Whole genome assays for DNA methylation

1. Whole genome assays for DNA methylation Feb 8 2006

2. DNA methylation short intro • DNA methylation reefers to the addition of a methyl group (-CH3) to some cytosines in genomic DNA • In mammals typically happens at CpG sites • Methylation patterns are maintained through cell division by the enzyme Dnmt1 (DNA methyltransferase 1)

3. DNA methylation short intro • Methylation status of CpGs 5’ to genes influences transcription • mCpGs  gene silenced • CpG  gene expressed (unless otherwise silenced) • May be caused by mCpG binding proteins or changes in chromatin structure • Modification is reversible and can’t be seen by looking at primary sequence alone

4. DNA methylation short intro • Different methylation patterns in different tissues results in different sets of genes being turned on • Interesting development/differentiation questions • Methylation in cancer goes haywire • Bulk genomic DNA becomes hypomethylated • Some CpG islands become hypermethylated

5. Relation to CpG islands • 5-methylcytosine (mC) is unstable and may spontaneously convert to thymine (T) • CpGs in promoters of genes are typically unmethylated while CpGs in bulk genomic sequence are typically methylated • Some evidence that methylation is a defense against forigen DNA • Genes inserted into transgenic organsims become silenced by methylation • This might be how the genome defends itself against repeats like alus

6. Relation to CpG islands • Over evolutionary time mCpGs in repeats become TpGs • Leaves islands of CpG 5’ of transcribed genes

7. Methylation maps • How do you query the methylation status of the genome? • Look at 3 recently published methods • All consist of some method for selecting methylated or unmethylated sequence + some method of detection • Webber et. al, Aug 2005 (Immunopercipitation + microarray) • Rollins Et. al, Feb 2006 (Digestion + sequencing) • Richard Myers lab, July 2005 (Digestion + Nimblegen array of ENCODE regions)

8. Immunopercipitation + microarray • Sonicate genomic DNA to produce 300-1000 bp fragments • Pull down methylated fragments with an mC antibody • Results in 90 fold enrichment of methylated DNA fragments over non-methlated fragments

10. Immunopercipitation + microarray • Label input DNA and immunopercipitated DNA with different flourescent dyes • Hybridize samples to a BAC tiling array covering the whole human genome (32,433 overlaping BACs ~80kb resolution)

12. Immunopercipitation + microarray • BAC glows red  hypomethylated • BAC glows green  hypermethylated

13. Immunopercipitation + microarray • Also tested specific CpG islands with a microarray consisting of ~12,000 CpG islands

14. Immunopercipitation + microarray • So what does this give you? • Blurry snapshot of the whole genome (at 80 kb resolution) • Better focused picture of methylation status of lots of CpG islands (at 300-1000 bp resolution)

15. Digestion + sequencing • Create libraries of methylated and unmethylated sequence domains • Methylated domains • Digest genomic DNA with mC sensitive restriction enzyme (mC blocks cutting) TaiI (ACGT), BstUI (CGCG), HhaI, (GCGC), HpaI, (CCGG) AciI, (CCGC & GCGG) • Any large fragments that remain are methylated

16. Digestion + sequencing • Unmethylated domains • Digest genomic DNA with E. coli enzyme complex called McrBC • Cuts at sites like (A/G)mC-N40-500-(A/G)mC • Large fragments left over are unmethylated • Don’t know if I believe that as strongly as the last silde (one enzyme, fewer sites but less specific)

17. Digestion + sequencing • Take end reads off of fragments in libraries generated as just described • Blat results back to genome • Methylated domains • 3501 • Lengths ~1kb – 9kb • Unmethylated domains • 4252 • Lengths ~2kb – 4.5 kb

18. Digestion + sequencing • What do you get from this? • Collection of long segments of genome that are methylated or unmethylated* • *Restriction enzyme digest so you don’t necessarily get pure methylated or unmethylated domains

19. Digestion + Nimblegen array • Done as part of the ENCODE project • Digest genomic DNA with mC sensitive restriction enzymes (cocktail of 6) • Amplify digested and undigested DNA • Label digested and undigested DNA with different fluorescent dyes • Hybridize 2 samples to Nimblegen tiling array covering the ENCODE regions

20. Digestion + Nimblegen array • Each spot on array represents 50 bp region with 11 bp overlap at ends • Value from each spot is ratio of signal from undigested/digested samples • The way this works is • If all CpGs on array probe are methylated restriction enzymes are blocked • DNA from digested sample hybridizes to array same as undigested sample • get same signal from digested sample as undigested sample  ratio of undigested/digested is ~1

21. Digestion + Nimblegen array • If some CpG on array probe is unmethylated enzymes can cut • DNA from digested sample can’t hybridize to array probe • Get weaker signal from digested sample than from undigested sample • Ratio of undigested/digested is large

22. Digestion + Nimblegen array • What do you get from this? • Methylation status at 50 bp resolution over 1% of human genome in 8 cancer lines* • *restriction digest so again… are missing some CpGs • However over 50 bp window might be able to figure out exactly which CpGs you are querying and to which you are blind (going to do this when I get a chance!) • Potential to tile whole human genome like this • Picture

24. Conclusion • DNA methylation appears to be important in several areas of biology • Techniques for mapping the methylome exist at various resolutions and are getting better • Some work has already been done and some data is available