1. BS222 – Genome Science Lecture 7
DNA methylation
Dr. Vladimir Teif

2. Module structure
Genomes, sequencing projects and genomic databases (VT) (Oct 9, 2018)
Sequencing technologies (VT) (Oct 11, 2018)
Genome architecture I: protein coding genes (VT) (Oct 16, 2018)
Genome architecture II: transcription regulation (VT) (Oct 18, 2018)
Genome architecture III: 3D chromatin organisation (VT) (Oct 23, 2018)
Epigenetics overview (PVW) (Oct 25, 2018)
DNA methylation and other DNA modifications (VT) (Oct 30, 2018)
NGS experiments and analysis I: the basics (VT) (Nov 1, 2018)
NGS experiments and analysis II: data integration (VT) (Nov 8, 2018).
Comparative genomics (JP, guest lecture) (Nov 13, 2018)
SNPs, CNVs, population genomics (LS, guest lecture) (Nov 15, 2018)
Histone modifications (PVW) (Nov 20, 2018)
Non-coding RNAs (PVW) (Nov 22, 2018)
Genome Stability (PVW) ) (Nov 27, 2018)
Transcriptomics (PVW) (Nov 29, 2018)
Year's best paper (PVW) (Dec 6, 2018)
Revision lecture (all lecturers; spring term)

3. [Short] history of DNA methylation
1944: Avery, Macleod, McCarty:
DNA methylation discovered in mammals
1948: It is mostly cytosine that is methylated in DNA
5-methylcytosine (5mC)
1955: Sinsheimer: 5mC is mostly in the context of CpGs
1980s: DNA methylation is involved in gene regulation
Cedar & Co: Methylated remains methylated,
Unmethylated remains unmethylated.
5mC in Genomic imprinting
5mC in X chromosome inactivation
Feinberg & Vogelstein: 5mC in cancer

4. Biochemistry of DNA methylation
DNA methylation is an enzymatic reaction
DNA methyltransferases: DNMT1, DNMT3a, DNMT3b
Unmodified DNA is a rather poor substrate for methylation
Hemi-methylated DNA (i.e., methylated on one strand)
has 100-fold better Km for this reaction

5. DNA methyltransferases

6. Recognition of hemi-methylated DNA
Arita et al., 2008, Nature

7. Maintenance of DNA methylation
Christman, 2002, Oncogene

8. Chemistry of DNA methylation
Dantas Machado et al. (2014) Brief Func Genom, 14, 61-73

9. Electrostatics of DNA methylation
Dantas Machado et al. (2014) Brief Func Genom, 14, 61-73

10. GC suppression
Methylated C residues can spontaneously deaminate to form T residues; hence CpG dinucleotides steadily mutate to TpG dinucleotides, which gives rise to the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency)

11. Methyl-cytosine deamination
GC suppression

12. DNA methylation vs de-methylation
Reif and Walter, 2001, Nature

13. How DNA methylation affects DNA-protein binding?
1) Prohibits TF binding to their methylated binding sites
2) Attracts 5mC-binding proteins -> recruit other proteins

14. DNA methylation can prohibit TF binding
Many TFs have a CpG inside their binding site. When this CpG gets methylated they can not bind DNA (or it is more difficult to bind)
Examples: CTCF, p53, Myc

15. Methyl-binding proteins bind 5mC
MBD = methyl binding domain
Bird A. (2002) Genes & Development 16, 6-21

16. 5mC roles in gene regulation
Reddington et al., Biochem J., 2013

17. Distribution of DNA methylation
70% of CpGs in mammalian genomes are methylated
CpG islands are usually protected from methylation

18. CpG islands G+C content greater than 50%
Ratio of observed to expected CpG greater than 0.6
25,000 CpG islands in the human genome
75% of human CpG islands are less than 850bp long
~50% of CpG islands are located in gene promoters
~25% of CpG islands lie in gene bodies
60-70% of human gene promoters contain CpG islands

19. CpG islands If a CpG island inside gene promoter is methylated,
this can switch off the corresponding gene
But CpG islands are usually protected from methylation.
Reif and Walter, 2001, Nature

21. Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved
Put a human chromosome in a mice cell
it recapitulates the same methylation
patterns of CpG islands as in human
Long et al. (2016), Nucleic Acids Res., 44, 6693–6706

22. Genomic imprinting Affects several dozen mammalian genes
Those genes are expressed from only one of the two parental chromosomes

23. Genomic imprinting

24. Imprinting Control Region (ICR) Differentially Methylated Region (DMR)
Bartolomei and Ferguson-Smith, 2011, CSHL Perspectives

25. Life cycle of methylation imprinting
Reif and Walter, 2001, Nature

26. Bartolomei and Ferguson-Smith, 2011, CSHL Perspectives

27. Methylation in embryonic development
Reif and Walter, 2001, Nature

28. Jullien et al., 2006, Plant Cell

29. Imprinting in different species

30. X chromosome inactivation
Happens in female mammals
One of two copies of X chromosome is inactivated
Discovered in 1961 by Mary Lyon
Image by Steffen Dietzel, 1996,
Dissertation an der Universität Heidelberg

31. DNA methylation and cancer
Baylin, 2005, Nature Clinical Practice

32. DNA methylation and cancer
TSG = tumour suppressor gene
Robertson, 2005,
Nature Rev Genet

33. Robertson, 2005,
Nature Rev Genet

34. Sporadic hypermethylation in cancer
Baylin, 2005, Nature Clinical Practice

35. [Incomplete] methylation summary

36. DNA hydroxymethylation (5hmC)
5hmC is an in many cases an antipode of 5mC:
5hmC is “active” gene mark, 5mC is “inactive”
5hmC is present at a given site 5mC is absent
5hmC destabilises nucleosomes, 5mC stabilises

37. DNA hydroxymethylation (5hmC)

38. Other DNA modifications
Hohli and Zhang, 2013, Nature

39. 5hmC vs 5mC dynamics during embryo develop-ment
Wu and Zhang, 2014, Cell

40. 5mC/5hmC in germ cell development
Wu and Zhang, 2014, Cell

41. DNA methylation and ageing
During ageing, all cells of the body undergo slow changes in methylation, with some sites becoming more methylated, while others undergo creeping demethylation.
Lamina associated domains undergo demethylation
Some CpG islands undergo de-novo methylation
Happens in all cells, but at a different rate for each tissue
Dor and Cedar (2018) The Lancet, 392,

42. Paternal germ line aging: DNA methylation age prediction from sperm
~150 genomic regions (~ 1 kb in size)
8 regions gain and 140 regions lose methylation with age
Can predict age with ~2 year error
Jenkins et al. (2018), BMC Genomics, 19, 763

43. NGS methods for DNA methylation
Bisulfite sequencing
Affinity purification (e.g. MeDIP)

44. Take home message DNA methylation, 5mC, CpG
DNA chemical modifications contain information in addition to the genome
MUST KNOW:
DNA methylation, 5mC, CpG
DNA methyltransferases: DNMT1, DNMT3a, DNMT3b
Maintenance vs de-novo methylation
GENOMIC IMPRINTING, X CHROMOSOME INACTIVATION
CpG islands, GC suppression, chemical structures
Differentially methylated region, imprinting control region
DNA methylation in cancer
Bisulfite sequencing, affinity purification