high-throughput bisulfite sequencing reveals relationships between gene expression and dna...
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High-throughput bisulfite sequencing reveals relationships
between gene expression and DNA methylation in the bivalve,
Crassostrea gigas
Mackenzie Gavery & Steven Roberts
University of Washington
School of Aquatic and Fishery Sciences
Seattle, WA USA
Open ScienceSlides, links and related materials can be found
at:
oystergen.es/norway
Background
DNA methylation in
invertebrates
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
G
DNA Methylation
Me
C
GC
G
Gene A
TF X
DNA Methylation
Me
C
GC
G
Gene A
TF X
DNA Methylation
VERTEBRATE
Gene A
TF X
VERTEBRATE
DNA Methylation
Gene A
TF X
VERTEBRATE
Gene A
INVERTEBRATE
DNA Methylation
Gene A
TF X
VERTEBRATE
Gene A
INVERTEBRATE
DNA Methylation
Gene A
TF X
VERTEBRATE
Gene A
?
INVERTEBRATE
DNA Methylation
Gene A
TF X
VERTEBRATE
Gene A
?
INVERTEBRATE
DNA Methylation
Gene A
TF X
VERTEBRATE
Gene A
?
INVERTEBRATE
DNA Methylation
Ancestral pattern
Gene A
TF X
VERTEBRATE
Gene A
?
INVERTEBRATE
DNA Methylation
Ancestral pattern
Important regulator of phenotype
DNA MethylationObjectives:
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)
> 2.5 million 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
Exon25%
Intron45%
Promoter4%
TE5%
Other22%
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 differential methylation (DM)
Part 3: ResultsIdentify differential methylation (DM)
DM7%
methylation same across tissues
93%
Part 3: ResultsIdentify differential methylation
DM7%
methylation same across tissues
93%
1/3 of all genes had DM
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:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
Tissue specific
methylation:
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
patterns change at fine-scale
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
patterns change at fine-scale
Role of methylation in
introns:
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
unknownRole of methylation in
introns:
patterns change at fine-scale
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
Role of methylation in inter-genic regions:
unknownRole of methylation in
introns:
patterns change at fine-scale
Gene function:
Expression:
Tissue specific
methylation:
Summaryunmethylated
inducible
low
housekeeping
high
methylated
Role of methylation in inter-genic regions:
unknownRole of methylation in
introns:
unknown
patterns change at fine-scale
Next Steps
GENES (DNA)
EPIGENOME(DNA methylation)
Explore relationships between DNA methylation and alternative splicing
Determine if DNA methylation mediates response to environmental stress in shellfish
Tool development: DNA tiling array (MeDIP-Chip)
Next Steps
GENES (DNA)
EPIGENOME(DNA methylation)
AcknowledgementsRoberts Lab: Steven Roberts
Samuel White Brent VadopalasEmma Timmins-SchiffmanClaire EllisLisa Crosson
Taylor Shellfish:Jonathan DavisMolly Jackson
website: oystergen.es/norway
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