Epigenetics Flashcards
Define Epigenetics. Now.
Epigenetics is the study of the changes in the regulation of gene activity and expression that are not dependent on DNA sequence. This can refer to stable changes that may or may not be heritable between cells.
What are the effectors of epigenetic effects?
DNA methylation, histone modification and non-coding (nc)RNA regulation.
What is DNA methylation?
The addition of repressive methyl groups to DNA bases. In higher animals this takes the form of cytosine methylation at CpG islands, which are most densely clustered near transcription start sites in order to better regulate a gene.
What organisms use DNA methylation?
Cytosine methylation is present in humans, drosophila and in plants but not in worms or yeast.
What is deamination?
Cytosine can be deaminated to form uracil, a process which is often photoinduced.
What are the consequences of deamination?
It creates a transition mutation (U paired with G) that is corrected by uracil glycosylase.
What happens when methyl-cytosine is deaminated, and what are the consequences?
5-methyl-cytosine deamination creates a thymine base, a different mismatch/transition mutation which is more likely to go unnoticed and so introduce mutation into one of the daughter strands during replication.
What are the three hypotheses of how DNA methylation represses genes?
- Unmethylated DNA naturally has a more open structure allowing for easier binding of transcription factors and other non-histone proteins, including the PIC.
- Methyl groups actually impede the binding of TFs to DNA.
- Methylated CpG islands recruit methyl-CpG-binding Proteins that inhibit transcription. This is the most subtantiated theory.
How do methyl-CpG binding proteins interact with the methyl groups?
With their aptly named Methyl-CpG Binding Domains (MBDs)
What families of proteins possess MBDs?
• Methyl CpG Binding Proteins (I know, change the name)
o Most sequence specific family
o Examples include MeCP1, MeCP2, MBD1, 2 and 4.
• SRA-SET and RING finger associated domain proteins
o Examples include UHFR1 and UHFR2
• Zinc Finger proteins such as Kaiso
What are the two types of DNA methylation addition?
De novo and maintenance
What is maintanence methylation?
This is performed directly after replication to ensure that methyl groups are present on the daughter strand also, otherwise all methylation patterns would be lost within a few generations.
What facilitates maintanence methylation?
Methyltransferase enzymes are recruited to hemimethylated CpG islands and add on the new methyl group to the corresponding Cytosine.
DNMT1 is the methyltransferase responsible for Maintenance Methylation.
What is De Novo Methylation?
The addition of new methylation to totally unmethylated CpG sites by de novo methylases such as DNMT3a and DNMT3b.
When does De Novo Methylation occur?
mostly during development, shortly after fertilisation when the original methylation patterns are stripped away
What is the mechanism of De Novo Methylation?
How this is directed in order to maintain methylation is the right places and remove it at sequences needed for development is unknown, but often appears to be directed by sequence specific DNA binding proteins that recruit the methyltransferases.
What do DNMTs use as a substrate?
DNMTs use S-adenosyl methionine (SAM) as a source of methyl groups, transferring the Me group from SAM to the cytosine in the CpG island.
What does DNMT2 do and why?
It methylates tRNA cytosines (hence also being known as NSun2 tRNA methyltransferase). The exact purpose of this is unclear but knockout studies show that it is absolutely necessary for proper protein synthesis.
What happens to mouse embryos that have both DNMT3a and b knocked out and why?
DNMT3a and b are required for initial reprogramming of the genome so the mutants for both methylases (E and F) are not born and do not even develop into anything mouse-shaped.
What happens to mouse embryos that have either DNMT3a or b knocked out and why?
Mutants with only 3a knocked out (A) are born but are stunted, whereas 3b knockout is (B) more serious and the embryo does not develop.
What happens to mouse embryos that have DNMT1 knocked out and why?
As it is a methylase that is used during replication but not for de novo methylation, the DNMT1 knockouts survive for longer than double DNMT3 knockouts.
DNMT -/- is still a recessive lethal mutation that prevents embryos from developing for longer than ten and a half days and causes highly abnormal morphology of what does result.
What is the agouti gene?
Mice have a gene called agouti which produces a yellow hair colour. It switches on cyclically to produce banded hair that looks brownish.
What is the a agouti mutation?
The a mutation knocks out the agouti yellow colour. This is a recessive mutation so still produces the agouti phenotype with one regular agouti gene (A)
What is the Avy gene (Agouti Viable Yellow)?
The agouti gene does not turn off, leading to entirely yellow mice.
What causes the Avy mutation?
