Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis thaliana...
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Transcript of Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis thaliana...
Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis
thaliana using whole genome tiling array
Linaria vulgaris flowers (Cubas et al., 1999)
DNA methylation
Tomato ripening mutant (Manning et al., 2006)
Genome defense against mobile elements
Regulation of gene activity
Symmetric cytosine methylation:
mCG
mCNG
Asymmetric cytosine methylation:
mCNN
Plant DNA methylation
Extent of CG methylation and methylation polymorphism among natural accessions
Inheritance of methylation polymorphisms
Any effect of methylation on gene expression
What we want to know
5’-C CGG-
3’-GGC C-
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 plantMspI
cuttingY Y N
CG-methylation and expression profiling
300ng genomic DNA Digest with either mspI or hpaII Label with biotin random primers Hybridize to AtTILE1F
Col♀ x Col♂ Van ♀ x Van ♂ Col ♀ x Van ♂Van ♀ x Col ♂
mRNA from 20ug totoal RNA Double-stranded cDNA synthesis Label with biotin random primers Hybridize to AtTILE1F
HpaII digestion
Random labeling
Random labeling
MspI digestion
* * *
* * *
* * *
A)
B)
Constitutive CG methylation
Hp
aII
Msp
IHp
aII
Msp
I
Col Van
inte
nsi
ty
Methylation polymorphisms
Hp
aII
Col Van
inte
nsi
ty
Msp
I
Hp
aII
Msp
I
HpaII digestion
* * *
* * *
A)
B)
Col genotype
Van genotype
MspI digestion
HpaII digestion
MspI digestion
* * *
A)
B)
Hp
aII
Col Van
inte
nsi
ty
Msp
I
Hp
aII
Msp
I
Sequence polymorphisms
*
*
Col genotype
Van genotype
Simultaneous genetic and epigenetic profiling
# of unique probes: 1,683,620
# of CCGG-containing probes: 54,519
model:
Intensity ~ genotype + enzyme + genotype x enzyme
Summary of sequence polymorphisms
FDR Calleda Falseb Sig-c Sig+c
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
Genome 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%
Genic distribution of SFPs
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.
Enzyme Genotype x enzyme
p-value HpaII > MspIa p-value Col-specificb Van-specificc
<0.01 2373 <0.01 1062 407
<0.05 4522 <0.03 2389 944
<0.1 6324 <0.05 3700 1515
Gened 3628 (20%) Gened 3498 (20%)
Total genee 17760 Total genee 17760
Promoterf 305 (6%) Promoterf 455 (9%)
Total promoterg 5041 Total promoterg 5041
Intergenich 1298 (16%) Intergenich 782 (9%)
Total intergenici 8264 Total intergenici 8264
Methylation polymorphisms are extensive
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 < 0.1) or genotype × enzyme
interaction (p < 0.05) eg cDNAs or promoters containing CCGG feature(s)h Intergenic features (excluding cDNAs or promoters) of enzyme effect (p < 0.1) or
genotype × enzyme interaction (p < 0.05)i Intergenic (excluding cDNAs or promoters) CCGG-containing features
Verification of methylation polymorphisms
Verification of methylation polymorphisms
bp
Genome distribution of constitutive and polymorphic methylation sites
bp
Co-methylation of pericentromere regions
Genic distribution of constitutive and polymorphic methylation sites
Correlation between gene size and constitutive CG methylation
ColColColVanVanVan
Col♂ x Van♀Col♂ x Van ♀
Van♂ x Col♀Van ♂ x Col ♀Van♂ x Col ♀
CC*GG
chromomethylase 2 (CMT2) exon19
0
1
2
3
4
5
6
hpaII mspI
log
inte
nsity
col van col♂xvan♀ van♂xcol♀
epiTyper
Full model:
Intensity ~ genotype + enzyme + genotype x enzyme
Genotype:
Additive (between parents)
Dominant (between F1 and mid-parent)
Maternal (between reciprocal F1s)
Inheritance of CG methylation polymorphism
Additive effect describes intensity difference between parent strains across enzyme treatments.
Additive effect
HpaII MspI
log
inte
nsi
ty
Col
Van Van
Col
HpaII MspI
log
inte
nsi
ty
Col
Van
Col
Van
SFP; Col has greater signal than Van.
