Characterizing epigenetic variation in the
Pacific oyster (Crassostrea gigas)
Claire Olson
School of Aquatic and Fishery Sciences
University of Washington
Committee Meeting April 24th 2014
Committee Meeting Outline
Overview of Master’s
thesis chapters
 Research Update
 Future steps
 Timeline and progress

Goals
•
•
Characterize distribution of DNA methylation
Identify potential functions of DNA methylation
Understanding epigenetic variation in
the oyster

Chapter 1: Characterizing
genome-wide DNA methylation
patterns in a single cell type
 Correlation
with gene expression
patterns

Chapter 2: DNA methylation
during oyster early development
 Heritability
variability
vs. Individual
Chapter 1: Genome-wide methylation
•
Determine genome-wide
methylation patterns
•
Whole Genome Bisulfite
Sequencing (WGBS)
•
•
•
Male gonad tissue: genome
wide scale, single base pair
resolution
Unmethylated C to U
(sequenced as T)
Differentiate between SNPs
from bisulfite conversion
CH3
A
C
CH3
G
C
T
C
A
G
Bisulfite treatment +
sequencing
CH3
A
C
CH3
G
T
T
C
A
G
Chapter 1: Genome-wide methylation
•
•
•
Whole Transcriptome
Shotgun Sequencing
Methylation vs expression
patterns in a single cell
type
Relationship between
gene expression and
promoter methylation
RNA
extracted
from gonad
tissue
AAAAA Enrich for mRNA
AAAAA
Create cDNA
library
Sequence on
Illumina
Gene expression data for
17,093 genes
Chapter 1: Genome-wide methylation
•
•
•
•
•
Coverage for 7.64 million CpGs
Overall 15% genome methylation
Methylation primarily in intragenic regions
No methylation in mitochondrial genome
Positive association between methylation status and
expression
•
Methylation involved in gene regulatory activity
Chapter 1: Genome-wide methylation
Distribution of methylation ratios
Distribution of Methylation
in the Oyster
Genome
DNA methylation is dispersed throughout
theLevels
oyster
genome
•
Histogram of methylation$score
5e+06
Histogram of methylation$score
20
•
4e+06
4e+05
n=2299785 CpG
10
n=1028202 CpG
5
0e+00
1e+05
1e+06
2e+05 2e+06
Density
3e+05
3e+06
15
n=493403 CpG
0.0
0.2
0.4
0.6
0.8
1.0
0.8
1.0
Proportion Methylation
0
0e+00
Frequency
Frequency
Exons
Introns
Promoter Regions
0.0
0.2
0.4
0.6
0.0
0.2
0.4
0.6
Proportion Methylation
Methylation Level
0.8
1.0
Chapter 1: Genome-wide methylation
Methylated CpGs
Non-methylated CpGs
mRNA
Exons
Introns
Distribution of methylation within
genomic regions
•
Methylation occurring predominantly in intragenic
regions (expressed portions and introns)
Exons
Introns
Methylated CpGs
All CpGs
Other
er&
3130
5065
1809
1842
Methylated+
586647
2886432 188472529 0.33739366 3.37393664
No+
Data+
278273
832569 39534356 0.07077234 0.70772336
122820
616223 27433172 0.04910943 0.4910943
1408456
5121236 297331540 0.53226737 5.32267372
5121236
558613125
Distribution of methylation within
Moderately+
Moderately+
genomic
regions
Methylated+
er&
Methylated Moderately+Methylated
UnmethylatedNo+Data
CGs
Size+of+regionPortion+of+oyster+genome
Exons
343032
61839
623331
106420
1134622 37121309 0.06645263 0.66452626
Introns
518734
287921
1493130
586647
2886432 188472529 0.33739366 3.37393664
Transposable+Elements
38532
120699
395065
278273
832569 39534356 0.07077234 0.70772336
Promoter+Regions 45241
46353
401809
122820
616223 27433172 0.04910943 0.4910943
Other
277587
523351
2911842
1408456
5121236 297331540 0.53226737 5.32267372
Total
5121236
558613125
Methylated+
hylated
