8 - diagnosis and treatment of epigenetic alterations Flashcards
tissue and cell type profiling
bisorphite sequencing –> looks at histone modification and methylation of whole tissues
DNMTi
DNA methylation transferase inhibition
removes hypermethylation of tumour suppressor genes
cancer treatment
5-azacytidine
DNMTi
successful epigenetic therapy
DNMT enzyme binds to 5-azacytidine and decreases its activity
disadvantages of DNMTi therapy alone
lacks specificity
resistance common
unsuccessful in solid tumours
function of HDAC inhibitors
inhibit histone deacetylation
hyper-acetylation –> promotion of tumour suppressor genes
promote cell cycle arrest and apoptosis
examples of HDAC inhibitors
vorinostat
belinostat
romidepsin
combination treatments
combine HDACi with DNMTi
combine DNMTi with cytotoxic drugs
combination of HDACi with DNMTi
increased expression of silenced genes and antitumour apoptotic response
combination of DNMTi with cytotoxic drugs aim
resensitize cancers to the standard agents
discovery using next generation sequencing about chromatin cancer mutations
more than 50% of human cancers harbour mutations in enzymes involved in chromatin organisation
cancer cell immune evasion using epigenetics
- tumour cells use epigenetic process to escape chemotherapy and host immune surveillance
challenges to cancer epigenetic research:
- order of events of abnormal gene silencing in cells
- role of chromatin modifications in mediating these processes and targeting DNA methylation changes
- maintenance of gene silencing in cancer cells
- epigenetic biomarkers have low sensitivity –> need for multi-gene panels
- epigenetic therapies lack specificity –> adverse side effects and of target effects
illumina 450K methylation array
epigenome interrogation technique
covers DNA methylation sites at single-nucleotide resolution
almost genome wide
disadvantages of bisulfite sequencing
difficult to optomise
base calling software –< cant cope with lack of ‘C’ signal
over-exaggerates at methylated sites
doesnt provide information about methylation sites of individual alleles
cant accurately quantify methylation (semi-quantitative)
‘gold-standard’ for gene-specific methylation analysis
sequencing of cloned bisulfite PCR amplicons
pros of sequencing of cloned bisulfite PCR amplicons
- PCR production cloned into plasmid vectors and clones sequenced
- Highly quantitative
- Gives molecule-specific information
cons of sequencing of cloned bisulfite PCR amplicons
time consuming
expensive
process of target-specific bisulfite-Pyrosequencing
Real-time sequencing method for analysis of short-medium length DNA sequences
o Nucleotides added sequentially to reaction
o Pyrophosphate released during nucleotide incorporation detected as light produced by enzyme substrate cascade system
pros of target-specific bisulfite-Pyrosequencing
o Fast and reliable o Cost-effective o Targets specific regions of genome o Provides single nucleotide read-out o Reproducible
cons of target-specific bisulfite-Pyrosequencing
o Not high throughput
o Small fragments (max 100bp)
features of whole-genome bisulfite sequencing
- Whole genome coverage
- Costly
- Time-consuming –> need to resequence genome many times
- Requires extensive bioinformatics
- Limited scalability per run
method of whole-genome bisulfite sequencing
Measures single-base cytosine methylation levels (measures ratio of methylated molecules rather than enrichment)
RRBS
Reduced representation bisulfite sequencing
pros/cons of RRBS
pros:
- Reduces sequencing costs
- Increases read-depth at CpG-rich regions
- Provides single nucleotide read out
Cons:
- Short fragments can create mapping problems
- Biased towards repeat sequences, CpG sequences
- Can miss under-methylated regions
Target enrichment:
–> ‘sure select xt human methyl-seq’
The first comprehensive methylation discovery system
probes independent of methylation state
determines methylation state of all methyl sites in region
pros and cons of target enrichment
Pros:
o reduced sequencing costs
o targeting CpG rich regions of genome shores and DMRs
o can create custom targets
cons: o library preparation is time-consuming o 3ug DNA required o not truly genome wide o automation not supported
process of methylated DNA immuno-precipitation (MeDIP)
o monoclonal antibody specific for 5meC
o immunoprecipitation of single stranded molecules containing one or more methylated cytosine
pros and cons of methylated DNA immuno-precipitation (MeDIP)
pros
o reduces sequencing costs (5Gb required)
o no bias to one specific sequence
o can be adapted for different cytosine residues e.g. 5-hmc
cons
o don’t get base pair resolution (max res. 150bp)
o cant target specific regions e.g. gene body shores etc.
o hard to detect under-methylated regions
o methylation levels hard to determine
pacific biosciences single-molecule DNA:
sequencing base modification directly
- use polymerase kinetics
- kinetic characteristics time between two successive base incorporations are altered by the presence of a modification increased space between fluorescence pulse interpulse duration
SMRT stands for
single molecule real time sequencing
pros and cons fo SMRT
Pros:
- longer reads –> allows phasing of halotypes, repeat regions etc.
