1. 1 H2AX: functional roles and potential applications

2. 2 the role of  -H2AX in homeostasis uses of  -H2AX in the understanding of DNA DSB formation the role of -H2AX in disease uses of  -H2AX to repair in cancer treatment

3. 3 Introduction Fig1. H2AX – 2~25% of mammalian H2A – N/C terminal modification: acetylation, biotinylation, phosphorylaton, methylation, ubiquitination

4. 4 Introduction Fig1. Unique C terminal tail – Serine four AAs from C terminal end: omega-4 – omega-4 and surrounding motif highly conserved H2AX p  H2AX

5. 5 Introduction Fig1. PI3K kinase family

6. 6 Introduction

7. 7 Fig1. Anti-γ-H2AX antibody available – Multiple foci detected in nucleus – Foci may represent DNA damage – Potential biomarker for clinical use

8. 8 H2AX as a key regulator of the DNA damage response

9. 9 Proteins recruited by γ-H2AX – 53BP1, MDC1, RAD51, BRCA1, MRE11/RAD50/NBS1(MRN) complex – Ubiquitin ligase cascade (RNF8-RNF168-UBC13) – Cohesin – FA/BRCA – TIP60-UBC13

10. 10 H2AX as a key regulator of the DNA damage response MRN complex – binds to DSB and initiate repair – End-processing of DSB : makes single strand DNA at the broken end

11. 11 H2AX as a key regulator of the DNA damage response BRCA1 – Repairs DNA DSB by homologous recombination with RAD51 FA/BRCA pathway – DNA repair pathway

12. 12 H2AX as a key regulator of the DNA damage response H2AX Y142 – Constitutive phosphorylation by WSTF – Dephosphorylaion by EYA1 or EYA3 after DSB – Dephosphorylaion allows MDC1 binding

13. 13 H2AX as a key regulator of the DNA damage response Checkpoint response – H2AX is required for G 2 -M checkpoint activation at low dose of ionizing radiation – Maintain response while repairing DNA or induce apoptosis if not repairable

14. 14 H2AX roles in disease H2AX null mice shows – Reduced isotype switching to IgG – Radiation sensitivity – Increased chromosomal abnormality – Spermatogenesis defect: male sterility

15. 15 H2AX roles in disease H2AX/p53 double KO – Increased cancer susceptibility including T/B cell lymphoma – Short lifespan as early as 6 weeks H2AX/ATM double KO – Embryonic lethality with chromosomal aberration – Hypersensitive to ROS

16. 16 H2AX roles in disease Mutation on H2AX locus 11q23 leads – Acute myeloid leukemia – Acute lymphoblastic leukemia – Colorectal cancer – Head and neck squamous cell carcinoma

17. 17 H2AX roles in disease H2AX and cancer susceptibility – G/A SNP 417bp upstream H2AX correlated to non-Hodgkins lymphoma – Downregulated H2AX in human gastrointestinal stromal tumor cells

18. H2AX roles in disease H2AX and aging – γ-H2AX increase w/o intentional damage in aging cells – Caused by dysfunctional telomeres and non- telomeric DNA DSB 18

19. γ-H2AX as a biomarker H2AX can be efficient biomarker – H2AX commonly used for genome integrity examination, drug development, translational studies – Antibodies commercially available 19

20. γ-H2AX as a therapeutic target No drugs targeting H2AX or PI3 kinases for H2AX developed – PI3 kinase inhibitor available (AstraZeneca/KuDos) H2AX might not be good target – ubiquitous to all cells – Structural function for chromatin integrity – Relatively long half-life – Similarity to other H2As 20

21. γ-H2AX as a therapeutic target Practical target: inhibition of H2AX phosphorylation – Peptide inhibitor for chemotherapeutic agent or treat with radiation therapy – Caffeine and wortmannin inhibits PI3K activity – Side effect: may affect noncancerous cells 21

22. γ-H2AX as a indicator of environmental health risks Testing possible DNA-damaging compounds – cigarette smoke, polycyclic aromatic compounds, dinitrobenzo[e]pyrene, norethindrone, chromium, crude oil, electromagnetic fields, microwaves from mobile phones, extreme heat… Testing environmental factors – radiation induced bystander effect, biological effects of high charge and energy ions during space exploration, terrorism threats from dirty bombs 22

23. Conclusions γ-H2AX is sensitive indicator of DNA DSB – detection of genotoxic stress – monitoring cancer and its therapeutic progress – Studying cellular response to DNA damage 23