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

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

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

DNA methyltransferases http://pdb101.rcsb.org/motm/139

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

Maintenance of DNA methylation Christman, 2002, Oncogene

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

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

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

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

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

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

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

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

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

Genomic imprinting

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

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

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

DNA methylation and cancer Baylin, 2005, Nature Clinical Practice

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

Robertson, 2005, Nature Rev Genet

Sporadic hypermethylation in cancer Baylin, 2005, Nature Clinical Practice

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, 777-786

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

NGS methods for DNA methylation https://www.slideshare.net/abizarl/dna-methylation-understanding-the-language-of-dna-20130806

NGS methods for DNA methylation https://www.slideshare.net/abizarl/dna-methylation-understanding-the-language-of-dna-20130806

NGS methods for DNA methylation https://www.slideshare.net/abizarl/dna-methylation-understanding-the-language-of-dna-20130806

DNA methylation microarrays (profile a limited number of loci)

DNA methylation methods timeline

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

Bisulfite sequencing data analysis workflow Wreczycka et al. (2017), J Biotechnol., 261, 105-115

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

Methylation data in genome browsers https://genestack-user-tutorials.readthedocs.io/tutorials/Methylation_profiling/

Methylation data in genome browsers https://www.epigentek.com/catalog/epinext-high-sensitivity-bisulfite-seq-kit-illumina-p-3637.html

Methylation data in genome browsers https://software.broadinstitute.org/software/igv/interpreting_bisulfite_mode

Manhattan plot: all genome at once Manhattan plot: all genome at once. The outstanding points are associated with the disease or trait of interest https://en.wikipedia.org/wiki/Manhattan_plot

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

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

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

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

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 http://jonfwilkins.blogspot.co.uk/2011/01/genomic-imprinting-v-dna-methylation.html

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

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

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

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

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

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