1600 1620 siwanon jirawatnotai
TRANSCRIPT
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Siwanon Jirawatnotai, Ph.D.Siriraj Laboratory of Systems Pharmacology,Dept of Pharmacology, Siriraj Medical School
A Direct Role of Cyclin D1 in DNA Repair
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Cyclin D1 is a cell cycle protein that drives mammalian cell division
gallus.reactome.org
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A function of cyclin D1 in DNA repair uncovered by proteomic screenings
TAP Mass-spec MCF-7, ZR-75-1
UMSCC-2
GRANTA 519
HT29
Jirawatnotai, Nature 2011
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Cyclin D1 is required for cell survival after DNA damage
0 h 16 h
sicont
siD1-B0-4
041
-6061
-80
81-10
0>1
000
20
40
60
80 sicont
siD1-B
Tail length (pixels)P
erce
nt o
f ce
lls
0-40
41-60
61-80
81-10
0>1
000
20
40
60
Tail length (pixels)
Per
cent
of
cells
***
*
***0 h 16 h
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Pierce A .,et al. 1999
sicon
t
siRAD51
siD1-A
siD1-B
siD1-C
0
20406080
100120
Per
cent
***
sicon
t
siD1-A
+ D1
020406080
100120
Perc
ent
***
Interaction between cyclin D1 and RAD51 facilitates efficient homologous recombination
Valerie et al., 2003 Oncogene
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Cyclin D1 directly interacts with RAD51
SP49
Z138C
Granta 519
H2009
ZR-75-1
HT-29
IP RAD IgG WCL
WB: cyclin D1
MCF7
In vivo bindingIn vitro binding
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N-terminus domain of cyclin D1 and C-terminus domain of RAD51 are require for the interaction
***
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Interaction between cyclin D1 and RAD51 facilitates RAD51 loading to damage foci
0-2 3-9 10-19
0
20
40
60
80
100 4 h
Foci/nucleus
Per
cent
of
cells
***
******
***
**
0-2 3-9 10-19
0
20
40
60
80
100 0 h
Foci/nucleus
Per
cent
of c
ells
0-2 3-9 10-19
0
20
40
60
80
100 2 h
Foci/nucleus
Per
cent
of
cells
*
******
******
*
0-2 3-9 10-19
0
20
40
60
80
100 8 hsicont siD1-A
siD1-C
Foci/nucleus
Perc
ent
of c
ells
******
******
****
0-2 3-9 10-19
0
20
40
60
80
100 6 h
Foci/nucleus
Per
cent
of c
ells
******
******
******
sicont
siD1-A
siD1-C
DAPI RAD51 -H2AX Merge
sicont
siD1-A
siD1-C
+ DNA damage
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Cyclin D1 localizes at damaged DNA
IgG
RAD51E2F
40
2
4
6
Before DNA breakAfter DNA break
Fold
enr
ichm
ent
IgG
RAD51E2F
40
2
4
6
8
Fold
enr
ichm
ent
siD1
IgG
Cyclin
D1
E2F4
0
5
10
15
Before DNA breakAfter DNA break
Fold
enr
ichm
ent
IgG
Cyclin
D1
E2F4
05
10152025
Fold
enr
ichm
ent
siD1
Pierce A .,et al. 1999
Targeted ChIP
DAPI - H2AX cyclin D1 Merge
Immunofluorescence
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Interaction between cyclin D1 and BRCA2
B2-
1
B2-
3
B2-
4
B2-
5
B2-
6
B2-
7
B2-
8B
2-9
B2-
2
GS
T
Cyclin D1 binding to BRCA2 in vitro(GST-pull-down)
BRCA2+/-H2AX+
siD1-A
sicont
Esashi, et al. 2005
Input GST
RAD51 binding to BRCA2 in vitro(GST-pull-down)
Esashi, et al. 2005Cyclin A/CDK2 phosphorylation
of BRCA2
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Nocodazole D1
GST-B2-9
Roscovitin +----+--
++++
Cyclin A
GST-B2-9
RAD51
Cyclin D1
GST B2-9
pull-down
293 cells
Phospho BRCA2 S3291 Kinase assay
Kinase assay
Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2
Cyclin D1 expression helps recruiting RAD51 to C-ter of BRCA2
