achim tresch computational biology
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‘Omics’ - Analysis of high dimensional Data. Achim Tresch Computational Biology. Epigenetics. Slides: Doug Brutlag , Stanford University School of Medicine http://biochem158.stanford.edu/Epigenetics.html. Epigenetics. - PowerPoint PPT PresentationTRANSCRIPT
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Achim TreschComputational Biology
‘Omics’ - Analysis of high dimensional Data
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Slides: Doug Brutlag, Stanford University School of Medicinehttp://biochem158.stanford.edu/Epigenetics.html
Epigenetics
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• C.H. Waddington coined the term epigenetics to mean above or in addition to genetics to explain differentiation.
• How do different adult stem cells know their fate?– Myoblasts can only form muscle cells– Keratinocytes only form skin cells – Hematopoetic cells only become
blood cells– But all have identical DNA
sequences.
Epigenetics
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Epigenetics
• Modern definition is non-sequence dependent inheritance.
• How can identical twins have different natural hair colors?
• How can a single individual have two different eye colors?
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Mosaicism: One Eye, two Colors
• How can identical twin liter mates show different coat colors?
• How can just paternal or maternal traits be expressed in offspring? This is called genetic imprinting.
• How can females express only one X chromosome per cell?
• How can acquired traits be passed on to offspring?
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Paula Vertino, Henry Stewart Talks
The ‘epigenetic’ code
DNA Methylation & Histone
Modifications
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Paula Vertino, Henry Stewart Talks
Methylation of Cytosine in DNA
cytosine 5-methyl cytosine
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Paula Vertino, Henry Stewart Talks
Methylation of Cytosine in DNA
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• CpG dinucleotides are partially methylated in higher vertebrates
• Human genome: only ~4% of all cytosines are methylated, but ~ 70%-80% 5mCpG
• Spontaneous deamination transforms CpG to TpG or CpA
• Estimated rate (after DNA-repair)[1]: 5.8*10-13 (5.8*10-17) 1/s*sites
Wikipedia: Deamination, Thymine, 5-methylcytosine
cytosine - uracil
5-methylcytosine - thymine
[1] Shen et al. (1993) Nucl. Acids Res.
DNA Methylation (Biochemistry)
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Methylation of Cytosine in DNA
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Me
DNA methylation and Histones
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Maintenance of Cytosine Methylation
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Maintenance of Cytosine Methylation
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Alex Meissner, Henry Stewart Talks
Maintenance of Cytosine Methylation
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Maintenance of Cytosine Methylation
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Functions of cytosine methylation
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Alex Meissner, Henry Stewart Talks
DNA Methylation and Cell Differentiation
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Alex Meissner, Henry Stewart Talks
DNA Methylation and Cell Differentiation
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Nuclear transplantation: Differentiated Cells can become Totipotent
DNA Methylation and Cell Differentiation
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Paula Vertino, Henry Stewart Talks
Methylation Changes During Development
Met
hyla
tion
leve
l
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Paula Vertino, Henry Stewart Talks
Methylation Changes During Development
Met
hyla
tion
leve
l
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Paula Vertino, Henry Stewart Talks
Methylation Changes During Development
Met
hyla
tion
leve
l
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© 2013 American Society of Plant Biologists
TTCGCCGACTAA TTCGCCGAuTAA
•DNA methylation– bisulfite sequencing
•Histone modification • chromatin immunoprecipitation (ChIP)
DNA Methylation and Histone Marks
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GREEN = H3K27me3PURPLE =
methylcytosine
Using next-generation sequencing, epigenetic modifications can be identified genome-wide: EPIGENOMICS and METHYLOMICS
DNA Methylation and Histone Marks
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http://www.39kf.com/uploadfiles/image/15902/TXT-20081228163836878.gif
DNA methylation and Gene Expression
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• Methylation in mammals is mainly targeted at CpG dinucleotides
• CpGs are either unmethylated or methylated on both strands
• Hemi-methylated CpGs are rare
• DNA methyltransferases (DNMTs) bind hemi-methylated sites and modify the remaining position
• Thus the epigenetic information is inherited to daughter cells
Adapted from: http://www.diagenode.com/en/applications/bisulfite-conversion.php Lars Feuerbach
Epigenomics
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© 2013 American Society of Plant Biologists
Bisulfite treatment
TTCGCCGACTAA
No treatment
TTCGCCGACTAA
TTCGCCGAuTAA
Methyl-cytosine
When DNA is bisulfite treated, unmethylated cytosine is converted to uracil. Methylcytosine is not affected.