A retrotransposon called PS1A slightly upstream of the agouti promoter, transcription of which interferes with the cyclical transcription of the agouti gene, locking it on.
What was initially expected from an Avy/a agouti mutant and why?
they would all be pure yellow as the Avy is dominant
What do Avy/a agouti mutants actually produce?
Despite being genetically identical the offspring have a whole range of coat colours from pure yellow to the normal colouring that was called pseudo-agouti.
What causes the varied phenotype of Avy/a agouti mutants?
Different levels of methylation on the retrotransposon in the different individuals. The more heavily methylated the transposon the less it was transcribed and so the less it caused the Avy yellowness.
What is partially responsible for the level of methylation of the PS1A transposon in Avy/a mutants?
The level of de novo methylation of the retrotransposon.
What happened when the mothers of the agout Avy/a mutants were supplemented with methyl donors such as SAM?
The level of pseudo-agouti phenotypes increased dramatically, but the level of transposon methylation did not. It is thought that the effect may be indirect, perhaps through histone modification.
What does maternal rat licking and grooming (LG) behaviour cause?
a chain reaction that eventually leads to decreased DNA methylation of the cortisol receptor in adult life, which leads to the rats being more resistant to stress and trauma.
What hormone levels were found to be different in adult rats whose mothers provided showed good LG behaviour?
cortisol, corticotrophin releasing hormone and adrenocorticotrophin were all different in ways that lower stress response.
How is rat LG behaviour passed on?
Female rats with high LG behaviour mothers became attentive mothers themselves, and vice versa.
Cross-fostering shows that the neuroplasticity of the young brain allows them to become more chilled out rats with higher LG behaviour themselves.
What is bisulphite PCR used for?
Measuing the level of methylation on a given area of DNA.
how does bisulphite PCR measure methylation levels?
- bisulphite treatment deaminates only UNMETHYLATED Cs to Us
- PCR used to amplify, and converts all Us to Ts as only dTNPs used
- Sequence of the PCR result compared to the original sequence. Cytosines still present in PCR were methylated in original.
What is used to measure the protein association with DNA, particularly used for identifying histone modifications?
Chromatin Immunoprecipitation (ChIP)
How does ChIP work?
- Use formaldehyde to cross-link proteins to DNA
- Sonicate DNA to shear strands that are not protected by proteins
- Purify the DNA using bead-localised antibodies specific to proteins
- Reverse cross-linking to dissociate the proteins
- Sequence DNA and identify in the genome, sometimes using microarrays
What is imprinting?
When the expression of a gene is different depending on whether the chromosome it is on is the paternally or maternally inherited one.
What organisms use imprinting?
These parent-of-origin effects are only present in placental mammals and are thought to have evolved 150 million years ago.
How many genes are thought to be imprinted in mice and in humans?
100 in mice and perhaps half that in humans
What are common examples of genetic disorders that involves imprinting?
Prader-Willi syndrome and Angelman Syndrome, which are each caused by mutations in the same gene but on the paternal and maternal chromosome respectively.
What are the IGF2 and H19 genes?
They are imprinted genes at closely spaced loci on chromosome 11. IGF2 is a hormone that promotes growth and body size, H19 is a long ncRNA that does the opposite.
In what ways are IGF2 and H19 imprinted and why?
In order to maintain dosage control the paternal version of IGF2 is expressed and the maternal one silenced, and vice versa for H19 which is expressed on the maternal chromosome and silenced on the paternal one.
What regulates the IGF2/H19 imprinting cluster?
The genes are separated by an insulator region which is methylated in order to allow transcription of Igf2 on the paternal chromosome and prevent H19 expression. The methylation is not present on the maternal chromosome so the opposite happens.
How does the IGF2/H19 insulator region methylation state control the imprinting?
methyl groups prevent the binding of four copies of the CTCF protein on the paternal chromosome, whereas they are present on the maternal chromosome. CTCF presence prevents a downstream enhancer from getting past the H19 gene, so it binds there and cannot activate the IGF2 gene. No CTCF means the enhancer only binds the IGF2.
What two genetic diseases are associated with the IGF2/H19 imprinting system?
Beckwith-Weidemann Syndrome
Silver-Russell Syndrome
What is Beckwith-Weidemann Syndrome?
When both maternal and paternal copies of Igf2 are expressed, leading to massive overgrowth of the child and an assortment of growth disorders, including often being born with parts of the intestine outside of the skin.
Can be caused by mutation or uniparental disomy.
What is BWS often associated with?
In over half of BWS cases there is also faulty imprinting at the adjacent but separately regulated Kcnq1 locus.
