David G. Johnson, Ph.D.Professor

Department of CarcinogenesisUniversity Of Texas MD Anderson Cancer Center

Science Park - Research Division

E2F1 Functions as an Accessibility Factor for DNA Repair

Regulation of E2F Transcriptional Activity

RbS Phase GenesE2F

Cyclin D/cdk 4/6Cyclin E/cdk 2

Rb

S Phase GenesE2F

P

PP

P

Repression

Activation

DP

DP

CdkInhibitors

corepressors

coactivators

G0,early G1

late G1,S phase

E2F Target GenesDNA Replication Cell Cycle Regulation Transcription FactorsDNA polymerase α Cyclins A, B, E E2F1, 2, 3aPCNA Cdk1, 2 c-mycReplication protein A Cdc25 c-mybReplication factor C4 Bub1 B-mybCdc6 p107 NFATc1, c3Mcm2, 3, 4, 5, 6, 7 p18INK4c HMG2, 4Orc1 p19INK4d Homeobox A10, 7, 9Topoisomerase p19ARF Enhancer of zeste

Nucleotide Biosynthesis Apoptosis DNA repairRibonucleotide reductase p73 Rad51Thymidine kinase Apaf1 BRCA1DHFR Caspase 3, 7 Uracil DNA gylcosylaseThymidylate synthase ASK1 MSH2, 6Deoxycytidine kinase AMPKα2 DNA polymerase δ

The E2F FamilyCyclin Abinding

DNAbinding

Dimer-ization

Markedbox

Rb familybinding

E2F1

E2F2

E2F3a

E2F3b

E2F4

E2F5

E2F6

E2F7

E2F8

Transactivation

E2F1 Responds to DNA damage

1) E2F1 is unique among the E2F family in that it is stabilized in response to a variety of DNA damaging agents, much like p53 (Blattner, 1999; Hofferer, 1999).

2) This involves phosphorylation of E2F1 at serine 31 by the ATM or ATR kinasesand E2F1 binding to 14-3-3τ (Lin, 2001; Wang, 2004).

3) E2F1 is also acetylated in response to double-strand breaks, but not UV-induced damage, and this is associated with E2F1-mediated induction of p73 gene expression and the promotion of apoptosis (Pediconi, 2003; Ianari, 2004).

4) Serine 31 phosphorylation also creates a binding site for one of the BRCT domains in TopBP1. This represses E2F1’s transcriptional activity independent of Rb and suppresses E2F1-mediated apoptosis (Liu 2004; Liu, 2004).

DNAbinding

Dimer-ization

Markedbox

Rb familybinding

E2F1

Transactivation

PSerine 31 phosphorylation

Ac Ac AcLysine 117, 121, 125 Acetylation

14-3-3τ & TopBP1binding

Binding to TopBP1 also “Sequesters”E2F1 at sites of DNA damage

untreated

doxorubicin

Hoechst33258

E2F1 TopBP1 Overlay

Lui et al,. 2004

E2F1

tubulin

-UVB 4 h 8h 12h 18h 24h

E2F1 Suppresses UVB-Induced apoptosis

E2f1 Status Does not Affect p53 Induction or Activation of MAP Kinases in Response to UV

UVB - - - + + + (1 h)

E2F1 -/- +/+ 1.1 -/- +/+ 1.1

ERK 1ERK2

p-ERK 1P-ERK2

p38

p-p38

β-tubulin0123456789

10

no UVB 3 hr

% p

-p53

(S18

) /

10 m

m e

pide

rmis

Wild typeE2f1-/-

Inactivation of E2f1 Reduces DNA Repair Efficiency

Transgenic Expression of E2F1 Stimulates DNA Repair and Suppresses

Apoptosis Following UV Irradiation

Acknowledgements

Ruifeng (Ray) Gou and Jie (Claire) Chen

E2F1 Suppresses Apoptosis and Promotes Cell Survival in Response to UV Irradiation

MTT Viability AssayApoptosis Assay

siCon siE2F1 siCon siE2F1

UV0 h

6 h

24 h

UV0 h

6 h

24 h

Anti-6-4 PP Anti-CPD

Knockdown of E2F1 Reduces DNA Repair Efficiency of UV Photoproducts

E2F1 Deficiency Does Not Alter the Expression of NER Factors

E2F1 Accumulates at Sites of UV-induced Damage

XPC GFP-E2F1 Merge+DAPI

p62 GFP-E2F1 Merge+DAPI

UVC50 J/m2

8um30min

E2F1 Co-localizes with XPC and p62 (TFIIH) and Associates with XPA in Response to UV Irradiation

NHFInput

UVB 500 J/m2 - +

IB:E2F1

IB:XPA

IP: IgG E2F1 E2F1UVB: + - +500 J/m2

IB:E2F1

IB:XPA

E2F1 Localization to Sites of UV Damage Requires ATR

E2F1 Serine 31 is Required for Localization to Sites of UV Damage

Knockdown of E2F1 Impairs Recruitment of NER Factors to Sites of UV Damage

Histone Acetylation and Chromatin Relaxation in Response to UV-induced DNA Damage

• Experiments by the Frieberg (1991) and Sancar (2000) groups demonstrated that nucleosomes can inhibit NER.

• Smerdon and coworkers originally demonstrated that UV irradiation increased histone acetylation, which stimulated DNA repair (1982-1986).

• Other groups have demonstrated that p53, ING1/2, and p300 participate in a pathway that promotes repair by inducing histone H4 acetylation, chromatin relaxation, and increased accessibility to NER factors.

