1
Q
What is epigenetics?
A
- Reversible regulation of gene expression
- Mediated principally through changes in DNA methylation and chromatin structure
- Occurs independently of DNA sequence
- Refers to chemical modifications that control gene accessibility but DO NOT change underlying genetic code
2
Q
What does epigenetic regulation determine?
A
How much RNA is made and when/where it is synthesised
3
Q
Genetic imprinting
A
- Usually 2 functional copies of each gene is inherited
- Imprinted genes are silenced through epigenetic mechanism
- Result: only one working copy is inherited
4
Q
Paternal imprinting
A
Allele from father is switched off, therefore, only 1 functioning allele remains
5
Q
How does chromatin compaction influence activity of DNA transcription?
A
- Heterochromatin = transcriptionally silent
- Euchromatin = transcriptionally active
6
Q
What is a nucleosome?
A
- DNA wound around a histone core
- Made of a nucleosome core particle and links DNA
7
Q
What is a histone core?
A
- Octamer of 4 histone proteins: H2A, H2B, H3 and H$
- Each histone protein has a structured domain, histone fold and unstructured N-terminal trail
8
Q
What do histone tails do?
A
- Provide a site for covalent modifcations - acetlyation/methylation
- Determines interaction of histone with other proteins which can alter chromatin structure
9
Q
What are ncMRNAs and what role do they have?
A
- Do not translate into proteins
- Role in transcription of protein coding transcripts into RNA and translation into functional proteins
10
Q
What role do ncMRNAs have in cancer?
A
- Long non-coding RNA (lncRNA) reveal diverse gene expression profiles in benign and metastatic tumours
- Small non-coding RNAs or microRNA (miRNA) are capable of reprogramming multiple oncogenic cascades, therefore, can be used as target agents
11
Q
How do Histone deacetylase inhibitors (HDACi) work?
A
- HDACi’s upregulate intrinsic and extrinsic apoptosis pathways through the induction of pro-apoptotic proteins Bmf, Bim, TRAIL and DRS respectively
- This results in histone hyperacetylation, which stabilises the p53 protein, promoting cell cycle arrest and expression of pro-apoptotic genes
- Prevents tumour angiogenesis - HIF-1α proangiogenic transcription is hyper acetylated by HDACi’s, resulting in its degradation
12
Q
What is the most common DNA methyltransferase (DNMT) used in epigenetic therapy?
A
- 5-azacytidine
- Nucleotide analogue is incorporated into RNA/DNA, resulting in rapid loss of DNA methyltransferase activity because the enzyme becomes irreversibly bound to 5-azacytidine
13
Q
How do DNA methyltransferase (DNMT) inhibitors work?
A
- Interacts with DNMTs, thus inhibiting DNN methylation in subsequent rounds of DNA synthesis
- Reactivates genes that were epigenetically silenced
14
Q
TET2 Mutations In Acute Myeloid Leukemia (AML)
A
- TET2 mutations associated with decreased levels of 5hmc and TET2
- Poor prognosis in intermediate risk AML
15
Q
What is CIMP?
A
- CpG islan methylator phenotype on cancer
- Cancers with high degrees of methylation - clinically and aetiologically distinct group - epigenetic instability
16
Q
How is the methylator phenotype an alternative drive of tumorigenesis?
A
- DNA hypermethylation silences tumour suppressor genes - accruement of mutations - cancer
17
Q
MLH1 in cancer
A
- Mismatch repair gene - frequently mutated in familial colon cancer
- Microsatellite instability phenotype (multiple genetic alterations)
18
Q
AML and DNA Methyltransferase 3 Alpha (DNMT3A)
A
- Recurrent mutations
- Expresses dominant-negative effects
- AML cells wth R882h mutation - profound reduction of de novo methyltransferase activity
19
Q
Which enzymes catalyse acetylation?
A
- Histone Acetyltransferase (HAT) and Histone Deactylase (HDAC)
20
Q
What does histone acetylation do?
A
- Reduces affinity of tail for adjacent nucleosomes, thus relaxing higher-ordered chromatin structure
- Removes the positive charge of the histone tails, thus, reducing affinity for the negatively charged phospahte groups of DNA
- Increases access of transcription factors to DNA through structural changes in nucleosomes or nucleosomal arrays
- Acetylated histones are specifically recognised by other proteins, such as bromodomain in transcription factors and HATS
21
Q
How does the epigenetic profile differ in cancer cells to normal, healthy cells?
A
- Cancer cells have abnormal DNA methylation profiles characterised by genome-wide hypermethylation and promoter region hypermethylation
22
Q
Hypermethylation in cancer cells
A
- Promoter hypermethylation - selective gene suppression including tumour suppressor genes
- Can result in loss of function mutations in genes encoding DNA demethylases (TET1, 2 and 3), or overexpression of genes encoding DNA methyltransferases
23
Q
How would DNA methylation make a good biomarker?
A
- Easy to detect with high degree of sensitivity
- DNA methylation is more stable than RNA or protein based markers
- DNA mutations and DNA methylation is reflected in cell free circulating DNA (circDNA) released from tumour into blood, so it is the ideal candidate for the basis of a blood-based cancer diagnostic test
24
Q
Role of DNA hypomethylation in cancer cells
A
- Genome instability
- May result in activation of protooncogenes or retroviruses
- Results from loss of function mutations from DNMT3A
25
How do mutations in the epigenome cause cancer?
