Epigenetics Flashcards
Definition for epigenetic
- Structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states.
- Additional, heritable layer of information, on top of the DNA nucleotide sequence, that influences the expression level of a subset of genes in a cell.
- Study of changes in the regulation of gene activity and expression that are not dependent on gene DNA sequence.
Gene expression is controlled by the epigenetic mechanisms of:
- Histone modification
- DNA methylation
- Non-coding or interfering RNA’s
Which part of the histone can undergo modification
The N-terminal tails of the core histones protrude from the nucleosomes and can undergo modifications.
What are some posttranslational covalent modifications that can happen to the histone tail.
- Acetylation
- Methylation
- Phosphorylation
- Ubiquitination
- Sumoylation
What is the the effect of acetylation on the DNA conformation.
Acetylation is negatively charged thus it repels the neg charge of DNA resulting in the loosening of histones and the associated DNA , accessible to the transcription factors-transcriptional activation.
Function of histone transferase (HAT)
Catalyse the addition of an acetyl group at the lysine residue
Function of histone deacetylase (HDACs)
Catalyse the removal of acetyl group from the lysine side chain of histone
Function of Histone methyltransferases
Transfer methyl groups onto the lysine or arginine residues of the H3 and H4 histone proteins
Where is DNA methylated?
Addition of a methyl group (CH3) to DNA at the site of a cytosine (C) base
Which DNA methyltransferases is responsible for maintenance of methylation after DNA replication.
DNMT1
Which DNA methyltransferases is responsible for de novo methylation.
DNMT3a & DNMT3b
Explain the phenomena of agouti mice. (Blonde and brown identical mice)
These two mice are genetically identical and exactly the same age. Each mouses mother received a different, specialised diet, which switched on or off chemical clusters called methyl groups that reside near genes. Flipping these genetic switches created differences in size, fur colour and health in the otherwise identical offspring.
What is the mechanism by which mature ncRNA molecules act.
It acts through partial complementary to one or more messenger RNA (mRNA) molecules thus preventing it from being translated into protein. Therefore it will down-regulate gene expression.
What makes Epigenetics a suitable target for therapies.
- Epigenetic marks are reversible
- Epigenetic proteins which establish or erase marks are attractive druggable targets that can be addressed through small-molecule inhibitors.
Epigenetic’s is crucial for which processes ?
- Cellular differentiation
- Embryonic development
Define parthenogenesis
Is the development of a female gamete into a new individual without being fertilised by a male gamete.
Can mammals go through parthenogenesis?
McGrath & Solter (1984) constructed fertilised zygotes that contained either two male or two female pronuclei. Development failed soon after implantation. Indicating that diploidy alone is not sufficient for normal embryonic growth and development.
What happens to a androgenic zygote (diploid paternal)
- Extra embryonic tissue (placenta) develops extensively
- Embryo physically regarded
What happens to a gynogenic zygote (diploid maternal)
- Embryo physically well developed but small.
- Extra embryonic tissue (placenta) poorly developed.
What does the fact that diploidy alone is not sufficient for normal embryonic growth and development mean?
That the maternal and paternal genome contributions are therefore functionally non-equivalent and possession of both parental genomes are essential for development.
Imprinting defined
Specific genes are inherited in such a way that one form is functional, while the other non-functional and that this is conditioned during gametogenesis
Epimutations can lead to…..
Both genes being active (too much protein product) OR both genes being inactivated (too little protein product)
What is re-established imprinting ?
When imprinting is wiped from the chromosome so that the correct parental Epigenetic pattern can be put in place to ensure the correct imprinting pattern is given by the female and male.
What are the two events that take place during Epigenetic reprogramming
- Primordial germ cells (PGCs) undergo imprinting erasure to establish correct imprinting for gametes.
- Early embryo (32-64 cell stage) erase epimutations to return a cell to pluripotency.
Imprinted genes are often important in ?
Growth and development of the foetus.
What are some modifications that happen to human chromosome 15 to ensure imprinting ? (Closed conformation)
- DNA cytosine methylation, histone H3 tail methylation at lysine 9.
- Recruitment of histone deacetylating enzymes and deacetylayed histones.
What are some modifications that happen to human chromosome 15 to ensure imprinting ? (Open conformation)
- Absent DNA cytosine 5-methylation
- Tails of histones H3 and H4 are lysine 4-methylated and acetylated respectively.
- Recruitment of histone acetyltransferase (HAT) to the domain on the paternal chromosome.
Clustering of imprinted genes
- Clusters may contain 3-10 imprinted genes.
- Human Chrs 2, 6, 7, 9, 11, 12 ,15 and 17 contain imprinted clusters.
What are imprinting control regions (ICR)
When grouping of several imprinted genes within a cluster allows them to be controlled by a common regulatory elements acting cis, these regulatory region are called imprinting control regions.
Example: XIC on X-chromosome which controls X-inactivation.
Examples of imprinting disorders examples
- Prayer-willi/angelman syndromes
- Beckwith -wiedemann / Silver-Russell syndromes
Similarities and differences between prader-willi and angelman syndromes.
- Both disorders are caused by genetic deletion and uniparental disomy for a domain on chromosome 15q.
- The difference between the two is the parental origin which leads to different clinical phenotypes.
Spontaneous deletion of chromosome region 15q11-1 in the case of angelman syndrome.
- Deletion On chromosome from mother
- No UBESA expression
- 65-75% of cases
Spontaneous deletion of chromosome region 15q11-1 in the case of Prader-Willi syndrome.
- Deletion on chromosome from father.
- Thus there is no expression of SNPRN
- (70-75% of patients)
What is the genetic mechanism that lead to imprinting disorders.
- Spontaneous ‘de novo’ deletion
- Uniparental disomy
UPD in 15q11-1 in the case of Prader-Willi syndrome.
- Lack of paternal contribution
- No SNRPN expression
- (20-25%)
UPD for 15q11-1 in the case of angelman syndrome
- Lack of maternal contribution
- No UBE3A expression
- (2% of patients)
Steps of Southern blot analysis
- DNA is cleaved, electrophoresis is used to separate DNA
- DNA fragments are blotted onto nitrocellulose filter
- Filter is exposed to radioactive probe
- Filter is exposed to photographic film, film is developed nn
How does southern blotting work to detect imprinting disorders.
Restriction enzyme does not cleave when chromosome is methylated thus is UPD has occurred no cleaving will happen thus one band will be present ( different imprinting disorder. Have different bands size)
Techniques that can be used to detect imprinting disorders
- Souther blotting
- Methylation -specific MLPA
- Methylation-specific PCR -bisulfites conversion
- Reduced representation bisulfite sequencing
- ChiP-Seq (NGS)
How does methylation -specific MLPA.
- If DNA is unmethylated, RE (Hhal) will digest probe and allele will not be amplified.
- If DNA is methylated, RE will not digest and allele will be amplified.
ChiP-Seq process
- Cell nucleus
- Cross link Protein and shear DNA
- Add protein specific antibody
- Immunoprecipitation and purify complexes
- Reverse cross links, Purify DNA and prepare for sequencing
- Sequence DNA fragment and map to genome
