Date post: | 04-Jul-2015 |
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Epigenetic mechanisms as a source of phenotypic plasticity 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
High-throughput bisulfite sequencing
Discussion/Future Directions
Outline
Epigenetics Heritable changes in trait or phenotype, caused by a
mechanism other than mutation to the DNA sequence
Most well understood epigenetic mechanism is DNA methylation
Me
C
G C
G
Regulates gene expression
Development
Tissue-specific expression
Environmental response
DNA methylation: invertebrates Only a handful of species have been evaluated
Model invertebrates lack DNA methylation
Most: intermediate methylation
Primarily in exons
Important regulatory functions – honey bee (e.g. Kucharski et al., 2008; Elango et al., 2009; Lyko et al., 2010)
CpG O/E
Predicted degree of DNA methylation
Mea
sure
d d
egre
e of
DN
A m
ethyl
atio
n
Enr
ichm
ent
leve
l in
MB
D l
ibra
ry
Summary of Previous Results
(Gavery & Roberts, 2010)
(Rob
erts
& G
aver
y, 2
011)
CpG O/E
Predicted degree of DNA methylation
Mea
sure
d d
egre
e of
DN
A m
ethyl
atio
n
Enr
ichm
ent
leve
l in
MB
D l
ibra
ry
Summary of Previous Results
(Gavery & Roberts, 2010)
(Rob
erts
& G
aver
y, 2
011)
CpG O/E
Predicted degree of DNA methylation
Mea
sure
d d
egre
e of
DN
A m
ethyl
atio
n
Enr
ichm
ent
leve
l in
MB
D l
ibra
ry
(Gavery & Roberts, 2010)
(Rob
erts
& G
aver
y, 2
011)
Summary of Previous Results
CpG O/E
Predicted degree of DNA methylation
Mea
sure
d d
egre
e of
DN
A m
ethyl
atio
n
Enr
ichm
ent
leve
l in
MB
D l
ibra
ry
Summary of Previous Results
(Gavery & Roberts, 2010)
(Rob
erts
& G
aver
y, 2
011)
Current Research: High-resolution methylation analysis
Pooled adult gill tissue
High-throughput bisulfite sequencing
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis
Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis
Workflow: - methyl binding domain enrichment
MBD
MBD
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis
– Illumina library – Bisulfite conversion
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis T T C G C
T T C G U
T T C G T
bisulfite
PCR
– Illumina library – Bisulfite conversion
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis T T C G C
T T C G U
T T C G T
bisulfite
PCR
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis T T C G C
T T C G U
T T C G T
bisulfite
PCR
Workflow: Reduce genome
Prepare gDNA library for methylation analysis
Data Analysis
T T C G C
T T C G U
T T C G T
bisulfite
PCR
– BSMAP software
– Reference:
- de novo contigs
- characterized genes
Visualization: hexokinase
0 bp 10,960 bp
MBD-mapped reads
CG
Visualization: hexokinase
0 bp 10,960 bp
MBD-mapped reads
CG
Visualization: hexokinase
0 bp 10,960 bp
mid mCG
low mCG
mCG
CG
Visualization: hexokinase
0 bp 10,960 bp
mid mCG
low mCG
mCG
CG
Visualization: hexokinase
0 bp 10,960 bp
mid mCG
low mCG
mCG
CG
Visualization: hexokinase
0 bp 10,960 bp
mid mCG
low mCG
mCG
CG
Visualization: hexokinase
0 bp 10,960 bp
CG
mCG
cDNA - gill
cDNA - larvae
Visualization: hexokinase
0 bp 10,960 bp
CG
mCG
cDNA - gill
cDNA - larvae
Visualization: hexokinase
0 bp 10,960 bp
CG
mCG
cDNA - gill
blastn EST
Visualization: hexokinase
0 bp 10,960 bp
CG
mCG
cDNA - gill
blastn EST
1384 bp
Nanostring: nCounter®
Screening of multiple sample types, multiple loci
0
20
40
60
80
A B C D A B m f
gill larvae gonad
% m
ethy
lati
on
hexokinase CG loci 1384bp
Targeted Bisulfite Sequencing
0%
25%
50%
75%
100%
1 2 3 4 5 6 7
% m
ethy
lati
on
position (bp)
hexokinase CG loci 1,287 – 1,435
gill larvae sperm
1384 1446 1454 1458 1369 1356 1285
Visualization: hexokinase
0 bp 10,960 bp
CG
mCG
cDNA - gill
blastn EST
Visualization: hsc70
0 bp 2,552 bp
CG
mCG
mid mCG
low mCG
Visualization: LAP
0 bp 5,494 bp
CG
mCG
mid mCG
low mCG
Visualization: gnrhr
0 bp 13,058 bp
CG
mCG
mid mCG
low mCG
Results: summary Using this technique we can characterize the methylation
status of individual CG loci at a genome wide scale
Patterns suggest that methylation may be involved in gene regulation
Provides a proof-of-concept in non-model species
Conclusions/Future Directions:
DNA methylation plays an important role in regulating gene expression in C. gigas
It will be important to understand how epigenetic mechanisms will be affected by the environment
Future work will test these possible mechanisms and how they will be affected by environmental stress
Acknowledgements
Samuel White (UW, SAFS)
Joth Davis (Taylor Shellfish Farms)
Robin Harper & Phillipa Webster (Nanostring)
email: [email protected] website: students.washington.edu/mgavery