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.
