How to measure DNA methylation Alix Groom
Introduction
• Which methods are applicable for research question • Where to look for methylation
Research Question
Illumina arrays
MeDIP-chip
MeDIP-seq
Bisulphite sequencing (NGS)
Genome-wide approaches
Research Question
No prior candidates
Qiagen Pyrosequencing
Sequenom EpiTYPER
Bisulphite sequencing
VeraCode BeadXpress
Analyse DNA methylation of target region
Candidates identified
Research Questionwhat to look at
• What changes in DNA methylation are important methylation at single CpG sites? mean across several CpG sites?
v
multiple independent CpGs
array technology VeraCode
multiple linked CpGs
Pyrosequencing EpiTYPER
MSP
single CpG
Pyrosequencing EpiTYPER
MSP
global analysis
repetitive elements (LINE-1)
Pyrosequencing-LUMA
unmethylated methylated adapted from Siegmund KD, Methods 2002 27:170-178
Shores, Shelves and the Open Sea
> 200bp in length GC percentage >50% Observed/expected CpG ratio >60%
CpG island
Shore
Up to 2kb from CpG island
Shelf
2-4 kb from CpG island
Open Sea
Isolated CpGs in the genome (Sandoval et al.)
Sandoval J, Epigenetics 2011 June; 6(6):692-702
Where to look CpG Islands (CGI)
gene CGI
gene expression
gene CGI
gene expression repressed X unmethylated
methylated
• 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
Where to look CpG Shore d
iffe
ren
tial
exp
ress
ion
differential methylation
T-DMR within 300bp TSS T-DMR 300-2000bp from TSS
Log ratios all genes >2kb TSS
• 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
Irizarry RA, Nature Genetics 2009 Feb;41(2):178-86
Where to look CpG Shelf/CpG open sea
Sandoval J, Epigenetics 2011 June; 6(6):692-702
• Infinium HumanMethylation450 BeadChip • distribution of hypomethyalted CpGs in HCT-116
Functional genomic distribution CpG content and neighbourhood context
Where to look exonic/intronic regions
Brenet F, PLoS One 2011 Jan 18;6(1):e14524
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
Exon 1
Methylation in introns and downstream exons highly correlated
and unassociated with the magnitude of gene expression
methylation surrounding the TSS is tightly linked to transcriptional silencing
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
TSS
Findings of Brenet et al. (using DNA methylation quantified from all transcripts annotated in the Refseq database
Where to look exonic/intronic regions
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