Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis thaliana using whole genome tiling array

1
Global analysis of genetic, epigenetic and
transcriptional polymorphisms in Arabidopsis
thaliana using whole genome tiling array
2
DNA methylation

• Genome defense against mobile elements
• Regulation of gene activity

Linaria vulgaris flowers (Cubas et al., 1999)
Tomato ripening mutant (Manning et al., 2006)
3
Plant DNA methylation

• Symmetric cytosine methylation
• mCG
• mCNG
• Asymmetric cytosine methylation
• mCNN

4
What we want to know

• Extent of CG methylation and methylation
  polymorphism among natural accessions
• Inheritance of methylation polymorphisms
• Any effect of methylation on gene expression

5
Enzyme methylome approach
5-CCGG- 3-GGCC- 5-CmCGG- 3-GGCmC- 5-mCmCGG- 3-GGCmCm- 5-mCCGG- 3-GGCCm-
HpaII cutting Y N N Rare in plant
MspI cutting Y Y N Rare in plant
6
CG-methylation and expression profiling
Col? x Col?
Van ? x Van ?
Col ? x Van ?
Van ? x Col ?

• 300ng genomic DNA
• Digest with either mspI or hpaII
• Label with biotin random primers
• Hybridize to AtTILE1F

• mRNA from 20ug totoal RNA
• Double-stranded cDNA synthesis
• Label with biotin random primers
• Hybridize to AtTILE1F

7
Constitutive CG methylation

A)
HpaII digestion

Random labeling

B)
MspI digestion
Random labeling
HpaII
HpaII
intensity
MspI
MspI
Col
Van
8
Methylation polymorphisms

A)
Col genotype

HpaII digestion

MspI digestion
B)
Van genotype
HpaII digestion
HpaII
intensity
HpaII
MspI
MspI
MspI digestion
Col
Van
9
Sequence polymorphisms

