NSA 2011
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DNA Methylation & Epigenetic Regulation in the Pacific Oyster Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences
description
Transcript of NSA 2011
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