- Mutations in genes affecting the nucleosome - remodelling complex and chromodomain helicase DNA binding (CHD) protein family can result in cancer
- These mutations perturb the transcription of genes involved in the control of proliferation and specification of cell fate
- Disrupts DNA repair as the NRC is responsible for providing access of repair proteins to DNA
26
What 2 physical changes are caused by DNA methylation?
- Displaces transcriptional factors
| - Attracts methyl-binding proteins
27
What are the 3 main types of methylated bases in DNA?
C5, N4, N6 methylcytosine
28
What are the roles of DNA methylation?
- Long term silencing of genes
- Silencing of repetitive elements
- X-chromosome inactivation
- Establishment and maintenance of imprinted genes
29
What is passive demethylation?
- Occurs when maintenance methyltransferases are inactive during cell cycle following DNA replication
- Results in retention of the unmethylated state of the newly synthesised DNA strand
30
What is active demethylation?
- Involves enzymes (demthylase) that can occur independent of DNA replication
31
How is DNMT1 involved in DNA methylation?
- Maintenance methylase
- Maintains pattern of DNA methylation after DNA replication
- Requires hemi-methylated DNA substrate
- Will accurately reproduce pattern of DNA methylation on new strand
32
How is DNMT3A and B involved in DNA methylation?
- De novo methylases
- Will add methly groups to CpG dinucleotides, previously unmethylated on both strands
- Re-establish methylation process
33
What is the mechanism of DNA methylation?
- Methyl groups transferred by S-Adenosyl methionine
- Reaction catalysed by DNA methyltransferases (DNMT) or methylases
- S-Adenosyl methionine is then converted into S-adenosyl homocysteine
34
What does histone methylation do?
- Recurits silencing or regulating proteins that bind to mehylated histones
- Chromodomain containing proteins interact with methylated histone tails
35
Where are methyl groups added and how many times?
- Lysine/arginine residues of histone tails
- Arginine residues methylated once or twice
- Lysine residues methylated up to three times
36
Where does C-5 cytosine methylation occur?
- Occurs in the sequenc 5'-CG-3'
| - Cytosine is followed immediately by a Guanine-CpG dinucleotide
37
Where do CpG dinucleotides occur in genome?
- Occur in low abundance throughout genome
| - Tend to concentrate in CpG islands - found in 50% of promoter regions of genes
38
Where are CpG dinucleotides usually methylated and where does this occur?
- Methylated in non-promoter regions
- Methylationn within promoter regions - transcriptional silencing
- Methylation pf CpG islands - dysregulates gene transcription through inhibition of transcription factor binding either directly or via histone acetylation
39
3 pros of sequencing base modifications directly?
- Longer reads, allowing phasing of haplotyples, repeat regions
- Sequence base modifications directly
- Sequence info and base modifications
40
4 cons of sequencing base modifications directly?
- Need good quality DNA
- 5 μg required
- x250 coverage needed for 5mc + 5hmc
- Technology still being optimised
41
Chromatin modification assays - 3 points
- Histones bound tightly to DNA - closed
- Histones can be displace by TFs, RNA polymerase - open proteins
- Histone marks, along with other assays of open chromatin are currently the only reliable indicators of the locations and activities of regulatory elements
42
What does a chromatin immunoprecipitation (ChIP) assay do and how does it work?
- Assesses changes in chromatin structure
- Uses formaldehyde to cross link DNA and protein
- Using fragmented chromatin - immunoprecipitation with specific antibody
- Analysis of bound DNA once DNA is released
43
How are ATAC-seqs (Assay for Transposase-Accessible Chromatin using sequencing) useful?
- Transposons incorporate into genomic regions free of nucleosome (open chromatin)
- Enrichment of sequences from certain loci in the genome indicates absence of DNA binding proteins or nucleosome in the region
- Regions of genome where DNA was accessible will contain significantly more sequencing reads - PEAK
44
How does Methylated DNA immunoprecipitation (MeDIP) work?
- Uses an antibody raised against DNA methylation
Pros:
- Reduces sequencing costs
- No bias to specific sequence
- Can be adapted for different cytosine residues
Cons:
- Cannot target specific regions
- Hard to detect undermethylated regions
- Methylation levels hard to determine
45
How is bisulphite modifcation used?
- Bisulphite modification converts non-methylated cytosines in the DNA to uracils and then thymines during DNA amplification by PCR
- DNA sequencing and methylation sensitive primers (MSPs) are used to analyses bisulphite treated DNA
1. Denaturation (incubated at 95 degrees, with fragment genome DNA)
2. Conversion (incubated with sodium bisulphite at 65 degrees and a low pH) - deaminates cytosine residue in fragmented DNA
3. Desulphonation (incubated at high pH for 15 mins)
46
Pros and cons of whole genome bisulphite sequencing?
Pros:
- Whole genome coverage
Cons:
- Costly and time consuming
- Requires extensive bioinformatics
- Limited scalability per run
47
3 cons of direct bisulphite sequencing
- Difficult to optimise
- Does not provide info about methylation patterns of individual alleles
- Cannot accurately quantify methylation
48
Sequencing of cloned bisulphite PCR amplifications
- 'Gold standard' for gene-specific methylation analysis
- Time consuming an expensive
- PCR products cloned into plasmid vectors and >20 clones sequenced
- Highly quantitative ans gives molecule specific info
49
What is Reduced representation bisulfite sequencing (RRBS)?
- Solves problem of wastefulness as only interested in CpGs
- Chop up genome with methylation sensitive enzymes, cutting where these is a CpG
- Able to enrich the genome for methylated and unmethylated regions
Epigenetics
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