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Epigenetics in Context: from Ecology to Evolution (ESF-EMBO). San Feliu de Guixols, Spain. Sept 18 - 23, 2011
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Mackenzie Gavery & Steven Roberts
University of Washington
School of Aquatic and Fishery Sciences
Seattle, WA USA
Exploring the role of DNA methylation as a source of phenotypic variation in
Crassostrea gigas
Background: oysters
Results: characterization of DNA methylation in Crassostrea gigas
Discussion: functional role
Outline
Oyster: Biology
egg sperm
free swimming larvae (2 weeks)
2-3 years
juvenile
Oysters: Economic value
(FAO Fishery Statistics, 2006)
Major Producers of Crassostrea gigas
Oysters: Threats
DNA methylationAn epigenetic mechanism found in plants and
animals
In animals: occurs primarily in a CpG context
Function: gene regulation
Can be affected by environmental factors
Me
C
GC
G
DNA methylation: invertebratesOnly a handful of species have been evaluated
Model invertebrates lack DNA methylation
Most: 30 – 60 % methylation
Primarily in exonic regions
Important regulatory functions – honey bee (e.g. Kucharski et al., 2008; Elango et al., 2009; Lyko et al., 2010)
Characterization of DNA methylation in oysters
Describe distribution of methylation
Elucidate functional significance
Results
in silico analysis
Genome wide methylation analysis
in silico approachPrinciple:
Methylated cytosines are highly mutable
C T
Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG)
m
CpG observed
CpG expectedCpG O/E low = methylated
in silico approachPrinciple:
Methylated cytosines are highly mutable
C T
Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG)
m
CpG observed
CpG expectedCpG O/E high = unmethylated
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
Results: in silico
Gavery & Roberts, 2010
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
Results: in silico‘h
ouse
keep
ing’
‘indu
cibl
e’
Gavery & Roberts, 2010
=methylated CpG
Summary of Results:Genes with differing regulatory requirements have
different levels of DNA methylation
‘housekeeping’, ubiquitously expressed = methylated
‘inducible genes’ = unmethylated
Results
in silico analysis
Genome wide methylation analysis
MBD-seq
YY
YMBD
MBD
MBD
Methyl-binding domain isolated - genome sequencing
CpG O/E(modified from Gavery and Roberts 2010)
Predicted degree of DNA methylation
MBD-seq: Results
CpG O/E(modified from Gavery and Roberts 2010)
Predicted degree of DNA methylation
Mea
sure
d de
gree
of D
NA
met
hyla
tion
En
rich
men
t le
vel
in M
BD
lib
rary
(unp
ublis
hed)
MBD-seq: Results
CpG O/E(modified from Gavery and Roberts 2010)
Predicted degree of DNA methylation
Mea
sure
d de
gree
of D
NA
met
hyla
tion
En
rich
men
t le
vel
in M
BD
lib
rary
(unp
ublis
hed)
MBD-seq: Results
CpG O/E(modified from Gavery and Roberts 2010)
Predicted degree of DNA methylation
Mea
sure
d de
gree
of D
NA
met
hyla
tion
En
rich
men
t le
vel
in M
BD
lib
rary
(unp
ublis
hed)
MBD-seq: Results
Summary of Results:Experimental analysis confirms in silico results
Genes with differing regulatory requirements have different levels of DNA methylation
‘housekeeping’ ubiquitously expressed = methylated
‘inducible genes’ = unmethylated
Discussion:
Why?
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
duci
ble’
Gavery & Roberts, 2010
Discussion: Functional Role
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
duci
ble’
Gavery & Roberts, 2010
Conventional transcription of genesrequired for essential functioning
Discussion: Functional Role
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
duci
ble’
Gavery & Roberts, 2010
Discussion: Functional Role
Increased variation in environmental response genes
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
duci
ble’
Gavery & Roberts, 2010
Discussion: Functional Role
TFTFIncreased variation in environmental response genes
a) alternative splicing
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
duci
ble’
Gavery & Roberts, 2010
Discussion: Functional Role
Increased variation in environmental response genes
a) alternative splicingb) sequence variation
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’Discussion: Functional Role
Gavery & Roberts, 2010
Increased variation in environmental response genes
a) alternative splicingb) sequence variationc) transient methylation
‘indu
cibl
e’
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’Discussion: Functional Role
Gavery & Roberts, 2010
Increased variation in environmental response genes
a) alternative splicingb) sequence variationc) transient methylation
‘indu
cibl
e’
Conventional transcription of genesrequired for essential functioning
The distribution of DNA methylation may function to promote variation in environmental response genes Planktonic larvae
Sessile
Variable environments
Discussion: Functional Role
Conclusions/Future Directions:
Oysters have a functioning DNA methylation system
Ubiquitously expressed and inducible genes have different levels of methylation – indicating a functional roleFuture work:
Test the hypothesis that the DNA methylation system functions to enhance random variation in aquatic invertebrates
Investigate epigenetic effects of synthetic estrogens in oysters
Acknowledgements
Samuel White (UW, SAFS)
Joth Davis (Taylor Shellfish Farms)
US Environmental Protection Agency
Graduate School, UW
email: [email protected]: students.washington.edu/mgavery