A)
Col genotype

B)
Van genotype
MspI
HpaII
intensity
MspI
HpaII
Col
Van
10
Simultaneous genetic and epigenetic profiling
of unique probes 1,683,620 of
CCGG-containing probes 54,519 model Intensity
genotype enzyme genotype x enzyme
11
Summary of sequence polymorphisms
FDR Calleda Falseb Sig-c Sigc
13.05  211220  29007  58628  152592 
6.22  173611  11363  33227  140384 
2.74  153401  4431  23326  130075 
1.16  138552  1698  17742  120810 
0.51  126499  678  14131  112368 
0.22  116122  272  11448  104674 
0.09  106817  104  9347  97470 
Called significant features False false
positives based on permutation Sig- Van greater
signal Sig Col greater signal
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Genome distribution of SFPs
13
Genic distribution of SFPs
  CDc Intron UTRs Promoterd Downstreame Intergenic  Total
SFPa 23180 19806 5130 30190 32158 50539 161003
Featureb  526407 301947 105260 429585 452681 593757 2409637
Percentage 4.40 6.56 4.87 7.03 7.10 8.51 6.68
aThe number of SFPs within each annotation
category.  bThe number of features within
each annotation category. cCoding sequences.
dThe sequences from transcriptional start to
upstream 1kb. eThe sequences from
transcriptional stop to downstream 1kb.
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Methylation polymorphisms are extensive
Enzyme Genotype x enzyme Genotype x enzyme
p-value HpaII gt MspIa p-value Col-specificb Van-specificc
lt0.01 2373 lt0.01 1062 407
lt0.05 4522 lt0.03 2389 944
lt0.1 6324 lt0.05 3700 1515
Gened 3628 (20) Gened 3498 (20) 3498 (20)
Total genee 17760 Total genee 17760 17760
Promoterf 305 (6) Promoterf 455 (9) 455 (9)
Total promoterg 5041 Total promoterg 5041 5041
Intergenich 1298 (16) Intergenich 782 (9) 782 (9)
Total intergenici 8264 Total intergenici 8264 8264
a Features of constitutive CG methylation bc
Features of Col- or Van-specific methylation df
cDNAs or promoters with feature(s) of enzyme
effect (p lt 0.1) or genotype enzyme
interaction (p lt 0.05) eg cDNAs or promoters
containing CCGG feature(s) h Intergenic
features (excluding cDNAs or promoters) of enzyme
effect (p lt 0.1) or genotype enzyme interaction
(p lt 0.05) i Intergenic (excluding cDNAs or
promoters) CCGG-containing features
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Verification of methylation polymorphisms
16
Verification of methylation polymorphisms
17
Genome distribution of constitutive and
polymorphic methylation sites
18
Co-methylation of pericentromere regions
19
Genic distribution of constitutive and
polymorphic methylation sites
20
Correlation between gene size and constitutive CG
methylation
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epiTyper
Col Col Col Van Van Van Col? x Van? Col? x Van ?
Van? x Col? Van ? x Col ? Van? x Col ?
CCGG
chromomethylase 2 (CMT2) exon19
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Inheritance of CG methylation polymorphism
Full model Intensity genotype enzyme
genotype x enzyme Genotype Additive (between
parents) Dominant (between F1 and
mid-parent) Maternal (between reciprocal F1s)
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Additive effect
Additive effect -
Additive effect
log intensity
log intensity
Col
Van
Van
Col
F1v
F1v
F1c
F1v
F1c
F1c
F1v
F1c
Col
Col
Van
Van
HpaII MspI
HpaII MspI
SFP Col has greater signal than Van.
Van duplication or deletion in Col Van has
greater signal than Col
Additive effect describes intensity difference
between parent strains across enzyme treatments.
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Dominant effect
Dominant effect
Dominant effect -
log intensity
log intensity
Col
Col
Col
F1v
F1c
Col
F1c
F1v
F1v
F1c
F1c
F1v
Van
Van
Van
Van
HpaII MspI
HpaII MspI
Increased F1 hybridization compared with expected
from mid-parent
Reduced F1 hybridization compared with expected
from mid-parent
Dominant effect describes intensity difference
between mid-parent (average of parents dashed
line) and average of F1 hybrids across enzyme
treatments.
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Maternal effect
Maternal effect
Maternal effect -
log intensity
log intensity
F1v
F1c
F1v
Col
Col
F1c
Van
Van
Col
Col
F1c
F1c
Van
Van
F1v
F1v
HpaII MspI
HpaII MspI
Random variation Col-mother F1 with greater
signal than Van-mother F1
Random variation Van-mother F1 with greater
signal than Col-mother F1
Maternal effect describes intensity difference
between reciprocal F1 hybrids across enzyme
treatments.
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Enzyme effect
Enzyme effect
Enzyme effect -
log intensity
log intensity
Col
Van
F1c
F1v
Van
F1c
Van
Col
F1v
F1c
F1v
Van
F1c
Col
Van
Col
F1v
HpaII MspI
HpaII MspI
Constitutive CG methylation HpaII samples have
greater signal
Normalization and/or preferential labeling of
short fragment MspI samples have greater signal
Enzyme effect describes intensity difference
between HpaII and MspI enzyme treatment across
genotypes.
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Additive x enzyme interaction
Additive x enzyme effect
Additive x enzyme effect -
log intensity
log intensity
Col
Van
F1c
F1v
F1v
F1c
Van
Col
Van
Col
Col
Van
F1v
F1c
F1c
F1v
HpaII MspI
HpaII MspI
Col-specific methylation
Van-specific methylation
Additive x enzyme effect describes differential
enzyme sensitivity between parent strains.
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Dominant x enzyme interaction
Dominant x enzyme effect
Dominant x enzyme effect -
log intensity
log intensity
F1v
F1c
Col
F1c
F1v
Col
F1v
F1v
F1c
F1c
Van
Col
Col
Van
Van
Van
HpaII MspI
HpaII MspI
Col-dominant methylation
Van-dominant methylation
Dominant x enzyme effect describes differential
enzyme sensitivity between mid-parent (average of
parents dashed line) and average of F1 hybrids.
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Maternal x enzyme interaction
Maternal x enzyme effect
Maternal x enzyme effect -
log intensity
log intensity
F1v
Col
Van
F1c
F1v
Van
Col
Van
Col
Van
F1c
F1v
F1c
F1v
Col
F1c
HpaII MspI
HpaII MspI
Col-mother hybrid specific methylation
Van-mother hybrid specific methylation
Maternal x enzyme effect describes differential
enzyme sensitivity between reciprocal F1 hybrids