What imprinting occurs at the Kcnq1 locus?
This is a large locus with 8 protein coding genes, controlled by the methylation state of the promoter of an ncRNA within the locus. The ncRNA transcript represses transcription of all eight genes.
Paternal hypomethylation of this promoter allows expression of the ncRNA and hence the locus is silenced. On the maternal chromosome the promoter is hypermethylated, the ncRNA is not transcribed and the genes are transcribed.
What is Silver-Russell Syndrome?
When mutation or maternal uniparental disomy causes both H19 genes to be expressed, leading to dwarfism.
How do histone modifications control gene expression?
By causing the chromatin structure to open or close resulting in more or less or less access to the promoters for TFs and transcription machinery.
Briefly describe the organisation of DNA.
- Octamer formation (beads on a string), 10nm
- coiling beads into 30nm fibre
- Euchromatin, long looping structures of coil, 300nm
- Heterochromatin, mitotic chromosome arm, 700nm
What are the most common forms of histone modification?
The most common kinds of modification are acetylation and methylation, followed by phosphorylation and ubiquitination.
Which residue is the most important in histone tail modification?
H3K9, as it can be acetylated or methylated with different results.
Describe the modifiable tails present on each histone
Histones have loops on their C and N terminal ends. Only H2A and B have loops on both ends that can be modified. H3 and H4 have only a long modifiable N-terminal chain, though H3 also has a small loop that protrudes from the main body of the histone. H1b can also be slightly modified, but these are not thought to have any large impact.
How are histone modification conserved during replication?
This is unknown, histone proteins are believed to be distributed to the daughter strand randomly. However it may be that the existing histones on the parent strand are somehow used as a template.
What are the rarer histone modifications?
SUMOylation is the addition of Small Ubiquitin-like Modifier proteins (SUMOs) to H2A and H2B histones. They are thought to be transcriptionally repressive.
Biotinylation is the addition of biotin, which has been found to be present at transcriptionally silent chromatin regions.
ADP-Polyribosylation is found on histone H1. It takes the form of the addition of ADP-ribose polymers which are thought to be involved in maintaining unmethylated CpG islands.
What does histone acetylation do?
Addition of acetyl (CH3CO) to histones generally stimulates transcription.
What residues can be acetlyated?
This happens exclusively on Lysine residues.
There are three acetylable residues on histone H3; H3K9, K14 and K18.
There are a further four on H4; K5, K8, K12 and K16.
Histones H2A and H2B can also be acetylated.
What adds histone acetylations, and what families of proteins are there of this class of enzyme?
Lysines are acetylate by Histone Acetyltransferases (HATs)
There are three families of HAT:
• MYST
• GNAT
• p300
What removes histone acetylations
Acetylations are removed by Histone Deacetylases (HDACs). There are four families of HDAC, simply labelled class I-IV.
By what mechanisms does histone acetylation increase transcription?
Negating DNA binding charge on histone lysine tails.
Several regulatory proteins also bind specifically to acetylated histones, offering another mechanism for upregulating transcriptional activity.
How do proteins that recognise histone acetylation work?
They bind to the acetylated loop by bromodomains that recognise a four amino acid sequence. Since this is not enough to develop proper target specificity the proteins (such as Gcn5p) are suspected to also interact with other regions as well.
Where and in what form can histone methylation occur?
This can occur on lysine or arginine residues on the histones, primarily H3 and H4 loops. Residues can be mono, di or trimethylated – the latter being the most important modification.
What effect does histone methylation have?
The modifications can stimulate or repress transcription. K4 methylation tends to be activation and K9 repressive.
What adds histone methylation and how?
Histone Methyltransferases (HMTs), such as Suv39H or G9a, or histone lysine methyltransferases (HKMTs) are responsible for adding the methyl marks using their catalytic SET domains.
What removes histone methylation?
Histone Demethylases
How does histone methylation effect transcription?
Methyl groups recruit chromatin remodeller proteins that can open or close the structure enzymatically.
What is the action of the G9a HMT?
G9a methylates H3K9. This mark is recognised by HP1 which recruits DNMT1 to methylate the CpG islands at that promoter.
What is a notable difference between G9a and Suv39H histone methylation?
G9a tends to mono and dimethylate whereas Suv39H performs trimethylation.
What proteins recognise histone methylation and how?
Effector proteins such as HP1 and polycomb repressors bind to the methylated lysines by chromodomains, a common and conserved motif of around 60 residues.
What is histone methylation responsible for?
