To begin to evaluate a possible connection between Eya-mediated tyrosine dephosphorylation of H2AX Y142 and modulation of the apoptotic response, we examined the function of this phosphotyrsoine mark in the context of the DNA damage response. FLAG-tagged H2AX Y142F mutant was phosphorylated on S139 in response to damage, although at levels significantly lower than FLAG-tagged wild-type H2AX. () Time course anlysis of S139 phoshorylation of H2AX Y142F in response to 10Gy IR in 293T human embryonic kidney cells revealed consistently reduced levels compared to wild type between 1 and 8 hours (Supplementary Fig. 8
). Thus, while Y142 phosphorylation does not function as a pre-requisite for S139 phosphorylation in the DNA damage response [24
], it may play a significant role in promoting or maintaining serine phosphorylation by DNA-damage response kinases.
Figure 5 H2AX Y142 phosphorylation discriminates between apoptotic and repair responses to DNA-damage. (a) S139 phosphorylation of H2AX Y142F is present but reduced in comparison to wild type H2AX after 5Gy IR. (b) Affinity purification performed on nuclear extract (more ...)
It has been established that a key function of H2AX S139 phosphorylation is to provide a docking site for DNA repair factors near or at DNA double strand breaks [18
]. These factors include Mediator of DNA Damage Checkpoint protein 1 (MDC1) which has been shown to bind directly to phosphorylated S139 of H2AX at the sites of double strand breaks [24
] based on tandem BRCT1 repeats within the C-terminus of MDC1 [25
]. MDC1 functions in the recruitment of a set of ancillary repair factors including MRE11, RAD50, NBS1 (the MRN complex), 53BP1 and BRCA1 [26
], although these factors are not wholly dependent on MDC1 and γH2AX for recruitment to breaks [28
]. Because an intact H2AX COOH-terminal tyrosine has been found to be required for MDC1-H2AX interaction and productive DNA repair [24
], it was of particular interest to determine whether persistent phosphorylation of Y142 in the absence of Eya could negatively impact MDC1 recruitment to the tail of γH2AX. We first generated peptides corresponding to the C-terminal tail of H2AX with phosphorylation of both S129 and Y142, or of S139 alone. Peptides lacking any phosphorylation marks or where tyrosine 142 was mutated to alanine failed to interact with MDC1, consistent with previously published reports (Supplementary Fig. 9
]. Affinity purification of nuclear extract from irradiated 293T cells with each peptide revealed that, in the absence of Y142 phosphorylation, a set of DNA repair factors including MDC1, MRE11 and Rad50 were bound to the S139 phosphorylated H2AX peptide (). Intriguingly, when phosphorylated tyrosine 142 was present with phosphoserine 139, binding of these factors was greatly reduced; instead, the established pro-apoptotic factor JNK1 was now present (). The stress-response kinase JNK1, activated by DNA damage and initiating a pro-apoptotic program, has been recently shown to translocate into the nucleus upon activation where it phosphorylates substrates including H2AX S139, an event critical for DNA degradation mediated by caspase-activated DNase (CAD) in apoptotic cells [10
]. In agreement with our peptide purification experiments, we were able to detect a robust interaction between transfected wild-type H2AX and endogenous JNK1 in 293T cells in response to high-dose radiation; this interaction was markedly reduced in the case of the H2AX Y142F mutant ().
To further confirm the specificity of these phosphorylation-dependent interactions we performed peptide competition assays. The H2AX tail peptide phosphorylated on S139 alone was able to effectively compete for binding of MDC1 in a peptide pull-down assay, while the free peptide bearing both S139 and Y142 phosphorylation marks competed away interaction with JNK1 (Supplementary Fig. 10
Based on our previous data that loss of Eya phosphatase results in increased tyrosine phosphorylation of H2AX, we predicted that depleting Eya in 293T cells would result in decreased binding of MDC1 to H2AX in response to DNA damage. We knocked down Eya3 using specific siRNA and subsequently tested for MDC1-H2AX interaction by co-immunoprecipitation. As predicted, loss of Eya3 resulted in complete loss of this interaction in comparison to untransfected cells treated with 10Gy IR (Supplementary Fig. 11
It was of particular interest to identify proteins containing SH2 and PTB phosphotyrosine-binding domains that could bind directly to H2AX phospho-tyrosine 142 under conditions of genotoxic stress. We tested a set of known nuclear proteins containing these domains for binding to tyrosine-phosphorylated H2AX (Supplementary Table 1
, partial list) and found that, while most exhibited no interaction, the PTB-domain protein Fe65 [29
], a co-factor for several cell-surface receptors that has been shown to translocate to the nucleus during DNA damage response and suggested to exert a pro-apoptotic role [30
], bound specifically to wild type γH2AX under DNA damage conditions, but not to the γH2AX Y142F mutant (). Significantly, we found that Fe65 protein interacted with endogenous JNK1 by co-immunoprecipitation in 293T cells treated with the DNA-damage agent etoposide (), consistent with the idea that Fe65 helps to mediate JNK1 recruitment to γH2AX. Co-immunoprecipitation experiments demonstrated that the second PTB domain on Fe65 may be key for the interaction between Fe65 and tyrosine phosphorylated H2AX (Supplementary Fig. 12a
). GST pull-down assays using purified recombinant protein of Fe65 PTB domains 1 and 2 also revealed a direct interaction between PTB2 and the H2AX present in purified HeLa histones (Supplementary Fig. 12b
). We postulated that Fe65 may function as an adaptor protein, binding directly to the phosphotyrosine reside on γH2AX via PTB2 and facilitating the recruitment of pro-apoptotic factors such as JNK1. To test this, we knocked down endogenous Fe65 in 293T cells using specific siRNAs (Supplementary Figure 2
) and assessed the interaction between H2AX and JNK1 in response to genotoxic stress by co-immunoprecipitation. While control siRNA had no effect on the ability of H2AX to co-immunoprecipitate JNK1, knockdown of Fe65 strongly inhibited this interaction ().
To confirm the function of tyrosine 142 phosphorylation in regulation of the apoptotic response, we transfected H2AX
mouse embryonic fibroblasts (MEFs) [32
] with either wild type or Y142F H2AX expression constructs. When these cells were subjected to high-dose ionizing radiation, cells expressing H2AX Y142F displayed a reduced apoptotic response in comparison to cells expressing wild-type H2AX (~6-fold decrease) (). These data suggested to us that lack of H2AX Y142 phosphorylation promotes a damage repair response instead of an apoptotic response to DNA damage, in part by promoting successful recruitment of MDC1 and associated repair factors. The presence of Y142 phosphorylation in wild type-H2AX transfected MEF cells is proposed to lead to the recruitment of pro-apoptotic factors such as JNK1 to H2AX, while inhibiting the recruitment of the damage repair complex, directly promoting apoptotic response to genotoxic stress.