Genome-Wide DNA Methylation Assays

Nadia Khan, Rick Smith, and Anna KupermanEpigenetics 2012

Introduction• Most Genome Wide Approaches

were adapted from technologies originally developed for detecting methylation at the level of a single gene

• Advantages of a Genome Wide Approach– Scale of information

• Whole chromosome• Whole genome

– Wider regulatory networks– Facilitates comparative and

population level analysis

Tollefsbol 2009

Many methylation methods can be grouped into major categories based upon their general principles

Concept of Methylation Sensitive Endonuclease Assays

• Use a restriction enzyme(s) that is methylation specific and separate the unmethylated from the methylated

• Unmethylated regions enzyme-sensitive Methylated regions enzyme-resistant

• Identify multiple de novo methylated areas across genomes vs. one specific area– 2D gel and scintillation counting– Array

Laird 2010

Restriction Landmark Genomic Scanning (RLGS)

• Cleave genome into pieces based upon restriction landmarks (sites)

• Radioactively label cleaved ends• Separate using 1D and 2D gel electrophoresis• Quantitate signal by amount of fluoresence in

gel– Intensity = copy number of the restriction site

Tollefsbol 2009

• NotI:• Radioactively

labels ends• LEAST specific

• EcoRV: • MORE specific

• HinfI:• MOST specific

Eng et al. 2000

RLGS in-use

Takamiya, et al. 2009

Microarray Coupling of Methylated CpG Island Amplification (MCAM)

• Cleave genomic DNA with SmaI (methylation-sensitive)

• Cut received pieces again with XmaI; create sticky ends

• Amplify pieces using PCR• Hybridize onto a microarray• Analyze fluorescence reads and identify

corresponding genomic address

Tollefsbol 2009

Tollefsbol 2009

MCA

M in

-use

Estecio 2007

Sodium Bisulfite Methods

• Bisulfite Sequencing• Targeted and Whole Genome approaches• Bisulfite Libraries

Bisulfite sequencing

• m-C are resistant to bisulfite conversion• Compare with unconverted reference sequence to infer methylation pattern• Allows for single base resolution, but technically challenging genome wide

Tollefsbol 2011

Whole Genome Approach

• Pair Bisulfite Conversion with Next Generation Sequencing (NGS)– Massively parallel sequencing• Roche 454, Illumina, SOLiD platforms• Quick, relatively cheap, large scale analysis

Tollefsbol 2011

Pyrosequencing

Whole Genome Approach

• Pair Bisulfite Conversion with Next Generation Sequencing (NGS)– Massively parallel sequencing

• Roche 454, Illumina, SOLiD platforms• Quick, relatively cheap, large scale analysis

• Tenable for relatively small genomes– Arabidopsis thaliana

• Significant challenges for mammalian genomes– Reduced complexity of the genome– Short sequence reads– Solutions

• Longer sequence reads• Targeted approaches

Cokus et al. 2008; Tollefsbol 2011

Targeted Bisulfite Sequencing

• Reduced Representation Bisulfite Sequencing (RRBS)

• Molecular Inversion Probes (MIP)– Padlock Probes

Targeted Bisulfite Sequencing

• Reduced Representation Methylation Sequencing (RRMS) – Enrichment for CG-rich regions via Msp1 digestion

(5‘-CCGG-3‘)– NGS– Disadvantages: mostly un-methylated DNA

Meissner 2005; Jeddeloh 2008

Targeted Bisulfite Sequencing

Deng et al. 2009

Padlock Probes

Bisulfite Libraries• Applications and Advantages• Coverage of relevant genome regions • Facilitates large comparative study• Multiplex Sequencing• High sensitivity• Whole library amplification

• Pair with NGS or Array

Gu et al. 2011

Biological Affinity Based Methods• Basic Concept: Use antibodies that are specific

for 5meC or proteins that bind preferably to methylated genomic DNA to profile DNA methylation patterns. These patterns are detected through microarrays or parallel high through-put sequencing.

