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