- sequences base modifications directly
- sequence information and base modification
cons:
- need good quality ds DNA
- 5ug required
- X250 coverage needed for hmC and h-hMc
- Technology still being optomised
use of chromatin modification assays
- Histone marks along with other assays of open chromatin are presently the only reliable indicators of locations and activities of regulatory elements
open chromatin
euchromatin
histones bound loosely to DNA
binding sites accessible for transcription factors –> active
closed chromatin
heterochromatin
histones packaged tightly
process of chromatin immunoprecipitation (ChIP)
Antibody attached to a DNA binding protein is used to capture DNA bound to the protein in a living cell
o DNA and protein cross-linked in the cell using formaldehyde
o Crosslinked chromatin sheared yielding short fragments of DNA and protein complexes
o Antibodies capture fragments containing DNA binding protein
o DNA ‘released’ and analysed –> PCR
- Creates pool of sequences highly enriched in binding sites for a particular protein
o Requires availability of excellent antibodies that can detect the protein in its in vivo context
ATAC-SEQ stands for
(Assay for Transposase-Accessible Chromatin using Sequencing)
how does ATAC-SEQ assay work
- Transposons incorporate preferentially into genomic regions free of nucleosomes
- Enrichment of sequences from certain loci in the genome indicates absence of DNA-binding proteins of nucleosome in the region
- Regions of the genome where DNA was accessible during the experiment will contain significantly more sequencing reads = Peak
steps involved in ATAC-SEQ assay
- Cell preparation
- Transposase reaction
- Library amplification
- Sequencing on an illumina platform
- Bioinformatic analysis
3 scales of chromatin structure
primary
secondary
tertiary
what does primary chromatin structure encompass
DNA methylation sequence features DNA-bound factors nucleosome positions modification DNA accessiblity
what does secondary chromatin structure encompass
local structures formed by nucleosome-nucleosome interactions
what does tertiary chromatin structure encompass
promoter-enhancer 3D contacts
mega-base scale domains
where are majority of GWAS variants found
regulatory regions (promoters/enhancers) not in protein-coding regions
original hypothesis
SNP caused by change in amino acid sequence
causes change in protein product
alternative hypothesis
SNP caused by DNA methylation
causes changes in gene expression
use of mQTL
used to identify functional consequences of genetic risk factors
results overlapped with GWAS results
why can GWAS not identify the specific causal variant
GWAS identifies regions
there could be two distinct causal variants in the same LD block
3 categories of laboratory techniques to interrogate the epigenome
- DNA methylation analysis
- analysis of DNA/protein interaction
- chromatin accessibility and conformation assays
methods of DNA methylation analysis
- bisulfite conversion (most common)
- high resolution melt (HRM) analysis
- methylation DNA immunoprecipitation (MeDIP)
which techniques is analysis of DNA/protein interaction
chromatin immunoprecipitation (ChIP)
general process of bisulfite conversion
identifies 5mC
converts unmethylated cytosines to uracil
5mC is not converted –> identified through PCR
quantifies levels of methylation
COBRA
Combined bisulfite restriction analysis
- type of bisulfite conversion
- Useful for analysing specific regions of DNA for methylation
- Requires restriction enzymes
High resolution melt (HRM) analysis
- Qualitative analysis of DNA fragment’s melt curve following bisulfite conversion and PCR amplification
method of HRM analysis
PCR target region to amplify
increase temperature of amplicon (DNA strands separate)
obtain melt temperature and melt curve profile
compare to standard DNA samples to determine relative levels of methylation
advantages of HRM analysis
Simple, cost-effective, fast
Methylation DNA immunoprecipitation (MeDIP)
- Genome-wide
- DNA isolated from cells and sheared using sonication (application of sounds energy)
- Antibodies specific to DNA fragments isolate methylated regions (5mC)
- Methylated DNA identified using high-resolution DNA microarrays or next gen sequencing
- Allows quantification of enriched methylated DNA fragments
limitation of ChIP
requires a large number of cells for reliable measurements
use of nuclease enzymes in chromatin accessibility assays
euchromatin (open) is digested
nucleases are unable to digest heterochromatin (closed)
therefore DNA is left available for examination
