How telomeres protect chromosome ends from engaging the ... · How telomeres protect chromosome...

How telomeres protect chromosome endsHow telomeres protect chromosome endsfrom engaging the DNA damage response

Sandy Chang, M.D., Ph.D.

Dept. of Laboratory MedicineDept. of Laboratory MedicineYale University School of Medicine

NIANIAJanuary 18, 2011

Mammalian telomeres

t l

telomereuncapping

telomeredysfunction

absence of t l

DNA d m

telomerase

DNA damageresponse

p53 losswt p53

cancer initiation

pp

apoptosissenescence

Deng et al., Nat Rev Cancer2008

n t at onsenescence

Consequence of natural telomere attrition: multiple non-reciprocal chromosomal translocations in tumors with short telomeres

G6G6

Breast tumor TD 574

T(18; 6; 5)T(6; 12)

T(18; 6; 5)T(6; 12) T(18; 6; 5)T(6; 12)

T(6;2)

DAPI image SKY image

Artandi et al., Nature 2000

Massive end-to-end chromosome fusions in the absence of Trf2

Dysfunctional telomeres promote genomic instability

Six subunits in shelterin interacts with the t-loop: TRF1,TRF2,TIN2,RAP1,TPP1 and POT1

telomerase

POT1-Protection of Telomere 1

Required for telomere end protection

Negative regulator of telomere length

POT1 structure

M P t1 N- OB1 OB2Mouse Pot1a

74% 73% 71%

TPP1 BD

N- OB1 OB2Mouse Pot1b

74% 81% 73%

TPP1 BD

OB1 OB2N-Human POT1

74% 81% %

TPP1 BD

Lei et al., Nature Struc and Mol Biol 2004

Conditional Deletion of Mouse Pot1a

frtfrtlo

xP

ATG

4

loxP

targeting

ADT

NotIneo 4 5 vector

B BDT

frtfrt loxP

targeted allele Pot1recneo 5

B B

ATG

4

7 kbS S

11 8 kblo

xP

Cre recombinase

7 kb 11.8 kb

null allelePot1Δ

1 re

c/Δ

B C D Δ/Δ

1 Δ

/Δ

rec/Δ

+/+

C

Pot1

mPot1aCmPot1a

Pot1

a-m

yc

Pot1

a+/+

Pot1

Pot1

Pot1

Pot1

Pot1

Δ/Δ

Pot1

rec/Δ

BGAPDHcontrol

Pot1rec/Δ MEF

*

P PP

Pot1rec/Δ MEF

Elevated DNA damage response in Pot1a deleted cells

A10 Gy γ-IR, Pot1rec/Δ Pot1Δ/+ Pot1rec/Δ

AX

Pot1Δ/Δ

A

γH

2A

C53BP1 telomere mergeB magnified

AdCIRPot1Δ/+ Pot1rec/ΔPot1rec/Δ

AdC

ATM-Ser1981-PChk2-PChk2

- + +-AdCreγ-IR

- +AdCre

t1Δ

/Δ, p

53-/-

Chk2γ-H2AX-P

γ-tubulin

Pot

A B

Pot1a loss leads to multiple cytogenetic aberrations and malignant transformation

A

f db

B

a b c d a c

d

e

b

C D

Pot1rec/Δ sarcomaPot1aΔ/Δ , p53-/- sarcoma

Pot1aΔ/Δ , p53-/-

MMTVcre - pot1aF/Fbreast tumor

Acceleration of MMTV-Cre+ ; Pot1aFF breast tumorsin the absence of p53

1.0 cre-pot1af/f

MMTVcre - pot1aF/F breast tumor

1.0 cre- pot1f/f p53f/

MMTVcre-pot1f/f-p53 mutant breast tumors

l

Cre- Pot1aFF

n=21al

Cre- Pot1aFF p53FF

n=22al

0.5

cre+pot1af/f.

