NSA Feb 2010: DNA Methylation Patterns & Epigenetic Regulation in the Pacific Oyster
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Transcript of NSA Feb 2010: DNA Methylation Patterns & Epigenetic Regulation in the Pacific Oyster
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DNA Methylation Patterns & Epigenetic Regulation in the
Pacific Oyster Mackenzie Gavery & Steven Roberts
University of WashingtonSchool of Aquatic and Fishery Sciences
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Background: epigenetics, DNA methylation
Results: characterization of DNA methylation in Pacific oysters
Implications
Future directions
Outline
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GENES (DNA)
TRAITS
color
growth
disease resistance
ENVIRONMENT
nutritionpathogens
toxins
Background:
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GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growth
disease resistance
ENVIRONMENT
nutritionpathogens
toxins
Background:
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GENES (DNA)
EPIGENOME(DNA methylation)
TRAITS
color
growth
disease resistance
ENVIRONMENT
nutritionpathogens
toxins
Background:
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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
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Effects of disruptions:
tumor promotion
alteration of development
inhibition of reproduction
Compounds that impact normal epigenetic functions:
Endocrine disruptors: estrogen, BPA, pesticides
DNA methylation
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Characterization of DNA methylation in Pacific oysters:
describe distribution of methylation
elucidate functional significance
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Results
Methylation Specific PCR
Bisulfite sequencing
In silico analysis
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Results
Methylation Sensitive PCR
Bisulfite sequencing
In silico analysis
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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)
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Results
Methylation Sensitive PCR
Bisulfite sequencing
In silico analysis
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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
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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
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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
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Implications:evidence suggests DNA methylation plays a
regulatory role in Pacific oysters
implications for immune/stress responses
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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
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Implications: Selective BreedingSelective breeding can contribute to improved &
predictable performance in oysters
Understanding genetic and epigenetic influences will increase predictability
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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
=
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Implications: Nutritiondiet can modify traits by affecting DNA
methylation.
Waterland & Jirtle, Molecular and Cellular Biology, 2003
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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
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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
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AcknowledgementsUW, SAFS
Dr. Steven Roberts
Samuel White
Lisa Crosson
Emma Timmins-Schiffman
Taylor Shellfish Farms
Joth Davis
NSA-PCS
NOAA Aquaculture Program