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: m CG m CNG  Asymmetric cytosine methylation: m CNN 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 YNN Rare in plant MspI cutting YYN

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 MspI digestion * * * A) B) Constitutive CG methylation HpaII MspI HpaII MspI ColVan intensity

Methylation polymorphisms HpaII ColVan intensity MspI HpaII MspI HpaII digestion * * * A) B) Col genotype Van genotype MspI digestion HpaII digestion MspI digestion * * *

A) B) HpaII ColVan intensity MspI HpaII MspI 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 FDRCalled a False b Sig- c Sig+ c 13.05% % % % % % % Called: significant features False: false positives based on permutation Sig-: Van greater signal Sig+: Col greater signal

Genome distribution of SFPs

CD c IntronUTRsPromoter d Downstream e Intergenic Total SFP a Feature b Percentage4.40%6.56%4.87%7.03%7.10%8.51%6.68% Genic distribution of SFPs a The number of SFPs within each annotation category. b The number of features within each annotation category. c Coding sequences. d The sequences from transcriptional start to upstream 1kb. e The sequences from transcriptional stop to downstream 1kb.

EnzymeGenotype x enzyme p-valueHpaII > MspI a p-valueCol-specific b Van-specific c < < < < < < Gene d 3628 (20%)Gene d 3498 (20%) Total gene e 17760Total gene e Promoter f 305 (6%)Promoter f 455 (9%) Total promoter g 5041Total promoter g 5041 Intergenic h 1298 (16%)Intergenic h 782 (9%) Total intergenic i 8264Total intergenic i 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

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

Col Van Col♂ x Van♀ Van♂ x Col♀ CC*GG chromomethylase 2 (CMT2) exon19 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 intensity Col Van Col HpaII MspI log intensity Col Van Col Van SFP; Col has greater signal than Van. Van duplication or deletion in Col; Van has greater signal than Col F1c F1vF1c 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 effect Col Van Col F1c F1v F1c F1v HpaII MspI Increased F1 hybridization compared with expected from mid-parent log intensity Dominant effect + Col Van Col Dominant effect - F1c F1v F1cF1v HpaII MspI log intensity 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 F1vF1c log intensity 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 intensity 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 Van Enzyme effect + Van F1c F1v F1c F1v log intensity HpaII MspI Col 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 intensity Enzyme effect -

Additive x enzyme effect describes differential enzyme sensitivity between parent strains. Additive x enzyme interaction Additive x enzyme effect + log intensity Van Col Van HpaII MspI Col-specific methylation Van-specific methylation F1c F1v F1c F1v log intensity Van Col Van F1c 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 interaction Col Van Col F1c HpaII MspI Dominant x enzyme effect + F1v Van F1c F1v Col Van Col F1c HpaII MspI F1v Van F1c F1v log intensity Col-dominant methylation Van-dominant methylation log intensity 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 intensity Van Col Van HpaII MspI F1c F1v F1c F1v log intensity Van Col Van HpaII MspI F1c F1v F1c F1v Maternal x enzyme effect -

additivedominant maternal enzyme Significance of main effects

additive χ enzymedominant χ enzymematernal 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

effectGOatermp-valueGOatermp-value addenz Col > VanVan > Col GO: protein folding7.84E-05GO: intracellular signaling cascade1.72E-03 GO: regulation of flower development5.05E-03GO: photosynthesis2.76E-03 GO: microtubule-based movement8.56E-03GO: defense response5.82E-03 GO: ubiquitin-dependent protein catabolic process1.27E-02GO: metal ion transport1.24E-02 GO: multicellular organismal development1.50E-02GO: lignin biosynthetic process2.49E-02 GO: ribosome biogenesis and assembly2.03E-02GO: potassium ion transport2.91E-02 GO: protein metabolic process2.16E-02GO: response to gibberellin stimulus4.85E-02 GO: protein amino acid dephosphorylation2.73E-02 GO: double fertilization forming a zygote and endosperm2.98E-02 GO: cell redox homeostasis3.39E-02 GO: aging4.67E-02 domenz F1 hybrids > parentsc parents > F1 hybridsc GO: leaf morphogenesis2.13E-04GO: ribosome biogenesis and assembly6.60E-03 GO: nucleotide-sugar metabolic process4.21E-04GO: response to bacterium1.36E-02 GO: lipid transport2.96E-03GO: response to sucrose stimulus2.31E-02 GO: regulation of stomatal movement8.79E-03GO: vesicle-mediated transport2.59E-02 GO: polysaccharide biosynthetic process9.77E-03GO: regulation of progression through cell cycle2.60E-02 GO: chlorophyll biosynthetic process2.03E-02GO: regulation of transcription3.60E-02 GO: root development2.11E-02GO: transport4.08E-02 GO: response to heat2.33E-02 GO: flower development4.08E-02 GO: photosynthesis4.11E-02 GO: cell redox homeostasis4.13E-02 GO: sucrose catabolic process using beta-fructofuranosidase4.41E-02 GO: organ morphogenesis4.49E-02 matenz Col-mother F1 > Van-mother F1dVan-mother F1 > Col-mother F1d GO: photosynthesis1.17E-03GO: ATP synthesis coupled proton transport1.09E-02 GO: chlorophyll biosynthetic process1.22E-03GO: protein amino acid dephosphorylation1.11E-02 GO: response to heat1.76E-02GO: toxin catabolic process1.14E-02 GO: response to light stimulus2.87E-02GO: membrane fusion2.60E-02 GO: amino acid metabolic process3.38E-02GO: regulation of flower development2.92E-02 GO: defense response to bacterium3.50E-02GO: ethylene mediated signaling pathway4.07E-02 GO: mRNA processing4.44E-02 Gene set enrichment in genic CG methylation polymorphisms

Col methylation > Van methylation c Col-mother F1 expression > Van-mother F1 expression d GO e termp-valueGO e termp-value BP a GO: protein folding*7.84E-05GO: translation2.13E-32 GO: regulation of flower development5.05E-03GO: protein folding*2.09E-30 GO: microtubule-based movement*8.56E-03GO: ribosome biogenesis and assembly*2.82E-15 GO: ubiquitin-dependent protein catabolic process1.27E-02GO: microtubule-based movement*1.14E-11 GO: multicellular organismal development1.50E-02GO: nucleosome assembly1.88E-09 GO: ribosome biogenesis and assembly*2.03E-02GO: response to heat4.49E-09 MF b GO: heat shock protein binding*1.67E-03GO: structural constituent of ribosome6.21E-32 GO: microtubule motor activity*7.56E-03GO: microtubule motor activity*2.75E-13 GO: unfolded protein binding*1.27E-02GO: RNA binding1.34E-12 GO: protein disulfide oxidoreductase activity1.90E-02GO: unfolded protein binding*1.44E-12 GO: FK506 binding*2.59E-02GO: peptidyl-prolyl cis-trans isomerase activity*6.31E-10 GO: peptidyl-prolyl cis-trans isomerase activity*3.19E-02GO: GTP binding1.63E-08 GO: heat shock protein binding*1.99E-08 GO: FK506 binding*3.02E-06 Maternal methylome could be important for reciprocal F1 gene expression