Evolution of DNA Methylation Sylvain Forêt Evolution, Ecology and Genetics Research School of Biology
May 11, 2015
Evolution of DNA Methylation
Sylvain ForêtEvolution, Ecology and Genetics
Research School of Biology
History
1942 1944 1953
The double helix
Watson, Crick, Wilkins
DNA is the supportof genetic information
Avery, MacLeod, McCarty
1942 1944 1953
The double helix
Watson, Crick, Wilkins
DNA is the supportof genetic information
Avery, MacLeod, McCarty
Epigenetics
Waddington
From Genomes to Cells
AATAATAACGCTATCGGTTATTGTATCATTATTGTATATATTCGTAGTCGGTTTCCGAACGCGAAACGAAACGGACGTGTTTCTCTCTGCTCTCCGAGTAAAGACTTTATCGCTGTGAATAAAATATTATAGTGTAAAAATTTATTAAATAAAATAAAAATATTGTAGCGTAAAGATTATTAACGAATTTTATAAGTCATATAATAAATTCCAATTCCAAGAAGAGGAAATTTTTTAATCTTTAAAATTTTGTAAACTTTGAATCGCTGTATCTCCGAAGATAATAGCGATACCTACGATTTTCTGATGTGATTGCGATCGGGAAACGTTGCTCTTTCGATTTAATCGTTATTCCACGTCGAGAACTATCGTTGAAATGAACGCTATCGTTTAAATATGAGAAGAATCTTTGGAATTAAACTTGTGCAAACTTTCGACTGCTGTATCTCCAAAGATACAGTAGCGATATGTACGATTTTTTGAGATAGTTGCGAGCGGGAAACATTGCTCTTTATAATGTGGAAAAGGAAAAATAAAAAACTTAACCAAATGTAAACGATTAGATCGATTTAAACTGATACTGATAATTGCAATGAAAATAACCGGAAAATTAAGAAGATAAACGAAAGAAATCTTTGAATCTTTTTTTACGCGTCTTATAAATTAATAATAATGAAAAAAAAATTAAAAAAAATTAATTATCGAAAATATTATCGATAAAAAAAAATTAAATCATTAAATTTTAAAGAAACGAAAAATAACGAAACGAATCTAACGGAGTAATATATTAACGAGTGATAAAATGTAAAAATAAAATGAAAATTAATAATACCTTATGAATATTATAGTGAATAAAATAGCGAACGTAAATCGTGTCGATTGGTATTGTATACGTCTCGGTTAAACGTACTCGTTTTCCAAATACGAAACGAAACACGTGTTCCTTCCATTGTATTTATATACGTATAGACTAAACGTAGTCGGTTTTCGAACACGGAACGGGACGGACGTATTCCTTTCCGCTCTCTCGATAAAAATTTTATCGATATACGATATATTAATATTAAAATATAAATAATTGTTAAATAAATAAAATATGGAAGTGTAAGAATATCGAGTGTAATCATCATTAATACGTAAATCGATAAAAAGAAGAATTAAATATTTATCGAATACGAGGATAAAATGAAGAACGAAACGAGCCGAAACGTAAAAAAAAATAATCCTTTAACCGATAAATACTTAGAAATTAAAAAAAAATAATATAATAATTAAAATCGATAAATAAAATAAAAAATTCGAATTATTCGATTCTTTTCTTTTATTTCATTTTCAATTTACATATATTTTTTCTTTTTCTCTTACATTTTAAATATTAAAATATGTATTATCGTATTAAA
AATAATAACGCTATCGGTTATTGTATCATTATTGTATATATTCGTAGTCGGTTTCCGAACGCGAAACGAAACGGACGTGTTTCTCTCTGCTCTCCGAGTAAAGACTTTATCGCTGTGAATAAAATATTATAGTGTAAAAATTTATTAAATAAAATAAAAATATTGTAGCGTAAAGATTATTAACGAATTTTATAAGTCATATAATAAATTCCAATTCCAAGAAGAGGAAATTTTTTAATCTTTAAAATTTTGTAAACTTTGAATCGCTGTATCTCCGAAGATAATAGCGATACCTACGATTTTCTGATGTGATTGCGATCGGGAAACGTTGCTCTTTCGATTTAATCGTTATTCCACGTCGAGAACTATCGTTGAAATGAACGCTATCGTTTAAATATGAGAAGAATCTTTGGAATTAAACTTGTGCAAACTTTCGACTGCTGTATCTCCAAAGATACAGTAGCGATATGTACGATTTTTTGAGATAGTTGCGAGCGGGAAACATTGCTCTTTATAATGTGGAAAAGGAAAAATAAAAAACTTAACCAAATGTAAACGATTAGATCGATTTAAACTGATACTGATAATTGCAATGAAAATAACCGGAAAATTAAGAAGATAAACGAAAGAAATCTTTGAATCTTTTTTTACGCGTCTTATAAATTAATAATAATGAAAAAAAAATTAAAAAAAATTAATTATCGAAAATATTATCGATAAAAAAAAATTAAATCATTAAATTTTAAAGAAACGAAAAATAACGAAACGAATCTAACGGAGTAATATATTAACGAGTGATAAAATGTAAAAATAAAATGAAAATTAATAATACCTTATGAATATTATAGTGAATAAAATAGCGAACGTAAATCGTGTCGATTGGTATTGTATACGTCTCGGTTAAACGTACTCGTTTTCCAAATACGAAACGAAACACGTGTTCCTTCCATTGTATTTATATACGTATAGACTAAACGTAGTCGGTTTTCGAACACGGAACGGGACGGACGTATTCCTTTCCGCTCTCTCGATAAAAATTTTATCGATATACGATATATTAATATTAAAATATAAATAATTGTTAAATAAATAAAATATGGAAGTGTAAGAATATCGAGTGTAATCATCATTAATACGTAAATCGATAAAAAGAAGAATTAAATATTTATCGAATACGAGGATAAAATGAAGAACGAAACGAGCCGAAACGTAAAAAAAAATAATCCTTTAACCGATAAATACTTAGAAATTAAAAAAAAATAATATAATAATTAAAATCGATAAATAAAATAAAAAATTCGAATTATTCGATTCTTTTCTTTTATTTCATTTTCAATTTACATATATTTTTTCTTTTTCTCTTACATTTTAAATATTAAAATATGTATTATCGTATTAAA
