Lecture 1 Flashcards
What is the outcome of pluripotent cell going down different cell lineages?
Loss of its pluripotency
Each lineage will have the same genotype but different genotypes
What did Muller discover in the 1930’s?
White eye mutation in drosophila, an unusual patchy phenotype
Something is influencing expression of the white gene - as fly is able to make the red gene still
Mutants made using X-ray
How was the phenotype seen by Muller arising?
An inversion produces position effect variegation
White+ gene produces red facets
Inversion places White+ close to heterochromatin, heterochromatin spreads and silences the gene= producing white facets
All due to changes in chromatin packagaing
Outline Brink’s work from 1958
Had two alleles, Rst and Rr, for maize with distinct leaf phenotype.
Rst leads to stippled leaf pattern
Closing homozygotes for both alleles creates stippled F1 ( appears stippled is dominant)
Would expect RrRr F2 to not be stippled but it is (Rst interaction has caused paramutation - changed phenotype of another allele)
The RrRr F2 also now has ability to pass on the stippled phenotype
What is different about the Rst and Rr mutants in the F0’s?
How does this change during breeding?
Rst is methylated whereas Rr is not
All alleles acquire DNA methylation due to interaction with methylated Rst
What epi-mutant was found by Linnaeus?
The peloria mutant of Linaria vulgaris
How was the peloria mutant different?
The flowers had radial symmetry rather then bi-lateral symmetry
What cause was put forward by Coen for the cause of the peloria phenotype?
How was this disproved?
What was found to be the cause?
A mutation in its cycloidea gene (as seen in Antirrhinum majus)
Sequencing of the gene showed no mutations
The cycloidea gene in peloria is heavily methylated, switching off the gene
What was shown in the trans-generational stress response?
Mothers who were pregnant during famine, shown during the hongerwinter of 1944, had children of lower birthweight, especially if the famine occurred early in the gestation period, and these children had a higher rate of obesity and diabetes in later life. The offspring of these children also gave birth to low weight offspring with the same higher incidence of diabetes and obesity even though they were not pregnant during a famine. DNA methylation was shown to be changed due to grandmothers/mothers nutrition.
What is Riggs’ definition of epigenetics?
The study of mitotically and/or meiotically heritable change sin gene expression that cannot be explained by changes in DNA sequence
What is requires for epigenetic changes?
1) Mechanisms to create specific “expression states” that result in differential gene expression
2) Mechanisms that allow these expression states to be maintained during cell division and development
What is a transient change in gene expression?
Signal changes expression state of the gene, but removing this signal causes the expression to revert to its original state
What is seen in epigenetic re-programming of gene expression?
Signal changes expression state of the gene, and removing this signal does not caue the expression to revert to its original state
What are the key players in epigenetic re-programming?
DNA methylation
Histone acetylation
Non-coding RNA’s
(Lots of interplay seen between these players)
Where does DNA methylation occur?
What is it associated with?
On cytosine residues in many eukaryotes
Generally with transcriptional inhibition (when located close to promoter sequences)
Which other nucleotide can be methylated?
Adenine (but not covered due to early state of the field)
What are the main roles of methylation:
in plants and vertebrates?
in fungi?
in invertebrates
Gene expression control - methylation of then gene (plants, vertebrates and invertebrates)
Gene defence - methylation of transposable elements (plants, vertebrates and fungi)
How is cytosine methylated?
S-adenosyl methionine (SAM) donor, donates methyl group (to become a SAH) which is added to cytosine by a DNA methyltransferase to create 5-methyl-cytosine
What two ways could DNA methylation be affecting gene expression?
1) Blocking binding of a transcription factor
2) Recruiting proteins that later chromatin structure and interfere with transcription
Why are Xenopus oocyte’s good expression systems?
