Aquaculture 2013, Nashville TN

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The document characterized DNA methylation in the Pacific oyster (Crassostrea gigas). Results showed DNA methylation is present and predictive analysis aligned with experimental measurements. High-throughput bisulfite sequencing of gill tissue revealed methylation in exons, introns, and intergenic regions. Methylation levels correlated negatively with gene expression. Comparisons between tissues identified differentially methylated regions, with half in gene bodies. Methylation may distinguish housekeeping from inducible genes and have a role in tissue-specific functions.

DNA methylation as a source of
epigenetic regulation in the Pacific oyster
(Crassostrea gigas)

Mackenzie Gavery & Steven Roberts
University of Washington, School of Aquatic and Fishery Sciences

Outline
 Background
 Epigenetics
 DNA methylation
 Results
Characterization of DNA
methylation in Pacific oysters

 Discussion & Future Directions

Background
color disease resistance
growth

TRAITS

temperature
pathogens
nutrition

GENES (DNA)
ENVIRONMENT

Background
color disease resistance
growth

TRAITS

temperature
pathogens
nutrition
EPIGENOME
(DNA methylation)

GENES (DNA)
ENVIRONMENT

DNA Methylation
 Invertebrates?
 Model invertebrates lack DNA methylation
 Distribution & function unclear

DNA Methylation
 Invertebrates?
 Model invertebrates lack DNA methylation
 Distribution & function unclear

 Objectives:
 Characterize DNA methylation in C. gigas
 Gain an understanding of the functional role

Part 1
 Approach
 In silico analysis
 Experimental analysis: MBD-Seq

Measured degree of DNA methylation
(Roberts & Gavery, 2011)
Enrichment level in MBD library
Part 1: Results

CpG O/E
(Gavery & Roberts, 2010)
Predicted degree of DNA methylation

Part 2
 Approach
 High-throughput bisulfite sequencing:
 Gill tissue
 Additional resources:
 RNA-seq data: gill tissue (Zhang et al, 2012)

genomic DNA

Part 2: Results
 >250,000 CG dinucleotides

Part 2: Results
0bp 200,000bp

CG

genes

exons
ex

%methylation
100%

0%

scaffold 86
(Galaxy Trackster)

Part 2: Results
 Distribution in genomic elements

Part 2: Results
 Distribution in genomic elements

exon
17%

unannotated
48%

intron
35%

Part 2: Results
 Relationship with expression

Part 2: Results
 Relationship with expression
DNA methylation/gene

Gene expression (Deciles)

RNA-Seq data (Zhang et al., 2012)

Part 3
 Approach:
 High-throughput bisulfite sequencing:
 Gill tissue

Part 3
 Approach:
 High-throughput bisulfite sequencing:
 Gill tissue
 Male gamete (sperm) tissue

0bp
Part 3: Results 6,000bp

CG

genes

%methylation: gill

%methylation: sperm

Part 3: Results
 Identify differentially methylated regions (DMR)

Part 3: Results
 Identify differentially methylated regions (DMR)
 100bp windows
 DMR >75% difference between tissues

Part 3: Results
 >200,000 regions were evaluated

Part 3: Results
 >200,000 regions were evaluated

DMR
7%

methylation
same across
tissues
93%

Part 3: Results
 >200,000 regions were evaluated
 half of DMR in gene
DMR
bodies
7%
 genes with DMR had
significantly less
methylation

methylation
same across
tissues
93%

Summary
methylated unmethylated

Gene function:

Summary
methylated unmethylated

Gene function: housekeeping inducible

Summary
methylated unmethylated

Gene function: housekeeping inducible

Expression:

Summary
methylated unmethylated

Gene function: housekeeping inducible

Expression: high low

Summary
methylated unmethylated

Gene function: housekeeping inducible

Expression: high low

Tissue specific
methylation:

Summary
methylated unmethylated

Gene function: housekeeping inducible

Expression: high low

Tissue specific conserved
tissue specific
methylation: across tissues

Next Steps
 Explore relationships between DNA methylation and
alternative splicing
 Annotate intergenic regions of the C. gigas genome

EPIGENOME
(DNA methylation)

GENES (DNA)

Conclusions

EPIGENOME
(DNA methylation)

GENES (DNA)

Conclusions
color disease resistance
growth

TRAITS

temperature
pathogens
nutrition
EPIGENOME
(DNA methylation)

GENES (DNA)
ENVIRONMENT

Acknowledgements
 Roberts Lab:
Samuel White
Caroline Storer
Emma Timmins-Schiffman
Claire Ellis
Lisa Crosson
 Taylor Shellfish:
Jonathan Davis
Molly Jackson

email: mgavery@uw.edu
website: students.washington.edu/mgavery

This document summarizes research characterizing DNA methylation in the Pacific oyster Crassostrea gigas. High-throughput bisulfite sequencing was used to analyze DNA methylation patterns at high resolution. Several genes were found to have different levels and patterns of methylation across tissues and developmental stages. The results provide evidence that DNA methylation plays an important regulatory role and may be involved in environmental responses in C. gigas. Future work will investigate how epigenetic mechanisms are affected by environmental stressors.

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