AATAATAACGCTATCGGTTATTGTATCATTATTGTATATATTCGTAGTCGGTTTCCGAACGCGAAACGAAACGGACGTGTTTCTCTCTGCTCTCCGAGTAAAGACTTTATCGCTGTGAATAAAATATTATAGTGTAAAAATTTATTAAATAAAATAAAAATATTGTAGCGTAAAGATTATTAACGAATTTTATAAGTCATATAATAAATTCCAATTCCAAGAAGAGGAAATTTTTTAATCTTTAAAATTTTGTAAACTTTGAATCGCTGTATCTCCGAAGATAATAGCGATACCTACGATTTTCTGATGTGATTGCGATCGGGAAACGTTGCTCTTTCGATTTAATCGTTATTCCACGTCGAGAACTATCGTTGAAATGAACGCTATCGTTTAAATATGAGAAGAATCTTTGGAATTAAACTTGTGCAAACTTTCGACTGCTGTATCTCCAAAGATACAGTAGCGATATGTACGATTTTTTGAGATAGTTGCGAGCGGGAAACATTGCTCTTTATAATGTGGAAAAGGAAAAATAAAAAACTTAACCAAATGTAAACGATTAGATCGATTTAAACTGATACTGATAATTGCAATGAAAATAACCGGAAAATTAAGAAGATAAACGAAAGAAATCTTTGAATCTTTTTTTACGCGTCTTATAAATTAATAATAATGAAAAAAAAATTAAAAAAAATTAATTATCGAAAATATTATCGATAAAAAAAAATTAAATCATTAAATTTTAAAGAAACGAAAAATAACGAAACGAATCTAACGGAGTAATATATTAACGAGTGATAAAATGTAAAAATAAAATGAAAATTAATAATACCTTATGAATATTATAGTGAATAAAATAGCGAACGTAAATCGTGTCGATTGGTATTGTATACGTCTCGGTTAAACGTACTCGTTTTCCAAATACGAAACGAAACACGTGTTCCTTCCATTGTATTTATATACGTATAGACTAAACGTAGTCGGTTTTCGAACACGGAACGGGACGGACGTATTCCTTTCCGCTCTCTCGATAAAAATTTTATCGATATACGATATATTAATATTAAAATATAAATAATTGTTAAATAAATAAAATATGGAAGTGTAAGAATATCGAGTGTAATCATCATTAATACGTAAATCGATAAAAAGAAGAATTAAATATTTATCGAATACGAGGATAAAATGAAGAACGAAACGAGCCGAAACGTAAAAAAAAATAATCCTTTAACCGATAAATACTTAGAAATTAAAAAAAAATAATATAATAATTAAAATCGATAAATAAAATAAAAAATTCGAATTATTCGATTCTTTTCTTTTATTTCATTTTCAATTTACATATATTTTTTCTTTTTCTCTTACATTTTAAATATTAAAATATGTATTATCGTATTAAA
AATAATAACGCTATCGGTTATTGTATCATTATTGTATATATTCGTAGTCGGTTTCCGAACGCGAAACGAAACGGACGTGTTTCTCTCTGCTCTCCGAGTAAAGACTTTATCGCTGTGAATAAAATATTATAGTGTAAAAATTTATTAAATAAAATAAAAATATTGTAGCGTAAAGATTATTAACGAATTTTATAAGTCATATAATAAATTCCAATTCCAAGAAGAGGAAATTTTTTAATCTTTAAAATTTTGTAAACTTTGAATCGCTGTATCTCCGAAGATAATAGCGATACCTACGATTTTCTGATGTGATTGCGATCGGGAAACGTTGCTCTTTCGATTTAATCGTTATTCCACGTCGAGAACTATCGTTGAAATGAACGCTATCGTTTAAATATGAGAAGAATCTTTGGAATTAAACTTGTGCAAACTTTCGACTGCTGTATCTCCAAAGATACAGTAGCGATATGTACGATTTTTTGAGATAGTTGCGAGCGGGAAACATTGCTCTTTATAATGTGGAAAAGGAAAAATAAAAAACTTAACCAAATGTAAACGATTAGATCGATTTAAACTGATACTGATAATTGCAATGAAAATAACCGGAAAATTAAGAAGATAAACGAAAGAAATCTTTGAATCTTTTTTTACGCGTCTTATAAATTAATAATAATGAAAAAAAAATTAAAAAAAATTAATTATCGAAAATATTATCGATAAAAAAAAATTAAATCATTAAATTTTAAAGAAACGAAAAATAACGAAACGAATCTAACGGAGTAATATATTAACGAGTGATAAAATGTAAAAATAAAATGAAAATTAATAATACCTTATGAATATTATAGTGAATAAAATAGCGAACGTAAATCGTGTCGATTGGTATTGTATACGTCTCGGTTAAACGTACTCGTTTTCCAAATACGAAACGAAACACGTGTTCCTTCCATTGTATTTATATACGTATAGACTAAACGTAGTCGGTTTTCGAACACGGAACGGGACGGACGTATTCCTTTCCGCTCTCTCGATAAAAATTTTATCGATATACGATATATTAATATTAAAATATAAATAATTGTTAAATAAATAAAATATGGAAGTGTAAGAATATCGAGTGTAATCATCATTAATACGTAAATCGATAAAAAGAAGAATTAAATATTTATCGAATACGAGGATAAAATGAAGAACGAAACGAGCCGAAACGTAAAAAAAAATAATCCTTTAACCGATAAATACTTAGAAATTAAAAAAAAATAATATAATAATTAAAATCGATAAATAAAATAAAAAATTCGAATTATTCGATTCTTTTCTTTTATTTCATTTTCAATTTACATATATTTTTTCTTTTTCTCTTACATTTTAAATATTAAAATATGTATTATCGTATTAAA