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Significance of main effects
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Significance of genotype x enyzme effects
additive ? enzyme
dominant ? enzyme
maternal ? enzyme
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Correlation of constitutive CG methylation and
absolute gene expression
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Correlation of polymorphic CG methylation and
gene expresson variation
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Gene set enrichment in genic CG methylation
polymorphisms
effect GOa GOa term p-value GOa term p-value
addenz Col gt Van Col gt Van Col gt Van Col gt Van Van gt Col Van gt Col Van gt Col
addenz GO0006457 protein folding protein folding 7.84E-05 GO0007242 intracellular signaling cascade 1.72E-03
addenz GO0009909 regulation of flower development regulation of flower development 5.05E-03 GO0015979 photosynthesis 2.76E-03
addenz GO0007018 microtubule-based movement microtubule-based movement 8.56E-03 GO0006952 defense response 5.82E-03
addenz GO0006511 ubiquitin-dependent protein catabolic process ubiquitin-dependent protein catabolic process 1.27E-02 GO0030001 metal ion transport 1.24E-02
addenz GO0007275 multicellular organismal development multicellular organismal development 1.50E-02 GO0009809 lignin biosynthetic process 2.49E-02
addenz GO0042254 ribosome biogenesis and assembly ribosome biogenesis and assembly 2.03E-02 GO0006813 potassium ion transport 2.91E-02
addenz GO0019538 protein metabolic process protein metabolic process 2.16E-02 GO0009739 response to gibberellin stimulus 4.85E-02
addenz GO0006470 protein amino acid dephosphorylation protein amino acid dephosphorylation 2.73E-02    
addenz GO0009567 double fertilization forming a zygote and endosperm double fertilization forming a zygote and endosperm 2.98E-02    
addenz GO0045454 cell redox homeostasis cell redox homeostasis 3.39E-02    
addenz GO0007568 aging aging 4.67E-02      
domenz F1 hybrids gt parentsc F1 hybrids gt parentsc F1 hybrids gt parentsc   parents gt F1 hybridsc parents gt F1 hybridsc parents gt F1 hybridsc
domenz GO0009965 leaf morphogenesis leaf morphogenesis 2.13E-04 GO0042254 ribosome biogenesis and assembly 6.60E-03
domenz GO0009225 nucleotide-sugar metabolic process nucleotide-sugar metabolic process 4.21E-04 GO0009617 response to bacterium 1.36E-02
domenz GO0006869 lipid transport lipid transport 2.96E-03 GO0009744 response to sucrose stimulus 2.31E-02
domenz GO0010119 regulation of stomatal movement regulation of stomatal movement 8.79E-03 GO0016192 vesicle-mediated transport 2.59E-02
domenz GO0000271 polysaccharide biosynthetic process polysaccharide biosynthetic process 9.77E-03 GO0000074 regulation of progression through cell cycle 2.60E-02
domenz GO0015995 chlorophyll biosynthetic process chlorophyll biosynthetic process 2.03E-02 GO0045449 regulation of transcription 3.60E-02
domenz GO0048364 root development root development 2.11E-02 GO0006810 transport 4.08E-02
domenz GO0009408 response to heat response to heat 2.33E-02    
domenz GO0009908 flower development flower development 4.08E-02    
domenz GO0015979 photosynthesis photosynthesis 4.11E-02    
domenz GO0045454 cell redox homeostasis cell redox homeostasis 4.13E-02    
domenz GO0019575 sucrose catabolic process using beta-fructofuranosidase sucrose catabolic process using beta-fructofuranosidase 4.41E-02    
domenz GO0009887 organ morphogenesis organ morphogenesis 4.49E-02      
matenz Col-mother F1 gt Van-mother F1d Col-mother F1 gt Van-mother F1d Col-mother F1 gt Van-mother F1d Van-mother F1 gt Col-mother F1d Van-mother F1 gt Col-mother F1d Van-mother F1 gt Col-mother F1d
matenz GO0015979 photosynthesis photosynthesis 1.17E-03 GO0015986 ATP synthesis coupled proton transport 1.09E-02
matenz GO0015995 chlorophyll biosynthetic process chlorophyll biosynthetic process 1.22E-03 GO0006470 protein amino acid dephosphorylation 1.11E-02
matenz GO0009408 response to heat response to heat 1.76E-02 GO0009407 toxin catabolic process 1.14E-02
matenz GO0009416 response to light stimulus response to light stimulus 2.87E-02 GO0006944 membrane fusion 2.60E-02
matenz GO0006520 amino acid metabolic process amino acid metabolic process 3.38E-02 GO0009909 regulation of flower development 2.92E-02
matenz GO0042742 defense response to bacterium defense response to bacterium 3.50E-02 GO0009873 ethylene mediated signaling pathway 4.07E-02
matenz GO0006397 mRNA processing mRNA processing 4.44E-02      
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Maternal methylome could be important for
reciprocal F1 gene expression
Col methylation gt Van methylationc Col methylation gt Van methylationc Col-mother F1 expression gt Van-mother F1 expressiond Col-mother F1 expression gt Van-mother F1 expressiond
GOe term p-value GOe term p-value
BPa GO0006457 protein folding 7.84E-05 GO0006412 translation 2.13E-32
BPa GO0009909 regulation of flower development 5.05E-03 GO0006457 protein folding 2.09E-30
BPa GO0007018 microtubule-based movement 8.56E-03 GO0042254 ribosome biogenesis and assembly 2.82E-15
BPa GO0006511 ubiquitin-dependent protein catabolic process 1.27E-02 GO0007018 microtubule-based movement 1.14E-11
BPa GO0007275 multicellular organismal development 1.50E-02 GO0006334 nucleosome assembly 1.88E-09
BPa GO0042254 ribosome biogenesis and assembly 2.03E-02 GO0009408 response to heat 4.49E-09
MFb GO0031072 heat shock protein binding 1.67E-03 GO0003735 structural constituent of ribosome 6.21E-32
MFb GO0003777 microtubule motor activity 7.56E-03 GO0003777 microtubule motor activity 2.75E-13
MFb GO0051082 unfolded protein binding 1.27E-02 GO0003723 RNA binding 1.34E-12
MFb GO0015035 protein disulfide oxidoreductase activity 1.90E-02 GO0051082 unfolded protein binding 1.44E-12
MFb GO0005528 FK506 binding 2.59E-02 GO0003755 peptidyl-prolyl cis-trans isomerase activity 6.31E-10
MFb GO0003755 peptidyl-prolyl cis-trans isomerase activity 3.19E-02 GO0005525 GTP binding 1.63E-08
MFb GO0031072 heat shock protein binding 1.99E-08
MFb GO0005528 FK506 binding 3.02E-06
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