Genome Wide DNA Methylation Assays
Epigenetics - 2011
John Blazeck and Amanda M. Lanza February 3, 2011
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Introduction and Background Methylation sensitive endonuclease-based methods
Restriction landmark genomic scanning (RLGS) Methylation sensitive fingerprinting (MSRF) Methylation sensitive representational difference analysis (MS-
RDA) Differential methylation hybridization (DMH) Help Assay Methylated CpG island amplification coupled to microarray
(MCAM) Methylation-specific digital karyotyping (MSDK) McrBC-based methods
Sodium bisulfite treatment-based methods Biological affinity-based methods
Introduction and Background Gene or region specific DNA methylation
analysis is based on techniques that differentially recognized 5-methylcytosine from cytosine Methylation-sensitive restriction enzymes Bisulfite-mediated DNA conversion Antibodies or methylated DNA binding protein
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Methylation-sensitive restriction enzymes
Restriction enzymes (REs) Cut specific DNA sequences. Ex. HpaII
cuts CCGG sequence
Methylation-sensitive REs Normally implies that RE can cut
unmethylated DNA, but is inhibited by cytosine methylation (Ex. HpaII)
McrBC is the opposite Can be used to distinguish between
methylated and unmethylated C Genome-scale data sets collected using
2-D gel electrophoresis, PCR analysis, or sequence analysis after digesting DNA with a methylation-sensitive RE
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Restriction landmark genomic scanning (RLGS) Digestion with NotI that cuts only unmethylated DNA, and then radiolabeled Digested with EcoRV (not methylation impaired, and a more frequent cutter) 1D gel electrophoresis Digested with HinfI (not methylation impaired and an even more frequent cutter) in
gel, and then 2D gel electrophoresis
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Open Rectangle = unmethylated NotI site Closed rectangle = methylated NotI site Star = radiolabeled DNA end
RLGS Other combinations of REs have been employed Provides excellent and reproducible results Very laborious Identification of differentially methylated regions is difficult but
getting easier Only finds differentially methylated regions in the NotI site
(GCGGCCGC)
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Methylation sensitive fingerprinting (MSRF)
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For identifying differential methylation between two tissues (tumor vs. normal cells)
Digest aliquots of tumor/normal DNA with either only MseI (TTAA) or MseI and BstUI (CGCG) MseI cuts gDNA once per ~140bps, but cuts CpG islands once per ~1000bp BstUI is methylation sensitive and appears once per ~5000 to 10000bp in regular gDNA and
once per ~90bp in CpG islands
PCR amplification of digested DNA with short, arbitrary primers (10mers) that often contain the CGCG sequence on their 3’ end. Example:
Run DNA on gel No methylation = No band Methylation = band Hypermethylation = brighter band Hypomethylation = less bright band
Fragments can be cut out of gel, reamplified, cloned, and sequenced Note: HpaII (CCGG) can be used instead of BstUI
(MSRF) Example of gel
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Methylation sensitive representational difference analysis (MS-RDA)
Isolated DNA fragments methylated differently between samples Start with HpaII (CCGG) methylation sensitive digest – cuts
unmethylated CCGG sites Ligate a universal adaptor to the digest – ligates to different parts of
the genome depending on where gDNA has been cut PCR amplify with universal adaptor primer Perform multiple (2-4) rounds of this competitive PCR amplification Gel resulting fragments and reamplify/sequence differences to find
differential methylation patterns Drawbacks:
Shrinks genome to ~1/9 to ~1/23 Doesn’t find many differential methylations Assumes local gDNA areas have same
methylation pattern
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Differential methylation hybridization (DMH)
Allows for determination of methylation status of large number of CpG islands and for differential determination between tissues
