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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

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


Replication




Hemi-methylated DNA


Replication




Hemi-methylated 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


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


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


● Cavalier-Smith and Szathmary (1995)– Focussed on genetic elements


● 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.


● 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


Download - Evolution of DNA Methylation - Sylvain Forêt

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