mor

sur

vial

0.5

cre+/+ p53f/fcre+pot1f/fp53f/+cre+pot1f/fp53f/f

mor

sur

vial

Cre+ Pot1aFF

n=21

ree

surv

iva

ree

surv

iv

0 0

% o

f tu

0 0

% o

f tun=23

% tu

mor

fr Cre+ Pot1aFF p53F/+

n=31Cre+ Pot1aFF p53FF

n=25

% tu

mor

f

0 10 20 300.0

Time ( months)0 5 10 15 20

0.0

Monthmonths

%

months

Pot1aF/F

Pot1aΔ/Δ

tail tumor

Pot1a

Ling Wu

Massive telomere amplifications in MMTV-Cre+ Pot1aΔ/Δ breast tumors

POT1a is required for telomere end protection

POT1a

POT1a

TIFs

p53

Genomic instability

tumorigenesis

Pot1Δ/Δ

GA

Conditional deletion of mouse Pot1b

Frt Frt

DT Lox p Lox p NotItargeting vector

wt allele11KB

ATGA

3

3 4

4Neo

11KB

Cre recombinase

recombined allele:Pot1brec

Neo

7 8KB

3 4

Cre recombinase

null allele: Pot1b-/-

7.8KB

B C DB C

Pot1b transcript

D

wt: 11Kbt t 7 8kb Pot1b transcript

GPDHloading control

mutant:7.8kb

Generating Pot1b null alleles

5’ TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG(TTAGGG) 3’

TIN2 TPP1

5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG(TTAGGG)n-3’3’-AAGCCCAAGCCCAAGCCCAAGCCC

POT1bTRF1 TRF2/Rap1 telomerase

5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG(TTAGGG)n-3’

TIN2 TPP1

( )3’-AAGCCCAAGCCCAAGCCCAAGCCC

POT1bTRF1 TRF2/Rap1 telomerase

Deletion of Pot1b in the setting of telomerase deficiency results in diminished lifespan y p

100

ve

-/+ mTR+/- n=9

Pot1b -/- mTR +/+ n=12

Pot1b

50

% m

ice

aliv

male Pot1b -/- mTR+/- n=12

Pot1b-/- mTR-/- n=6

female Pot1b-/- mTR +/- n=13

0 100 200 3000

days400

days

A Pot1b-/- mTR+/+ Pot1b-/- mTR+/- Pot1b-/- mTR-/-WT

Complete bone marrow failure in Pot1b-/- mTR+/- mice

Pot1b / mTR / Pot1b mTR Pot1b mTRWT

BWT Pot1b-/- mTR+/-

32% 15.2%69.9%1.1%

CD

11b

2%27% 1.4%31.4%

Gr-1

Depletion of hematopoietic progenitor/stem cells in Pot1b-/- mTR+/- bone marrow

WT (6 months) 2 months 4 months 6 monthsLin- Lin- Lin- Lin-

2.04% 0.23% 0.39% 0.06%1.34% 0.13% <0.01% <0.01%

C-K

it

Sca-1

Eric Wang

1500

A15

Peripheral blood defects in Pot1b-/- mTR+/- mice

0

500

1000

Plat

elet

X103 /μ

L

0

5

10R

BC

X106 μ

L

5.0

7.5

WB

CX1

03 / μL

00

5.0

7.5

% B

last

s

0.0

2.5

0.0

2.5

%

a bB C

AD

5.2%3.2% 12.6%0.1%WT Pot1b-/- mTR+/-

c d 7-AA

8.8%83.0% 70.9%16.4%

Annexin V

Pot1b-/- mTR +/- mice displays phenotypes resemblingDyskeratosis congenita (DC)

b l ki• abnormal skin pigmentation

Pot1b-/- mTR+/+ Pot1b-/- mTR+/-WT

• nail dystrophy

• BM failure

WT Pot1b-/- mTR+/+

Dyskeratosis congenita

Dyskeratosis congenita is a telomere disease

Dyskerin is a pseudouridine synthase; modifies rRNA, spliceosomal RNA, using a H/ACA small guide RNAs

•Mutated in X-linked dyskeratosis congenita (Collins)

•aplastic anemia

•pulmonary fibrosis, hyperpigmentation, oral leukoplakia, nail dystrophy

•very short telomeresy

•TERC or TERT are mutated in autosomal dominant DC (Dokal, Greider)

