Genome Wide DNA Methylation Assays

Epigenetics - 2011

John Blazeck and Amanda M. Lanza February 3, 2011

2

  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

3

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

4

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

5

  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)

6

Methylation sensitive fingerprinting (MSRF)

7

  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

8

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

9

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

10

DMH Example

11

  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)

12

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

13

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

14

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

15

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

16

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

36

Hydroxymethylation - single locus analysis

37

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

38