• 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
• 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
• 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