Van duplication or deletion in Col; Van has greater signal than Col
F1c
F1v
F1c
F1v F1c
F1v
F1c
F1v
Additive effect + Additive effect -
Dominant effect describes intensity difference between mid-parent (average of parents; dashed line) and average of F1 hybrids across enzyme treatments.
Dominant effectC
ol
Van
Van
Col
F1c
F1v F1c
F1v
HpaII MspI
Increased F1 hybridization compared with expected from mid-parent
log
inte
nsi
ty
Dominant effect +
Col
Van
Van
Col
Dominant effect -
F1c
F1v
F1c
F1v
HpaII MspI
log
inte
nsi
ty
Reduced F1 hybridization compared with expected from mid-parent
Maternal effect describes intensity difference between reciprocal F1 hybrids across enzyme treatments.
Maternal effect
F1v F1c
Maternal effect +
HpaII MspI F
1vF1c
log
inte
nsi
ty
F1c
HpaII MspI
F1v
F1c F1v
Random variation; Col-mother F1 with greater signal than Van-mother F1
Random variation; Van-mother F1 with greater signal than Col-mother F1
log
inte
nsi
ty
Col
Van Col
Van
Col
Van Col
Van
Maternal effect -
Enzyme effect describes intensity difference between HpaII and MspI enzyme treatment across genotypes.
Enzyme effect
HpaII MspI
Col
Col
Van
Enzyme effect +V
an
Van
F1c
F1v
F1c
F1v
log
inte
nsi
ty
HpaII MspI
Col
Col
Van
Van
F1c
F1v
F1c
F1v
Constitutive CG methylation; HpaII samples have greater signal
Normalization and/or preferential labeling of short fragment; MspI samples have greater signal
log
inte
nsi
ty
Enzyme effect -
Additive x enzyme effect describes differential enzyme sensitivity between parent strains.
Additive x enzyme interaction
Additive x enzyme effect +
log
inte
nsi
ty
Van
Col
Col
Van
HpaII MspI
Col-specific methylation Van-specific methylation
F1c
F1v
F1c
F1v
log
inte
nsi
ty
Van
Col
Col
VanF
1c
F1v
F1c
F1v
HpaII MspI
Additive x enzyme effect -
Dominant x enzyme effect describes differential enzyme sensitivity between mid-parent (average of parents; dashed line) and average of F1 hybrids.
Dominant x enzyme interactionC
ol
Van
Col
F1c
HpaII MspI
Dominant x enzyme effect +F
1v
Van F1c F1v
Col
Van ColF
1c
HpaII MspI
F1v
Van F1c F1vlo
g in
ten
sity
Col-dominant methylation Van-dominant methylationlo
g in
ten
sity
Dominant x enzyme effect -
Maternal x enzyme effect describes differential enzyme sensitivity between reciprocal F1 hybrids
Maternal x enzyme interaction
Maternal x enzyme effect +
Col-mother hybrid specific methylation
Van-mother hybrid specific methylation
log
inte
nsi
ty
Van
Col
Col
Van
HpaII MspI
F1c
F1v
F1c
F1v
log
inte
nsi
ty
Van
Col Col
Van
HpaII MspI
F1c
F1v
F1c
F1v
Maternal x enzyme effect -
additive dominant
maternal enzyme
Significance of main effects
additive χ enzyme dominant χ enzyme maternal χ enzyme
Significance of genotype x enyzme effects
Correlation of constitutive CG methylation and absolute gene expression
Correlation of polymorphic CG methylation and gene expresson variation
effect GOa term p-value GOa term p-value
addenz
Col > Van Van > Col
GO:0006457 protein folding 7.84E-05 GO:0007242 intracellular signaling cascade 1.72E-03
GO:0009909 regulation of flower development 5.05E-03 GO:0015979 photosynthesis 2.76E-03
GO:0007018 microtubule-based movement 8.56E-03 GO:0006952 defense response 5.82E-03
GO:0006511 ubiquitin-dependent protein catabolic process 1.27E-02 GO:0030001 metal ion transport 1.24E-02
GO:0007275 multicellular organismal development 1.50E-02 GO:0009809 lignin biosynthetic process 2.49E-02
GO:0042254 ribosome biogenesis and assembly 2.03E-02 GO:0006813 potassium ion transport 2.91E-02
