nsa feb 2010: dna methylation patterns & epigenetic regulation in the pacific oyster

DNA Methylation Patterns & 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

Implications

Future directions

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 at CpG sites in animals

regulates gene expression

influenced by the environment

Effects of disruptions:

tumor promotion

alteration of development

inhibition of reproduction

Compounds that impact normal epigenetic functions:

Endocrine disruptors: estrogen, BPA, pesticides

DNA methylation

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 CpG methylated

(homology to neuromedin-u receptor)

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

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 sequences from GigasBase

sequences were grouped by Gene Ontology term

an average predicted methylation status was determined

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 0.5 0.55 0.6 0.65 0.7

CpGo/e

Regulation of Gene Expression

high mid low

Predicted DNA Methylation

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 0.5 0.55 0.6 0.65 0.7

CpGo/e

Regulation of Gene Expression

high mid low

Predicted DNA Methylation

Implications:evidence suggests DNA methylation plays a

regulatory role in Pacific oysters

implications for immune/stress responses

Implications: Environment

Site C Site A5

5.5

6

6.5

7

7.5

8

est

imate

d %

DN

A m

eth

ylati

on P = 0.03

Low High

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

=

Implications: Nutritiondiet can modify traits by affecting DNA

methylation.

Waterland & Jirtle, Molecular and Cellular Biology, 2003

Future DirectionsMethod evaluation/development:

challenges associated with non-model species

new approaches:

Whole genome bisulfite sequencing (BS-seq)

Methylated DNA immunoprecipitation (MeDIP)

MeDIP-seq

MeDIP-chip

SummaryCharacterization of DNA methylation in Pacific oyster

suggests a role in gene regulation, specifically genes with inducible expression

DNA methylation could be an important mechanism contributing to phenotypic variation in oysters

Important evaluate & develop methods and tools to evaluate epigenetic mechanisms in bivalves

AcknowledgementsUW, SAFS

Dr. Steven Roberts

Samuel White

Lisa Crosson

Emma Timmins-Schiffman

Taylor Shellfish Farms

Joth Davis

NSA-PCS

NOAA Aquaculture Program