Phospho BRCA2 S3291
BRCA2
Cyclin D1
Cyclin B1
Cyclin A2
RAD51
CDK4
b-Actin
Async. G2/M (nocodazole)
- D1 Ro - D1 Ro
Cyclin B1 long exp
293 cells
Cyclin D1 expression prevents B2-9 phosphorylation by CDK2
Lysate +++-
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G2/M
siD1
D1 IR Ros-
Phospho BRCA2 S3291
Cyclin D1
Cyclin A2
CDK4
Cyclin B1
RAD51
HeLa cells
Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2
Depletion of cyclin D1 by siRNA increases S3291 phosphorylation
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IP: B2-9
G2
IR (5 Gy) - + - +
Cyclin A2
Cyclin D1
BRCA2S3291
Asynchronous
GST B2-9
Cyclin D1 is recruited to C-terminus of BRCA2 upon DNA damage
WB
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In vitro Competition assay
rHA-Cyclin D1
GST-BRCA2 C-Ter (B2-9) - + + + + + - + + + + +
rCyclin - - - -
+ + + + + + + + + + + +
DA
WB: HA
GST
Cyclin D1 controls phosphorylation of C-terminus BRCA2 by competing with cyclin A for BRCA2 binding
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Cyclin D1 cooperates with DNA damage to inhibit BRCA2 phosphorylation at Ser3291
Nocodazole
- + - + - + - + - +
IR - - - - - - + + + +
D1 - - - - + + - - + +
Ros - - + + - - - - - -
1 2 3 4 5 6 7 8 9 10
S3291 Ph
cyclin D1
BRCA2
CDK4
actin
IB:γH2AX
cyclin D1
IB:GST
GST pulldown
B2-9
B2-9 S
3291
E0.5 1 2 4 6
Hour after IR
actin
IB:
S3291 Ph
BRCA2
cdc25a
No
IR
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aa 20-90 of cyclin D1 is required for BRCA2-C-terminal binding
full-
leng
th
20-2
95
91-2
95
full-
leng
th
20-2
95
91-2
95
B2-5 B2-9 inp
IP
WB: Cyclin D1
full-
leng
th
WB:GST
B2-9 binding domain of cyclin D1 (aa 20-91) is required to prevent CDK2/Cyclin A2 phosphorylation on B2-9
D1 1-295 1-90
IB:GST
cyclin D1
B2-9 kinase assay
cyclin A-CDK2
0 nM 10 nM 20 nM 40 nM0
20
40
60
80
100
120
D1
Concentration of recombinant cyclin D1
Rel
ativ
e p
hosp
hory
latio
n le
vel
cyclin D1
Δ1-90
BSABSA
BSA BSA
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Cyclin box domain of cyclin D1 is required for RAD51 and BRCA2 binding
Cyclin box
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CDK4/C
yclin
D1
CDK2/C
yclin
A2
Shot exp.
Long exp.
Shot exp.
Long exp.
Shot exp.
Long exp.
GST-B2-5
GST-B2-9
GST-pRb
GST-B2-9
GST-pRb
GST-B2-5
WB: GST
Cyclin D1 that is in complex with CDK4 does not compete with CDK2/cyclin A
Shot exp.
Long exp.
Kinase assay
CDK4/Cyclin D1 CDK2/Cyclin A2
CDK2/Cyclin A2 CDK4/Cyclin D1
GST-B2-9
GST-Cyclin D1
GST-Cyclin A
WB: GST
0 Gy 2 Gy 4 Gy 6 Gy
1
10
100
0 µM1 µM3 µM
Perc
ent s
urvi
val
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Cyclin D1 depletion hypersensitizes Rb-negative cancers to IR treatment
Jirawatnotai, Nature 2011
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Cyclin D1 promotes RAD51 localization on damage DNA, and HR
Jirawatnotai, Oncogene, in press
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No DNA damageSevere DNA damage
Jirawatnotai, Cancer Research, 2012
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Thanks
• Chonvara Chalermrujinanant• Gunya Sittithumcharee• Peter Sicinski, DFCI
– Wojtek Michowski– Lisa Becks
• Fumiko Esashi (University of Oxford)
• David Livingston lab, DFCI– Yiduo Hu
• Steve Gygi lab, HMS– Joshua Elias
• John Quackenbush, CCCB, DFCI– Yaoyu Wang
• Bing Xia (University of Medicine and Dentistry• of New Jersey)