Biomimicry

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Genetic and epigenetic control of cis and trans
regulatory variationJustin BorevitzEcology
EvolutionUniversity of Chicagohttp//borevitzlab
.uchicago.edu
2
Universal Whole Genome Array
DNA
RNA
Gene/Exon Discovery Gene model correction Non-codi
ng/ micro-RNA
Chromatin Immunoprecipitation ChIP chip
Alternative Splicing
Methylation
Antisense transcription
Polymorphism SFPs Discovery/Genotyping
Transcriptome Atlas Expression levels Tissues
specificity
Comparative Genome Hybridization
(CGH) Insertion/Deletions Copy Number
Polymorphisms
RNA Immunoprecipitation RIP chip
Allele Specific Expression
Control for hybridization/genetic
polymorphisms to understand TRUE expression
variation
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Which arrays should be used?
cDNA array
Long oligo array
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Which arrays should be used?
Gene array
Exon array
Tiling array
35bp tile, 25mers 10bp gaps
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Which arrays should be used?
SNP array
How about multiple species? Microbial
communities?
Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica,
15 virus,
Ressequencing array
Tiling/SNP array 2007 250k SNPs, 1.6M
tiling probes
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Genomic profile of cellular systems responding to
the environment
ORFa
Transcriptome Atlas
ORFb
start
AAAAA
deletion
M
M
M
M
M
M
SFP
M
M
M
M
M
M
SNP
SNP
SFP
SFP
conservation
Chromosome (bp)
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Talk Outline
Talk Outline
Van x Col Copy Number Variation (Indels) Single
Feature Polymorphisms Methylation
Polymorphisms Expression Polymorphisms gene,
exon additive dominant Haplotype Diversity (si
hay tiempo) Dr Xu Zhang (borevitzlab.uchicago.ed
u)
Van x Col Copy Number Variation (Indels) Single
Feature Polymorphisms Methylation
Polymorphisms Expression Polymorphisms gene,
exon additive dominant Haplotype Diversity (si
hay tiempo) Dr Xu Zhang (borevitzlab.uchicago.ed
u)
8
The experiment
Col? x Col?
Van ? x Van ?
Col ? x Van ?
Van ? x Col ?

• parental strains and reciprocal F1 hybrids
• mRNA from total RNA genomic DNA

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Potential Deletions
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Deletions vs duplications
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Natural Variation on Tiling Arrays
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Distribution of indels along chromosomes
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Potential Deletions
gt500 potential deletions 45 confirmed by Ler
sequence 23 (of 114) transposons Disease
Resistance (R) gene clusters Single R gene
deletions Genes involved in Secondary
metabolism Unknown genes
14
Potential Deletions Suggest Candidate Genes
FLOWERING1 QTL
Chr1 (bp)
Flowering Time QTL caused by a natural deletion
in FLM
(Werner et al PNAS 2005)
15
SFPs and CCGG Methylome
Genomic DNA
Col
HpaII digestion
Random labeling
Col
Genomic DNA
MspI digestion
Random labeling
Genomic DNA
Van
HpaII digestion
Random labeling
Van
Genomic DNA
MspI digestion
Random labeling
Full model Intensity genotype enzyme
genotype x enzyme
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SFP detection on tiling arrays
Xu Zhang
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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 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
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Verification of methylation polymorphisms
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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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Co-methylation of pericentromere regions
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Genic distribution of constitutive and
polymorphic methylation sites
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Correlation between gene size and constitutive CG
methylation
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Double-stranded random labeling
AAAAA
Random reverse transcription
AAAAA
Double-stranded cDNA
Random priming
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Whole genome tiling array

• High density and resolution 1.6M unique probes
  at 35bp spacing
• Without bias toward known transcripts

Genetic hybridization polymorphisms could affect
the estimation of gene expression
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Transcription and splicing
Chromosomal DNA
Exon 1
Exon 2
Exon 3
Intron 1
Intron 2
Transcription
Nuclear RNA
RNA splicing
Messenger RNA
Exon 1
Exon 2
Exon 3
Exon 1
Exon 3
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The linear model
Gene probe Intensity additive dominant
maternal e
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The pattern of gene expression inheritance
Mean gene intensity
paternal
Maternal
Col dominant
over dominant
F1v dominant
F1c dominant
Van dominant
Col Van F1v F1c
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Gene expression variation between genotypes
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Additive, dominant and maternal effects of gene
expression
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The pattern of gene expression inheritance
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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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Default expression status of exon and intron

• Exons correction for gene expression
• corrected by gene mean
• corrected by a gene median
• splicing index (Meanexon/Meangene)
• Introns direct comparison

Exon/intron probe Intensity additive dominant
maternal e
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Differential exon splicing
Exon probe Intensity additive dominant
maternal e
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Differential intron splicing
Intron probe Intensity additive dominant
maternal e
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Some dominant effect in differential intron
splicing in F1 hybrids
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Comparison for enrichment in known alternatively
spliced exons
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Experimental determined FDR for differential
splicing
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Photosynthesis related genes
Differrentially spliced genes which are located
in chloroplast thylakoid
AT5G38660 APE1 (Acclimation of Photosynthesis to
Environment) mutant has altered acclimation
responses
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Generalized tiling array HMM
(by Jake Byrnes)

• 3-state HMM
• Discrete distribution for emission probability
• Transition probability counts for probe spacing
• Baum-Welch parameter estimation

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de novo transcriptome variation
Xu Zhang
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Comparison of annotation-based analysis and HMM
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Comparison of annotation-based analysis and HMM
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Array Haplotyping

• What about Diversity/selection across the genome?
• A genome wide estimate of population genetics
  parameters, ?w, p, TajimaD, ?
• LD decay, Haplotype block size
• Deep population structure?
• Col, Lz, Bur, Ler, Bay, Shah, Cvi, Kas, C24, Est,
  Kin, Mt, Nd, Sorbo, Van, Ws2
• Fl-1, Ita-0, Mr-0, St-0, Sah-0

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Array Haplotyping
Inbred lines Low effective recombination due to
partial selfing Extensive LD blocks
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SFPs for reverse genetics
14 Accessions 30,950 SFPs
http//naturalvariation.org/sfp
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Chromosome Wide Diversity
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Diversity 50kb windows
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Tajimas D like 50kb windows
RPS4
unknown
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R genes vs bHLH
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http//borevitzlab.uchicago.edu
http//borevitzlab.uchicago.edu
Arabidopsis Yan Li Megan Dunning Joy
Bergelson Magnus Nordborg Paul Marjoram Aquilegia
Christos Noutsos Scott Hodges Tall Grass
Prairie Geoff Morris Mike Miller
Arabidopsis Yan Li Megan Dunning Joy
Bergelson Magnus Nordborg Paul Marjoram Aquilegia
Christos Noutsos Scott Hodges Tall Grass
Prairie Geoff Morris Mike Miller
Arrays Xu Zhang Shinhan Shiu Ivan Baxter
Arrays Xu Zhang Shinhan Shiu Ivan Baxter