Regulation of environmental r esponses in the Pacific oyster, Crassostrea gigas

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

Agenda:Research updateProposed timeline

Environmental Response in Oysters

2009 2010 2011 2012 2013


2009 2010 2011 2012 2013

Cyclooxygenasecharacterization


2009 2010 2011 2012 2013

Gene expression: disease

resistance



2009 2010 2011 2012 2013

Whole transcriptome

Chapter I. CBP: Part D


resistance


Chapter I.RNA-Seq

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


742 novel contigs427 differentially expressed

Chapter I.RNA-Seq


2009 2010 2011 2012 2013

Whole transcriptome


resistance




2009 2010 2011 2012 2013

Chapter II. BMC Genomics

Frontiers

Whole transcriptome

DNA methylation:single gene/in silico


resistance



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


2009 2010 2011 2012 2013

Frontiers

Whole transcriptome



resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring

transgenerational (5-aza pesticide)

EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring

bringing it all together! meth & expression


EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


Ch III. PeerJtransgeneration

al (5-aza pesticide)

EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


Chapter IV. Brief Func Gen


EE2


resistance




Ch III. PeerJ

Chapter III: whole methylome

genomic DNA

ApproachHigh-throughput bisulfite sequencing

genomic DNA

ApproachHigh-throughput bisulfite sequencing


genomic DNA

ApproachHigh-throughput bisulfite sequencing RNA-Seq data


Gill tissue: > 2.5 million CG dinucleotides


scaffold 86 (Galaxy Trackster)

ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%


ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%



ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%



ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%



Distribution in genomic elements


Distribution in genomic elements

Exon25%

Intron45%

Promoter4%

TE5%

Other22%


Relationship with expression


RNA-Seq data (Zhang et al., 2012)

Relationship with expression

Gene expression (Deciles)

DNA

met

hyla

tion/

gene


Relationships with other gene specific attributes?


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)


PC1 (50.2%)

PC2

(26.

1%)


PC1 (50.2%)

PC2

(26.

1%)


Chapter III: summary

unmethylatedmethylatedChapter III: summary

Gene function:

unmethylatedmethylatedChapter III: summary

Gene function:

unmethylated

induciblehousekeeping

methylatedChapter III: summary

Gene function:

Expression:

unmethylated

induciblehousekeeping


Gene function:

Expression:

unmethylated

inducible

low housekeeping

high


Gene function:

Expression:Tissue

specific expression:

unmethylated

inducible

low housekeeping

high


Gene function:

Expression:Tissue


unmethylated

inducible

low housekeeping

high

methylated

variable conserved


Gene function:

Expression:Tissue


unmethylated

inducible

low housekeeping

high

methylated

variable conserved

A context dependent role of DNA methylation in bivalves (Gavery & Roberts 2014)

Chapter IV.


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


2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring



EE2


resistance




Ch III. PeerJ



2009 2010 2011 2012 2013

BMC Genomics

Frontiers

Whole transcriptome


Nanostring



EE2


resistance


Authorea link


Ch III. PeerJ


https://www.authorea.com/users/6045/articles/6321

https://www.authorea.com/users/6045/articles/6321

Agenda:Research updateProposed timeline

Proposed Timeline:Draft proposal to

reading committee: April 21st

Proposed Timeline:

Mem Day

Spring End

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)



EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f



EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f

10m / 5f - control



EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f

10m / 5f - control6m / 7f / 1? – EE2



EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f

10m / 5f - control6m / 7f / 1? – EE2

EE2 control0%

20%40%60%80%

100%

30 40

65 63

18 12HUFM



EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f

10m / 5f - control6m / 7f / 1? – EE2



EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(n=120/tx)7m / 3f

10m / 5f - control6m / 7f / 1? – EE2



EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)


(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

Transgenerational ExperimentDo methylation patterns persist across

generations?Expsure to either:

5-azacytidineVinclozolin

Spawned and raised larvaeParents sampled: nanostring assay

GrowoutSamples 18 months later (gametes)Findings: size difference, no meth difference by

Nanostring

Regulation of environmental r esponses in the Pacific oyster, Crassostrea gigas

Documents

expression analysis

oystersgene expression

dgene expression

meth expression chapter

oysters3environmental

d10environmental response

oysters chapter

gene discovery