The Dynamic Alterations of H2AX Complex during DNA Repair Detected by a Proteomic Approach Reveal the Critical Roles of Ca2+/Calmodulin in the Ionizing Radiation-induced Cell Cycle Arrest

Abstract

By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca 2؉ -binding proteins were found in the H2AX complex, the roles of Ca 2؉ were examined. The results indicate that Ca 2؉ /CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

“…MS forms the basis of many strategies for the proteomic characterization of proteins and protein mixtures. It has been successfully used by several groups in the analysis of histones [84][85][86][87][88][89][90][91][92][93]. FIGURE 1 summarizes the general experimental design that modern mass spectromists use most frequently for histone analysis using MS-based methodologies.…”

A Novel Proteomics Approach for the Discovery of Chromatin-associated Protein Networks

“…A common approach in these methods is to drastically reduce, or entirely remove, genomic DNA in the cellular extract by various mechanical or enzymatic means, thereby enhancing the solubility of the protein of interest. Such procedures have provided insight into the interaction partners of a number of histone variants and their interaction partners in both mammalian (16,17) and yeast cells (18). However, these methods drastically reduce the number of associated proteins purified with the bait of interests and hence provide an incomplete picture of the physical interactions for any given bait.…”

Calreticulin expression in the clonal plasma cells of patients with systemic light-chain (AL-) amyloidosis is associated with response to high-dose melphalan

“…62 Recently calreticulin was identified as a component of a histone-associating protein complex recruited immediately after radiation injury, suggesting that the nuclear presence of calreticulin could have stoichiometric implications with respect to mediating chromatin structure. 63,64 Given its pleotropic character, there may be multiple mechanisms by which calreticulin expression is linked to increased sensitivity to drug and radiation injury. Moreover, calreticulin surface expression on apoptotic cells is an "eat me" signal and plays a key role in anthracycline-related tumor-cell immunogenicity, enabling a T cell-mediated immune response to tumors in mice.…”