Genome-Wide DNA Methylation Assays
Nadia Khan, Rick Smith, and Anna Kuperman
Epigenetics 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
Genome-wide DNA Methylation Assays
Nadia
Rick
Anna
Methylation Sensitive Endonuclease- based
Sodium Bisulfite Treatments Biological Affinity- based Methods
1. Restriction Landmark Genomic Scanning (RLGS)
1. Whole and targeted
1. Methylated DNA Immunoprecipitation (MeDIP)
2. Microarray coupling of Methylated-CpG island amplification
2. Bisulfite Libraries
2. Methylated CpG island recovery assay
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 (methylationsensitive)
• 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
MCAM in-use
Estecio 2007
Genome-wide DNA Methylation Assays
Nadia
Rick
Anna
Methylation Sensitive Endonuclease- based
Sodium Bisulfite Treatments Biological Affinity- based Methods
1. Restriction Landmark Genomic Scanning (RLGS)
1. Whole and targeted
1. Methylated DNA Immunoprecipitation (MeDIP)
2. Microarray coupling of Methylated-CpG island amplification
2. Bisulfite Libraries
2. Methylated CpG island recovery assay
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
Padlock Probes
Deng et al. 2009
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
Genome-wide DNA Methylation Assays
Nadia
Rick
Anna
Methylation Sensitive Endonuclease- based
Sodium Bisulfite Treatments Biological Affinity- based Methods
1. Restriction Landmark Genomic Scanning (RLGS)
1. Whole and targeted
1. Methylated DNA Immunoprecipitation (MeDIP)
2. Microarray coupling of Methylated-CpG island amplification
2. Bisulfite Libraries
2. Methylated CpG island recovery assay
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
Methylated CpG
Unmethylated CpG
Figure 9.8 Tollefsbol 2009
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 2009
From 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 5hydroxymethylcytosine
• 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
References
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Deng J et al. 2009. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat
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Gu H et al. 2011. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling.
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Meissner A et al. 2005. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis.
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