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Regulation of environmental responses in the Pacific oyster,
Crassostrea gigas
Mackenzie Gavery
University of Washington
School of Aquatic and Fishery Sciences
Committee Meeting: April 15th, 2014
Agenda:
Research update
Proposed timeline
Environmental Response in Oysters
2009 2010 2011 2012 2013
Environmental Response in Oysters
2009 2010 2011 2012 2013
Cyclooxygenasecharacterization
Environmental Response in Oysters
2009 2010 2011 2012 2013
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Environmental Response in Oysters
2009 2010 2011 2012 2013
Whole transcriptome
Chapter I. CBP: Part D
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I.RNA-Seq
HIGH IMPACTLOW IMPACT
HIGH IMPACTLOW IMPACT
742 novel contigs427 differentially expressed
Chapter I.RNA-Seq
Ultra-short read technology is a powerful tool for gene discovery and expression analysis in organisms with limited genomic resources
HIGH IMPACTLOW IMPACT
742 novel contigs427 differentially expressed
Chapter I.RNA-Seq
Environmental Response in Oysters
2009 2010 2011 2012 2013
Whole transcriptome
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Environmental Response in Oysters
2009 2010 2011 2012 2013
Chapter II. BMC Genomics
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
DNA metabolism
cell cycle and proliferation
RNA metabolism
protein metabolism
death
other metabolic processes
cell organization and biogenesis
other biological processes
transport
stress response
developmental processes
cell-cell signaling
signal transduction
cell adhesion
0.45 0.50 0.55 0.60 0.65 0.70CpGO/E
Chapter II. in silico analysis‘h
ouse
keepin
g’
‘induci
ble
’
Predicted degree of DNA methylation
DNA metabolism
cell cycle and proliferation
RNA metabolism
protein metabolism
death
other metabolic processes
cell organization and biogenesis
other biological processes
transport
stress response
developmental processes
cell-cell signaling
signal transduction
cell adhesion
0.45 0.50 0.55 0.60 0.65 0.70CpGO/E
‘house
keepin
g’
‘induci
ble
’
DNA methylation detected by targeted bisulfite sequencing and methylation specific PCR
DNA methylation patterns are different between functionally distinct classes of genes
Chapter II. in silico analysis
Predicted degree of DNA methylation
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
transgenerational (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
bringing it all together! meth & expression
transgenerational (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
bringing it all together! meth & expression
Ch III. PeerJtransgeneration
al (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
bringing it all together! meth & expression
Chapter IV. Brief Func Gen
transgenerational (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Ch III. PeerJ
Chapter III: whole methylome
genomic DNA
Approach
High-throughput bisulfite sequencing
genomic DNA
Approach
High-throughput bisulfite sequencing
Chapter III: whole methylome
genomic DNA
Approach
High-throughput bisulfite sequencing
RNA-Seq data
Chapter III: whole methylome
Gill tissue: > 2.5 million CG dinucleotides
Chapter III: whole methylome
scaffold 86 (Galaxy Trackster)
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
Chapter III: whole methylome
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Chapter III: whole methylome
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Chapter III: whole methylome
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Chapter III: whole methylome
Distribution in genomic elements
Chapter III: whole methylome
Distribution in genomic elements
Exon25%
Intron45%
Promoter4%
TE5%
Other22%
Chapter III: whole methylome
Relationship with expression
Chapter III: whole methylome
RNA-Seq data (Zhang et al., 2012)
Relationship with expression
Gene expression (Deciles)
DN
A m
eth
yla
tion/g
ene
Chapter III: whole methylome
Relationships with other gene specific attributes?
Chapter III: whole methylome
Relationships with other gene specific attributes?
