Siwanon Jirawatnotai, Ph.D.Siriraj Laboratory of Systems Pharmacology,Dept of Pharmacology, Siriraj Medical School

A Direct Role of Cyclin D1 in DNA Repair

Cyclin D1 is a cell cycle protein that drives mammalian cell division

gallus.reactome.org

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

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

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

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

N-terminus domain of cyclin D1 and C-terminus domain of RAD51 are require for the interaction

***

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

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

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

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 +++-

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

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

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

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

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

Cyclin box domain of cyclin D1 is required for RAD51 and BRCA2 binding

Cyclin box

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

Cyclin D1 depletion hypersensitizes Rb-negative cancers to IR treatment

Jirawatnotai, Nature 2011

Cyclin D1 promotes RAD51 localization on damage DNA, and HR

Jirawatnotai, Oncogene, in press

No DNA damageSevere DNA damage

Jirawatnotai, Cancer Research, 2012

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)