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