Epigenetics Flashcards
What is epigenetics?
- 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
What does epigenetic regulation determine?
How much RNA is made and when/where it is synthesised
Genetic imprinting
- Usually 2 functional copies of each gene is inherited
- Imprinted genes are silenced through epigenetic mechanism
- Result: only one working copy is inherited
Paternal imprinting
Allele from father is switched off, therefore, only 1 functioning allele remains
How does chromatin compaction influence activity of DNA transcription?
- Heterochromatin = transcriptionally silent
- Euchromatin = transcriptionally active
What is a nucleosome?
- DNA wound around a histone core
- Made of a nucleosome core particle and links DNA
What is a histone core?
- Octamer of 4 histone proteins: H2A, H2B, H3 and H$
- Each histone protein has a structured domain, histone fold and unstructured N-terminal trail
What do histone tails do?
- Provide a site for covalent modifcations - acetlyation/methylation
- Determines interaction of histone with other proteins which can alter chromatin structure
What are ncMRNAs and what role do they have?
- Do not translate into proteins
- Role in transcription of protein coding transcripts into RNA and translation into functional proteins
What role do ncMRNAs have in cancer?
- 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
How do Histone deacetylase inhibitors (HDACi) work?
- 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
What is the most common DNA methyltransferase (DNMT) used in epigenetic therapy?
- 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
How do DNA methyltransferase (DNMT) inhibitors work?
- Interacts with DNMTs, thus inhibiting DNN methylation in subsequent rounds of DNA synthesis
- Reactivates genes that were epigenetically silenced
TET2 Mutations In Acute Myeloid Leukemia (AML)
- TET2 mutations associated with decreased levels of 5hmc and TET2
- Poor prognosis in intermediate risk AML
What is CIMP?
- CpG islan methylator phenotype on cancer
- Cancers with high degrees of methylation - clinically and aetiologically distinct group - epigenetic instability
How is the methylator phenotype an alternative drive of tumorigenesis?
- DNA hypermethylation silences tumour suppressor genes - accruement of mutations - cancer
MLH1 in cancer
- Mismatch repair gene - frequently mutated in familial colon cancer
- Microsatellite instability phenotype (multiple genetic alterations)
AML and DNA Methyltransferase 3 Alpha (DNMT3A)
- Recurrent mutations
- Expresses dominant-negative effects
- AML cells wth R882h mutation - profound reduction of de novo methyltransferase activity
Which enzymes catalyse acetylation?
- Histone Acetyltransferase (HAT) and Histone Deactylase (HDAC)
What does histone acetylation do?
- 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
How does the epigenetic profile differ in cancer cells to normal, healthy cells?
- Cancer cells have abnormal DNA methylation profiles characterised by genome-wide hypermethylation and promoter region hypermethylation
Hypermethylation in cancer cells
- 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
How would DNA methylation make a good biomarker?
- 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
Role of DNA hypomethylation in cancer cells
- Genome instability
- May result in activation of protooncogenes or retroviruses
- Results from loss of function mutations from DNMT3A
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
What 2 physical changes are caused by DNA methylation?
- Displaces transcriptional factors
- Attracts methyl-binding proteins
What are the 3 main types of methylated bases in DNA?
C5, N4, N6 methylcytosine
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
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
What is active demethylation?
- Involves enzymes (demthylase) that can occur independent of DNA replication
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
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
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
What does histone methylation do?
- Recurits silencing or regulating proteins that bind to mehylated histones
- Chromodomain containing proteins interact with methylated histone tails
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
Where does C-5 cytosine methylation occur?
- Occurs in the sequenc 5’-CG-3’
- Cytosine is followed immediately by a Guanine-CpG dinucleotide
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
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
3 pros of sequencing base modifications directly?
- Longer reads, allowing phasing of haplotyples, repeat regions
- Sequence base modifications directly
- Sequence info and base modifications
4 cons of sequencing base modifications directly?
- Need good quality DNA
- 5 μg required
- x250 coverage needed for 5mc + 5hmc
- Technology still being optimised
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
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
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
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
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
- Denaturation (incubated at 95 degrees, with fragment genome DNA)
- Conversion (incubated with sodium bisulphite at 65 degrees and a low pH) - deaminates cytosine residue in fragmented DNA
- Desulphonation (incubated at high pH for 15 mins)
Pros and cons of whole genome bisulphite sequencing?
Pros:
- Whole genome coverage
Cons:
- Costly and time consuming
- Requires extensive bioinformatics
- Limited scalability per run
3 cons of direct bisulphite sequencing
- Difficult to optimise
- Does not provide info about methylation patterns of individual alleles
- Cannot accurately quantify methylation
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
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
