Gene-specific Methylation Analyses

1. Gene-specific Methylation Analyses Fade Gong & Nam Nguyen

2. Introduction • DNA methylation in mammals occurs at CpG sites, where it serves many functions: • Self vs. non-self identification (such as in viral/bacterial infection) • Target for further regulatory events; examples: • Maintenance methyltransferases (functions after replication on hemi-methylated DNA) • Proteins involved in chromatin folding • CpG islands are elements of most promoters, which typically downregulate transcription in the methylated state

3. Principles of DNA Methylation Analyses • Single-gene methylation analyses • Sensitivity of restriction enzymes for methylated CpG sites • Bisulfite-mediated conversion of DNA • 2. Genome-wide methylation analyses • Anti-methylcytidine Ab • HPLC (high-performance liquid chromatography)/mass spectrometry

4. Overview: Single-gene methylation analyses • Methylation-sensitive restriction enzymes • Southern-blot hybridization • Bisulfite conversion of DNA • Sequencing • Methylation-specific PCR (MSP) • Real-time MSP • COBRA • Pyrosequencing • MassARRAY

5. Southern-blot Hybridization Restriction endonucleases: CpG-sensitive • the majority are active on CpG and are inhibited by presence of CMepG (we will focus on such an example: HpaII) (Bird & Tollefsbol)

6. Southern-blot Hybridization (cont’d) Steps for Southern blotting: • Run DNA sample on gel • treated with HpaII, in our example • run against untreated control (as in lanes 3 and 4, here) • Transfer to sticky membrane, such as nitrocellulose • Probe with oligonucleotide, conjugated to radioactive label • Expose to x-ray film (Bird)

7. Southern-blot Hybridization (cont’d) Advantages • Relatively quantitative • Low cost Disadvantages • Low flexibility in selection of DNA region (i.e. must be isolated via other restriction enzymes that don’t degrade the fragment of interest) • Large amount of high-quality DNA required • Not all CpG’s presented are subjected to the assay (depends on selectivity of the restriction enzymes)

8. Polymerase Chain Reaction (PCR) Review: (McGraw-Hill Companies)

9. Bisulfite-mediated Conversion of DNA

10. Bisulfite Sequencing PCR products are then sequenced and compared to the original sequence. CpG sites that remained CpG represent the presence of a 5-methylcytosine • Advantages: • Provides detailed information about all CpG sites • Requires smaller sample of DNA Disadvantages: Labor-intensive

11. Combined Bisulfite Restriction Analysis (COBRA) COBRA is based on the appearance or disappearance of a restriction site after bisulfite conversion (Images are from wiki page of combined bisulfite restriction analysis) Characteristics: Advantage: quantification; small amount DNA sample; cheap Disadvantage: CpG site regions are limited (Specific restriction site)

12. Methylation-specific PCR (MSP) • MSP takes advantage of changes in DNA sequence caused by bisulfite treatment • Primers are designed that contain CpG sites and can thus select for expansion of methylated or unmethyledCpG’s (Image was redrawn from the wiki page of Bisulfite sequencing)

13. MSP (cont’d) • Advantages: • Simple technique • Flexibility • Cheap • Disadvantages: • Optimal number of PCR cycles & annealing temperatures • Appropriate negative control • Not a quantification method

14. Real-time MSP Overview: quantitative PCR (qPCR)/ Real-time PCR • Double-stranded DNA-binding dyes as reporters (SYBR Green) • Fluorescent reporter probe method (MethyLight: a Taqman probe) (Image from Life technology website)

15. Real-time MSP (cont’d) Comparison between qPCR and PCR Typical amplification plot for qPCR (Images are from Qiagen website)

16. Real-time MSP (cont’d) Experiment Procedure: Bisulfiteconversion; design methylation- and unmethylation-specific primers Results analysis: Comparing amplification of test samples with standard samples that contain known numbers of DNA molecules. Characteristics: High flexibility; accuracy; quantification; small amount samples; low cost Methylight has higher specificity than SYBR Green based real-time MSP

17. Pyrosequencing Overview: ((PPi = pyrophosphate!)) (Nature methods, 2005)

18. Pyrosequencing for CpGMethylation • Characteristics • Advantage: • Small amount of DNA • Flexibility • Accuracy • Quantification, etc. • Disadvantage: • Suitable primers • Expensive • Specific instruments (Nature methods, 2005)

19. MassARRAY

20. MassARRAY Procedure: Bisulfite conversion PCR In vitro transcription RNase digestion Mass Spectrometry Readout: Mass of products with C or T (16Da) Base Specific RNases RNase A: digest pyrimidine (Uracil) (SEQUENOM, Product Preview Note, 2005)

21. MassARRAY • Characteristics: • Advantage: • Small amount of DNA • Flexibility • Quantification • Disadvantage: • Expensive • Specific instruments

22. References Bird, A. P., Southern, E. M. “Use of Restriction Enzymes to Study Eukaryotic DNA Methylation”. J. Mol. Biol. 118 (1978). 27-37. Critical Factors for Successful Real-Time PCR. Qiagen, Real-Time PCR Brochure, 2010. Ehrich, M. et al., Introduction to DNA Methylation Analysis Using the MassARRAY System. SEQUENOM Preview Note, 2005. Herman, James G. “Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands”. Proc, Natl. Acad. Sci. USA. 93 (1996). 9821-9826. Pyro Q-CpGTM: quantitative analysis of methylation in multiple CpG sites by Pyrosequencing. Nature Methods 2005, 2. doi:10.1038/nmeth800 TaqMan® Chemistry vs. SYBR® Chemistry for Real-Time PCR. Life Technologies, qPCR Education. Tollefsbol, Trygve. Handbook of Epigenetics: The New Molecular and Medical Genetics. Oxford, UK: Elsevier Inc., 2011. 125-134. Print. Wikipedia: Bisulfite sequencing http://en.wikipedia.org/wiki/Bisulfite_sequencing Wikipedia: Combined bisulfite restriction analysis http://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis