Genome-Wide DNA Methylation Assays

1. Genome-Wide DNA Methylation Assays Nadia Khan, Rick Smith, and Anna Kuperman Epigenetics 2012

2. 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

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

4. Concept of MethylationSensitiveEndonucleaseAssays • Use a restrictionenzyme(s) thatismethylationspecificand separatetheunmethylatedfromthemethylated • Unmethylatedregions enzyme-sensitiveMethylatedregions enzyme-resistant • Identifymultiplede novomethylatedareasacrossgenomesvs. onespecificarea • 2D gel and scintillationcounting • Array Laird 2010

5. RestrictionLandmarkGenomicScanning (RLGS) • Cleavegenomeintopiecesbaseduponrestrictionlandmarks (sites) • Radioactivelylabelcleavedends • Separateusing 1D and 2D gel electrophoresis • Quantitatesignalbyamount of fluoresence in gel • Intensity = copynumber of therestrictionsite Tollefsbol 2009

6. NotI: • Radioactivelylabelsends • LEASTspecific • EcoRV: • MOREspecific • HinfI: • MOSTspecific Enget al. 2000

7. RLGS in-use Takamiya, et al. 2009

8. MicroarrayCoupling of MethylatedCpG Island Amplification (MCAM) • Cleavegenomic DNA withSmaI (methylation-sensitive) • CutreceivedpiecesagainwithXmaI; createstickyends • Amplifypiecesusing PCR • Hybridizeonto a microarray • Analyzefluorescencereads and identifycorrespondinggenomicaddress Tollefsbol 2009

9. Tollefsbol 2009

10. MCAM in-use Estecio 2007

12. Sodium Bisulfite Methods • Bisulfite Sequencing • Targeted and Whole Genome approaches • Bisulfite Libraries

13. 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

14. 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 Tollefsbol2011

15. Pyrosequencing

16. 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; Tollefsbol2011

17. Targeted Bisulfite Sequencing • Reduced Representation Bisulfite Sequencing (RRBS) • Molecular Inversion Probes (MIP) • Padlock Probes

18. 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

19. Targeted Bisulfite Sequencing Padlock Probes Deng et al. 2009

20. 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

22. 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-CpGisland recovery assay Laird 2010

23. 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 UnmethylatedCpG Figure 9.8 Tollefsbol 2009

24. 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

25. 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

26. Methylated DNA Immunoprecipitation (MeDIP) Methylated CpG UnmethylatedCpG Figure 9.7 Tollefsbol 2009

27. 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

28. Methylated-CpGIsland 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

29. Methylated-CpG Island Recovery Assay (MIRA) Figure 9.9 Tollefsbol 2009 From activemotif.com

30. 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

31. 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: MethylCollectorTM Ultra

32. Biological Affinity Assays • Why are they good? • Quick and efficient genome-wide assessment of DNA methylation • Disadvantages: • Do not give information on individual CpGdinucleotides • Require experimental or bioniformatic adjustment for changing CpG density at different regions of genome Laird 2010

33. 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

34. 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

35. References Cross SH et al. 1994. Purification of CpG islands using a methylated DNA binding column. Nat. Genet. 6(3):236-44. Deng J et al. 2009. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol. 27(4):341-2. Estecio MRH et al. 2007. High-throughput Methylation Profiling by MCA Coupled to CpG Island Microarray. Genome Research 17(10): 1529-536. Gu H et al. 2011. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nature Protocols 6:468–481. JeddelohJA et al. 2008. Reduced-representation methylation mapping. Genome Biology 9:231. Laird PW. 2012. Principles and Challenges of Genome-wide DNA Methylation Analysis. Nature Reviews Genetics 11:191-203. MeissnerA et al. 2005. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nuc. Acids Res. 33(18):5868-5877. Morris MR et al. 2011. 6.Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma. Oncogene 30(12):1390-401. Rauch TA and Pfeifer GP. 2009. Chapter 9: Methods for Assessing Genome Wide DNA methylation. In: Handbook of Epigenetics : The New Molecular and Medical Genetics. ed. Tollefsbol T. Academic Press. Rauch TA et al. 2006. MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells. Cancer Res. 66(16)7939-47. Rauch TA et al. 2009. A human B cell methylome at 100 base pair resolution. Proc. Natl. Acad. Sci. 106(3):671-8. Takamiya et al. 2009. The Application of Restriction Landmark Genome Scanning Method for Surveillance of Non-Mendelian Inheritance in F1 Hybrids. Comparative and Functional Genomics 2009: 1-7. TostJ. 2009. Epigenetics. Caister Academic Press. Weber M et al. 2005. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 37(8):853-62.