MseI (TTAA) digestion to cut non-CpG island DNA
Ligation to linkers to allow for PCR amplification
Separation into two aliquots and methylation-sensitive BstUI (CGCG) digest of one aliquot
PCR amplification to form two “amplicon” pools
Hybridization of two amplicon pools to CGI array (has lots of BstBU sites on it) to find hypermethylated regions
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DMH Example
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Normal tissue versus two lines of breast cancer tissue
Breast cancer tissue has hypermethylated DNA. Therefore, it’s MseI/BstBU amplicon pool hybridizes more to the array than normal tissue’s (B’ and C’ versus A’)
Help Assay
Same process as DMH, but uses HpaII (CCGG) instead of BstBU (CGCG)
Has an extra amplicon pool using and HpaII’s methylation–INsensitive isoschizomer MspI (CCGG)
Example below of differing digestion patterns (real HELP assay uses PCR amplification followed by hybridization to microarrays)
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Methylated CpG island amplification coupled to microarray (MCAM)
Methylation sensitive SmaI (CCCGGG) digestion to form blunt ends Cuts unmethylated SmaI sites and leaves them unable to ligate to PCR-adaptors
Methylation insensitive Xma (CCCGGG), an isoschizomer, digestion to leave sticky ends for ligation to PCR adaptors
PCR amplification, labeling, hybridization, and data analysis
White circles = unmethylated Filled circles = methylated
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Methylation-specific digital karyotyping (MSDK) Cut DNA with methylation sensitive AscI
(GGCGCGCC) Cuts at unmethylated DNA (white circles)
Adaptor ligation to gDNA fragments Fragment shortening with NlaIII enzyme Fragment capturing on streptavidin beads Separated into two aliquots Each aliquot ligated with different adaptor.
Both adaptors harbor the same MmeI site. MmeI digestion cuts 17basepairs away from
it’s recognition site, forming complementary ends in each aliquot
Therefore, aliquots combined and ligated together to form ditags.
PCR amplification NlaIII Digestion Ditags ligated together to form concatemers
several hundred bp long Sequencing analysis
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McrBC-based methods
McrBC specifically cleaves methylated DNA, instead of unmethylated DNA (as all previously mentioned enzymes do)
Can be used for similar restriction enzyme-based differential methylation analyses
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Two Key Points Methylation sensitive enzymes can
differentiate between methylated or unmethylated DNA by their ability to cut or not cut the DNA
“(Their) main drawback is that they can provide only limited methylation profile analysis since there is no restriction enzyme that cleaves appropriately within all CpG islands” -Rauch and Pfeifer
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Whole Genome Bisulfite Sequencing MALDI-TOF MS Padlock Probes Golden Gate & Infinium Assays Pyrosequencing
Bisulfite Sequencing – Whole Genome Approach Bisulfite treatment converts cytosine to uracil
(but not methyl-C) Detect uracil across genome for specific tissues Parallel sequencing: Roche 454, ABI SOLiD,
produce short reads Challenge is accurately mapping reads back to
genome High coverage (>10x) Advanced computer algorithms Comparison to public human genome sequence
MALDI-TOF MS Bisulfite-coverted gDNA PCR amplification using T7 RNA polymerase
promoter – form ssRNA in vitro Digest with ribonuclease A, digests at uracil residues
Quantitative detection of cleaved products using TOF MS
Method easily scaled to high-throughput format
MALDI-TOF MS
http://www.sequenom.com/Home/Products---Services/Genetic-Analysis/Applications/EpiTYPER-DNA-Methylation-Analysis/How-it-Works
Padlock Probes Capture arbitrary set of
sequencing targets from bisulfite treated gDNA Padlock probes used to
amplify short gDNA regions
Prepare for multiplex sequencing
Golden Gate/Infinium Assay Golden Gate developed by Illumina
Infinium is improved assay, higher throughput Start with bisulfite-converted DNA
Hybridize with two sets of site specific primers, methylated & unmethylated, primer extension