•NOP10, a small ribonucleoprotein, is mutated in autosomal recessive DC (Dokal)

•Heterozygous mutation in TINF2 recently observed in DC e e o ygous u a o ece y obse ed Cpatients (Savage, Dokal)

Increased G-strand overhang and progressive telomere shortening in the absence of Pot1b

A B

35 42 1 35 3510 41 35 42 1 35 3510 41passage

Overhang(CCCTAA)4 (TTAGGG)4

Total telomere (CCCTAA)4

Overhang (TTAGGG)4

Total telomere

liver MEFsliver MEFs

Increased chromosome fusion and telomere shortening in Pot1b-/-

mTR+/- cells

mTR

+/-D

Pot

1b-/-

m

E

Pot1b-/- mTR+/- MEFs initiate a DNA damage response at telomeres

+/-m

TR+/

-

TTAGGG γ-H2Ax merge

100Pot1b+/- mTR+/-

Pot

1bm

TR+/

+

25

50

75

Pot1b , mTR

Pot1b-/-, mTR+/+

Pot1b-/-, mTR+/-

% o

f tot

al c

ells

Pot

1b-/-

TR+/

-

0 1-3 3-5 >60

number of TIFs per cell

Pot

1b-/-

mT

Pot1b is required to modulate ss overhang formation

1b

Pot1b loss

1b

Pot1b lossInsufficient telomerase

Increased overhang generation

Adapted from Ramiro and Karlseder, Nature 2007

Accelerated telomere shorteningIncreased DDR at telomeresp53-dependent apoptosis

Role of repressor activator protein 1 (RAP1) in telomere end protection

γγH2AXH2AX//TRF1TRF1

TRF2+/+

TRF2TRF2

RAP1RAP1

TRF1TRF1


TRF2TRF2TRF1TRF1

TIN2TIN2 TPP1TPP1 POT1POT15’3’


TRF2-/-

TRF1TRF1

TIN2TIN2 TPP1TPP1 POT1POT15’3’TIN2TIN2 TPP1TPP1 3

Celli, GB et al., (2006) Nature Cell Biology

Removal of TRF2 from telomeres results in DDR at telomeres and massive chromosome end fusions


TRF2-/-

TRF1TRF1TRF1TRF1

TIN2TIN2 TPP1TPP1 POT1POT15’3’

Removal of TRF2 results in rapid disappearance of Rap1 from telomeres.

In yeast, Rap1 is involved in protecting telomeres from NHEJ independent from TRF2-like homologs.

What are the roles for mammalian Rap1 in telomere end protection?

Knockdown of Rap1 by shRNA does not work well

TRF2TRF2TRF1TRF1

TIN2TIN2 TPP1TPP1 POT1POT15’ 3’TIN2TIN2 TPP1TPP1

ptfR

ap1

trans

crip

kno

ckdo

wn

of

%

Knockdown of RAP1 transcripts using 5 shRAP1 constructs. 1 2 3 4 5

O’Connor , JBC 2004

Strategy to study the in vivo function of Rap1

XX

RAP1RAP1

3’

TRF2 binding mutantTRF2 binding mutantTRF1TRF1

TIN2TIN2 TPP1TPP1 POT1POT15’

XX

TIN2TIN2 TPP1TPP1

Replacement of endogenous TRF2 ith R 1 bi diTRF2 with Rap1 binding

deficient mutant

shRNA mediated knockdown of TRF2 results in massive telomere fusions

Deng et. al., Nature 2009

Characterization of the TRF2-Rap1 interaction

Isothermal titration Calorimetry

Kd=16.5 nM

Ming Lei, U of Michigan

Purification and crystallization of the TRF2RBM and Rap1RCT complex

Crystal of the TRF2RBM-Rap1RCT complexgel-filtration SDS-PAGE

Crystal Structure of the TRF2RBM-Rap1RCT Complex at 1.95 A resolution

The TRF2RBM-Rap1RCT interface

TRF2: L288 Rap1: I318, F336

ITC analysis of the TRF2RBM-Rap1RCT interface

Yeast two-hybrid analysis of the TRF2RBM-Rap1RCT interface

Rap1 I318R TRF2 L288R

A single point mutation insidethe hydrophobic pocket issufficient to disrupt the ability of TRF2 to bind Rap1.