ted No+Data
Unmethylated
Unmethylated+ Transposable
7327 0.09379335 Unmethylated+
Elements
Methylated
egions&
9263 0.20324297 Sparsely
Moderately+
1322 0.33423416
Methylated
Methylated+
5129 0.19931096 Heavily
No+Data+
8188 0.27502267 No Data
No+Data+
Unmethylated+
Methylated+
Exons
Moderately+
Introns&
Methylated+
Moderately+
Introns
No+Data+
Methylated+
thylated+
Methylated+
Unmethylated+
derately+
thylated+
methylated+
+Data+
No+D
Methylated
Exons
0.30233153
Introns
0.17971461
Transposable+Elements
0.04628085
Promoter+Regions
0.0734166
Other
0.05420313
Totals
Methylated Moderately+Methylated
UnmethylatedNo+Data
CGs
Size+of+regionPortion+of+oyster+genome
Exons
343032
61839
623331
106420
1134622 37121309 0.06645263 0.66452626
Introns
518734
287921
1493130
586647
2886432 188472529 0.33739366 3.37393664
Transposable+Elements
38532
120699
395065
278273
832569 39534356 0.07077234 0.70772336
Promoter+Regions 45241
46353
401809
122820
616223 27433172 0.04910943 0.4910943
Other
277587
523351
2911842
1408456
5121236 297331540 0.53226737 5.32267372
Total
5121236
558613125
861766
Methylated
Exons
0.30233153
Introns
0.17971461
Transposable+Elements
0.04628085
Promoter+Regions
0.0734166
Other
0.05420313
Totals
Introns&
Methylated+
Moderately+
Methylated+
Moderately+
Methylated+
Unmethylated+
Unmethylated+
Unmethylated+
Promoter&Regions&
Methylated+
Moderately+
Methylated+
Unmethylated+
No+Data+
Methylated+
Moderately+
Methylated+
Moderately+
Methylated+
Unmethylated+
Unmethylated+
Unmethylated+
No+Data+
No+Data+
No+Data+
Other&
Moderately+
Methylated+
Unmethylated+
No+Data+
Other&
Methylated+
Moderately+
Methylated+
Unmethylated+
No+Data+
Other
861766
Other&
Methylated
Exons
0.30233153
Introns
0.17971461
Transposable+Elements
0.04628085
Promoter+Regions
0.0734166
Other
0.05420313
Totals
Moderately+Methylated
UnmethylatedNo+Data
Methylated+
0.05450185 0.54937327 0.09379335
0.09974979 0.51729263 0.20324297
Moderately+
0.14497177
0.47451322 0.33423416
Methylated+
0.07522115
0.65205129 0.19931096
0.10219232
0.56858188 0.27502267
Unmethylated+
No+Data+
Introns&
Transposable&Elements&
Methylated+
Methylated+
Methylated+
Moderately+
Methylated+
Moderately+
Methylated+
Moderately+
Methylated+
Unmethylated+
Unmethylated+
Unmethylated+
No+Data+
No+Data+
No+Data+
Promoter&Regions&
Methylated+
Moderately+
Methylated+
Unmethylated+
No+Data+
No+Data+
No+Data+
Transposable&Elements&
Methylated+
Moderately+
Methylated+
Methylated+
Methylated Moderately+Methylated
UnmethylatedNo+Data
CGs
Size+of+regionPortion+of+oyster+genome
No+Data+
No+Data+
Exons
343032
61839
623331
106420
1134622 37121309 0.06645263
0.66452626
Introns
518734
287921
1493130
586647
2886432 188472529 0.33739366 3.37393664
Transposable+Elements
38532
120699
395065
278273
832569 39534356 0.07077234 0.70772336
Promoter+Regions 45241
46353
401809
122820
616223 27433172 0.04910943 0.4910943
Other
277587
523351
2911842
1408456
5121236 297331540 0.53226737 5.32267372
Total
5121236
558613125
Exons&
Unmethylated+
Introns&
Methylated+
Promoter&Regions&
Methylated+
Moderately+
Methylated+
No+Data+
Exons&
Transposable&Elements&
Methylated+
861766
Moderately+Methylated
UnmethylatedNo+Data
0.05450185 0.54937327 0.09379335
0.09974979 0.51729263 0.20324297
0.14497177 0.47451322 0.33423416
0.07522115 0.65205129 0.19931096
0.10219232 0.56858188 0.27502267
Promoter
Regions
Moderately+Methylated
UnmethylatedNo+Data