Not only is histone phosphorylation associated with transcriptional regulation, it also seems to serve greater purposes involving the cell cycle and apoptosis, chromatin condensation, DNA repair and regulates developmental genes.
Where can histones be phosphorylated?
All histones, even H1 can be phosphorylated, most commonly the serine and threonine residues on H2A and B and H3. H3 serine phosphorylation is associated with gene activation and mitosis.
How does histone phosphorylation act?
It is uncertain how histone phosphorylation acts but it is likely that is functions through ‘cross-talk’ with other histone modifications, possibly serving to mediate recruitment and release of effector proteins that bind to the histone tails.
What is ubiquitin?
A short, 76AA protein that is commonly covalently attached to other proteins as a marker by ubiquitin ligase.
How are histones ubiquitinated?
Histones are only ever monoubiquitinated. The ubiquitination is added by ubiquitin ligases and removed by deubiquitinating peptidases (DUBS).
What effects do H2A and H2B ubiquitination have?
Histone H2B modification appears to be crucial to upregulation of the initiation and elongation stages of transcription, whereas H2A ubiquitination is associated with gene silencing.
Why is histone ubiquitination relevant in cancer research?
Monoubiquitinated H2A is dramatically downregulated in prostate cancer tumours and the same goes for H2B in breast cancer tumours.
The protein product of the tumour suppressor gene BRCA1 contains a ubiquitin ligase domain, and mutation in that gene are associated with an 87% risk of breast cancer and a 44% risk of ovarian cancer.
Why must Thaliana arabidopsis retain tight control of when it flowers?
To prevent its offspring from being produced too late and dying during the winter.
What must the regulation of arabidopsis flowering induction genes be sensitive to?
What the temperature is, but how long it has spent in the warm.
What protein controls flowering in arabidopsis?
Flowering is inhibited by the Flowering Locus C (FLC) protein.
How is the FLC gene regulated?
The FLC gene relies on histone acetylation for transcription.
After a period of cold weather the FLD gene is activated, which produces a deacetylase enzyme. This removes the acetyl groups from the FLC gene which causes the structure to close and transcription to stop, so the repression of flowering is lifted.
What provides the measurement of time spent in the warm?
How much of the H3K27 is still trimethylated at the distal region of the FLC gene, a mark which is induced by the cold after a delay and decays slowly.
This is as opposed to the H3K27Me3 levels in the nucleation region which increase immediately when vernalised but quickly decay.
This is called Bistable Expression
What is polycomb repression?
A mode of repression performed by a particular group of proteins (polycomb proteins) that is used to permanently silence genes over infinite cell generations
What is polycomb repression used on, and how does this effect its rarity?
Genes involved in many processes including development of different cell types (particularly unneeded Hox genes), cell cycle control, senescence, X-inactivation, and stem cell differentiation.
Because in each cell it is used to repress all other differentiation pathway genes it is very common, repressing thousands of genes in each cell.
What is the opposite of polycomb repression?
A counteracting set of proteins called the trithorax proteins create long term stable expression for the essential Hox genes in a cell.
What proteins are involved in polycomb repression?
Most PC proteins are involved in one of two different protein complexes which perform the repression. These complexes are named Polycomb Repression Complex 1 and 2, though PRC2 does in fact initiate the process.
What initiates polycomb repression?
PRC2 is thought to be recruited by repressor proteins bound to the DNA.
What is the action of PRC2?
It contains HDACs that act on the histones to close their structure and also contains a core protein called EZH2 which possesses a methyltransferase SET domain. This di and trimethylates H3K27 on all the histones across the target genes.
What is the role of PRC1
The methylated H3K27 is recognised by and recruits PRC1 by its dimeric Pc subunits. It is these Pc subunits binding to neighbouring nucleosomes that leads to chromatin compaction.
PRC1 does also possess a ubiquitin ligase domain that acts on H2A preventing Pol II elongation. PRC2 remains bound to the DNA to maintain the methylation during this process.
Why is X-inactivation necessary?
One of the X-chromosomes must be permanently inactivated to prevent getting a double dose of the gene products from it.
Which chromosome is turned off, and how is this decided?
Which chromosome is inactivated is random within the proper embryonic cells but the paternal chromosome is preferentially switched off in extra-embryonic tissues.
Selection mechanism is unclear, but it is known that the two chromosomes come together and join by a particular sequence before drifting apart and the inactivation process beginning on one of them.
What controls X-inactivation?
A single 100kb locus on the chromosome called the x-inactivation centre. This codes for the Xist ncRNA and its antisense transcript Tsix.
Xist coats only the target chromosome, ‘painting’ it as a signal for polycomb repression.
