1. Replication timing changes during transcriptional induction of Ptn
RIVERA-MULIA J. C.1, TREVILLA-GARCÍA C.1, CORTES-GUTIERREZ M.2, DENNIS J. H.1, HIRATANI I.3, GILBERT D. M.1
1Department of Biological Science, Florida State University, Tallahassee, FL, USA. 2Lieber Institute for Brain Development, Baltimore, MD, USA.
3Biological Macromolecules Laboratory, National Institute of Genetics, Mishima, Japan. "
FUTURE DIRECTIONS"
"
• Analyze replication-timing after removing the selectable
marker in mESCs."
• Analyze replication-timing of PtnP+/- and NFR+/- mES cell
lines."
• Generate homozygous mutant cell lines."
• Analyze replication-timing in homozygous mutants mESCs."
• Analyze replication-timing during NPC differentiation."
FIGURE 1. Genome context of the Ptn gene and its replication-
timing during mESC to NPC differentiation. "
FIGURE 2. RT change of Ptn is coincident with its transcriptional
induction and subnuclear repositioning. "
Mtpn% Chrm2% Ptn% Dgki%
5.0
0.0
-5.0
35.5M 36.0M 36.5M 37.0M
Nucleosome
Occupancy
NFR
36778K 36779K 36780K 36781K 36782K 36783K
5.0
0.0
-5.0
Nucleosome
Occupancy
NPCsmESCs
FIGURE 3. Nucleosome occupancy was determined by hybridization of
mononucleosomes against reference genomic DNA."
FIGURE 5. (A) qPCR screening of HygroR mESC colonies. (B) FISH
probes. (C) Screening of HygroR mESC colonies by FISH."
-1
0
1
Dup
C3-5k
A6-5k
A7-5K
A12-5K
B2-5k
B8-5K
B11-5k
A3-5K
B5-5k
A5-5k
B10-5k
A2-5
D3Cx
A1-5k
A11-5k
C2-5k
B9-5
C1-5k
C4-5
B12-5k
B4-5k
A8-5k
A9-5k
A10-5K
B7-5K
C7-5
B1-5k
B3-5k
Del
qPCR Screening of G418R colonies
CNV
A!
C!
WT Ptn+/-
RG-RRG-RG
1
2 3
4
1
2
3
4
B!
0
50
100
D3 B3-5K
RG-RG RG-R
Nuclei
FIGURE 6. Replication timing analysis of Ptn+/- cell line. (A) BrdU
pulse-labeled cells were separated in G1, S-phase (S1-S4) and G2 by
flow cytometry. (B) BrdU labeled DNA from distinct cell cycle fractions
was used to test the replication timing by PCR. (C) Replication-timing
specific hybridization. "
G1
S1 S2 S3 S4 G2
PROPIDIUM IODIDE
CELLCOUNT
A! B!
DNA extraction/sonication
Immunoprecipitation
with anti-BrdU antibody
mESC D3
Ptn domain switches from late to early replication (LtoE)"
Transcriptional induction and su-bnuclear repositioning "
A nucleosome-free region (NFR) upstream of Ptn in NPCs"
AIM"
To address the longstanding question of whether
transcriptional induction is necessary and/or sufficient for the
replication-timing establishment."
-2
0
2
34.0 35.0 36.0 37.0 38.0 39.0
mESCs EBM-3 EBM-6 NPCs
Chromosome 6 %
Mtpn% Chrm2% Ptn% Dgki% Creb3l2%
Log2(Early/Late)
LateEarly
Chromosome 6 (Mb)
METHODS"
Targeted deletions in Ptn domain"
a
b c
Ptn NFR
LoxP
FRT
HA5’ HA3’
DTA
Targeting vector"
DTA
Flpe + Ganc
HA5’ HA3’
Hyg-TK
Transfection in mESCs
+
Hygro
Targeted allele"
Hyg-TK
Clean deletion"
FIGURE 4. (A). Targeted deletions: a) Large deletion of 38 kb
including the Ptn promoter and the NFR, b) Ptn promoter (1.8kb) and
c) NFR (1kb). (B). Targeting strategy."
A!
B!
RESULTS"
Ptn DgkiNFR
38Kb Deletion
HyTK
Targeted allele"
WT allele"
Deletion of 38kb in the Ptn domain"
Replication timing change in the targeted allele"
Mitoch
Hba-a1
Hbb-b1
Pou5f1
Mmp15
Zfp42
Dppa2
Mash1
WT allele
Mutant allele
Ptn$
100&
&
&
&50&
&
&
&&0&
Ptn%mRNA%
%Rela8ve%to%Ac8n%
%%%Early%
Replica8on%
0.0&
1.5&
3.0&
mESC% 3% 4% 5% 6% 7% 8% 9%
mRNA% Early%Replica8on%
mESC&&&&&&3&&&&&&&&&&4&&&&&&&&&5&&&&&&&&&&6&&&&&&&&&&7&&&&&&&&&8&&&&&&&&&&&9&&
E L E L E L E L E L E L E L E L
Days%of%in$vitro$differen8a8on%
G1% Early% Late% G2/M%
S3Phase&
BrdU&Labeling&
BrdU&Labeling&
Ptn&
Late%probe%
Early%probe%
Ptn&
Late%probe%
WT&mESC& Ptn&+/3&mESC&
M& G2& Late&S& G1&
%Probesignals
BrdU&labelling&(h)&
WT&mESC&
Early&S&
0.0&
20.0&
40.0&
60.0&
80.0&
100.0&
0& 2& 4& 6& 8& 10& 12& 14& 16&
Early%probe$
Late%probe$
Ptn&probe!
0.0&
20.0&
40.0&
60.0&
80.0&
100.0&
0& 2& 4& 6& 8& 10& 12& 14& 16&
M& G2& Late&S& G1&
Ptn&+/3&mESC&
Early&S&
Early%probe$
Late%probe$
Ptn&WT! Mutant&Ptn!
BrdU&labelling&(h)&
C!
G1 S1 S2 S3 S4 G2 G1 S1 S2 S3 S4 G2
CONCLUSIONS"
"
• We engineered a targeted deletion of 30kb kb including
the Ptn promoter and the NFR."
• We have engineered targeted deletions of PtnP and NFR
(not shown)."
• The targeted deletion of 38kb changes the replication
timing of Ptn domain."
INTRODUCTION"
Duplication of the genome occurs in a very precise order
referred as its replication timing program (RT). RT changes
dynamically during development and is coordinated with
transcriptional competence. "
The Pleiotrophin (Ptn) is an oncogene over-expressed in
gliomas. Ptn gene switches from late to early replication
(LtoE) during the differentiation of mouse embryonic stem
cells (mESCs) to neural precursor cells (NPCs). "
RT change of Ptn is coincident with its transcriptional
induction, suggesting that transcriptional activity may be
involved in the mechanisms that regulate the replication-
timing establishment during differentiation."
16hr BrdU
8hr BrdU
16hr BrdU
8hr BrdU