GO:0019538 protein metabolic process 2.16E-02 GO:0009739 response to gibberellin stimulus 4.85E-02
GO:0006470 protein amino acid dephosphorylation 2.73E-02
GO:0009567 double fertilization forming a zygote and endosperm 2.98E-02
GO:0045454 cell redox homeostasis 3.39E-02
GO:0007568 aging 4.67E-02
domenz
F1 hybrids > parentsc parents > F1 hybridsc
GO:0009965 leaf morphogenesis 2.13E-04 GO:0042254 ribosome biogenesis and assembly 6.60E-03
GO:0009225 nucleotide-sugar metabolic process 4.21E-04 GO:0009617 response to bacterium 1.36E-02
GO:0006869 lipid transport 2.96E-03 GO:0009744 response to sucrose stimulus 2.31E-02
GO:0010119 regulation of stomatal movement 8.79E-03 GO:0016192 vesicle-mediated transport 2.59E-02
GO:0000271 polysaccharide biosynthetic process 9.77E-03 GO:0000074 regulation of progression through cell cycle 2.60E-02
GO:0015995 chlorophyll biosynthetic process 2.03E-02 GO:0045449 regulation of transcription 3.60E-02
GO:0048364 root development 2.11E-02 GO:0006810 transport 4.08E-02
GO:0009408 response to heat 2.33E-02
GO:0009908 flower development 4.08E-02
GO:0015979 photosynthesis 4.11E-02
GO:0045454 cell redox homeostasis 4.13E-02
GO:0019575 sucrose catabolic process using beta-fructofuranosidase 4.41E-02
GO:0009887 organ morphogenesis 4.49E-02
matenz
Col-mother F1 > Van-mother F1d Van-mother F1 > Col-mother F1d
GO:0015979 photosynthesis 1.17E-03 GO:0015986 ATP synthesis coupled proton transport 1.09E-02
GO:0015995 chlorophyll biosynthetic process 1.22E-03 GO:0006470 protein amino acid dephosphorylation 1.11E-02
GO:0009408 response to heat 1.76E-02 GO:0009407 toxin catabolic process 1.14E-02
GO:0009416 response to light stimulus 2.87E-02 GO:0006944 membrane fusion 2.60E-02
GO:0006520 amino acid metabolic process 3.38E-02 GO:0009909 regulation of flower development 2.92E-02
GO:0042742 defense response to bacterium 3.50E-02 GO:0009873 ethylene mediated signaling pathway 4.07E-02
GO:0006397 mRNA processing 4.44E-02
Gene set enrichment in genic CG methylation polymorphisms
Col methylation > Van methylationc Col-mother F1 expression > Van-mother F1 expressiond
GOe term p-value GOe term p-value
BPa
GO:0006457 protein folding* 7.84E-05 GO:0006412 translation 2.13E-32
GO:0009909 regulation of flower development 5.05E-03 GO:0006457 protein folding* 2.09E-30
GO:0007018 microtubule-based movement* 8.56E-03 GO:0042254 ribosome biogenesis and assembly* 2.82E-15
GO:0006511 ubiquitin-dependent protein catabolic process 1.27E-02 GO:0007018 microtubule-based movement* 1.14E-11
GO:0007275 multicellular organismal development 1.50E-02 GO:0006334 nucleosome assembly 1.88E-09
GO:0042254 ribosome biogenesis and assembly* 2.03E-02 GO:0009408 response to heat 4.49E-09
MFb
GO:0031072 heat shock protein binding* 1.67E-03 GO:0003735 structural constituent of ribosome 6.21E-32
GO:0003777 microtubule motor activity* 7.56E-03 GO:0003777 microtubule motor activity* 2.75E-13
GO:0051082 unfolded protein binding* 1.27E-02 GO:0003723 RNA binding 1.34E-12
GO:0015035 protein disulfide oxidoreductase activity 1.90E-02 GO:0051082 unfolded protein binding* 1.44E-12
GO:0005528 FK506 binding* 2.59E-02 GO:0003755 peptidyl-prolyl cis-trans isomerase activity* 6.31E-10
GO:0003755 peptidyl-prolyl cis-trans isomerase activity* 3.19E-02 GO:0005525 GTP binding 1.63E-08
GO:0031072 heat shock protein binding* 1.99E-08
GO:0005528 FK506 binding* 3.02E-06
Maternal methylome could be important for reciprocal F1 gene expression