Transcript Lect11_DNAMethylation

ChIP-seq Downstream Analysis
Xiaole Shirley Liu
STAT115, STAT215, BIO298, BIST520
Human TF Binding Distribution
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Most TF binding sites are outside promoters
How to assign targets?
Binding in promoter (how far)?
Nearest distance?
Number of binding?
Other knowledge?
• Still open Q
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Higher Order Chromatin Interactions
Interactions ~ follows
exponential decay with
distance
Lieberman-Aiden et al, Science 2009
How to Assign Targets for Enhancer
Binding Transcription Factors?
• Regulatory potential: sum of binding sites
weighted by distance to TSS with exponential
decay
• Rank1 of genes based on binding
TSS
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How to Assign Targets for Enhancer
Binding Transcription Factors?
• Regulatory potential: sum of binding sites
weighted by distance to TSS with exponential
decay
• Rank1 of genes based on binding
• Rank2 of genes based on expression
– Differential expression upon TF perturbation
– Gene expression correlation in cohorts
• Rank ordered list of targets: Rank1 * Rank2
• Only minority of binding sites is functional in a
condition
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BETA
• Binding expression target analysis
• Is TF an activator, repressor or both?
– Do genes with higher regulatory potential show more
up/down expression than random genes
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BETA
• Binding expression target analysis
• Is TF an activator, repressor or both?
– Do genes with higher regulatory potential show more
up/down expression than random genes
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Functional analysis of targets?
How can a factor be both activator and repressor?
Collaborating transcription factors
Motif analysis on the binding sites near up vs down
genes separately
ER
TF??
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Summary
• ChIP-seq identifies genome-wide in vivo
protein-DNA interaction sites
• MACS for ChIP-seq peak calling
– Shift reads, Poisson with dynamic λ
• ChIP-seq QC: FDR, fold, conservation, etc
• Functional analysis of ChIP-seq data:
– Target identification
– Activator / repressor function
– Motif analysis
Break
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Epigenetics
Xiaole Shirley Liu
STAT115, STAT215, BIO298, BIST520
Epigenetics
• Heritable changes in gene function that occur
without a change in the DNA sequence
– How come not all the motif sites are bound by the
factor?
– How come TF binding only regulate some of the
nearby genes?
Epigenetics
• Functionally relevant changes to the genome that
do not involve a change in the nucleotide
sequence
• The study of heritable (transgenerational)
changes in gene activity that are not caused by
changes in the DNA sequence
• The study of stable, long-term alterations in the
transcriptional potential of a cell that are not
necessarily heritable
The Making of a Queen
Larvae
From Ting Wang, Wash U
Queen
Worker
Agouti Mice and DNA Methylation
In Human
• Nature vs nurture
• Zygotic twins: same DNA different epigenome
• North American Ice Storm of 1998
Epigenetic Landscape
Conrad Hal Waddington
(1905–1975)
Developmental biologist
Paleontologist
Geneticist
Embryologist
Philosopher
Founder for systems biology
Components
• DNA-methylation
• Nucleosome
position and
histone
modifications
• Chromatin
accessibility
• Higher order
chromatin
interactions
Break
DNA Methylation
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DNA Methylation Distribution in
Mammalian Genomes
• In human somatic cells, 60%-80% of all CpGs
(~1% of total DNA bases) are methylated
– Most methylation is found in “repetitive”
elements
• “CpG islands”, GC-rich regions that possess a high
density of CpGs, remain methylation-free
– The promoter regions of ~70% of genes have
CpG islands
Two classes of DNA methyltransferases
(DNMTs)
Jones and Liang, 2009
Nature Review Genetics
Inheritance of DNA Methylation
DNA Methylation Detection
• Bisulfite sequencing
– Unmethyl C  T, sequence 30X
– High resolution, quantitative, but expensive!
– RRBS enriches CpG rich regions for sequencing
ACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGC
CGGGTTTATTTGCTTTTTTATGGGC
TGGGTTTATTTGCTTTTTTATGGGC
TGGGTTTATTTGCTTTCCTATGGGC
CGGGCTTATTTGCTTTCCTATGGGC
CGGGCTTATTTGCTTTCCTATGGGC
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60% methylated
0/5
0% methylated
BS-seq Methylation Call
• Bismark: Krueger & Andrews, Bioinfo 2011
– Create additional sequence in the BWA index to
account for the C -> T conversion
• Methylation or genome sequence difference?
+ ACGAACGCAACG
ACGAATGCAATG
+ ACGAATGCAATG
- TGCTTGTGTTGT
ACGAATGCAATG
TGCTTACGTTAC
Analysis Issues
• Methylation level within a short distance is
consistent: smoothing across bigger regions
• Most regions are either mostly methylated or
mostly unmethylated (dichotomy)
• Estimate tumor purity
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DNA Methylation Controls
Gene Expression
• Methylation at CpG islands often repress nearby
gene expression
• Many highly expressed genes have CpG
methylation on their exons
Some genes could
be imprinted, so
maternal and
paternal copies
have different
DNA methylation
Large Unmethylated Canyons
• 1,104 “Canyons” with > 3.5 Kb in the mouse HSC
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Jeong et al. Nature Genetics. 2014
DNA Methylation in Cancer
• Prevalent misregulation of DNA methylation in
cancer: global hypomethylation and CpG island
hypermethylation
• Methylation variable regions in cancer
DNA Demethylation
• Recently, another type of DNA methylation
called hydroxyl methylation (hmC) is found
• hmC is an intermediate step between mC and C.
• TET family of proteins are important for DNA
demethylation
• Mutation in TET is linked to many cancers
Acknowledgement
• Evan Johnson, BU
• Ting Wang, Wash U
• Wei Li, Baylor
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