1. DNA methylation and TF binding: Integrative data analysis
BS312 – Genome Bioinformatics
Lecture 6
DNA methylation and TF binding: Integrative data analysis
Dr. Vladimir Teif

2. DNA methylation (5mC) 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

3. 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

4. DNA methyltransferases

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

6. Maintenance of DNA methylation
Christman, 2002, Oncogene

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

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

9. 5mC roles in gene regulation
Reddington et al., Biochem J., 2013

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

11. 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

12. 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

14. 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

15. DNA methylation lingo
CpG Canyons are large conserved regions of low DNA methylation

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

17. Genomic imprinting

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

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

20. 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

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

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

23. Robertson, 2005,
Nature Rev Genet

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

25. 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,

26. 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

27. NGS methods for DNA methylation

28. NGS methods for DNA methylation

29. NGS methods for DNA methylation

30. DNA methylation microarrays (profile a limited number of loci)

31. DNA methylation methods timeline

32. We mostly use these two methods:
Bisulfite sequencing
MeDIP (similar to ChIP-seq)

33. Bisulfite sequencing data analysis workflow
Wreczycka et al. (2017), J Biotechnol., 261,

34. Methylation of individual CpGs
 value = nMethylated/nTotal
Usually when we say that a given CpG is methylated me mean  value > 0.8
 value = 0.5 may mean that it is methylated in one allele and unmethylated in another allele
BISMARK

35. Methylation data in genome browsers

36. Methylation data in genome browsers

37. Methylation data in genome browsers

38. Manhattan plot: all genome at once
Manhattan plot: all genome at once. The outstanding points are associated with the disease or trait of interest

39. How DNA-protein binding affects DNA methylation?
1) Enhances or prohibits DNA methylation at this site
2) Sets boundaries to limit DNA methylation spreading
Wiehle L., …. Teif V.B., in preparation

40. CTCF locally inhibits DNA methylation and sets borders for methylation spreading
NucTools
Wiehle L., …. Teif V.B., in preparation

41. Differentially methylated regions (DMR)
DMRcaller
Wiehle L., …. Teif V.B., in preparation

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

43. DNA methylation and TF binding
Many TFs have 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

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

45. Calculating profiles of TF and nucleosome occupancy with respect to DNA methylation
FMR = Fully Methylated Region (most genomic regions)
LMR = Low Methylated Region (regulatory regions, e.g. enhancers)
UMR = Un-methylated Region (CpG islands, promoters, etc)
Teif et.al., Genome Res 2014

46. How DNA methylation is related to nucleosome positioning?
1) Methylation stiffens DNA  stabilises the nucleosome
2) Enzymes that add/remove DNA methylation may not have access to DNA inside the nucleosome
3) Over evolutionary timescales co-existing patterns of DNA methylation and nucleosomes have evolved

47. DNA methylation is enriched inside the nucleosome, increasing to nucleosome ends
Teif et.al., Genome Res 2014

48. DNA methylation & gene expression
Lund et al., (2014) Genome Biology, 15:406

49. Take home message DNA methylation, 5mC, CpG DNA methylation in cancer
From individual CpGs to differentially methylated regions to integration with TF binding and gene expression
MUST KNOW:
DNA methylation, 5mC, CpG
GENOMIC IMPRINTING, X CHROMOSOME INACTIVATION
CpG islands, CpG shores,
Differentially methylated region, imprinting control region
DNA methylation in cancer
NGS methods: WGBS, RRBS, MeDIP