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TMS exam > 8 - diagnosis and treatment of epigenetic alterations > Flashcards

8 - diagnosis and treatment of epigenetic alterations Flashcards

1
Q

tissue and cell type profiling

A

bisorphite sequencing –> looks at histone modification and methylation of whole tissues

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2
Q

DNMTi

A

DNA methylation transferase inhibition

removes hypermethylation of tumour suppressor genes

cancer treatment

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3
Q

5-azacytidine

A

DNMTi
successful epigenetic therapy
DNMT enzyme binds to 5-azacytidine and decreases its activity

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4
Q

disadvantages of DNMTi therapy alone

A

lacks specificity
resistance common
unsuccessful in solid tumours

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5
Q

function of HDAC inhibitors

A

inhibit histone deacetylation
hyper-acetylation –> promotion of tumour suppressor genes
promote cell cycle arrest and apoptosis

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6
Q

examples of HDAC inhibitors

A

vorinostat
belinostat
romidepsin

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7
Q

combination treatments

A

combine HDACi with DNMTi

combine DNMTi with cytotoxic drugs

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8
Q

combination of HDACi with DNMTi

A

increased expression of silenced genes and antitumour apoptotic response

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9
Q

combination of DNMTi with cytotoxic drugs aim

A

resensitize cancers to the standard agents

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10
Q

discovery using next generation sequencing about chromatin cancer mutations

A

more than 50% of human cancers harbour mutations in enzymes involved in chromatin organisation

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11
Q

cancer cell immune evasion using epigenetics

A
  • tumour cells use epigenetic process to escape chemotherapy and host immune surveillance
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12
Q

challenges to cancer epigenetic research:

A
  • 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
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13
Q

illumina 450K methylation array

A

epigenome interrogation technique
covers DNA methylation sites at single-nucleotide resolution
almost genome wide

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14
Q

disadvantages of bisulfite sequencing

A

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)

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15
Q

‘gold-standard’ for gene-specific methylation analysis

A

sequencing of cloned bisulfite PCR amplicons

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16
Q

pros of sequencing of cloned bisulfite PCR amplicons

A
  • PCR production cloned into plasmid vectors and clones sequenced
  • Highly quantitative
  • Gives molecule-specific information
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17
Q

cons of sequencing of cloned bisulfite PCR amplicons

A

time consuming

expensive

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18
Q

process of target-specific bisulfite-Pyrosequencing

A

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

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19
Q

pros of target-specific bisulfite-Pyrosequencing

A 
o	Fast and reliable
o	Cost-effective 
o	Targets specific regions of genome 
o	Provides single nucleotide read-out 
o	Reproducible
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20
Q

cons of target-specific bisulfite-Pyrosequencing

A

o Not high throughput

o Small fragments (max 100bp)

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21
Q

features of whole-genome bisulfite sequencing

A
  • Whole genome coverage
  • Costly
  • Time-consuming –> need to resequence genome many times
  • Requires extensive bioinformatics
  • Limited scalability per run
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22
Q

method of whole-genome bisulfite sequencing

A

Measures single-base cytosine methylation levels (measures ratio of methylated molecules rather than enrichment)

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23
Q

RRBS

A

Reduced representation bisulfite sequencing

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24
Q

pros/cons of RRBS

A

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
25
Q

Target enrichment:

–> ‘sure select xt human methyl-seq’

A

The first comprehensive methylation discovery system

probes independent of methylation state

determines methylation state of all methyl sites in region

26
Q

pros and cons of target enrichment

A

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
27
Q

process of methylated DNA immuno-precipitation (MeDIP)

A

o monoclonal antibody specific for 5meC

o immunoprecipitation of single stranded molecules containing one or more methylated cytosine

28
Q

pros and cons of methylated DNA immuno-precipitation (MeDIP)

A

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

29
Q

pacific biosciences single-molecule DNA:

A

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
30
Q

SMRT stands for

A

single molecule real time sequencing

31
Q

pros and cons fo SMRT

A

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
32
Q

use of chromatin modification assays

A
  • Histone marks along with other assays of open chromatin are presently the only reliable indicators of locations and activities of regulatory elements
33
Q

open chromatin

A

euchromatin

histones bound loosely to DNA

binding sites accessible for transcription factors –> active

34
Q

closed chromatin

A

heterochromatin

histones packaged tightly

35
Q

process of chromatin immunoprecipitation (ChIP)

A

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

36
Q

ATAC-SEQ stands for

A

(Assay for Transposase-Accessible Chromatin using Sequencing)

37
Q

how does ATAC-SEQ assay work

A
  • 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
38
Q

steps involved in ATAC-SEQ assay

A
  1. Cell preparation
  2. Transposase reaction
  3. Library amplification
  4. Sequencing on an illumina platform
  5. Bioinformatic analysis
39
Q

3 scales of chromatin structure

A

primary
secondary
tertiary

40
Q

what does primary chromatin structure encompass

A 
DNA methylation
sequence features
DNA-bound factors
nucleosome positions
modification 
DNA accessiblity
41
Q

what does secondary chromatin structure encompass

A

local structures formed by nucleosome-nucleosome interactions

42
Q

what does tertiary chromatin structure encompass

A

promoter-enhancer 3D contacts

mega-base scale domains

43
Q

where are majority of GWAS variants found

A

regulatory regions (promoters/enhancers) not in protein-coding regions

44
Q

original hypothesis

A

SNP caused by change in amino acid sequence

causes change in protein product

45
Q

alternative hypothesis

A

SNP caused by DNA methylation

causes changes in gene expression

46
Q

use of mQTL

A

used to identify functional consequences of genetic risk factors
results overlapped with GWAS results

47
Q

why can GWAS not identify the specific causal variant

A

GWAS identifies regions

there could be two distinct causal variants in the same LD block

48
Q

3 categories of laboratory techniques to interrogate the epigenome

A
  • DNA methylation analysis
  • analysis of DNA/protein interaction
  • chromatin accessibility and conformation assays
49
Q

methods of DNA methylation analysis

A
  • bisulfite conversion (most common)
  • high resolution melt (HRM) analysis
  • methylation DNA immunoprecipitation (MeDIP)
50
Q

which techniques is analysis of DNA/protein interaction

A

chromatin immunoprecipitation (ChIP)

51
Q

general process of bisulfite conversion

A

identifies 5mC
converts unmethylated cytosines to uracil

5mC is not converted –> identified through PCR

quantifies levels of methylation

52
Q

COBRA

A

Combined bisulfite restriction analysis

  • type of bisulfite conversion
  • Useful for analysing specific regions of DNA for methylation
  • Requires restriction enzymes
53
Q

High resolution melt (HRM) analysis

A
  • Qualitative analysis of DNA fragment’s melt curve following bisulfite conversion and PCR amplification
54
Q

method of HRM analysis

A

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

55
Q

advantages of HRM analysis

A

Simple, cost-effective, fast

56
Q

Methylation DNA immunoprecipitation (MeDIP)

A
  • 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
57
Q

limitation of ChIP

A

requires a large number of cells for reliable measurements

58
Q

use of nuclease enzymes in chromatin accessibility assays

A

euchromatin (open) is digested

nucleases are unable to digest heterochromatin (closed)

therefore DNA is left available for examination

TMS exam (22 decks)

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