Spatial
e.g. number of exons, length of mRNA
Gene expression (Zhang et al 2012):
Mean expression level across 9 tissues
Variation in expression between tissues (%CV)
Chapter III: whole methylome
PC1 (50.2%)
PC
2 (
26.1
%)
Chapter III: whole methylome
PC1 (50.2%)
PC
2 (
26.1
%)
Chapter III: whole methylome
Chapter III: summary
unmethylatedmethylatedChapter III: summary
Gene function:
unmethylatedmethylatedChapter III: summary
Gene function:
unmethylated
induciblehousekeeping
methylatedChapter III: summary
Gene function:
Expression:
unmethylated
induciblehousekeeping
methylatedChapter III: summary
Gene function:
Expression:
unmethylated
inducible
low
housekeeping
high
methylatedChapter III: summary
Gene function:
Expression:
Tissue specific
expression:
unmethylated
inducible
low
housekeeping
high
methylatedChapter III: summary
Gene function:
Expression:
Tissue specific
expression:
unmethylated
inducible
low
housekeeping
high
methylated
variable conserved
Chapter III: summary
Gene function:
Expression:
Tissue specific
expression:
unmethylated
inducible
low
housekeeping
high
methylated
variable conserved
A context dependent role of DNA methylation in bivalves (Gavery & Roberts 2014)
Chapter IV.
Chapter III: summary
Summary of Chapters:I. Transcriptomic approaches to
understanding environmental response
II. First characterization of DNA methylation
in oysters (in silico analysis)
III. DNA methylation & gene expression:
whole genome analysis
IV. Perspective/Review: DNA methylation in
bivalves
Environmental Response in Oysters
2009 2010 2011 2012 2013
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
bringing it all together! meth & expression
transgenerational (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Chapter I. CBP: Part D
Chapter II. BMC Genomics
Ch III. PeerJ
Chapter IV. Brief Func Gen
Environmental Response in Oysters
2009 2010 2011 2012 2013
BMC Genomics
Frontiers
Whole transcriptome
DNA methylation:single gene/in silico
Nanostring
bringing it all together! meth & expression
transgenerational (5-aza pesticide)
EE2
Gene expression: disease
resistance
Cyclooxygenasecharacterization
Authorea link
Chapter I. CBP: Part D
Ch III. PeerJ
Chapter IV. Brief Func Gen
Agenda:
Research update
Proposed timeline
Proposed Timeline:
Draft proposal to reading committee: April 21st
Proposed Timeline:
Mem
Day
Spring
End
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank)
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank)
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control6m / 7f / 1? – EE2
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank)
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control6m / 7f / 1? – EE2
EE2 control0%
20%
40%
60%
80%
100%
30 40
6563
18 12
H
U
F
M
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank)
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control6m / 7f / 1? – EE2
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control6m / 7f / 1? – EE2
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?
EE2 (n=50/tank
control (n=50/tank)
Day 0(n=10)
Day 7(n=15/tx)
Day 35(n=15/tx)
Day 60(n=120/tx)
7m / 3f
10m / 5f - control6m / 7f / 1? – EE2
Estrogen Experiment
Identifying differential methylation in day 7 females
Approach: MBD-Chip
Results:
45 differentially methylated regions
42 in genes, 3 upstream of genes
38 different genes identified
E.g.: CRHR, serotonin receptor, acetylcholine receptor, various DNA binding proteins
Next steps: validate DMRs by pyrosequencing
Appendices:Gene expression:
1. Characterization of cycloxygenase in C. gigas
2. Gene expression in disease resistant oysters
DNA methylation:
4. Nanostring report
5. Transgenerational Experimental Design
6. EE2 experiment: phenotypic data & DNA methylation results
Prostaglandins in oyster immunity
Prostaglandins in oyster immunity
Sequenced full length gene
Phylogenetic analysis
Tissue Distribution
Immune Response
Gene Expression in Disease Resistant oysters
Crassostrea virginica
Resistant and Naïve population
Immune Related Genes
Major Findings:
Resistant population appeared to be ‘primed’ with higher level of baseline expression of genes prior to exposure
Nanostring Project
Targeted methylation analysis
nCounter system
Methylation at 48 target genes
About 30% of the targets performed well
Major findings
Identified first differential methylation between tissue types
Transgenerational Experiment
Do methylation patterns persist across generations?
Expsure to either:
5-azacytidine
Vinclozolin
Spawned and raised larvae
Parents sampled: nanostring assay
Growout
Samples 18 months later (gametes)
Findings: size difference, no meth difference by Nanostring