Transcript Document

Chromatin-guided interpretation
of variation in a disease cohort.
• Methylome variability associated w/specific chromatin states
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–
–
–
Methylation patterns primarily driven by tissue of origin
Little inter-individual variability, in hemi-methylated probes
Strong and weak enhancers most variable across individuals
Bi-modal and multi-modal probes depleted for promoter regions
• Methylome variability strongly genotype driven
– 55,000 methyl-QTLs, including 85% of most-variable probes
– methylQTL signal much stronger than for eQTLs or chromatinQTLs
– Depleted for promoter regions, enriched for enhancer regions
• Methylation changes associated with Alzheimer’s disease.
– Global hypermethylation signature across 7000 top-ranked probes.
– Enriched for brain-specific enhancers, depleted for promoter regions
– Motif enrichments for brain regulators in AD-associated probes.
Genetic and epigenetic data in 750 Alzheimer’s patients/controls
Chromatin States
PC / Repressed
Low signal
Heterochromatin
Repetitive
Active Promoter
Inactive/poised promoter
Promoter Flanking
Active Enhancer
Weak enhancer
Weak gene bodies
Strong gene bodies
Bradley Bernstein
• ROS & MAP studies:
 Patients join study
cognitively normal
in middle age.
 Patients followed
for 10+ years with
cognitive
evaluations.
 Brain samples
(Dorsolateral
prefrontal cortex,
DLPFC) donated
post-mortem.
750 individuals (~50% w/AD) • Integrate:
Genotypes
imputed with
1000Genomes
Project
 AD Phenotypes
 Methylation and
genotype (DeJager
Lab)
486,000
methylation
probes
Philip DeJager
 Reference
chromatin map
(Bernstein Lab).
Methylome driven primarily by tissue of origin.
Consistent between individuals, technologies and
studies.
Correlation heatmap
DLPFC,
this study.
Mean R2 ≈ 0.99
CD4+, Rakyan et
al., 2010, 27k array
CD4+, this study
Probe Standard Deviation
Profile of methylome reveals little
variation.
Mean methylation signal
4
Each state has a unique methylation profile
Average
Methylation level
Inter-individual
Variance
Active TSS flanking
Inactive/poised promoter
Inactive/poised promoter
Strong promoter
Strong promoter
CNV/Rep
CNV/Rep
Heterochromatin
Heterochromatin
Low signal
Low signal
PC Repressed
PC Repressed
Methylation
Strong promoters – little methylation, little variance.
Strong gene bodies – high methylation.
Polycomb repressed areas – high variability.
Per−CpG Methylation Variance
0.004
Active TSS flanking
0.003
Active enhancer
Two
highest
0.002
Active enhancer
1.0
Weak enhancer
0.8
Weak enhancer
0.6
Weak transcribed/
active proximal
0.4
Weak transcribed/
active proximal
0.2
Strong transcription
0.0
Strong transcription
0.001
Strong gene bodies
Weak gene bodies
Weak enhancer
Active Enhancer
Promoter Flanking
Inactive/poised promoter
Active Promoter
Repetitive
Heterochromatin
Low signal
PC / Repressed
DNA methylation probe variance by chromatin state
0.000
DNA methylation by chromatin state
Chromatin-guided interpretation
of variation in a disease cohort.
• Methylome variability associated w/specific chromatin states
–
–
–
–
Methylation patterns primarily driven by tissue of origin
Little inter-individual variability, in hemi-methylated probes
Strong and weak enhancers most variable across individuals
Bi-modal and multi-modal probes depleted for promoter regions
• Methylome variability strongly genotype driven
– 55,000 methyl-QTLs, including 85% of most-variable probes
– methylQTL signal much stronger than for eQTLs or chromatinQTLs
– Depleted for promoter regions, enriched for enhancer regions
• Methylation changes associated with Alzheimer’s disease.
– Global hypermethylation signature across 7000 top-ranked probes.
– Enriched for brain-specific enhancers, depleted for promoter regions
– Motif enrichments for brain regulators in AD-associated probes.
Autosomal Probes (2831)
Discovering variable probes through devonvolution
Individuals
Correcting for covariates
Bij = Rij + x0 + x1(age) + x2(gender) + x3(study) + x4(batch)
Enriched in areas of
low regulatory activity
*
Strong transcription
* *
*
Weak transcribed/
active proximal
*
* *
Weak enhancer
* **
Active enhancer
0.1
Active TSS flanking
0.3
Inactive/poised promoter
Strong promoter
CNV/Rep
Heterochromatin
0.4
Low signal
PC Repressed
Multimodal probes -- distribution
All probes
Bimodal
Trimodal
Bimodal+Trimodal
***
0.2
Depleted in active
promoters
*
* *
0.0
Chromatin-guided interpretation
of variation in a disease cohort.
• Methylome variability associated w/specific chromatin states
–
–
–
–
Methylation patterns primarily driven by tissue of origin
Little inter-individual variability, in hemi-methylated probes
Strong and weak enhancers most variable across individuals
Bi-modal and multi-modal probes depleted for promoter regions
• Methylome variability strongly genotype driven
– 55,000 methyl-QTLs, including 85% of most-variable probes
– methylQTL signal much stronger than for eQTLs or chromatinQTLs
– Depleted for promoter regions, enriched for enhancer regions
• Methylation changes associated with Alzheimer’s disease.
