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How to measure DNA methylation
Alix Groom
Introduction
• Which methods are applicable for research question
• Where to look for methylation
Research Question
Research Question
No prior candidates
Genome-wide approaches
Candidates identified
Analyse DNA methylation
of target region
Illumina arrays
Qiagen Pyrosequencing
MeDIP-chip
Sequenom EpiTYPER
MeDIP-seq
Bisulphite sequencing
Bisulphite sequencing (NGS)
VeraCode BeadXpress
Research Questionwhat to look at
• What changes in DNA methylation are important
methylation at single CpG sites?
mean across several CpG sites?
global analysis
single CpG
multiple independent CpGs
multiple linked CpGs
v
repetitive elements
(LINE-1)
Pyrosequencing-LUMA
unmethylated
methylated
Pyrosequencing
EpiTYPER
MSP
array technology
VeraCode
Pyrosequencing
EpiTYPER
MSP
adapted from Siegmund KD, Methods 2002 27:170-178
Shores, Shelves and the Open Sea
CpG island
> 200bp in length
GC percentage >50%
Observed/expected CpG ratio >60%
Open Sea
Shore
Shelf
Isolated CpGs in the
genome (Sandoval et
al.)
Up to 2kb
from CpG
island
2-4 kb from
CpG island
Sandoval J, Epigenetics 2011 June; 6(6):692-702
Where to look CpG Islands (CGI)
gene expression
CGI
gene
X
CGI
gene expression repressed
unmethylated
methylated
gene
• within/near ~ 40% promoters
• aberrant methylation CGIs in tumours
• usually unmethylated in ‘normal’ cells
Where to look CpG Shore
• CHARM analysis
• greatest tissue differential methylation
outside CGI
T-DMR
6% CGI
76% shore
18% > 2kb from CGI
Irizarry RA, Nature Genetics 2009 Feb;41(2):178-86
differential expression
Where to look CpG Shore
• analysed gene expression in 5 primary livers and brains
• 2,041 gene/T-DMR pairs for brain vs liver
• gene expression strongly correlated with T-DMR at CpG shores
differential methylation
T-DMR within 300bp TSS
T-DMR 300-2000bp from TSS
Log ratios all genes >2kb TSS
Irizarry RA, Nature Genetics 2009 Feb;41(2):178-86
Where to look CpG Shelf/CpG open sea
Functional genomic distribution
CpG content and neighbourhood context
• Infinium HumanMethylation450 BeadChip
• distribution of hypomethyalted CpGs in HCT-116
Sandoval J, Epigenetics 2011 June; 6(6):692-702
Where to look exonic/intronic regions
Brenet et al. undertook genome-wide analyses of DNA methylation
and gene expression
• determine how the pattern of intragenic
methylation correlates with transcription
• assess the relationship between methylation of
exonic and intronic portions of the gene body
Methods used:
• STAMP : identify DNA fragments and ABI SOLiD
sequencing
•gene expression using Illumina Human Ref8
microarrays
•other methods to check data including
qPCR Methylight
deep sequencing using 454 Titanium Sequencer
HumanMethylation27 arrays
Brenet F, PLoS One 2011 Jan 18;6(1):e14524
Where to look exonic/intronic regions
Findings of Brenet et al. (using DNA methylation quantified from all transcripts
annotated in the Refseq database
methylation surrounding
the TSS is tightly linked to
transcriptional silencing
TSS
Methylation in introns and
downstream exons highly correlated
and unassociated with the magnitude
of gene expression
Exon 1
DNA methylation downstream of the TSS, in the region
of the first exon, is much more tightly linked to
transcriptional silencing than is methylation in the
upstream promoter region
Brenet F, PLoS One 2011 Jan 18;6(1):e14524
Summary
• Method to analyse DNA methylation dependent on research question
• Range of genome wide and locus specific methods available
• Selecting region to analyse
CpGs cluster in CGI
shores/shelves
CpG position in relation to gene
References
• Siegmund KD et al, Methods 2002 27:170-178
Analysis of complex methylation data
• Sandoval J et al, Epigenetics 2011 June; 6(6):692-702
Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome
• Irizarry RA et al, Nature Genetics 2009 Feb;41(2):178-86
The human colon cancer methylome shows similar hypo- and hypermethylation at
conserved tissue-specific CpG island shores
• Brenet F et al, PLoS One 2011 Jan 18;6(1):e14524
DNA methylation of the first exon is tightly linked to transcriptional silencing
How to measure DNA methylation
Alix Groom