Ligate & Amplify the DNA fragments using ddNTPs Detect 2 fluorophores; methylated & unmethylated
Measure ratio of 2 fluorophores Quantitatively determine likelihood that a given CpG
is methylated
Pyrosequencing
Bisulfite converted DNA as template PCR amplify target region (up to 350bp)
Primers complimentary to converted DNA seq One primer carries biotin residue at 5’ end
Capture single stranded fragments using streptavidin
Luminescent sequencing reaction Base pair incorporation releases pyrophosphate Sequential addition of single nucleotides
Determine methylation frequency by comparing C/T incorporation ratios
http://www.nature.com/nprot/journal/v2/n9/full/nprot.2007.314.html
Whole Genome Affinity-based methods MeDIP MAC MIRA
Affinity Based Methods Purify DNA fragments that bind specific
proteins (like antibodies) Requires:
Proteins that specifically bind methylated or unmethylated DNA
Method for recovery/separation
Methylated DNA Immunoprecipitation (MeDIP) gDNA is sheared to 300-1000-bp
(sonication or other methods) Denature to ssDNA Incubated DNA with 5-methylcytosine Ab Recover 5mC antibody (magnetic beads
conjugated to anti-mouse-IgG) Purify DNA, analyze with microarray or
Sequencing
MeDIP Limited by:
Quality/cross reactivity of 5mC Ab Sensitivity of Ab, needs many 5mC Produces only short sequencing reads
MBD-Affinity Column (MAC) Methyl-CpG binding domain (MBD) proteins
naturally bind methylated DNA seq Fragment gDNA (large amount) Run through an affinity column with MBDs
Retains methylated sequences Elute with high salt buffer
Fluorescently label elute, hybridize to microarray to detect sequences
Methylated-CpG Island Recovery Assay (MIRA) MBD2b has highest affinity for methylated DNA
Can form heterodimers with MBD3L1 Fragmented gDNA is incubated with tagged
MBD2b & MBD3L Purify MBD-DNA complexes with coated magnetic
beads Analyze the fragments using microarray or
NextGen Seq
MIRA Advantages:
Works on dsDNA Requires a min of 2 methylated residues Small amount of DNA (
Hydroxymethylation Generated by oxidation of 5-methylcytosine (5mC) to 5
-hydroxymethylcytosine (5hmC) via TET1 hydroxylase Recently found to be abundant in mammalian cells, with highest levels
found in neuronal cells in central nervous system Unsure of its regulatory roles (if it has them) There’s currently no genome-wide method for finding 5hmC
Like 5-methylcytosine, converted to uracil by bisulfite However, MeDIP and MIRA are in fact specific for 5mC A subtractive analysis could be used, but this wouldn’t be at a single base resolution and
hasn’t been attempted yet
For single locus analysis, EpiMark™ 5-hmC and 5-mC Analysis Kit from New England Biolabs Uses a Glucosylation Reaction with T4 β-glucosyltransferase Followed by a MspI or HpaII digest
Cut same sequence HpaII won’t cut any methylation MspI will cut regular methylation, but not a glucosylated methyl group
PCR analysis to find differences in hydroxymethylation
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Hydroxymethylation - single locus analysis
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References 1. Hatada, I., Y. Hayashizaki, S. Hirotsune, H. Komatsubara, et al., A GENOMIC SCANNING METHOD FOR HIGHER
ORGANISMS USING RESTRICTION SITES AS LANDMARKS. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(21), 9523-9527.
2. Jin, S.G., S. Kadam, and G.P. Pfeifer, Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Research, 38(11), 7.
3. Huang, T.H.M., D.E. Laux, B.C. Hamlin, P. Tran, et al., Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique. Cancer Research, 1997, 57(6), 1030-1034.
4. Ushijima, T., K. Morimura, Y. Hosoya, H. Okonogi, et al., Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(6), 2284-2289.
5. Huang, T.H.M., M.R. Perry, and D.E. Laux, Methylation profiling of CpG islands in human breast cancer cells. Human Molecular Genetics, 1999, 8(3), 459-470.
6. Khulan, B., R.F. Thompson, K. Ye, M.J. Fazzari, et al., Comparative isoschizomer profiling of cytosine methylation: The HELP assay. Genome Research, 2006, 16(8), 1046-1055.
7. http://www.neb.com/nebecomm/products/productE3317.asp
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