AATAATAACGCTATCGGTTATTGTATCATTATTGTATATATTCGTAGTCGGTTTCCGAACGCGAAACGAAACGGACGTGTTTCTCTCTGCTCTCCGAGTAAAGACTTTATCGCTGTGAATAAAATATTATAGTGTAAAAATTTATTAAATAAAATAAAAATATTGTAGCGTAAAGATTATTAACGAATTTTATAAGTCATATAATAAATTCCAATTCCAAGAAGAGGAAATTTTTTAATCTTTAAAATTTTGTAAACTTTGAATCGCTGTATCTCCGAAGATAATAGCGATACCTACGATTTTCTGATGTGATTGCGATCGGGAAACGTTGCTCTTTCGATTTAATCGTTATTCCACGTCGAGAACTATCGTTGAAATGAACGCTATCGTTTAAATATGAGAAGAATCTTTGGAATTAAACTTGTGCAAACTTTCGACTGCTGTATCTCCAAAGATACAGTAGCGATATGTACGATTTTTTGAGATAGTTGCGAGCGGGAAACATTGCTCTTTATAATGTGGAAAAGGAAAAATAAAAAACTTAACCAAATGTAAACGATTAGATCGATTTAAACTGATACTGATAATTGCAATGAAAATAACCGGAAAATTAAGAAGATAAACGAAAGAAATCTTTGAATCTTTTTTTACGCGTCTTATAAATTAATAATAATGAAAAAAAAATTAAAAAAAATTAATTATCGAAAATATTATCGATAAAAAAAAATTAAATCATTAAATTTTAAAGAAACGAAAAATAACGAAACGAATCTAACGGAGTAATATATTAACGAGTGATAAAATGTAAAAATAAAATGAAAATTAATAATACCTTATGAATATTATAGTGAATAAAATAGCGAACGTAAATCGTGTCGATTGGTATTGTATACGTCTCGGTTAAACGTACTCGTTTTCCAAATACGAAACGAAACACGTGTTCCTTCCATTGTATTTATATACGTATAGACTAAACGTAGTCGGTTTTCGAACACGGAACGGGACGGACGTATTCCTTTCCGCTCTCTCGATAAAAATTTTATCGATATACGATATATTAATATTAAAATATAAATAATTGTTAAATAAATAAAATATGGAAGTGTAAGAATATCGAGTGTAATCATCATTAATACGTAAATCGATAAAAAGAAGAATTAAATATTTATCGAATACGAGGATAAAATGAAGAACGAAACGAGCCGAAACGTAAAAAAAAATAATCCTTTAACCGATAAATACTTAGAAATTAAAAAAAAATAATATAATAATTAAAATCGATAAATAAAATAAAAAATTCGAATTATTCGATTCTTTTCTTTTATTTCATTTTCAATTTACATATATTTTTTCTTTTTCTCTTACATTTTAAATATTAAAATATGTATTATCGTATTAAA
From Genomes to Organisms
FixedSimple structureVegetative reproductionImmortal
MotileComplex nervous systemComplex sensory structuresSexual reproductionShort lifespan
`
One per hiveLong-livedReproductive
Thousands per hiveShort-livedNon-reproductive
● These epigenetic changes are induced by environmental clues
● link between genome and environment● Epigenetic regulations are context dependent
interpretations of the genome:● conditional phenotypes
A Wide Variety of Roles
● Cellular differentiation during development● Differentiation between morphs● Many medical implications
– Cancer● Silencing of DNA repair and apoptosis genes● Activation of “proliferation” genes
– Other complex diseases● Diabetes, obesity
Histone modifications
DNA methylation
Epigenomics
DNA Methylation
DNA Methylation
● Addition of a methyl group onto a cytosine● Typically in the CG context (CpG)● Sometimes in CHH or CHG contexts
(especially in plants)
DNA Methylases
Some environmental / developmental signal
Cellular integration of the signal
Methylation of target sequences DNMT3 (de novo methylase)
A T C G T C T AT A G C A G A T