Big and easy to inject into to look for expression
Outline Kass’ work with Xenopus oocyte’s
Different DNA constructs injected into oocyte
With a Herpes Simplex tk promoter driving a reporter gene, a chloramphenicol acetyltransferase (CAT)
Injected the constructed with the promoter methylated or unmethylated and then measured the transcription of the reporter gene
Methylating the tk causes reduction in gene expression but not immediately, supports that this is due to the methylation recruiting proteins to alter expression.
Then carried out competition experiment; added the constructs again but with another DNA sequence that was either un-methylated or methylated The methylated DNA removed the repressive effect of methylating the tk promoter.
Methylates DNA forms a nucleosome structure that is relatively resistant to nuclease treatment, does not get digested by micrococcal nuclease.
Which methyl-cytosine binding protein is most studied?
What are the common features of methyl-cytosine binding proteins?
MECP2
Methylcytosine-binding domains and transcriptional repressor domains
Which proteins did Jones investigate and how?
What was seen?
MECP2, Sin3 and histone deacetylase
Ion exchange chromatography to separate out and then used antibodies for MECP2 and Sin3 to see which fraction they were in, and enzyme assay to see which fraction the deacetylase was in.
The three proteins are found in the same fraction; didn’t prove a physical interaction.
Then showed that they did interact by doing an immunoprecipitation of MECP2 and Sin3 and seeing that they were associated with the histone deacetylase
Inhibiting the deacetylase relieved the transcriptional repression
How does a methylated region of DNA inhibit transcription by recruiting other proteins?
A methylcytosine binding protein, such as MECP2, binds to the site and recruits a co-repressor, such as Sin3a, and a histone deatylase (HDAC). Resulting in changes to the chromatin structure.
Additionally histone lysine methyltranferases (HKMT’s) are recruited to change the chromatin structure.
What do mutations in MECP2 mutations cause?
Rett syndrome
Severe neurodevelopmental disorder in females, which kills males (X-linked)
How is Rett syndrome reversible?
In mice, expressing a functional MECP2 cures the syndrome
What happens when symmetrically methylated DNA is replicated?
The two daughters are hemimethylated - acquiring one methylated strand from the parent and one newly synthesis unmethylated strand
How are hemi-methylated symmetrical daughter sites dealt with?
They are substrates for maintenance DNA methyltransferase which methylates the other strand to return the gene to what it was in the parent
What happens when non-symmetrically methylated DNA is replicated?
One daughter is like the parent; hemi-methylated
Whereas the other is un-methylated
De novo methyltransferases re-establish the methylation pattern due to signals that specify this sequence (due non-coding RNA’s in plants)
What are imprinted genes?
Genes that have a parent origin methylation pattern
What happens to the methylation states if the paternal and maternal genome during development?
Directly after fertilisation the paternal genome is un-methylated, this also happens to the maternal genome, but slower. The two genomes are then re-methylated during embryo development.
What happens to the paternal and maternal methylation states during germ cell development?
The same de-methylation and then re-establishment, which is different depending on if maternal or paternal, this is essential for correct embryo development
Which enzymes carry out de novo methylation?
Dnmt3a and Dnmt3b
Which enzymes carry out maintenance methylation?
Dnmt1
How can de-methylation occur?
Which type is seen in which parental genome?
Active - enzymatic removal of methyl group (Paternal)
Passive - Failure to maintain during replication (Maternal)
Which enzymes carry out active de-methylation?
Ten-eleven translocation (TET) enzymes; TET1,2 and 3
Can convert T-methylcytosine to 5-hydroxymethylcytosine
What percentage of methylated cytosines are 5hmC?
What is it associated with?
10%
Associated with active gene expression
Where is 5hmC commonly seen?
In the brain - increasing over age
In cancer - where TET enzymes are more active
How is 5-hmC then made into cytosine?
Passive demethylation
Deamination; removal of amine group and then base excision repair(BES)
Glycosylation and then BES
Further action of TET enzymes
What is the study of Epigenetics according to Waddington?
How genotypes give rise to phenotypes during development