– Global hypermethylation signature across 7000 top-ranked probes.
– Enriched for brain-specific enhancers, depleted for promoter regions
– Motif enrichments for brain regulators in AD-associated probes.
Most epigenomic variability is genotype-driven
P-value (-log10P)
Manhattan plot of 450,000 methylation probes
Chromosome and genomic position
• Cutoff of 10-14 (10-2 after Benjamini-Hochberg correction).
• Prune for probes disrupted by SNP.
 140,000 CpGs associated with genotype at 1% FDR
 55,000 at Bonferoni-corrected P-value of 10-2
 From most variable 1% of CpGs: 85% are SNP associated.
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*
0.05
*
Strong transcription
0.20
Weak transcribed/
active proximal
*
Weak enhancer
0.10
Active enhancer
*
Active TSS flanking
Inactive/poised promoter
Strong promoter
CNV/Rep
Heterochromatin
Low signal
0.15
PC Repressed
SNP-associated CpGs depleted in
promoters, enriched in enhaners
*
All probes
meQTL probes
*
*
*
0.00
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Chromatin-guided interpretation
of variation in a disease cohort.
• Methylome variability associated w/specific chromatin states
–
–
–
–
Methylation patterns primarily driven by tissue of origin
Little inter-individual variability, in hemi-methylated probes
Strong and weak enhancers most variable across individuals
Bi-modal and multi-modal probes depleted for promoter regions
• Methylome variability strongly genotype driven
– 55,000 methyl-QTLs, including 85% of most-variable probes
– methylQTL signal much stronger than for eQTLs or chromatinQTLs
– Depleted for promoter regions, enriched for enhancer regions
• Methylation changes associated with Alzheimer’s disease.
– Global hypermethylation signature across 7000 top-ranked probes.
– Enriched for brain-specific enhancers, depleted for promoter regions
– Motif enrichments for brain regulators in AD-associated probes.
Global hyper-methylation in 1000s of AD-associated loci
P-value
QQ plot: Many loci with weak effects?
8
480,000 probes, ranked by Alzheimer’s association
6
4
Methylation
Observed (-logP)
10
Top 7000 probes
2
0
0
2
4
6
8
Expected (-logP)
10
Alzheimer’s-associated probes are hypermethylated
• Global effect across 1000s of probes
Alzheimer’s
Normal
– Rank all probes by Alzheimer’s association
– Observe functional changes down ranklist
– 7000 probes show shift in methylation
Complex disease: genome-wide effects
Hypermethylated probes
(repressed)
Estimating functionally enriched Alzheimer’s probes
0.00
PC Repressed 44109
Low signal 98450
Heterochromatin 2980
CNV/Rep 902
Strong promoter 109360
Inactive/poised promoter 46041
Active TSS flanking 25106
Active enhancer 14365
Weak enhancer 32342
Weak transcribed/low signal proximal to active regions 65825
Strong transcription 31433
−0.05
(Obs − Exp) / Total
0.05
Strong enhancers / TSS flanking regions
Heterochromatin
0
100000
200000
Probe Rank
300000
400000
Alzheimer’s prediction vs. likely biological pathways
NRSF
All probes, ranked by AD assoc. P-value
ELK1
All probes, ranked by AD assoc. P-value
CTCF
Predictive power: 7k probes + APOE
Regulatory motifs associated
with Alzheimer-associated probes
suggest potential pathways
We have not solved Alzheimer’s, but new insights gained
Chromatin basis of methylation variation in disease
• Methylome variability associated w/specific chromatin states
–
–
–
–
Methylation patterns primarily driven by tissue of origin
Little inter-individual variability, in hemi-methylated probes
Strong and weak enhancers most variable across individuals
Bi-modal and multi-modal probes depleted for promoter regions
• Methylome variability strongly genotype driven
– 55,000 methyl-QTLs, including 85% of most-variable probes
– methylQTL signal much stronger than for eQTLs or chromatinQTLs
– Depleted for promoter regions, enriched for enhancer regions
• Methylation changes associated with Alzheimer’s disease.
– Global hypermethylation signature across 7000 top-ranked probes.
– Enriched for brain-specific enhancers, depleted for promoter regions
– Motif enrichments for brain regulators in AD-associated probes.

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Reference epigenome maps: context for natural variation, disease
Enhancers: most variable, genotype-driven, disease-associated
Systems view of complex disease: 1000s of variants, weak effects
Opportunity: Interplay between genotype, epigenome, disease
Acknowledgements
• Kellis group:
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Manolis Kellis
Pouya Kheradpour
Luke Ward
Gerald Quon
• UCLA:
– Jason Ernst
• De Jager group:
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Gyan Srivastava
Lori Chibnik
Brendan Keenan
Phil De Jager
Joshua Shulman
Cristin Aubin
Towfique Raj
• Rush University:
– David Bennett