neurodevelopmental disorder affecting females (lethal in males)
• Caused my MeCP2 mutation
• MeCP2 located on the X-chromosome
• MeCP2 highly expressed in the neurons
• in Mice, MeCP2 K/O almost paralysed mice, if reintroduced, can be fixed (reversible)

Question 31

How are DNA methylation patterns inherited? (symmetrical)

Answer 31

• methylation commonly occurs in a CG context (symmetrical, antiparallel sequence on the other strand)
• when methylated strands are replicated this results in a hemi-methylated daughter cell hemi-methylated DNA is a substrate for maintenance DNA methyltransferase
• Othe cystein needs to tbe remethylated
• hemimethylated state becomes homomethylated state

Question 32

How are non-symmetrical DNA methylation states inherited?

Answer 32

• occur in plants, stem cells
• following DNA replication, have one like the parent and one unmethylated
• requires the action of a de novo DNA methyltransferase to re-establish methylation pattern (add methyl groups to an unmethylated template)
• must be signals that specify that this sequence should be de novo methylated

Question 33

What patterns mammalian developments

Answer 33

Two waves of epigenetic reprogramming during mammalian development

Question 34

Outline the two waves of epigenetic re-programming that occur durnig mammalian development

Answer 34

• DNA methylation patterns are dynamic during mammalian developemt
• Imprinted genes have parent-of-origin specific DNA methylation patterns
• paternal genes rapidly demethylated in the embryo during fertilisation
• maternal genes are demethylated slower

Question 35

What occurs in cells that go onto produce gametes?

Answer 35

Cells that will go onto produce gametes undergo de/re methylation v. early and this is essential for development

Question 36

What are teh dynamics of mammalian DNA methylation?

Answer 36

• De novo methylation can be demethylation or remethylation
  
  • can be active demethylation
  • passive demethylation
  • maintenance methylation (Dnmt1)
• Maintenance methylation
  
  • Dnmt1 interacts with the replication fork as soon as a hemi-methylated state is formed
• Demethylation
  
  • active (enzymatically removing methyl groups - paternal reprogramming)
  • passive (failiure to maintain during replication - maternal reprogramming)

Question 37

What is the process of active demethylation/

Answer 37

• Involves 5-hydrocymethylcytosine
• Ten-eleven translocation (TET) enzymes oxidise 5-methylcytosine to 5-hydroxymethylcytosine (5hmc)
• Tet1, 2, 3
• 10% of 5mc are 5hmc
• unsure as to whether anintermediate in removal of 5mc or a true epigenetic modification

Question 38

Outline the features of 5-hydroxymethylcytosine

Answer 38

• abundance is highest in brain and nervous system
• accumulates in the brain
• suggest it is an active mark
• relatively high levels in pluripotent/multipotent cells
• TET1 and TET2 mediated aquisition of 5hmcis involved in reprogrammin somatic cells to pluripotent cells

Question 39

What are the pathways for DNA demethylation in mammals?

Answer 39

• 5mC converted to 5hmc (TET proteins)
  
  • replcation -> passive demthylation
  • OR glycosylation - BER -> form 5 formylcytosine or 5-carbozylcytosine (levels of which are constantly low)
  • OR direct effectso of 5hmc leading to the displacement or recruitment of proteins