Rap1 F336R

Rap1 I318R TRF2 L288RTRF2 L288E

Co-IP analysis the TRF2-Rap1 Interface in mammalian cells

TRF2 is essential for Rap1 localization to telomeres

telomerestelomeres

The TRF2-Rap1 interaction not required for TRF2 telomere localization

telomeres

Minimal DNA damage response at telomeres in the absence of Rap1

TRF2, not Rap1, is required to repress the DDR at telomeres.

Rap1 is required to repress telomere HR

TTAGGG merge

25

53BP1-/-

53BP1-/-+shTRF

d2 FL

CCCTAA6R

10

15

20

chro

mos

ome

end

TRF

hTR

F2

TRF2

L286

Fl53BP1

shTR

f2FL5

3BP1

D1256

AD15

21R

S18

0

5

T-SC

E/sh

86R

TRF2

L2

Distinct roles for Rap1 and TRF2 in telomere end protection

TRF2TRF2

RAP1RAP1

TRF1TRF1 5’ TRF1TRF1 5’

TRF2 L286RTRF2 L286R

TRF1TRF1 5’

XX

TIN2TIN2 TPP1TPP1POT1POT1 3’



no DDR massive DDRrampant chrom fusions

minimal DDRfew chrom fusions

mTrf2shRNA/mTRF2 L286R

NHEJ repression rampant chrom fusions few chrom fusionstelomere HR

Rap1 is not required to repress DDR and NHEJ at telomeresRap1 repress HR at telomeres..

Repair of dysfunctional telomeres by the NHEJ pathway

O’Sullivan and Karlseder, Nature Reviews MCB 2010

Trf2 and Pot1a repress distinct DNA damage signaling pathways

TIN2 TPP1


TIN2 TPP1

POT1TRF1 TRF2/Rap1

ATM ATR

Possibility that TRF2 and POT1-TPP1 repress distinct DNA repair pathways at telomeres.

Denchi and de Lange, Nature 2007Guo et al; EMBO J 2007

Disruption of TRF2 leads to ATM-dependent telomere fusions through C-NHEJthrough C NHEJ

TIN2 TPP1


TIN2 TPP1

POT1TRF1MRN

ATMATR

Ku70/8053BP1

C-NHEJfactors

DNA-PK

Ligase 4

end-to-end chromosomefusionsDeng et al., Nature 2009

Dimitrova et al., MCB 2009

Denchi and de Lange, Nature 2007Guo et al; EMBO J 2007

Disruption of Pot1a-Tpp1 leads to ATR-dependent telomere fusions

TIN2


TIN2

TRF1TRF1 TRF2/Rap1

ATM ATR

? DNA damage signal

end-to-end chromosome fusions TIFsDenchi and de Lange, Nature 2007Guo et al; EMBO J 2007