Chromatin Relaxation in Response to UV Is Impaired by E2F1 Deficiency

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

Avera

ge N

ucl

eo

som

e S

ize

siRNA Con Con Con E2F1 E2F1 E2F1UV - 5min 30min - 5min 30min

Micrococcal Nuclease Assay

The GCN5 Histone Acetyltransferase (HAT) Accumulates at Sites of UV damage

UV - + - +

E2F1 Interacts with GCN5 Following UV Irradiation and Recruits GCN5 to Sites of Damage

Knockdown of GCN5 Impairs Recruitment of NER factors to Sites of UV Damage

B

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

Avera

ge N

ucl

eo

som

e S

ize

siRNA Con E2F1 GCN5UV - 5min 30min - 5min 30min - 5min 30min

Knockdown of GCN5 Prevents Chromatin Relaxation in Response to UV

siCon siE2F1 siGCN5 siCon siE2F1 siGCN5UV

0 h

6 h

24 h

UV

0 h

6 h

24 h

Anti-6-4 PP Anti-CPD

Knockdown of E2F1 or GCN5 Reduces DNA Repair Efficiency

Increased H3 K9 Acetylation in Response to UV Is Impaired by E2F1 or GCN5 Deficiency

Rapid Accumulation of Acetylated H3 K9 and H4 K16 at Sites of UV damage

Knockdown of E2F1 or GCN5 Impairs H3 K9 Acetylation at Sites of UV Damage

E2F1 and GCN5 Are Not Required for H4 K16 Acetylation at Sites of UV Damage

The DNA Damage Response Converts E2F1 Into an Accessibility Factor for DNA Repair

S Phase Genes

E2F1Transcriptional Regulation DP

coactivators

E2F1 DP

GCN5

TopBP1

Rad9

Hus1Rad1 CPD, 6-4 PP, DSB, other?

DNA Repair

Histone acetylation, increased access to basal transcription machinery

H3K9 acetylation, increased access to DNA repair machinery

ATM/ATR

X

P

DNA damage

E2F1 Localizes to Nuclear Foci in Response to Ionizing Radiation

Mock

IR

DAPI DAPIγH2AX E2F1

1 2 3 4 5 6 7 8 9 10 11 12

γH2AX

GAPDH

E2f1+/+ E2f1 -/-

Un 0h 2.5h 6h 12h 24h Un 0h 2.5h 6h 12h 24h

Inactivation of E2F1 Affects the Kinetics of H2AX Phosphorylation Following IR Exposure

γH2AX E2F1 Merge+DAPI� � �

NoTreatment

HUTreatment

NCSTreatment

E2F1 Foci Formation in Cells Treated with Hydroxyurea (HU) and Neocarzinostatin (NCS)

-- HU NCS -- HU NCS

p-Chk1Ser 345

E2f1+/+ E2f1-/-

Chk1

Increased Chk1 Activation in the Absence of E2F1

E2f1+/+

E2f1-/-

γH2AX Merge +DAPI

Accumulation of DNA damage in Untreated Primary E2f1-/- Cells

Cytogenetic Analysis of Primary Keratinocytes Indicates Genomic Instability in the Absence of E2F1

Genotype % aberrant met. % with breaks % with fragments % with fusionsWild type 13.0 10.3 0 4.6E2f1-/- 31.5 18.6 4.6 17.4

Future Directions

• Determine how E2F1 is recruited to sites of DNA damage.

• Determine how E2F1 recruits GCN5 to sites of DNA damage.

• Determine if and how E2F1 participates in the repair of double-strand breaks and perhaps other types of DNA damage.

• Determine if E2F1 associates with other chromatin modifiers and mediates other histone modifications in response to DNA damage.

The DNA Damage Response Converts E2F1 Into an Accessibility Factor for DNA Repair

S Phase Genes

E2F1Transcriptional Regulation DP

coactivators

E2F1 DP

GCN5

TopBP1

Rad9

Hus1Rad1 CPD, 6-4 PP, DSB, other?

DNA Repair

Histone acetylation, increased access to basal transcription machinery

H3K9 acetylation, increased access to DNA repair machinery

ATM/ATR

X

P

DNA damage

E2F1 S31A Knock in Scheme

pgK NeoCAT/SV40pA DT-A TK PyF

101

Sma1 Xba1 Sal1 SacII SacI

1 2 3

✶S31A

✶

Wild type E2f1 allele

AviII

pgK Neo

✶

Targeting vector

E2f1 S31A Knock-in allele

S31A

S31A

BamH1 BamH1

5´ Probe 3´ Probe

5´ Probe 3´ Probe

BamH1 BamH1AviII

AviII

E2f1 S31A Knock-in allele after Neo excision

BamH1 BamH1

3´ Probe5´ Probe

E2F1 Can Behave as Both an Oncogene and Tumor Suppressor Gene in Mouse Models

E2F1

MitogenicSignals

DNA Damage

DNA RepairProliferation

Oncogenesis Tumor Suppression

DNA damage converts E2F1 into a transcriptional repressor as well as a DNA repair factor

E2F1 DP

HATs

E2F1 DP

GCN5

TopBP1

Rad9

Hus1Rad1

P

Histone acetylation,Induction of transcription

E2F1 targetsATM/ATR

X

Histone acetylation,stimulation of repair

DNA damage

E2F1 targetsDNA damage

E2F1 DP

TopBP1P

Chromatin remodeling,transcriptional repressionBrg/Brm

SWI/SNF

Differences in Photoproduct Removal Are Not Due to Differences in DNA Synthesis

E2f1; p53 Double Knockouts are Hypersensitive to UV-induced Apoptosis

0

10

20

30

40

50

60

70

SBC

/10

mm

epi

derm

is

Dose (mJ/cm2)

0 10025 50

E2F1 +p53 DKO

E2F1 KO

Wildtype

p53 KO

Berton et al., 2005