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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
Background
Epigenetics
DNA methylation
Results
Characterization of DNA
methylation in Pacific oysters
Discussion & Future
Directions
Outline
GENES (DNA)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
temperature
Background
GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
Background
temperature
GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
Background
temperature
Me
C
GC
GGene A
TF X
Invertebrates?
DNA Methylation
Invertebrates?Model invertebrates lack DNA methylationDistribution & function unclear
DNA Methylation
Invertebrates?Model invertebrates lack DNA methylationDistribution & function unclear
DNA Methylation
Objectives:Characterize DNA methylation in C. gigasGain an understanding of the functional
role
Part 1
Part 1Approach
In silico analysis
Experimental analysis: MBD-Seq
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
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
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
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
Part 2
Part 2
genomic DNA
Approach
High-throughput bisulfite sequencing:
Gill tissue
Additional resources:
RNA-seq data: gill tissue (Zhang et al, 2012)
genomic DNA
Part 2Approach
High-throughput bisulfite sequencing:
Gill tissue
Additional resources:
RNA-seq data: gill tissue (Zhang et al, 2012)
>250,000 CG dinucleotides
Part 2: Results
scaffold 86 (Galaxy Trackster)
Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Part 2: Results
ex
CG
genes
exons
%methylation
0bp 200,000bp
100%
0%
scaffold 86 (Galaxy Trackster)
Distribution in genomic elements
Part 2: Results
Distribution in genomic elements
Part 2: Results
unan-notated
48%intron35%
exon17%
Part 2: ResultsRelationship with expression
RNA-Seq data (Zhang et al., 2012)
Part 2: ResultsRelationship with expression
Gene expression (Deciles)
DN
A m
eth
yla
tion/g
ene
Part 3
Approach:
High-throughput bisulfite sequencing:
Gill tissue
Part 3
Approach:
High-throughput bisulfite sequencing:
Gill tissue
Male gamete (sperm) tissue
Part 3
Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
Part 3: Results
genes
%methylation: gill
CG
%methylation: sperm
0bp 6,000bp
Part 3: ResultsIdentify differentially methylated regions
(DMR)
Part 3: ResultsIdentify differentially methylated regions
(DMR)
100bp windows
DMR >75% difference between tissues
Part 3: Results>200,000 regions were evaluated
Part 3: Results>200,000 regions were evaluated
DMR7%
methylation same across tissues
93%
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
Summary
Summaryunmethylatedmethylated
Gene function:
Summaryunmethylatedmethylated
Gene function:
Summaryunmethylated
induciblehousekeeping
methylated
Gene function:
Expression:
Summaryunmethylated
induciblehousekeeping
methylated
Gene function:
Expression:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Tissue specific
methylation:
Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
Role of methylation in
introns:
Tissue specific
methylation:
Gene function:
Expression:
Summaryunmethylated
inducible
low
tissue specific
housekeeping
high
conserved across tissues
methylated
unknownRole of methylation in
introns:
Tissue specific
methylation:
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:
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:
Next Steps
Explore relationships between DNA methylation and alternative splicing
Annotate intergenic regions of the C. gigas genome
Next Steps
GENES (DNA)
EPIGENOME(DNA methylation)
GENES (DNA)
EPIGENOME(DNA methylation)
Conclusions
EPIGENOME(DNA methylation)
TRAITS
color
growthdisease resistance
ENVIRONMENT
nutritionpathogens
temperature
GENES (DNA)
Conclusions
AcknowledgementsRoberts Lab:
Samuel White Caroline StorerEmma Timmins-SchiffmanClaire EllisLisa Crosson
Taylor Shellfish:Jonathan DavisMolly Jackson
email: [email protected]: students.washington.edu/mgavery
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