Proteomic dissection of cell type-specific H2AX-interacting protein complex associated with hepatocellular carcinoma

Abstract

The replacement histone variant H2AX senses DNA double-strand breaks (DSBs) and recruits characteristic sets of proteins at its phosphorylated (gamma-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 preneoplastic 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 multifunctional role of coordinating a variety of biological pathways involved in DNA damage recognition and DNA repair, apoptosis, nucleic acid metabolism, Ca(2+)-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 coimmunoprecipitation/ immunoblotting and confocal colocalization 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) polymerase 1, 14-3-3 zeta, coflin 1, and peflin 1, 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.

Figure 1. Expression of FLAG-tagged H2AX in stable QGY-7703 cells

(A). FLAG-tagged H2AX was detected only in the nuclear fraction, not in the cytoplasmic fraction. Equal amounts of protein (40μg) extracted from cytoplasm and nuclear fraction of cells stably expressing FLAG-tagged H2AX (FLAG-H2AX cells) were loaded onto a 12% SDS-polyacrylamide gel for Western blotting (WB) analysis. GAPDH and endogenous H2AX were detected and used as the control markers of the cytoplasmic and nuclear fractions, respectively. (B). FLAG-tagged H2AX and its phosphorylated form were expressed at a level comparable to that of its endogenous counterpart. Equal amounts of protein (15μg) extracted from the parental 7703 cells and the FLAG-H2AX cells were loaded onto a 15% SDS-polyacrylamide gel for Western blotting (WB) analysis using either anti-H2AX or anti-phospho-H2AX or anti-FLAG antibody. The bands appearing close to 15 or 25 kDa corresponded to the endogenous or FLAG-tagged H2AX, respectively.

Figure 2. AACT/SILAC-based MS analysis of H2AX complexes

(A) The distribution of H/L ratio of the identified proteins in the H2AX complex formed in HCC cells. The threshold for distinguishing specific interactors from non-specific contaminants was an H/L of 1.34. The sign of diamond, circle and square indicate those proteins with H/L over 1.34, suggesting specificity in associating with H2AX, while a triangle represents a protein with H/L less than 1.34 or a non-specific protein in the complex. The green square represents the bait protein. The red circles indicate proteins known previously to interact with H2AX in other types of cells. The blue diamonds suggest newly identified H2AX-interacting partners. (B) SDS-PAGE analysis of FLAG-immunoprecipitates as visualized by Coomassie Brilliant staining. The band of FLAG-tagged H2AX is indicated by arrow.

Figure 3. The distribution of HCC cell H2AX interacting proteins in different functional categories

The identified proteins were analyzed by bioinformatics tools using Entrez Gene and KEGG databases. A total of 102 proteins identified as H2AX-interacting partners were found to be associated with fourteen functional categories or biological processes. Proteins with multiple functions were assigned to the best known function.

Figure 4. The H2AX-associating pathway modules found in the HCC-specific H2AX interactome

The regulatory/signaling pathway modules of identified proteins were analyzed by DAVID and STRING 8.1 (http://www.string.embl.de/). (A) apoptosis and cell cycle, (B) DNA repair, (C) alcohol metabolism, (D) carbohydrate catabolism and cellular macromolecule metabolism, (E) stress response, (F) RNA processing and protein synthesis.

Figure 5. Immunochemical analyses of selected proteins identified as H2AX-interacting partners in HCC cells

The immunoprecipitated proteins from FLAG-QGY-7703 cells and FLAG-H2AX-QGY-7703 cells were analyzed by immunoblotting (IB). Anti-FLAG antibody was used to detect H2AX with nuleolin (NCL) as a loading control. Lane 1: the QGY-7703 cells expressing FLAG alone; lane 2: the QGY-7703 cells stably expressing FLAG-H2AX.

Figure 6. Confocal laser scanning analysis of co-localized endogenous interactions between γ-H2AX and either 14-3-3ζ, or PEF1, or PARP-1 or CFL1 respectively using

The parental QGY7703 cells were stained with TRITC for γ-H2AX (red), and with FITC for either PARP-1, or 14-3-3ζ, or PEF1 or CFL1 respectively (green). The nucleus was stained with Hoechst 33342 (blue). With a scale bar of 10 μm the merged images are indicated by white foci. All foci are indicated by white arrows. The confocal images of co-localized (A). γ-H2AX and PARP-1, (B). γ-H2AX and 14-3-3 ζ, (C). γ-H2AX and PEF1, and (D). γ-H2AX and CFL1.

Figure 7. Immunoblotting analysis of bleomycin (BLM)-induced changes in the H2AX binding to its interacting partners

(A) Time course-dependent BLM-induced phosphorylation of H2AX in HCC cells, and (B) Immunoprecipitates isolated from either untreated or BLM-stimulated cells (40 U/ml for 3 hours), respectively, were analyzed by immunoblotting (IB). The FLAG tag was detected and used as the loading control. Lane 1: from the untreated FLAG-H2AX-QGY-7703 cells; lane 2: from the BLM-stimulated FLAG-H2AX-QGY-7703 cells.

Figure 8. Comparative analysis of the H2AX binding with selected protein partners in paired hepatocytes (QSG-7701)versusHCC (QGY-7703) cells

Using the antibodies as indicated in the Figure, immunoprecipitates were obtained from QSG-7701 cells expressing FLAG alone, QSG-7701 cells expressing FLAG-H2AX, the QGY-7703 cells expressing FLAG alone and QGY-7703 cells expressing FLAG-H2AX respectively. In immunoblotting (IB) analysis, the FLAG tag was detected and used as the loading control. Lane1: from the FLAG-QSG-7701 cells; lane2: from the FLAG-H2AX-QSG-7701 cells; lane3: from the FLAG-QGY-7703 cells; lane4: from the FLAG-H2AX-QGY-7703 cells.