aquaculture 2013, nashville tn
TRANSCRIPT
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DNA methylation as a source of epigenetic regulation in the Pacific
oyster (Crassostrea gigas)
Mackenzie Gavery & Steven Roberts
University of Washington, School of Aquatic and Fishery Sciences
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Background
Epigenetics
DNA methylation
Results
Characterization of DNA
methylation in Pacific oysters
Discussion & Future
Directions
Outline
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GENES (DNA)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
temperature
Background
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GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
Background
temperature
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GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
Background
temperature
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Me
C
GC
GGene A
TF X
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Invertebrates?
DNA Methylation
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Invertebrates?Model invertebrates lack DNA methylationDistribution & function unclear
DNA Methylation
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Invertebrates?Model invertebrates lack DNA methylationDistribution & function unclear
DNA Methylation
Objectives:Characterize DNA methylation in C. gigasGain an understanding of the functional
role
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Part 1
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Part 1Approach
In silico analysis
Experimental analysis: MBD-Seq
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CpG O/E
Predicted degree of DNA methylation
Measu
red d
egre
e o
f D
NA
meth
yla
tion
En
rich
men
t le
vel in
MB
D lib
rary
(Gavery & Roberts, 2010)
(Rob
ert
s &
Gavery
, 2011
)
Part 1: Results
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CpG O/E
Predicted degree of DNA methylation
Measu
red d
egre
e o
f D
NA
meth
yla
tion
En
rich
men
t le
vel in
MB
D lib
rary
(Gavery & Roberts, 2010)
(Rob
ert
s &
Gavery
, 2011
)
Part 1: Results
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CpG O/E
Predicted degree of DNA methylation
Measu
red d
egre
e o
f D
NA
meth
yla
tion
En
rich
men
t le
vel in
MB
D lib
rary
(Gavery & Roberts, 2010)
(Rob
ert
s &
Gavery
, 2011
)
Part 1: Results
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CpG O/E
Predicted degree of DNA methylation
Measu
red d
egre
e o
f D
NA
meth
yla
tion
En
rich
men
t le
vel in
MB
D lib
rary
(Gavery & Roberts, 2010)
(Rob
ert
s &
Gavery
, 2011
)
Part 1: Results
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Part 2
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Part 2
genomic DNA
Approach
High-throughput bisulfite sequencing:
Gill tissue
Additional resources:
RNA-seq data: gill tissue (Zhang et al, 2012)
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genomic DNA
Part 2Approach
High-throughput bisulfite sequencing:
Gill tissue
Additional resources:
RNA-seq data: gill tissue (Zhang et al, 2012)
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>250,000 CG dinucleotides
Part 2: Results
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scaffold 86 (Galaxy Trackster)
Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
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Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
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Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
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Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
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Distribution in genomic elements
Part 2: Results
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Distribution in genomic elements
Part 2: Results
unan-notated
48%intron35%
exon17%
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Part 2: ResultsRelationship with expression
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RNA-Seq data (Zhang et al., 2012)
Part 2: ResultsRelationship with expression
Gene expression (Deciles)
DN
A m
eth
yla
tion/g
ene
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Part 3
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Approach:
High-throughput bisulfite sequencing:
Gill tissue
Part 3
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Approach:
High-throughput bisulfite sequencing:
Gill tissue
Male gamete (sperm) tissue
Part 3
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Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
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Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
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Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
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Part 3: ResultsIdentify differentially methylated regions
(DMR)
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Part 3: ResultsIdentify differentially methylated regions
(DMR)
100bp windows
DMR >75% difference between tissues
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Part 3: Results>200,000 regions were evaluated
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Part 3: Results>200,000 regions were evaluated
DMR7%
methylation same across tissues
93%
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Part 3: Results>200,000 regions were evaluated
DMR7%
methylation same across tissues
93%
half of DMR in gene bodies
genes with DMR had significantly less methylation
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Summary
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Summaryunmethylatedmethylated
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Gene function:
Summaryunmethylatedmethylated
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Gene function:
Summaryunmethylated
induciblehousekeeping
methylated
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Gene function:
Expression:
Summaryunmethylated
induciblehousekeeping
methylated
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Gene function:
Expression:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
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Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
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Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Tissue specific
methylation:
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Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Role of methylation in
introns:
Tissue specific
methylation:
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Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
unknownRole of methylation in
introns:
Tissue specific
methylation:
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Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Role of methylation in inter-genic regions:
unknownRole of methylation in
introns:
Tissue specific
methylation:
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Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Role of methylation in inter-genic regions:
unknownRole of methylation in
introns:
unknown
Tissue specific
methylation:
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Next Steps
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Explore relationships between DNA methylation and alternative splicing
Annotate intergenic regions of the C. gigas genome
Next Steps
GENES (DNA)
EPIGENOME(DNA methylation)
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GENES (DNA)
EPIGENOME(DNA methylation)
Conclusions
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EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
temperature
GENES (DNA)
Conclusions
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AcknowledgementsRoberts Lab:
Samuel White Caroline StorerEmma Timmins-SchiffmanClaire EllisLisa Crosson
Taylor Shellfish:Jonathan DavisMolly Jackson
email: [email protected]: students.washington.edu/mgavery