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H2AX:

functional roles

and

potential applications

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the role of g-H2AX in homeostasis

uses of g-H2AX in the understanding of DNA DSB formation

the role of -H2AX in disease

uses of g-H2AX to repair in cancer treatment

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Introduction

Fig1.

H2AX

– 2~25% of mammalian H2A

– N/C terminal modification: acetylation, biotinylation, phosphorylaton, methylation, ubiquitination

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Introduction

Fig1.

Unique C terminal tail

– Serine four AAs from C terminal end: omega-4

– omega-4 and surrounding motif highly conserved

H2AX

p

-g H2AX

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Introduction

Fig1.

PI3K kinase family

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Introduction

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Introduction

Fig1.

• Anti-γ-H2AX antibody available

– Multiple foci detected in nucleus

– Foci may represent DNA damage

– Potential biomarker for clinical use

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H2AX as a key regulator of the DNA damage response

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H2AX as a key regulator of the DNA damage response

• 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

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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

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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

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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

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H2AX as a key regulator of the DNA damage response

• Checkpoint response

– H2AX is required for G2-M checkpoint

activation at low dose of ionizing radiation

– Maintain response while repairing DNA or

induce apoptosis if not repairable

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H2AX roles in disease

• H2AX null mice shows

– Reduced isotype switching to IgG

– Radiation sensitivity

– Increased chromosomal abnormality

– Spermatogenesis defect: male sterility

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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

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H2AX roles in disease

• Mutation on H2AX locus 11q23 leads

– Acute myeloid leukemia

– Acute lymphoblastic leukemia

– Colorectal cancer

– Head and neck squamous cell carcinoma

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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

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

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γ-H2AX as a biomarker

• H2AX can be efficient biomarker

– H2AX commonly used for genome integrity

examination, drug development, translational studies

– Antibodies commercially available

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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

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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

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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 bombs22

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

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