• Types– MBD affinity column (MAC)– Methylated DNA Immunoprecipitation (MeDIP)– Methylated-CpG island recovery assay

Laird 2010

MBD Affinity Column (MAC)• Uses MeCP2, a member of a family of proteins that contain methyl-CpG

binding domains. • Developed by Cross et al in 1994. This is the first time affinity enrichment

was used to look at genome-wide methylation

Figure 9.8 Tollefsbol 2009

Methylated CpG

Unmethylated CpG

MBD Affinity Column (MAC)

• Advantages: this method is fast and efficient.• Limitations: Needs a large amount of starting

genomic DNA to pass through column purification

Figure 9.8 Tollefsbol 2009

Methylated DNA Immunoprecipitation (MeDIP)

• Introduced in 2005 by Weber et al• Uses an antibody that specifically binds to methylated

cytosines.• Fragmented DNA is incubated with the antibodies,

immunoprecipitated, and then enrichment is quantified.• Advantages: Efficient, sensitive, large-scale analysis of

genomic methylation• Limitations: need good quality 5meC antibodies and

denatured ssDNA, which can be difficult to obtain in CpG rich genes, is required for analysis

Figure 9.8 Tollefsbol 2009

Methylated DNA Immunoprecipitation (MeDIP)

Methylated CpG

Unmethylated CpG

Figure 9.7 Tollefsbol 2009

MeDIP• How can it be used?– Identifying genes involved

in cancer development• Ex: Morris et al were able

to shortlist genes involved in renal cell carcinoma (RCC) suppression by looking at promoter regions that were frequently methylated in RCC lines, but not in normal kidney cell lines.

Morris et al. 2011

Methylated-CpG Island Recovery Assay (MIRA)

• MBD2b/MBD3L1 complex has a high affinity to methylated DNA (higher than MBD2b on it’s own; MBD3L1 has no affinity)

• MIRA developed in 2006 by Rauch et al to have a better screen for methylation patterns in lung cancer tumors so can have a better early detection

• Advantages: Can be used to examine large number of genes simultaneously, works on dsDNA, only need a few hundred nanograms of genomic DNA

Figure 9.7 Tollefsbol 2009

Methylated-CpG Island Recovery Assay (MIRA)

Figure 9.9 Tollefsbol 2009From activemotif.com

MIRA• Rauch et al (2006)

were able to identify lung tumor suppressor genes

• Rauch et al (2009) were able to use MIRA to characterize a human B-cell methylome at 100 bp resolution

Rauch 2006

• A lot of these assays are commercially available

• MeDIP– MagMeDIP Kit TM (Diagenode), – Methylated-DNA IP Kit (Zymo Research) and

Methylamp™ – Methylated DNA Capture Kit (Epigentek)

• MIRA– Ex: MethylCollector TM Ultra

Biological Affinity Assays

• Why are they good? – Quick and efficient genome-wide assessment of

DNA methylation• Disadvantages: – Do not give information on individual CpG

dinucleotides– Require experimental or bioniformatic adjustment

for changing CpG density at different regions of genome

Laird 2010

Complications with 5-hydroxymethylcytosine

• 5-hydroxymethylcytosine has been discovered in mammalian DNA, and is produced by an enzymatic pathway involving TET1 hydroxylase.

• All 3 methods discussed are unable to distinguish between 5mC and 5hmC.

• However, one can distinguish 5-hmc by adding a glucose to the hydroxy-group (EpiMark Kit)

Tollefsbol 2009

The Future of Genome-Wide Methylation Assays

• As more data is experimentally collected about the methylome, there will be more and more need for analysis. Bioinformatics is beginning to play a big role.

• Increasing role of sequencing as opposed to arrays.

• Nanopore sequencing could directly allow sequencing of 5meC

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Research 17(10): 1529-536.Gu H et al. 2011. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling.

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