What is the role of Tsix?
Tsix is expressed on the both chromosomes prior to inactivation. While Tsix is still being transcribed Xist cannot be, and neither can RepA.
What is RepA?
A smaller ncRNA product within the Xist coding region that is transcribed into a stem loop structure.
What does RepA do?
It is co-transcriptionally loaded onto a PRC2 complex, which can then dissociate and rebind onto a particular site on the X-inactivation centre. However this cannot happen while Tsix is active.
What stops Tsix transcription, and so initiated lyonisation?
the production of Jpx from either chromosome (the only part of the regulation which acts in trans between the chromosomes) which knocks out Tsix transcription only on the chromosome that is to be inactivated.
What happens when Tsix transcription is knocked out by Jpx?
This allows RepA to be transcribed and the RepA/PRC2 complex to load onto the X-inactivation centre at the nucleation centre.
What happens when a RepA/PRC2 complex is formed at the nucleation centre?
Xist begins to be transcribed. Xist is also co-transcriptionally loaded with PRC2 complex
What happens when PRC2 is loaded with Xist?
This allows a transcription factor called YY1 to bind to another site in the nucleation centre as well as binding to the nascent Xist transcript. This results in the Xist acting as a rope between the PRC2, the YY1 and then Pol II as it is still being transcribed.
What happens when Xist is established as a tether between Pol II, PRC2 and YY1?
PRC2 then begins inactivating the chromosome by trimethylate H3K27. The process happens multiple times to produce several copies of this Xist-RepA-PRC2 unitthat each bind to strong polycomb marks of which there are about 150 across the chromosome.
The Xist is not actually needed at this point, as only more PRC2 complexes are needed to potentiate the repression.
What spreads the PRC2 repression during lyonisation?
The PRC2 complexes spread the inactivation marks out across the chromosome through the positive feedback of the methylation marks created.
There are also weak polycomb inactivation marks to which the complexes bind after the first round of inactivation, effectively ‘filling in the gaps’.
Where is ncRNA loading into PRC2 used as a mechnism?
co-transcriptional loading of an ncRNA is actually a common mechanism for recruiting polycomb repression complexes to areas for things such as lyonisation and silencing imprinted genes.
How is the PRCS tethered to the DNA when a long ncRNA is loaded onto it?
The DNA will have a PRC binding motif inside of a ncRNA transcript as well as a pause site in the gene that stops the Pol II from advancing past a certain point, ensuring that the ncRNA and hence the PRC2 complex is tethered to this area
What happens when a PRC2 is tethered to the DNA by a long ncRNA loaded onto it?
This ties the PRC2 to the areas so that other, as yet undefined proteins can bind to it.
It also allows the complex to repress the surrounding DNA, which is ended when the short-lived ncRNA is released and degraded.
What possible treatment does the x-inactivation mechanism provide for?
Using gene therapy to add an X-inactivation centre to one of the chromosome 21’s in the fibroblast of a Down’ Syndrome patient.
How was Xist added to Down’s Syndrome chromosomes? What effect did this have?
using a doxycycline induced promoter which, when activated, led to the inactivation of the edited chromosome via hypermethylation, ubiquitination and addition of Macro H2A histone variants into a chromosome 21 Barr Body.
What kinds of short nc RNA are there?
- miRNA
- small interfering (siRNA)
- PIWI interacting RNA
Why are miRNAs of interest to medical research?
Micro RNAs are particularly important in gene regulation, and are also of interest to medical research as abnormal expression of miRNAs is associated with a wide variety of cancers as well as Alzheimer’s disease.
What is a bivalent cell?
Cells found at the beginning point of two different possible lineages without having committed to either one.
In such cells where differentiation must occur quickly the RNA Pol II is often poised on the gene ready to transcribe as soon as the cell is signalled to differentiate.
What properties do bivalent cells have?
Their genes possess epi-marks both activating and repressing.
In such cells where differentiation must occur quickly the RNA Pol II is often poised on the gene ready to transcribe as soon as the cell is signalled to differentiate.
What patterns are seen in chromatin structure, epi-marks and global transcription during differentiation?
The chromatin in bivalent cells is highly dynamic, and tends to be generally more open in pluripotent cells with increasing density as the cell travels down the landscape.
Global transcription rates steadily decrease as the cell differentiates.
Specific types of methylations and acetylations are also seen to change in frequency with the change in potency.
What novel epigenetic mark was recently identified?
Wu et al (2016) showed that N-6 adenine methylation can be epigenetically silencing in mammals.