Disruption of Pot1a-Tpp1 leads to chromosome fusions independent of C-NHEJ

TIN2


TIN2

TRF1TRF1 TRF2/Rap1MRN

ATRATM

Ku70/8053BP1

C-NHEJfactors

Ku70/80DNA-PK

Ligase 4

end-to-end chromosome fusions

Chromosome fusions in the absence of Tpp1-Pot1a does not require 53BP1

60

70

80

used

53BP1+/+ 53BP1 /

*****

** ****

60

70

80

used

53BP1+/+ shTRF2 53BP1-/- TPP1ΔRD

pty RF2 RDΔ RDΔ c

0

10

20

30

40

50

% o

f end

s f53BP1+/+ 53BP1-/-

ty F2 RDΔ RDΔ

0

10

20

30

40

50

% o

f end

s fu

53BP1-/- shTRF2 53BP1-/- shTRF2-TPP1ΔRD

Empsh

TRF Δ

TPP1

Δ

shTRF2+

TPP1 vec

Emptysh

TRF2 RΔ

TPP1

RΔ

shTRF2+

TPP1 Em

Chromosome fusions in the absence of Tpp1-Pot1a does not require Lig4

30d Lig4-/- 53BP1-/-

Lig4-/- shTRF2 Lig4-/- shTRF2-TPP1ΔRD

10

20

omos

ome

ends

fuse

Lig4 53BP1ve

ctor

shTR

F2 RDΔ

TPP1

RDΔ

shTRF2+

TPP1

RD

ig4+s

hTRF2+

TPP1sh

TRF

0% c

hro

53BP1-/- shLig4 shTRF2-TPP1ΔRD shLigLig4-/- TPP1ΔRD

Chromosome fusions in the absence of Tpp1-Pot1a does notrequire Ku70

Ku--/-shTRF2-TPP1ΔRD

***

**

***

**

60

d

Ku-/- shTRF2 Ku-/-+TPP1ΔRD Ku-/- -shTRF2-TPP1ΔRD

20

40

% o

f end

s fu

sed

Ku70-/- shLig4 + Ku70-/-

vecto

rsh

TRF2 RDΔ

TPP1

RDΔ

shTRF2+

TPP1 vecto

rsh

TRF2 RDΔ

TPP1

RDΔ

shTRF2+

TPP1

0

Ku-/- -shLig-shTRF2 Ku-/--shLig4-TPP1ΔRD Ku-/--shLig4-shTRF2-TPP1ΔRD

s s

A-NHEJ mediated repair of DNA double strand breaks

Rass et al., NSMB 2009

Disruption of Pot1a-Tpp1 leads to alternative NHEJ mediated chromosome fusions

TIN2


TIN2

TRF1TRF1 TRF2/Rap1MRN

ATRATM

No chromosome fusions

CtIP

No chromosome fusions

shCtIP-TPP1RD

CtIP is required for A-NHEJ mediated repair of telomeres lacking Tpp1-Pot1a

TPP1ΔRD shTRF2-TPP1ΔRDshTRF2

Fs

7553BP1-/-

53BP1-/- + shCTIPd

shTRF2-TPP1RD

53B

P1-/-

MEF

25

50

% o

f end

s fu

sed

shCtIP-shTRF2

vecto

r RDΔ

shTRF2+

TPP1 vecto

rsh

TRF2 RDΔ

TPP1

RDΔ

shTRF2+

TPP1

0

hTR

hTR

shCtIP-TPP1RD shCtIP-shTRF2-TPP1RD shT

shT

Naturally shortened telomeres are repaired independent of the C-NHEJ pathway

G4 mTerc-/- 53BP1-/- miceG4 mTerc-/- 53BP1+/-mice

1.0

1.5

er m

etap

hase

BM

BP1-/-

BP1+/-

BP1-/-

BP1+/-

BP1-/-

0.0

0.5

fusi

ons

pe

/ B

G1 BP

G1-G2 B

G3-G4 B

P

G3-G4 BG4 53BP1+/-

10

15

taph

ase

ma

0

5

fusi

ons

per

me

lym

pho m

G4 53BP1-/-

G2 BP1+

/+

G2 BP1-/

-

G4 BP1+

/+

G4 BP1-/

-

0

MEFs

Distinct repair pathways depending on how telomeres are uncapped

TIN2 TPP1


TIN2 TPP1

POT1TRF1TRF1

MRN

ATM ATR

C-NHEJ A-NHEJ53BP1Ku70/80Lig4

fusions fusionsLig4

Distinct repair pathways depending on how telomeres are uncapped

TIN2


TIN2

TRF1TRF1 TRF2/Rap1

MRN

natural telomere attrition

ATM ATR

C-NHEJ A-NHEJ

CtIP

fusions fusions

Acknowledgements

Pot1aLing Wu

Asha Multani

Funding

NIAAsha Multani

Pot1bHua He

E i Y

NCISusan Komen

Kadoorie Charitable TrustEric Yang

Rap1Rekha Rai

Ming Lei, U of Michigan

A-NHEJRekha RaiRekha Rai

Hong ZhengPhilip Carpenter, UT Medical

School

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