E2F1 Functions as an Accessibility Factor for DNA Repair

David G. Johnson, Ph.D. Professor Department of Carcinogenesis University Of Texas MD Anderson Cancer Center Science Park - Research Division Regulation of Transcriptional Activity

corepressors G0, Rb early G1 E2F DP S Phase Repression

Cdk Cyclin D/cdk 4/6 P Inhibitors Cyclin E/cdk 2 P Rb P P

late G1, coactivators S phase E2F DP S Phase Genes Activation E2F Target Genes

DNA Replication Regulation Transcription Factors DNA polymerase α Cyclins A, B, E , 2, 3a PCNA Cdk1, 2 c- Replication A Cdc25 c- Replication factor C4 Bub1 B-myb Cdc6 p107 NFATc1, c3 Mcm2, 3, 4, 5, 6, 7 p18INK4c HMG2, 4 Orc1 p19INK4d A10, 7, 9 Topoisomerase p19ARF Enhancer of zeste

Nucleotide Biosynthesis DNA repair Ribonucleotide reductase Rad51 Thymidine kinase Apaf1 BRCA1 DHFR Caspase 3, 7 Uracil DNA gylcosylase Thymidylate synthase ASK1 MSH2, 6 Deoxycytidine kinase AMPKα2 DNA polymerase δ The E2F Family Transactivation Cyclin A DNA Dimer- Marked Rb family binding binding ization box binding E2F1

E2F2

E2F3a

E2F3b

E2F4

E2F6

E2F7

E2F8 E2F1 Responds to DNA damage

Transactivation 14-3-3τ & TopBP1 binding DNA Dimer- Marked Rb family binding ization box binding E2F1 P Ac Ac Ac Serine 31 Lysine 117, 121, 125 phosphorylation Acetylation

1) E2F1 is unique among the E2F family in that it is stabilized in response to a variety of DNA damaging agents, much like (Blattner, 1999; Hofferer, 1999). 2) This involves phosphorylation of E2F1 at serine 31 by the ATM or ATR kinases and 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 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). Binding to TopBP1 also “Sequesters” E2F1 at sites of DNA damage

untreated

doxorubicin

Hoechst E2F1 TopBP1 Overlay 33258 Lui et al,. 2004 -UVB 4 h 8h 12h 18h 24h E2F1 tubulin

E2F1 Suppresses UVB-Induced apoptosis E2f1 Status Does not Affect p53 Induction or Activation of MAP Kinases in Response to UV

UVB - - - + + + Wild type (1 h) E2f1-/- ERK 1 ERK2 10 9 p-ERK 1 8 P-ERK2 7 6 5 4 p38

% p-p53(S18) / 3 10 mm epidermis 2 p-p38 1 0 no UVB 3 hr β-tubulin

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

Apoptosis Assay MTT Viability Assay Knockdown of E2F1 Reduces DNA Repair Efficiency of UV Photoproducts

siCon siE2F1 siCon siE2F1 UV UV 0 h 0 h

6 h 6 h

24 h 24 h

Anti-6-4 PP Anti-CPD E2F1 Deficiency Does Not Alter the Expression of NER Factors E2F1 Accumulates at Sites of UV-induced Damage E2F1 Co-localizes with XPC and p62 (TFIIH) and Associates with XPA in Response to UV Irradiation

XPC GFP-E2F1 Merge+DAPI

UVC 50 J/m2 8um p62 GFP-E2F1 Merge+DAPI 30min

NHF IP: IgG E2F1 E2F1 Input UVB UVB: + - + 2 500 J/m2 -+ 500 J/m

IB:E2F1 IB:E2F1

IB:XPA 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

2

1.9

1.8

1.7

1.6

1.5

1.4

1.3

Average Nucleosome Size 1.2

1.1

1

siRNA Con Con Con E2F1 E2F1 E2F1 UV - 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 Knockdown of GCN5 Prevents Chromatin Relaxation in Response to UV

