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

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)

[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

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

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

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

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)

Methyl-cytosine deamination GC suppression

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

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 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 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

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

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

Bartolomei and Ferguson-Smith, 2011, CSHL Perspectives

Methylation in embryonic development Reif and Walter, 2001, Nature

Jullien et al., 2006, Plant Cell

Imprinting in different species http://what-when-how.com/genetics/dna-methylation-in-epigenetics-development-and-imprinting-genetics/

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

[Incomplete] methylation summary https://blogbiosyn.files.wordpress.com/2013/05/methylation-of-cysteines.jpg

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 hydroxymethylation (5hmC) http://www.blopig.com/blog/2015/06/investigating-structural-mechanisms-with-customised-natural-moves/

Other DNA modifications Hohli and Zhang, 2013, Nature

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

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

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 Bisulfite sequencing Affinity purification (e.g. MeDIP)

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