regulation of environmental r esponses in the pacific oyster, crassostrea gigas
DESCRIPTION
Regulation of environmental 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 Proposed t imeline. Environmental Response in Oysters. - PowerPoint PPT PresentationTRANSCRIPT
Regulation of environmental responses in the Pacific oyster,
Crassostrea gigas
Mackenzie Gavery University of Washington
School of Aquatic and Fishery SciencesCommittee Meeting: April 15th, 2014
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
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
keep
ing’
‘indu
cible
’
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
‘hou
seke
epin
g’‘in
ducib
le’
DNA methylation detected by targeted bisulfite sequencing and methylation specific PCRDNA 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
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
Exon25%
Intron45%
Promoter4%
TE5%
Other22%
Chapter III: whole methylome
RNA-Seq data (Zhang et al., 2012)
Relationship with expression
Gene expression (Deciles)
DNA
met
hyla
tion/
gene
Chapter III: whole methylome
Relationships with other gene specific attributes?Spatial
e.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 methylome
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
Estrogen ExperimentDoes DNA methylation mediate the phenotypic
effects of estrogen exposure in bivalves?
Estrogen ExperimentDoes 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 ExperimentDoes 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 ExperimentDoes 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 ExperimentDoes 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 ExperimentDoes 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
65 63
18 12HUFM
Estrogen ExperimentDoes 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 ExperimentDoes 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 ExperimentDoes 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 ExperimentIdentifying differential methylation in day 7
femalesApproach: MBD-Chip Results:
45 differentially methylated regions42 in genes, 3 upstream of genes 38 different genes identifiedE.g.: CRHR, serotonin receptor, acetylcholine
receptor, various DNA binding proteinsNext 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 report5. Transgenerational Experimental Design6. EE2 experiment: phenotypic data & DNA
methylation results
Prostaglandins in oyster immunityProstaglandins in oyster immunitySequenced full length genePhylogenetic analysisTissue DistributionImmune Response
Gene Expression in Disease Resistant oysters
Crassostrea virginicaResistant and Naïve population Immune Related GenesMajor Findings:
Resistant population appeared to be ‘primed’ with higher level of baseline expression of genes prior to exposure
Nanostring ProjectTargeted methylation analysisnCounter systemMethylation at 48 target genesAbout 30% of the targets performed wellMajor findings
Identified first differential methylation between tissue types