Genetic and epigenetic dissection of cis regulatory variation

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Genetic and epigenetic dissection of cis
regulatory variation Xu Zhang Borevitz Lab
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Arabidopsis thaliana found in diverse
environments
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• Gene expression variation
• Complex trait controlled by many genetic and
  non-genetic factors
• Among genetic factors, allelic variation (cis) is
  simple in inheritance in comparison to trans
• Genetic and environmental perturbation may result
  in different readout of allelic variation (cis x
  trans, cis x environment)

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Evidence of epigenetic variation contributing to
phenotypic variation
Linaria vulgaris flowers (Cubas et al., 1999)
Tomato ripening mutant (Manning et al., 2006)
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• Questions
• How common are genetic, epigenetic and gene
  expression polymorphisms among A. thaliana, any
  correlation pattern
• How common are genes affected by cis variation
• What are these cis variations
• Does any of the gene expression variation have
  some phenotypic effect

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• Outline
• The extent of natural genetic, epigenetic and
  transcriptional polymorphisms
• Genome-wide cis regulatory effects
• Mapping of ASE and cis x trans
• Components of gene expression regulation in
  phenotype regulation

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Test of additive inheritance and parental effect
in F1 reciprocal hybrids
additive dominant
maternal effect
intensity
Col
Col
Col
F1v
F1v
F1v
F1c
F1c
F1c
Van
Van
Van
Null hypothesis phenotype of offspring is at the
intermediate level of that of parents
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Experimental design
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

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A. thaliana whole genome tiling array AtTILE1

• 35bp spacing, 1.7million unique features
• Covers both transcribed and intergenic region
  without bias for annotation
• Genetic and expression information can be
  integrated into a single platform

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Enzyme methylome approach
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Genetic and CG methylation polymorphisms


A)
HpaII digestion


Random labeling


B)
MspI digestion

Random labeling
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Genetic Single Feature Polymorphisms
Total tested probes 1,683,620
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CG methylation polymorphisms
Total tested CCGG containing probes 54,519
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Genome distribution of constitutive and
polymorphic methylation sites
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Genic distribution of constitutive and
polymorphic methylation sites
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Correlation of constitutive CG methylation and
gene expression level
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Correlation of polymorphic CG methylation and
gene expression variation
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Additive inheritance of CG methylation
polymorphisms
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• Conclusion
• About the same magnitude of polymorphic CG
  methylation as constitutive CG methylation sites
• Polymorphic CG methylation likely plays a
  regulatory roles
• Inheritance of CG methylation polymorphism is
  largely additive

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• Outline
• The extent of natural genetic, epigenetic and
  transcriptional polymorphisms
• Genome-wide cis regulatory effects
• Mapping of ASE and cis x trans
• Components of gene expression regulation in
  phenotype regulation

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Allele specific expression in a heterozygous
system
allele A
allele B
Allele A and B are exposed to the same pool of
trans factors. Thus allelic expression should be
due to cis difference
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Variance partition of cis and trans effects
cis only
cis and composite trans
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AtSNPtile A SNP/tiling array

• 1.4M tiling probes at 35bp resolution
• 1M SNP probes for 250K SNPs
• each SNP 2 alleles x 2 strands
• allele A antisense strand GACCAATTTTGACCCTAGATC
  GCCA
• allele A sense strand CTGGTTAAAACTGGGATCT
  AGCGGT
• allele B antisense strand GACCAATTTTGAACCTAGATC
  GCCA
• allele B sense strand CTGGTTAAAACTTGGATCT
  AGCGGT

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Allelic variation measured as log allele ratio
Different allele proportion in target
Different total amount of target
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Experiment design
Col ? x Van ?
Van ? x Van ?
Van ? x Col ?
Col ? x Col ?

• 4 replicates for parental gDNA11 mix
• 4 replicates for parental mRNA11 mix
• 4 replicates for Col (mother) x Van F1s
• 4 replicates for Van (mother) x Col F1s

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The models
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Summary for 12,311 analyzed genes
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The effect size of cis and trans
cis vs parental difference trans vs
parental difference trans vs cis
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Regional difference in sequence polymorphism
between cis and trans genes
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Epigenetic modifications associated with cis and
trans effect genes
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Gene expression specificity for cis and trans
genes
expression level
expression specificity
gene length
Data from Schmid et al, 2005 63 diverse tissues
on Col wild type background
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• Conclusion
• Less cis than trans effect genes but larger cis
  than trans effect
• cis variation genes tend to located in
  polymorphic chromosome regions, and associated
  with repressive epigenetic modification
• trans effect genes tend to located in conserved
  chromosome regions, and associated with
  activating epigenetic modification

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• Outline
• The extent of natural genetic, epigenetic and
  transcriptional polymorphisms
• Genome-wide cis regulation effects
• Mapping of ASE and cis x trans
• Contribution of gene expression regulation to
  phenotype regulation

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Mapping of ASE in F1 hybrid panel
homo Col
homo Van
het
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ASE distributions in F1 panel
no cis
only cis
cis x trans
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Association by likelyhood distance
expected ASE distribution
ASE distribution of candidate SNP
scanning SNPs
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• Outline
• The extent of natural genetic, epigenetic and
  transcriptional polymorphisms
• Genome-wide cis regulation effects
• Mapping of ASE and cis x trans
• Components of gene expression regulation in
  phenotype regulation

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Monitoring of plant growth
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Thanks!
Borevitz Lab Justin Borevitz Yan Li Christos
Noutsos Geoff Morris Eleni Boikoglo Paul
Grabowski Traci Viinanen Whitney Panneton Eric
Hanss Maria Valdes Sam Betcher Shinhan
Shiu Evadne Smith Andy Cal
E E Wen-Hsiung Li Shen Yang Jake Byrnes