r esponses in the Pacific oyster Crassostrea gigas Mackenzie Gavery University of Washington School of Aquatic and Fishery Sciences Committee Meeting April 15 th 2014 Agenda Research update ID: 611406
Download Presentation The PPT/PDF document "Regulation of environmental" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Regulation of environmental responses in the Pacific oyster, Crassostrea gigas
Mackenzie Gavery
University of Washington
School of Aquatic and Fishery Sciences
Committee Meeting: April 15
th
, 2014Slide2
Agenda:
Research update
Proposed
t
imelineSlide3
Environmental Response in OystersSlide4
Environmental Response in OystersCyclooxygenasecharacterizationSlide5
Environmental Response in OystersGene expression: disease resistance
Cyclooxygenase
characterizationSlide6
Environmental Response in OystersWhole transcriptome
Chapter I
. CBP: Part D
Gene expression: disease resistance
Cyclooxygenase
characterizationSlide7
Chapter I.RNA-Seq
HIGH IMPACT
LOW IMPACTSlide8
HIGH IMPACTLOW IMPACT
742 novel
contigs
427 differentially expressed
Chapter I.
RNA-
SeqSlide9
Ultra-short read technology is a powerful tool for gene discovery and expression analysis in organisms with limited genomic resources
HIGH IMPACT
LOW IMPACT
742 novel
contigs
427 differentially expressed
Chapter I.
RNA-
SeqSlide10
Environmental Response in OystersWhole transcriptome
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part DSlide11
Environmental Response in OystersChapter II.
BMC Genomics
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part DSlide12
Chapter II. in silico analysis‘housekeeping’
‘inducible’
Predicted degree of DNA methylationSlide13
‘housekeeping’‘inducible’
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 methylationSlide14
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC GenomicsSlide15
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC GenomicsSlide16
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
t
ransgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC GenomicsSlide17
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
b
ringing it all together! meth & expression
t
ransgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC GenomicsSlide18
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
b
ringing it all together! meth & expression
Ch
III.
PeerJ
t
ransgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC GenomicsSlide19
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
b
ringing it all together! meth & expression
Chapter IV.
Brief
Func
Gen
transgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC Genomics
Ch
III.
PeerJSlide20
Chapter III: whole methylome
g
enomic DNA
Approach
High
-throughput bisulfite
sequencingSlide21
g
enomic DNA
Approach
High
-throughput bisulfite
sequencing
Chapter III: whole
methylomeSlide22
g
enomic DNA
Approach
High
-throughput bisulfite
sequencing
RNA-
Seq
data
Chapter III: whole
methylomeSlide23
Gill tissue: > 2.5 million CG dinucleotidesChapter III: whole methylomeSlide24
scaffold 86 (Galaxy Trackster)exCG
genes
exons
%methylation
0bp
200,000bp
100%
0%
Chapter III: whole
methylomeSlide25
exCGgenesexons
%methylation
0bp
200,000bp
100%
0%
scaffold 86
(Galaxy
Trackster
)
Chapter III: whole methylomeSlide26
exCGgenesexons
%methylation
0bp
200,000bp
100%
0%
scaffold 86
(Galaxy
Trackster
)
Chapter III: whole
methylomeSlide27
exCGgenesexons
%methylation
0bp
200,000bp
100%
0%
scaffold 86
(Galaxy
Trackster
)
Chapter III: whole
methylomeSlide28
Distribution in genomic elementsChapter III: whole methylomeSlide29
Distribution in genomic elementsChapter III: whole methylomeSlide30
Relationship with expression
Chapter III: whole
methylomeSlide31
RNA-Seq data (Zhang et al., 2012)Relationship with expressionGene expression (Deciles) DNA methylation/gene Chapter III: whole methylomeSlide32
Relationships with other gene specific attributes?Chapter III: whole methylomeSlide33
Relationships with other gene specific attributes?Spatiale.g. number of exons, length of mRNAGene expression (Zhang et al 2012):Mean expression level across 9 tissuesVariation in expression between tissues (%CV)
Chapter III: whole
methylomeSlide34
PC1 (50.2%) PC2 (26.1%) Chapter III: whole methylomeSlide35
PC1 (50.2%) PC2 (26.1%) Chapter III: whole methylomeSlide36
Chapter III: summarySlide37
unmethylated
methylated
Chapter III: summarySlide38
Gene function:
unmethylated
methylated
Chapter III: summarySlide39
Gene function:
unmethylated
inducible
housekeeping
methylated
Chapter III: summarySlide40
Gene function:
Expression:
unmethylated
inducible
housekeeping
methylated
Chapter III: summarySlide41
Gene function:
Expression:
unmethylated
inducible
l
ow
housekeeping
h
igh
methylated
Chapter III: summarySlide42
Gene function:
Expression:
Tissue specific expression:
unmethylated
inducible
l
ow
housekeeping
h
igh
methylated
Chapter III: summarySlide43
Gene function:
Expression:
Tissue specific expression:
unmethylated
inducible
l
ow
housekeeping
h
igh
methylated
variable
conserved
Chapter III: summarySlide44
Gene function:
Expression:
Tissue specific expression:
unmethylated
inducible
l
ow
housekeeping
h
igh
methylated
variable
conserved
A context dependent role of DNA methylation in bivalves (Gavery & Roberts 2014)
Chapter IV.
Chapter III: summarySlide45
Summary of Chapters:
Transcriptomic
approaches to understanding environmental response
First characterization of DNA methylation in oysters (
in
silico
analysis)
DNA
methylation & gene expression: whole genome analysis
Perspective/Review: DNA methylation in bivalvesSlide46
Environmental Response in Oysters
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
b
ringing it all together! meth & expression
t
ransgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Chapter I
. CBP: Part D
Chapter II.
BMC Genomics
Ch
III.
PeerJ
Chapter IV.
Brief
Func
GenSlide47
Environmental Response in Oysters
BMC Genomics
Frontiers
Whole transcriptome
DNA methylation:
s
ingle gene/
in
silico
Nanostring
b
ringing it all together! meth & expression
t
ransgenerational
(5-aza pesticide)
EE2
Gene expression: disease resistance
Cyclooxygenase
characterization
Authorea
link
Chapter I
. CBP: Part D
Ch
III.
PeerJ
Chapter IV.
Brief
Func
GenSlide48
Agenda:
Research update
Proposed timelineSlide49
Proposed Timeline:
Draft proposal to reading committee: April 21
stSlide50
Proposed Timeline:Mem DaySpring EndSlide51
Estrogen Experiment
Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?Slide52
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
)Slide53
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 / 3fSlide54
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 - controlSlide55
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
6m / 7f / 1? – EE2Slide56
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
6m / 7f / 1? – EE2Slide57
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
6m / 7f / 1? – EE2Slide58
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
6m / 7f / 1? – EE2Slide59
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
6m / 7f / 1? – EE2Slide60
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
pyrosequencingSlide61
Appendices:
Gene expression:
Characterization of
cycloxygenase
in C.
gigas
Gene expression in disease resistant oysters
DNA methylation:
Nanostring
report
Transgenerational
Experimental Design
EE2 experiment: phenotypic data & DNA methylation resultsSlide62
Prostaglandins in oyster immunity
Prostaglandins in oyster immunity
Sequenced full length gene
Phylogenetic analysis
Tissue Distribution
Immune ResponseSlide63
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 exposureSlide64
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 typesSlide65
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