Symmetricallymethylated DNA
A T C G T C T AT A G C A G A T
Replication
A T C G T C T AT A G C A G A T
A T C G T C T AT A G C A G A T
Symmetricallymethylated DNA
Hemi-methylated DNA
A T C G T C T AT A G C A G A T
Replication
A T C G T C T AT A G C A G A T
A T C G T C T AT A G C A G A T
Symmetricallymethylated DNA
Hemi-methylated DNA
A T C G T C T AT A G C A G A T
A T C G T C T AT A G C A G A T
Symmetricallymethylated DNA
DNMT1 (maintenance methylase)
Maintenance
RNA
De-Novo
xx
A Computational Approach
Methylation and Genome Composition
CpG CpGM
TpG
Methylation resultsin CpG under-representation
CpG Bias=GpG observedCpG expected
=nATGC×nCpGnC×nG
Microarray Meta-Analysis:Presence / Absence Calls
Presence / Absence in Body Parts
Acropora millepora
Credits: Eldon Ball
● Stage-specific libraries● Mapping onto genome● Counts per transcript● Tissue-specificity index
– N: Number of conditions– Xi: Counts in condition i– Xmax: Max count
Ubiquitous Specific
DNA Methylation:Molecular Methods
Capture-Based Methods
● MeDIP: Methylated DNA Immuno-Precipitation● MethylCap: uses Methyl-Binding Domain
(MBD) of the MeCP2 protein● Followed by
– Tiling array – Sequencing (MeDIP-seq, MethylCap-seq)
Bisulfite Convertion
Methylation
BisulfiteConversion
A T C A T C T C A C G A T C G A T
A T T A T T T T A C G A T T G A T
M
Sodium Bisulfite
Array-Based Methods
● Illumina Infinium
Array-Based Methods
● Illumina Infinium– Pros
● Cheap● “Small” data size (~ ½ million CpGs)
– Cons● Only covers a small proportion of all CpGs● Focuses mainly on CpG Islands● Only exists for Human
Bock et al, Nature Biotechnologies, 2010
Bisulfite Sequencing
A T C A T C T C A C G A T C G A T
A T T A T T T T A C G A T T G A T
M
Sodium Bisulfite
Whole Genome Bisulfite Sequencing
● Bisulfite conversion directly followed by sequencing
● Large amount of data:– Single base pair resolution– Single strand resolution
A T A C G T A AT A T G C A T T
A T A C G T A AT A T G C A T T
Methylated Hemi-methylated
Conversion Breaks Complementarity
A C A C G T G AT G T G C A C T
A T A C G T G A
T G T G C A T T
Bisulfite Conversion
Watson (+)
Crick (-)
(+)
(-)
A T A C G T G AT A T G C A C T
A C T G C A A TT G T G C A T T
(++)
(+-)
(--)
(-+)
Mapping
● Convert genome (C to T)– Genome– Reverse complement
● Convert reads– Forward reads (++ and -+ strands): C to T– Reverse reads (+- and – strands): G to A
● Map (three letter space)● Convert the alignments back to the original
sequences
Calling Methylated Bases
● Bisulfite conversion is only a partial reaction (between 99% and 99.9%)
● With large amount of data, small artifacts can become highly significant
Calling Methylated Bases
CpGs 30,000,000
mCpGs 25,000,000
Cs 1,000,000,000 (both strands)
Conversion rate 99.9%
Coverage per strand 10
Change of spurious mC 1%
Inferred mC 20,000,000
Homo sapiens
Calling Methylated Bases
CpGs 5,000,000
mCpGs 50,000
Cs 50,000,000 (both strands)
