NSA 2012 M.Gavery

Epigenetic mechanisms as a source of phenotypic plasticity in the Pacific oyster

Crassostrea gigas

Mackenzie Gavery & Steven Roberts

University of Washington

School of Aquatic and Fishery Sciences

  Background:

  Epigenetics

  DNA methylation

  Results: characterization of DNA methylation in Pacific oysters

  High-throughput bisulfite sequencing

  Discussion/Future Directions

Outline

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

G C

G

  Regulates gene expression

 Development

 Tissue-specific expression

 Environmental response

DNA methylation: invertebrates   Only a handful of species have been evaluated

  Model invertebrates lack DNA methylation

  Most: intermediate methylation

  Primarily in exons

  Important regulatory functions – honey bee (e.g. Kucharski et al., 2008; Elango et al., 2009; Lyko et al., 2010)

CpG O/E

Predicted degree of DNA methylation

Mea

sure

d d

egre

e of

DN

A m

ethyl

atio

n

Enr

ichm

ent

leve

l in

MB

D l

ibra

ry

Summary of Previous Results

(Gavery & Roberts, 2010)

(Rob

erts

& G

aver

y, 2

011)

CpG O/E


Mea

sure

d d

egre

e of

DN

A m

ethyl

atio

n

Enr

ichm

ent

leve

l in

MB

D l

ibra

ry


(Rob

erts

& G

aver

y, 2

011)


CpG O/E


Mea

sure

d d

egre

e of

DN

A m

ethyl

atio

n

Enr

ichm

ent

leve

l in

MB

D l

ibra

ry



(Rob

erts

& G

aver

y, 2

011)

Current Research:   High-resolution methylation analysis

  Pooled adult gill tissue

 High-throughput bisulfite sequencing

Workflow:   Reduce genome

  Prepare gDNA library for methylation analysis

  Data Analysis

  Reduce genome


  Data Analysis

Workflow: - methyl binding domain enrichment

MBD

MBD



  Data Analysis



  Data Analysis

– Illumina library – Bisulfite conversion



  Data Analysis T T C G C

T T C G U

T T C G T

bisulfite

PCR

– Illumina library – Bisulfite conversion



  Data Analysis T T C G C

T T C G U

T T C G T

bisulfite

PCR



  Data Analysis

T T C G C

T T C G U

T T C G T

bisulfite

PCR

– BSMAP software

– Reference:

- de novo contigs

- characterized genes

Visualization: hexokinase

0 bp 10,960 bp

MBD-mapped reads

CG


0 bp 10,960 bp

mid mCG

low mCG

mCG

CG


0 bp 10,960 bp

CG

mCG

cDNA - gill

cDNA - larvae


0 bp 10,960 bp

CG

mCG

cDNA - gill

blastn EST


0 bp 10,960 bp

CG

mCG

cDNA - gill

blastn EST

1384 bp

Nanostring: nCounter®

  Screening of multiple sample types, multiple loci

0

20

40

60

80

A B C D A B m f

gill larvae gonad

% m

ethy

lati

on

hexokinase CG loci 1384bp

Targeted Bisulfite Sequencing

0%

25%

50%

75%

100%

1 2 3 4 5 6 7

% m

ethy

lati

on

position (bp)

hexokinase CG loci 1,287 – 1,435

gill larvae sperm

1384 1446 1454 1458 1369 1356 1285


0 bp 10,960 bp

CG

mCG

cDNA - gill

blastn EST

Visualization: hsc70

0 bp 2,552 bp

CG

mCG

mid mCG

low mCG

Visualization: LAP

0 bp 5,494 bp

CG

mCG

mid mCG

low mCG

Visualization: gnrhr

0 bp 13,058 bp

CG

mCG

mid mCG

low mCG

Results: summary   Using this technique we can characterize the methylation

status of individual CG loci at a genome wide scale

  Patterns suggest that methylation may be involved in gene regulation

  Provides a proof-of-concept in non-model species

Conclusions/Future Directions:

  DNA methylation plays an important role in regulating gene expression in C. gigas

  It will be important to understand how epigenetic mechanisms will be affected by the environment

  Future work will test these possible mechanisms and how they will be affected by environmental stress

Acknowledgements

  Samuel White (UW, SAFS)

  Joth Davis (Taylor Shellfish Farms)

  Robin Harper & Phillipa Webster (Nanostring)

email: [email protected] website: students.washington.edu/mgavery

NSA 2012 M.Gavery

Technology

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