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Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG - Nature (2009)

Bottom Line: Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress.Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142).This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute School of Medicine, University of California, San Diego, California 92037, USA.

ABSTRACT
Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

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Eya interacts with H2AX in a DNA-damage dependent manner. (a) HA-tagged Eya1 or Eya3 interacts with FLAG-tagged H2AX in 293T cells in response to IR (5Gy), but not under basal conditions. (b) Co-immunoprecipitation experiments examining endogenous Eya3 protein using a specific Eya3 antibody recapitulated that interaction data for the tagged proteins. (c) Using sonicated chromatin as input, co-immunoprecipitation experiments showed that HA-Eya1/3 interacts with H2AX on chromatin. Immunostaining of 293T cells demonstrates that transfected, HA-tagged Eya1 (d) or Eya3 (e) localizes to DNA-damage induced foci coincident with γH2AX specifically after treatment with IR (5Gy, 1hr). Representative examples of foci formation are shown.
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Figure 2: Eya interacts with H2AX in a DNA-damage dependent manner. (a) HA-tagged Eya1 or Eya3 interacts with FLAG-tagged H2AX in 293T cells in response to IR (5Gy), but not under basal conditions. (b) Co-immunoprecipitation experiments examining endogenous Eya3 protein using a specific Eya3 antibody recapitulated that interaction data for the tagged proteins. (c) Using sonicated chromatin as input, co-immunoprecipitation experiments showed that HA-Eya1/3 interacts with H2AX on chromatin. Immunostaining of 293T cells demonstrates that transfected, HA-tagged Eya1 (d) or Eya3 (e) localizes to DNA-damage induced foci coincident with γH2AX specifically after treatment with IR (5Gy, 1hr). Representative examples of foci formation are shown.

Mentions: We therefore investigated a potential interaction between Eya and H2AX by co-immunoprecipitation assays using 293T embryonic kidney cells before and after exposing the cells to ionizing radiation to induce DNA damage. We could detect interactions between H2AX and wild-type Eya1 or Eya3 only under DNA damage conditions both using transfected, tagged expression constructs for Eya1/3 and H2AX (Fig. 2a), and when examining endogenous Eya3 and H2AX proteins with specific antibodies (Fig. 2b). Eya was capable of interacting with H2AX in the context of chromatin, based on co-immunoprecipitation experiments using fixed sonicated chromatin from 293T cells as input (Fig. 2c). In response to IR-induced double stranded DNA breaks, H2AX is phosphorylated by ATM/ATR PI3K-family kinases on chromatin forming long stretches of serine phosphorylated γH2AX flanking the break visible as γH2AX immunostained foci [18]. Endogenous Eya3 co-immunoprecipitated γH2AX in 293T cells after IR treatment (Fig. 2b, lower panel), and immunostaining of transfected HA-tagged Eya1 or Eya3 protein in 293T embryonic kidney cells revealed a clear co-localization of Eya with γH2AX foci after treatment with IR (Fig. 2d, e). These results suggest that in response to damage, Eya is recruited to H2AX foci that mark DNA double-strand breaks. To formally test this, we utilized the estrogen receptor-I PpoI system [19, 20], in which 4-hydroxytamoxifen (4-OHT) is used to induce activation of the eukaryotic homing endonuclease I-PpoI which then generates double stranded breaks at defined genomic loci, including a site on chromosome 1 within an intron of the DAB1 locus. ChIP analysis following 4-OHT induction of I-PpoI in 293T cells revealed that γH2AX and Eya3 were present at a 6 hour time point at a 4kb region flanking the IPpoI cut site, which is in consistent with a direct role for Eya in the cellular response to genotoxic stress (Supplementary Fig. 4).


Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG - Nature (2009)

Eya interacts with H2AX in a DNA-damage dependent manner. (a) HA-tagged Eya1 or Eya3 interacts with FLAG-tagged H2AX in 293T cells in response to IR (5Gy), but not under basal conditions. (b) Co-immunoprecipitation experiments examining endogenous Eya3 protein using a specific Eya3 antibody recapitulated that interaction data for the tagged proteins. (c) Using sonicated chromatin as input, co-immunoprecipitation experiments showed that HA-Eya1/3 interacts with H2AX on chromatin. Immunostaining of 293T cells demonstrates that transfected, HA-tagged Eya1 (d) or Eya3 (e) localizes to DNA-damage induced foci coincident with γH2AX specifically after treatment with IR (5Gy, 1hr). Representative examples of foci formation are shown.
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Related In: Results  -  Collection

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Figure 2: Eya interacts with H2AX in a DNA-damage dependent manner. (a) HA-tagged Eya1 or Eya3 interacts with FLAG-tagged H2AX in 293T cells in response to IR (5Gy), but not under basal conditions. (b) Co-immunoprecipitation experiments examining endogenous Eya3 protein using a specific Eya3 antibody recapitulated that interaction data for the tagged proteins. (c) Using sonicated chromatin as input, co-immunoprecipitation experiments showed that HA-Eya1/3 interacts with H2AX on chromatin. Immunostaining of 293T cells demonstrates that transfected, HA-tagged Eya1 (d) or Eya3 (e) localizes to DNA-damage induced foci coincident with γH2AX specifically after treatment with IR (5Gy, 1hr). Representative examples of foci formation are shown.
Mentions: We therefore investigated a potential interaction between Eya and H2AX by co-immunoprecipitation assays using 293T embryonic kidney cells before and after exposing the cells to ionizing radiation to induce DNA damage. We could detect interactions between H2AX and wild-type Eya1 or Eya3 only under DNA damage conditions both using transfected, tagged expression constructs for Eya1/3 and H2AX (Fig. 2a), and when examining endogenous Eya3 and H2AX proteins with specific antibodies (Fig. 2b). Eya was capable of interacting with H2AX in the context of chromatin, based on co-immunoprecipitation experiments using fixed sonicated chromatin from 293T cells as input (Fig. 2c). In response to IR-induced double stranded DNA breaks, H2AX is phosphorylated by ATM/ATR PI3K-family kinases on chromatin forming long stretches of serine phosphorylated γH2AX flanking the break visible as γH2AX immunostained foci [18]. Endogenous Eya3 co-immunoprecipitated γH2AX in 293T cells after IR treatment (Fig. 2b, lower panel), and immunostaining of transfected HA-tagged Eya1 or Eya3 protein in 293T embryonic kidney cells revealed a clear co-localization of Eya with γH2AX foci after treatment with IR (Fig. 2d, e). These results suggest that in response to damage, Eya is recruited to H2AX foci that mark DNA double-strand breaks. To formally test this, we utilized the estrogen receptor-I PpoI system [19, 20], in which 4-hydroxytamoxifen (4-OHT) is used to induce activation of the eukaryotic homing endonuclease I-PpoI which then generates double stranded breaks at defined genomic loci, including a site on chromosome 1 within an intron of the DAB1 locus. ChIP analysis following 4-OHT induction of I-PpoI in 293T cells revealed that γH2AX and Eya3 were present at a 6 hour time point at a 4kb region flanking the IPpoI cut site, which is in consistent with a direct role for Eya in the cellular response to genotoxic stress (Supplementary Fig. 4).

Bottom Line: Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress.Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142).This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute School of Medicine, University of California, San Diego, California 92037, USA.

ABSTRACT
Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

Show MeSH
Related in: MedlinePlus