O N
NH2
N
~O N
NH2
N
~
CH3
cytosine 5-methylcytosine
O N
NH2
N
~
CH3
O N
O
N
~uracil 5-methylcytosine
Bisulfite treatment
Bisulfite Sequencing
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© 2013 American Society of Plant Biologists
Bisulfite treatment
TTCGCCGACTAA
No treatment
TTCGCCGACTAA
TTCGCCGAuTAA
TTCGCCGACTAA TTCGCCGATTAA
Methyl-cytosine
After bisulfite treatment, unmethylated Cs are read as T and so differ in the treated and untreated samples.
By contrast, methyl-C is read as C and is the same as the reference sequence.
Bisulfite Sequencing
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RRBS-Seq• DNA is digested by MSP1 restriction enzyme which
cuts at CCGG sites• All DNA fragments start with CpG• Alignment is simplified as reads have to map to MSP1
restriction sites• Reads are enriched for CpG rich areas
http://www.neb.com/nebecomm/products/productR0106asp
CGGATGTTTTGTACTAGGATAACTATGC CGGAT
Reduced Representation Bisulfite Sequencing
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Reference
Read out
Standard alignment to the reference is not possible. Adapted alignment procedures have lower accuracy.
Alignment of BS converted reads
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Tools supporting the alignment of BS reads:- Bismark- BSMAP - BS Seeker
Simon Andrews, Bioinformatics 2011
Alignment of BS converted reads
Key concept:- Convert the reference genome in silico as bilufite
treatment does- Perform conversion for
+ strand and – strand- Then align reads against
both genomes
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Simon Andrews, Bioinformatics 2011
Alignment of BS converted reads
H = IUPAC character for the letters {A,C,T}
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Pearl-Necklace diagrams (lollipop plots)
Measure unmethylated Cs (#C)Measure methylated Cs (#5mC)Report the methylation ratio
CmCmC
#5#5#
Description of DNA methylation
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© 2013 American Society of Plant Biologists
Reprinted by permission from Macmillan Publishers Ltd: Zhong, S., Fei, Z., Chen, Y.R., Zheng, Y., Huang, M., Vrebalov, J., McQuinn, R., Gapper, N., Liu, B., Xiang, J., Shao, Y., and Giovannoni, J.J. (2013). Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol. [ in press].
Density of methylated DNA and other features in chromosomes of the tomato fruit
The Tomato Methylome
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Characterize deamination by repetitive sequences
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Evolution of CpG content in repetitive sequences
Peifer et al. (2008) Bioinformatics
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Evolution of CpG-rich promoters
•AT-rich promoters in bacteria
•Mixed promoters in worm and fly
•Increasing GC and CpG content in mosquito
•Small CpG islands in fish
•Broad CpG islands in humans
Khuu et al., PNAS, Sep. 2007
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Promoter Types in Humans
Weber et al., 2007, Nat. Genet.
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Model of CpG island evolution
Ancestral Genome
0,00%1,00%2,00%3,00%4,00%5,00%6,00%7,00%8,00%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Position on chromosome
CpG
freq
uenc
y
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Model of CpG island evolution
After 0.1 transversions
0,00%1,00%2,00%3,00%4,00%5,00%6,00%7,00%8,00%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Position on chromosome
CpG
freq
uenc
y
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Model of CpG island evolution
Observable genome
0,00%1,00%2,00%3,00%4,00%5,00%6,00%7,00%8,00%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Position on chromosome
CpG
freq
uenc
y
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CpG island definitions
Observable genome
0,00%1,00%2,00%3,00%4,00%5,00%6,00%7,00%8,00%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Position on chromosome
CpG
freq
uenc
y
CpG content TakeiJones Gardiner-Gardener
CpG island definition:
1. GC-content
2. Ratio observed over expected CpG frequency
3. Minimal Length
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•CpG dinucleotides are rare in the human genome
•CpG Islands are exceptions•Elevated GC content and
CpG frequency•50-60% of promoters are
CpG islands•Methylation level anti-
correlated to expression in HCP promoters
•Cause or consequence ?