Conversion rate 99.9%5
Coverage per strand 10
Change of spurious mC 1%
Inferred mC 500,000
Apis mellifera
The Null Hypothesis
C
C T
99.9%0.1%
The Null Hypothesis
C
C T
99.9%0.1%
Bernoulli process with probability “p”
N Bernoulli trials: Binomial process (N, p)
The Null Hypothesis
C
C T
99.9%0.1%
Bernoulli process with probability “p”
N Bernoulli trials: Binomial process (N, p)
Many p-values: adjust for multiple testing
ubiquitous specific
Forêt et al, 2009
CpG depletedmethylatedubiquitous
normal CpG contentnon-methylated
condition-specific
Lyko, Forêt et al, 2010
Vertebrates
Invertebrates
Feng et al, PNAS, 2010
Position-Dependent Effect
Portela and Esteller, Nature Biotechnologies, 2010
Summary
● Probable ancestral animal methylation landscape● Gene body (introns and exons), transposons
● Dense methylation landscapes are a vertebrate innovation
● Honey bee: lack of transposon methylation and scarce intronic methylation are also apomorphic
● Despite these differences in methylation levels, the targets of gene body methylation are broadly conserved
Other Important Topics
● D ifferential methylation● Dealing with complex samples
Concluding Thoughts
Major Transitions in Evolution
● Cavalier-Smith and Szathmary (1995)
1) from rep licating mo lecu les to popu lations of mo lecu les in compartments (protocells);
2) from independent genes to chromosomes;
3) from RNA as both an information carrier and enzyme to DNA as the carrier of information and proteins as the enzymes;
4) from prokaryotes to eukaryotes;
5) from asexual clones to sexual populations;
6) from single-cell eukaryotes to multicellular organisms with differentiated cells;
7) from solitary individuals to colonies with non- reproductive castes
Major Transitions in Evolution
● Cavalier-Smith and Szathmary (1995)– Focussed on genetic elements
Major Transitions in Evolution
● Cavalier-Smith and Szathmary (1995)– Focussed on genetic elements
● Other non-genetic heritable changes (Jablonka (1994), Jablonka & Lamb (2006))– Membranes
– Prions
– Self-sustaining metabolic cycles– Modified DNA bases– Other molecular marks attached to DNA
Role of non-genetic heritable changes
● Emergence of cells / chromosomes.– Transmission of epigenetic memory from the parent
to the daughter cells / chromosomes to put these in a state suitable for the environment.
Role of non-genetic heritable changes
● Transition to multicellularity.– Co-option of mechanisms to transmit information
about structure, state, and activity to daughter cells.– In order to maintain a coherent organism:
● Prevent de-differentiation● Early segregation of germ line
● Transition to social / colonial systems with division of labour or polyphenism– Phenotypic differentiation at the level of the whole
organism– Co-option of epigenetic mechanisms involved in
cellular differentiation
Role of non-genetic heritable changes