Epigenetics Flashcards
Define epigenetics
Factors (Information) that can be transmitted to progeny cells following cell division but are not directly attributable to the DNA sequence
What is CpG island methylation?
Methylated cytosine ( C ) where the 3’ carbon is linked to the 5’ carbon of guanine ( G ) by a phospodiester bond
Methylation occurs at the carbon 5 of cytosine to generate 5-Methylcytosine
What is SAM and what does it do?
S-adenosyl methionine
It methylates the cytosine produce 5-methylcytosine
What are the characteristics of CpG islands?
Occurance is 1 in 50
They are quite dense and not usually methylated
They are usually associated with promotor regions of a gene
They are transcriptionally competent
How do CpG islands behave in tumours?
Islands are methylated
Lead to chromosomal instability (hypomethylation)
Transcription may be lost (hypermethylation)
What are the exception to the CpG island rule?
Imprinted genes and X-chromosome inactivation in females
How is methylation maintained during replication?
During replication of parental strand the daughter strand not initially methylated
Daughter strand methylated by DNMT1
What are the three key mechanisms of methylation silencing genes?
Methylation within promoter regions prevents binding on specific transcription factors
As above but permits binding of proteins that suppress gene expression
Chromatin remodelling
What proteins are recruited by methylated DNA and what do they do?
MeCP1 AND MeCP2 which deacetylate histones
What is expressed on relaxed DNA (euchromatin)?
Non-methylated CpG islands and acetylated histone tails
What is expressed on condensed DNA (heterochromatin)?
Methylated CpG islands and non-acetylated histone tails
What histone modifications may take place?
Acetylation, methylation, ubiquitination, sumoylation and phosphorylation
What mechanisms lead to the loss of function of tumour suppression genes?
Loss of heterozygosity and mutation in the retained allele.
Homozygous deletion resulting in loss of both alleles.
Methylation of CpG islands leading to loss or reduced gene expression.
What is the role of p16 in cell cycle arrest?
- When RB1 is phosphorylated, it loses it’s affinity for E2F when then promotes progress through the cell cycle
- When RB2 is not phosphorylated, it binds to E2F and sequesters it which stops proliferation
- CDK4/6 binds to CCND1 to promote phosphorylation of RB1
- p16 sequesters CCND1 thus disallowing the phosphorylation of RB1
- Silencing of p16 may lead to uncontrolled cell proliferation
What is the degree of silencing by methylation dependent on?
Density of the methylation
What are the candidate gene approaches to methylation profiling?
Bisulphite sequencing Methylation specific PCR Methylation sensitive PCR Combined bisulphite restriction analysis Pyrosequencing
What are the genome wide approaches to methylation profiling?
Restriction landmark genomic scanning
Methods based on arbitrarily primed PCR
Differentially methylated hybridisation with CpG island array
Methylated DNA immunoprecipitation with tiling array
What are the candidate gene approaches to histone profiling?
Single gene chromatin immunoprecipitation
What are the genome wide approaches to histone profiling?
ChIP with tiling array
ChIP with whole genome sequencing
What are the candidate gene approaches to epigenetic unmasking?
DNA demethylating or histone acetylating drug with RT-PCR
Gene knockout with RT-PCR
siRNA with RT-PCR
What are the genome wide approaches to epigenetic unmasking?
DNA demethylating or histone acetylating drug with expression array
Gene knockout with expression array
siRNA with expression array
What does treatment with sodium bisulphite do?
It converts any C in the context of CG into a U unless it is methylated as it remains a CG
Primers are designed to non-specifically amplify methylated/unmethylated sodium bisulphite converted template DNA. PCR products are either sequenced directly, or individual molecules are sequenced after TA cloning.
What are the advantages of sodium bisulphite treatment?
Methylation can be characterized at single CpG resolution.
Quantitative
What are the disadvantages of sodium bisulphite treatment?
PCR primer design can be problematic due to reduced DNA sequence complexity following sodium bisulphite conversion.
Expensive thereby limiting genome wide applicability.
What is methylation sensitive PCR?
“Methylation” and “non-methylation” specific primers are used to interrogate the methylation status of small CpG-dense sequences, determined by the ability of each primer pair to achieve amplification of sodium bisulphite converted DNA by PCR
What are the advantages of methylation sensitive PCR?
Technically simple.
Considered to be the most sensitive methodological approach at a specific locus.
Quantification is possible using adapted methodologies
What are the disadvantages of methylation sensitive PCR?
Non-genome wide.
Resolution is reduced due the number of CpG sites within the primer sequences
What is methylation (restriction enzyme) sensitive PCR?
Genomic DNA is digested with a methylation sensitive restriction enzyme such as HpaI, SacII or NarI, and is used as a template for specific PCR amplification
What are the advantages of methylation (restriction enzyme) sensitive PCR?
Technically simple.
Potential single CpG resolution
What are the disadvantages of methylation (restriction enzyme) sensitive PCR?
Restriction enzyme site not always present within sequence
False positive and false negative results generated through inefficient “cutting” or insensitivity to the enzymes
Non-genome wide analysis
What is the evidence that methylation reinforces the silent state?
X-Chromosome inactivation in females:
Silencing appears to take place before the gene/chromosome becomes methylated
What is the evidence that for tumours methylation is responsible for gene silencing?
Need to demethylate a gene and then show it is
re-expressed
Drug: 5-Aza-2’ Deoxycytodine
Inhibits the action of the methylase enzyme DNMT1
