DNA Methylation & Epigenetic Regulation in the

Pacific Oyster Mackenzie Gavery & Steven Roberts

University of WashingtonSchool of Aquatic and Fishery Sciences

Background: epigenetics, DNA methylation

Results: characterization of DNA methylation in Pacific oysters

Current directions: method development

Implications

Outline

GENES (DNA)

TRAITS

color

growth

disease resistance

ENVIRONMENT

nutritionpathogens

toxins

Background:

GENES (DNA)

EPIGENOME(DNA methylation)

TRAITS

color

growth

disease resistance

ENVIRONMENT

nutritionpathogens

toxins

Background:

GENES (DNA)

EPIGENOME(DNA methylation)

TRAITS

color

growth

disease resistance

ENVIRONMENT

nutritionpathogens

toxins

Background:

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

GC

G

occurs in CpG in animals

functionsregulates gene expression

essential for development

genome stability

Characterization of DNA methylation in Pacific oysters

describe distribution of methylation

elucidate functional significance

Results

Methylation Specific PCR

Bisulfite sequencing

In silico analysis

Results

Methylation Sensitive PCR

Bisulfite sequencing

In silico analysis

Results: gene-targeted approachMethylation Sensitive PCR

5 stress related genes were examined

Identified CpG methylation in heat shock protein 70

Bisulfite sequencing136 bp fragment: 1 of 7 cytosines methylated

(homology to neuromedin-u receptor)

93 bp fragment: 1 of 2 cytosines methylated (homology to bromodomain adjacent to zinc finger domain)

Gavery & Roberts, 2010

Results

Methylation Sensitive PCR

Bisulfite sequencing

In silico analysis

Results

Methylation Sensitive PCR

Bisulfite sequencing

In silico analysis

predicted methylation status of 12,000 C. gigas genes

CpG observed

CpG expectedCpG o/e

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 = methylatedhigh = 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

Regulation of Gene Expression

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

Regulation of Gene Expression‘h

ouse

keep

ing’

‘hig

hly

regu

late

d’

Gavery & Roberts, 2010

Summary:oyster DNA is methylated

genes with differing regulatory requirements have different levels of DNA methylation

Current DirectionsMethod evaluation/development:

challenges with non-model species

MBD-isolated genome sequencing (MBD-seq)

Goal: MBD-seqgenome wide methylation analysis

evaluate in silico results

which genes are methylated?

which parts of the genome are methylated?

Work Flow: MBD-seqgenomic DNA

Work Flow: MBD-seq1. fragmentation

Work Flow: MBD-seq

2. enrichment

YY

YMBD

MBD

MBD

Work Flow: MBD-seq

3. library prep & sequencing

YY

YMBD

MBD

MBD

unmethylatedunmethylated

Work Flow: MBD-seq

genomic DNA – reference sequence

4. mapping

methylated methylated

Status: MBD-seqMBD isolation: complete

library prep and sequencing: in progress

methylated 22%

unmethylated 78%

Summarygenes with differing regulatory requirements have

different levels of DNA methylation

currently evaluating & developing methods and tools to evaluate epigenetic mechanisms in bivalves

Implications…

Implications: Environment

Endocrine disrupting compounds:

cause changes in DNA methylation patterns

associated with negative phenotypes

can be passed on for multiple generations

Implications: Selective BreedingSelective breeding can contribute to improved &

predictable performance in oysters

Understanding genetic and epigenetic influences will increase predictability

Implications: Hybrid Vigor

Heterosis (hybrid vigor)

mechanism not fully understood

epigenetic mechanisms have been proposed

better understanding will allow for greater control in predicting and manipulating gene expression in oysters

X

=

ConclusionElucidating the functional significance of DNA

methylation in aquatic invertebrates will improve our understanding of the interactions between the environment, gene expression, and organismal responses.

Acknowledgements

UW, SAFS

Dr. Steven Roberts

Samuel White

Taylor Shellfish Farms

Joth Davis

National Shellfisheries Association