CpG islands
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Caiafa and Zampieri,(2005) JCB
CpG islands and chromatin
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Histone modifications
• How to read the nomenclature:– Histone protein (H3)– Position in tail (K9)– Modification type (me3)
Füllgrabe et al., 2011, Oncogene
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Histone code
•Füllgrabe et al., 2011, Oncogene
H3K4me2-me3 Active transcription, near TSSs
H3K9me3 Heterochromatin
H3K9ac Euchromatin, near TSSs
H3K27me3 Polycomb marker, closes chromatin
H4K16ac Higher order chromatin, repeat methylation
H4K20me3 Heterochromatin
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Interplay
•Ceder&Bergman,2009,Nature Rev Genet
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Allele-unspecific DNA methylation
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Allele-specific DNA methylation
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Imprinting• Origin-of-allele-specific gene expression• Exception from Mendel’s inheritance rules• Mediated by methylation of imprinting control regions
University of Florida: http://www.peds.ufl.edu/divisions/genetics/teaching
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Allele specific – Histone modifications
Adapted from: http://genomebiology.com/content/figures/gb-2005-6-6-113-1-l.jpg
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Reference Methylomes – Laurent et al.
• Laurent data on human embryonic stem cells and fibroblasts
• 70% of all CpGs covered by at least 3 reads
Laurent et al. Genome Research 2010
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Reference methylomes – Molaro et al.
• Male germline methylome for human and chimpanzee
• Direct comparison to Laurent et al. data
Molaro et al. Cell 2011
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ENCODE, IHEC and Epigenome Roadmap
• One Genome many Epigenomes• Cataloguing epigenetic modifications in
different tissues
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Translation into NGS signals
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Translation of epigenetic signals
• Capture-seq– Chromatin Immunoprecipitation (ChIP)– Metylated DNA Immunoprecipitation (MeDIP)– MBD chromatography
• Conversion-seq– Bisulfite sequencing (methyl-seq)– Reduced representation bisulfite sequencing
(RRBS)– Ultra-deep amplicon sequencing
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Two signal types
• Coverage
EnrichmentSequencingMappingPeak calling
Sequence
PreparationSequencingSpecial mappingDecoding
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Enrichment-seq – Workflow I
Genome
Epigenetically modified regions
DNA LibraryPreparation
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Enrichment-seq – Workflow II
Enrichment
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Enrichment-seq – Workflow III
Mapping
Genome
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Enrichment-seq – Workflow IV
Genome
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Methylated DNA immunoprecipitation
•http://en.wikipedia.org/wiki/Methylated_DNA_immunoprecipitation
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Lutsik P et al. Nucl. Acids Res. 2011;nar.gkr312
BiQ Analyzer HT
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Allele-specific methylation analysis pipeline
Matthias Bieg et al., in preparation
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Summary• Epigenetics plays a key role in cell function
• Each cell type has its own epigenome
• Epigenetic modifications are can be converted to NGS signals
• Bioinformatic in depth analysis of epigenomes is still in its infancy
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References
•Laurent, L.; Wong, E.; Li, G.; Huynh, T. et al•“Dynamic changes in the human methylome•during differentiation”•Genome Research (2010) 20 320-331
•Molaro, A.; Hodges, E.; Fang, F.; Song, Q. et al.•“Sperm Methylation Profiles Reveal•Features of Epigenetic Inheritance•and Evolution in Primates”•Cell (2011) 146 1029-1041
•Lutsik,P.; Feuerbach, L. ; Arand, J.; Lengauer, T. et al.•“BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing”•NAR (2011)
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Environment can Influence Epigenetic Changes
Emma Whitelaw, Henry Stewart Talks
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Hongerwinter 1944• German’s blocked food to the Dutch in the winter of 1944.• Calorie consumption dropped from 2,000 to 500 per day for
4.5 million.• Children born or raised in this time were small, short in
stature and had many diseases including, edema, anemia, diabetes and depression.
• The Dutch Famine Birth Cohort study showed that women living during this time had children 20-30 years later with the same problems despite being conceived and born during a normal dietary state.