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1
H2AX:
functional roles
and
potential applications
2
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
3
Introduction
Fig1.
H2AX
– 2~25% of mammalian H2A
– N/C terminal modification: acetylation, biotinylation, phosphorylaton, methylation, ubiquitination
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
-g H2AX
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Introduction
Fig1.
PI3K kinase family
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Introduction
7
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
14
H2AX roles in disease
• H2AX null mice shows
– Reduced isotype switching to IgG
– Radiation sensitivity
– Increased chromosomal abnormality
– Spermatogenesis defect: male sterility
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
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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
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
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
19
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
21
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
23