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