2.6

2.4

2.2

2

1.8

1.6

1.4 Average Nucleosome Size Nucleosome Average

1.2

1

siRNA Con E2F1 GCN5 UV - 5min 30min - 5min 30min - 5min 30min Knockdown of E2F1 or GCN5 Reduces DNA Repair Efficiency

siCon siE2F1 siGCN5 siCon siE2F1 siGCN5 UV UV

0 h 0 h

6 h 6 h

24 h 24 h

Anti-6-4 PP Anti-CPD 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

Histone acetylation, Transcriptional coactivators increased access to basal Regulation E2F1 DP transcription machinery

S Phase Genes DNA ATM/ATR damage

GCN5

E2F1 DP H3K9 acetylation, P increased access to DNA Repair TopBP1 DNA repair machinery Rad9 X Rad1 Hus1 CPD, 6-4 PP, DSB, other? E2F1 Localizes to Nuclear Foci in Response to Ionizing Radiation

DAPIγH2AX DAPI E2F1

Mock

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

E2f1+/+ E2f1 -/-

Un 0h 2.5h 6h 12h 24h Un 0h 2.5h 6h 12h 24h γH2AX

GAPDH

1 2 3 4 5 6 7 8 9 10 11 12 E2F1 Foci Formation in Cells Treated with Hydroxyurea (HU) and Neocarzinostatin (NCS)

γH2AX E2F1 Merge+DAPI No Treatment

HU Treatment

NCS Treatment Increased Chk1 Activation in the Absence of E2F1

E2f1+/+ E2f1-/- -- HU NCS -- HU NCS

p-Chk1 Ser 345

Chk1 Accumulation of DNA damage in Untreated Primary E2f1-/- Cells

γH2AX Merge +DAPI

E2f1+/+

E2f1-/- Cytogenetic Analysis of Primary Keratinocytes Indicates Genomic Instability in the Absence of E2F1

Genotype % aberrant met. % with breaks % with fragments % with fusions Wild type 13.0 10.3 0 4.6 E2f1-/- 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

Histone acetylation, Transcriptional coactivators increased access to basal Regulation E2F1 DP transcription machinery

S Phase Genes DNA ATM/ATR damage

GCN5

E2F1 DP H3K9 acetylation, P increased access to DNA Repair TopBP1 DNA repair machinery Rad9 X Rad1 Hus1 CPD, 6-4 PP, DSB, other?

E2F1 S31A Knock in Scheme

5´ Probe 3´ Probe 1 2 3 Wild type E2f1 allele

BamH1 BamH1

S31A ✶ CAT/ PyF Targeting vector DT-A TK pgK Neo SV40pA 101

Sma1 Xba1 AviII Sal1 SacII SacI

5´ Probe S31A 3´ Probe ✶ E2f1 S31A Knock-in allele pgK Neo

AviII BamH1 BamH1

5´ Probe S31A 3´ Probe ✶ E2f1 S31A Knock-in allele after Neo excision

AviII BamH1 BamH1 E2F1 Can Behave as Both an Oncogene and in Mouse Models

Mitogenic DNA Damage Signals

E2F1

Proliferation DNA Repair

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

Histone acetylation, HATs Induction of transcription E2F1 DP E2F1 targets DNA damage ATM/ ATR Chromatin GCN5 SWI/SNF remodeling, transcriptional E2F1 DP Brg/Brm repression P TopBP1 Histone acetylation, TopBP1 stimulation of repair P Rad9 E2F1 DP X Rad1 Hus1 E2F1 targets DNA damage Differences in Photoproduct Removal Are Not Due to Differences in DNA Synthesis E2f1; p53 Double Knockouts are Hypersensitive to UV-induced Apoptosis

Berton et al., 2005 70

60 E2F1 + p53 DKO 50

E2F1 KO 40

30 Wild type 20

SBC/10 mm epidermis 10 p53 KO

0 0 25 50 100 Dose (mJ/c2m)