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Identification of aberrant gene expression Identification of aberrant gene expression 
associated with aberrant promoter associated with aberrant promoter 
methylation in primordial germ cells methylation in primordial germ cells 
between E13 and E16 rat F3 generation between E13 and E16 rat F3 generation 
vinclozolin lineagevinclozolin lineage
Y­h. Taguchi
 Department of Physics
Chuo University
Tokyo
Japan
http://www.biomedcentral.com/1471­2105/16/S18/S16/http://www.biomedcentral.com/1471­2105/16/S18/S16/
1. What is PCA based unsupervisedunsupervised FE?
2. What is transigenetational epigenetics? 
3.  Comparison  of  primordial  germ  cells 
between  E13  and  E16  rat  F3  generation 
vinclozolin lineage and biological validations 
of obtained gene set
4. Summary  
What is PCA based unsupervisedunsupervised FE?
 N features
Categorical 
multiclasses
In contrast to usual usage of PCA, not samples but
features are embedded into Q dimensional space.
PCA
PC1
samples
M samples
N × M Matrix X (numerical values)
PC2
PC1
+
+ +
+ +
++
+
+
+
++ +
+
+
No distinction 
between classes
Synthetic example
10 samples
10 samples
90 features 10 features
N(0)
N()
[N()+N(0)]/2
+:Top 10 outliers

Thus, extracting outliers 
selects features distinct 
between two classes in an 
unsupervised way.
Accuracy:(100 trials)Accuracy:(100 trials)
 89.5% (
 52.6% (
PC1
PC2
Normal μ:mean 
Distribution ½ :SD
The First example:
Transgenerational Epigenetics (TGE)
Phenotype transfers between generations 
without DNA modification
(also focused in “Cell Best of 2014Cell Best of 2014”)
F3 generation of F0 pregnant female exposed 
to endocrine disruptor
F0♀
F1♂ F2
F3
Abnormalities 
without inherited 
DNA exposed to 
endocrine disruptor?
Yes!
・ male infertility
  (Guerrero­Bosagna, PLoS ONE 2013)
・ anxiety behavior (Skinner, PLoS ONE, 2008)
・ mate preference (Skinner, BMC Genom., 2013)
・ various diseases  (Anway, Endocrinology, 2006) 
 (on prostate, kidney, immune system, testis, and 
tumor development)
・ reprogramming of primordial germ cells 
(Skinner, PLoS ONE, 2013)
・ stress responses ( Crews, PNAS, 2012)
However, understanding how TGE takes place still lacks.
Authors' conclusion : 
“A comparison between the germ cell 
differential DNA methylation regions 
and the differentially expressed genes 
indicated no significant overlap”
Significant overlaps observed would be Significant overlaps observed would be 
interesting....interesting....
Skinner, PLoS ONE, 2013:
Primordial germ cell in F3 generation at E13 
and E16, gene expression/promoter methylation
F2♀
F3
N'' common 
genes
Feature
Extraction
N' genes
Feature
Extraction
Promoter 
methylation
Vinclozolin 
treated
Control
E13 E16 E13 E16
Gene 
expression
Vinclozolin 
treated
Control
E13 E16 E13 E16
Our strategy.....Our strategy.....
N'  ←→ N''
  PP  
Total N genes
N〜104
Results.....Results.....
P=0.05
T­test based feature selectionsT­test based feature selections
No overlaps
(N' 300)≦
N〜104
Results.....Results.....
P=0.05
limma based feature selectionslimma based feature selections
N''=33 genes with RefSeq ID
No significant enrichments of  
GO BP, GO CC, GO MF, KEGG pathway, or PPI 
using DAVID, TargetMine, g:profiler,  or  STRING
N〜104
Results.....Results.....
P=0.05
SAM based feature selectionsSAM based feature selections
N''=30 genes with RefSeq ID
P=10­2
No significant enrichments of
 GO BP, GO CC, GO MF, KEGG pathway, or PPI 
using DAVID, TargetMine, g:profiler, or STRING
N〜104
Results.....Results.....
P=0.05
P=10­3
P=10­2
PCA based PCA based unsupervisedunsupervised FE FE
N''=48 genes with RefSeq ID
multiple significant enrichments of GO BP,  GO MF,  using 
DAVID and g:profiler
Seven PPIs (P=0.05) detected by STRING
N〜104
Results.....Results.....
P=0.05
P=10­3
P=10­2
PCA based PCA based unsupervisedunsupervised FE FE
N''=179 genes with RefSeq ID
DAVIDDAVID
g:profilerg:profiler
TargetMineTargetMine
We further validated that 22 genes among 48 
genes selected by PCA based unsupervisedunsupervised 
FE  when N'=1000 are related to either 
kidney, testis, prostate, tumor and immune 
system whose associated diseases were 
caused by TGE.
We also proposed that Chemokine signaling 
pathway as well as LRR proteins are deeply 
related to TGE.
Because of lack of time, we cannot discuss 
about it.
Summary
・PCA based unsupervisedunsupervised FE identified more genes 
associated  with    both  differential  gene  expression 
and  aberrant  promoter  methylation  between  E13 
and E16 rat F3 generation vinclozolin lineage
・ Genes  selected  by  PCA  based  unsupervisedunsupervised  FE 
were  associated  with  various  enrichments  of 
biological  terms/concepts  while  none  were 
associated  with  those  selected  by  t­test,  limma,  or 
SAM based FE.
・ These  suggested  definitely  the  superiority  of 
unsupervisedunsupervised method toward supervised methods.  
This study was supported by
Chuo university joint research grant 
“In silico drug discovery using FAMS”
 and 
KAKENHI  
“Initiative for High­Dimensional Data­
driven Science through Deepening of 
Sparse Modeling”

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