Title : The role of Gamma H2AX in apoptosis
Abstract:
Nowadays it is well established that γH2AX is the most sensitive biomarker to indicate loss of chromosomal integrity from DNA double-strand breaks in living cells. γH2AX denotes a specific phosphorylation at serine 139. This phosphorylation occurs rapidly after the generation of double-strand breaks, and extends along megabase-long domains of chromatin, each side of the break. γH2AX is induced not only by external agents that generate double strand breaks into the DNA of living cells, but also during cell functions mediated by DNA double-strand breaks, e.g., DNA repair, V(D)J recombination, meiotic recombination, class switching, and retroviral genome integration. The unequivocal role of γH2AX in DNA damage & repair signaling has been established bynumerous research publications for over 20 years. Curiously, γH2AX also forms during apoptosis, as the result of apoptosis-induced DNA fragmentation. One striking difference between the above-mentioned cellular pathways and apoptosis is that cells are destined to die after the activation of executional caspases, and the repair mechanisms are incapacitated by execution of the apoptotic program. To investigate this paradox, we have set out to experimentally approach several questions; Is γ-phosphorylation of H2AX in apoptosis a byproduct, or γH2AX has an integral distinct role in the apoptotic program? If yes, what is the role of γH2AX in apoptosis? And more interestingly, is there a common feature between the role of γH2AX in apoptosis and DNA damage & repair signaling? Our working hypothesis is that apoptosis and DNA-damage & repair signalling share common, initial, chromatin-related steps, but afterwards, the cellular pathways diverge. In a previous publication (Rogakou et al., 2000a) we have shown that, during the executional phase of apoptosis, γH2AX appears downstream of caspase activation. Quantitative analysis in 2D high-resolution histone gels indicate that the γ-phosphorylation accounts for 70 - 100% of the total H2AX. Surprisingly, immunocytochemistry on apoptotic cells fails to follow γH2AX induction, while immunoblotting is successful. This experimental evidence suggests that γH2AX epitope physically interacts with proteins during the course of apoptosis and becomes masked to antibodies used in immunocytochemistry. Further, we have developed a novel buffer to reveal the masked γ-epitope, designated REB. The restored immunofluorescent signal of γΗ2ΑΧ colocalizes with DNA. In addition, no changes to the DAPI pattern are observed after pretreatment, indicating that the γH2AX revealing protocol did not affect chromatin condensation in apoptotic cells. To identify novel factors that interact with γH2AX in apoptosis, we have developed a strategy that includes cell fractionation and selective extraction, based on REB. The extract was applied to a streptavidin column with an immobilized biotinylated c-terminal γH2AX peptide, phosphorylated at the reciprocal serine 139. Consecutive mass spectrometry analysis has identified novel factors that are candidates to physically interact with γH2AX during the execution phase of apoptosis. These novel γH2AX interactors that have been identified from apoptotic cells support our working hypothesis that these factors participate in fundamental mechanisms in both, apoptosis and DNA-damage & repair signalling, and differ on the 2nd class interactors
