The replacement histone variant H2AX senses DNA double-strand breaks (DSBs) and recruits characteristic sets of proteins at its phosphorylated (γ-H2AX) foci for concurrent DNA repair. We reasoned that the H2AX interaction network, or interactome formed in the tumor-associated DNA DSB environment such as in hepatocellular carcinoma (HCC) cells, where pre-neoplastic lesions frequently occur, is indicative of HCC pathogenic status. By using an in vivo dual-tagging quantitative proteomic method, we identified 102 H2AX-specific interacting partners in HCC cells that stably expressed FLAG-tagged H2AX at close to the endogenous level. Using bioinformatics tools for data-dependent network analysis, we further found binary relationships among these interactors in defined pathway modules, implicating H2AX in a multi-functional role of coordinating a variety of biological pathways involved in DNA damage recognition and DNA repair, apoptosis, nucleic acid metabolism, Ca2+-binding signaling, cell cycle, etc. Furthermore our observations suggest that these pathways interconnect through key pathway components or H2AX interactors. The physiological accuracy of our quantitative proteomic approach in determining H2AX-specific interactors was evaluated by both co-immunoprecipitation/ immunoblotting and confocal co-localization experiments performed on HCC cells. Due to their involvement in diverse functions, the H2AX interactors involved in different pathway modules, such as Poly(ADP-ribose) polymerase1, 14-3-3ζ, coflin1, and peflin1, were examined for their relative H2AX binding affinities in paired hepatocytes and HCC cells. Treatment with the DSB-inducing agent bleomycin enhanced binding of these proteins to H2AX, suggesting an active role of H2AX in coordinating the functional pathways of each protein in DNA damage recognition and repair