0.05450185 0.54937327 0.09379335
0.09974979 0.51729263 0.20324297
0.14497177 0.47451322 0.33423416
0.07522115 0.65205129 0.19931096
0.10219232 0.56858188 0.27502267
Exons&
861766
Other&
Methylated+
Moderately+
Methylated+
Unmethylated+
No+Data+
Moderately+
Methylated+
Met
Transposable&Eleme
Mod
Met
Unm
Unmethylated+
No+D
No+Data+
Chapter 1: methylation vs expression
Chapter 1: Summary
•
•
•
•
Characterization of methylation in a
single cell type
Methylation only observed in CpG
motifs
Lack of methylation in oyster mtDNA
15% overall genome methylation
•
•
•
•
Intermediate level
Methylation not variable between
tissue types
DNA methylation predominantly in
exons and introns
Likely association between
methylation status and gene
expression
Chapter 2: DNA methylation and
oyster development
•
•
Characterize methylation landscape
ID potential functions of DNA methylation throughout
various stages of oyster development
•
Sperm, eggs and Larvae
Chapter 2: Developmental methylation
•
Sperm and larvae methylome
•
•
•
2 males strip spawned, fertilized eggs from one female
Sperm & eggs frozen
Larvae collected 3 days and 5 days post-fertilization
Chapter 2: Developmental methylation
Male 1
Male 3
Sperm
(+ Eggs)
Tank 1
Day 3
Tank 3
Day 5
Chapter 2: Developmental methylation
•
•
Sperm: single cell type (removes bias of cell-specific
methylation)
Larvae: significant changes in tissue-specific gene
expression occurring
Chapter 2: Developmental methylation
Female
Male 1
Male 3
Larv1 Day 3
Larv1 Day 5
Larv3 Day 3
Larv3 Day 5
Female coverage
Male 1 coverage
Genes
Chapter 2: Developmental methylation
Larv1
Day 5
Male 3
40,654
common
loci
Larv1
Day 3
•
Methylation
profiles
among
sperm and
larvae
Male 1
•
Larv3
Day 3
Larv3
Day 5
Chapter 2: Developmental methylation
0.20
0.15
0.10
Larv1 Day
T1D55
MaleM11
Larv1 Day
T1D33
Larv3 Day
T3D55
MaleM33
T3D33
Larv3 Day
0.05
0.00
Height
0.25
0.30
0.35
CpG methylation clustering
Samples
Distance method: "correlation"; Clustering method: "ward"
Chapter 2: Developmental methylation
CpG methylation PCA Analysis
PC2
50
100
Larv1
T1D3Day 3
0
Larv3T3D3
Day 3
T3D5
Larv3
Day 5
MaleM3
3
Larv1 Day 5
-50
T1D5
M1 1
Male
-100
-50
0
PC1
50
Chapter 2: Developmental methylation
Male 1
Male 3
Larv1Day3
Larv1Day5
Larv3Day3
Larv3Day5
Chapter 2: Summary
•
Overall methylation levels similar for sperm and larvae
samples
•
•
~ 12%-17% genome methylation
Similar spermatozoa and larvae methylation profiles
Future steps
•
•
Examine hypo/hyper
methylated regions
from sperm and
larvae samples
Identification of
DMRs
MS Timeline
Courses to date
•
•
•
•
•
•
QSCI 482: Statistical Inference (Fall 2012)
FISH 510: Topics: Local Adaptation (Spring 2013)
FISH 521: Research Proposal Writing (Winter 2013)
FISH 522: Hot Topics (Fall 2012)
FISH 510: Topics: Endangered Species Act (Spring 2014)
Additional coursework:
•
•
•
•
•
FISH 541: Environmental Physiology (Fall 2012)
FISH 546: Bioinformatics (Winter 2013)
FISH 552: R Programming (Fall 2013)
FISH 554: Beautiful Graphics in R (Winter 2014)
TA experience
•
•
FISH 310: Biology of Shellfish (Spring 2013 and 2014)
FISH 546: Environmental Physiology (Fall 2013)
Understanding epigenetic variation in
the oyster

Chapter 1: Characterizing
genome-wide DNA methylation
patterns in a single cell type
 Correlation
with gene expression
patterns

Chapter 2: DNA methylation
during oyster early development
 Heritability
variability
vs. Individual