The actin-related protein hArp8 accumulates on the mitotic chromosomes and functions in chromosome alignment

The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hINO80-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis.     

Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein

The formation of γ-H2AX foci after DNA double strand breaks (DSBs) is crucial for the cellular response to this lethal DNA damage. We previously have shown that BRG1, a chromatin remodeling enzyme, facilitates DSB repair by stimulating γ-H2AX formation, and this function of BRG1 requires the binding of BRGI to acetylated histone H3 on γ-H2AX-containing nucleosomes using its bromodomain (BRD), a protein module that specifically recognizes acetyl-Lys moieties. We also have shown that the BRD of BRG1, when ectopically expressed in cells, functions as a dominant negative inhibitor of the BRG1 activity to stimulate γ-H2AX and DSB repair. Here, we found that BRDs from a select group of proteins have no such activity, suggesting that the γ-H2AX inhibition activity of BRG1 BRD is specific. This finding led us to search for more BRDs that exhibit γ-H2AX inhibition activity in the hope of finding additional BRD-containing proteins involved in DNA damage responses. We screened a total of 52 individual BRDs present in 38 human BRD-containing proteins, comprising 93% of all human BRDs. We identified the BRD of cat eye syndrome chromosome region candidate 2 (Cecr2), which recently was shown to form a novel chromatin remodeling complex with unknown cellular functions, as having a strong γ-H2AX inhibition activity. This activity of Cecr2 BRD is specific because it depends on the chromatin binding affinity of Cecr2 BRD. Small interfering RNA knockdown experiments showed that Cecr2 is important for γ-H2AX formation and DSB repair. Therefore, our genomewide screen identifies Cecr2 as a novel DNA damage response protein.     

Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin

Actin-related proteins (Arps), which share a basal structure with actin isoforms but possess different functions, have been identified in a wide variety of organisms. The Arps are classified into subfamilies based on the relatedness of their sequences and functions. Recently, several Arp subfamilies have been shown to be localized in the nucleus and included in protein complexes involved in the organization of chromatin structure, for example, in chromatin remodeling and histone acetyltransferase complexes. A member of the Arp6 subfamily in Drosophila, dArp6, is localized on centric heterochromatin together with heterochromatin protein 1 (HP1). We have identified the first examples of the Arp6 subfamily in vertebrates, novel human and chicken Arps, hArp6 and gArp6, respectively. They are closely related to each other (98% similar) and show apparent similarity to dArp6 (70%). In addition, the hArp6 gene possesses evolutionarily conserved exon/intron structures compared with genes for members of the Arp6 subfamily in invertebrates. Like Drosophila dArp6, gArp6 is expressed abundantly in the early developmental stages, when heterochromatin condensation and nuclear maturation occur. The finding of a conserved Arp6 subfamily in vertebrates will contribute to the understanding of molecular mechanisms of heterochromatin organization.     

The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (γ-H2AX) molecules form foci covering many megabases of chromatin. The formation of g-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of γ-H2AX foci formation, we analyzed the distribution of γ-H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that γ-H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with g-H2AX during mitosis. In addition, while γ-H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.     

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.     

Delayed kinetics of DNA double-strand break processing in normal and pathological aging

Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage-induced foci of phosphorylated histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ-H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ-H2AX foci than those taken from normal donors of comparable age. Further increases in γ-H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ-H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ-H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ-H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1

Phosphorylated histone H2AX (γ-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with γ-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether γ-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a γ-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-γ-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, γ-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.     

Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways

In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (gammaH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, gammaH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.     

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.     

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.     

ADP‐ribosylation of histone variant H2AX promotes base excision repair

Optimal DNA damage response is associated with ADP‐ribosylation of histones. However, the underlying molecular mechanism of DNA damage‐induced histone ADP‐ribosylation remains elusive. Herein, using unbiased mass spectrometry, we identify that glutamate residue 141 (E141) of variant histone H2AX is ADP‐ribosylated following oxidative DNA damage. In‐depth studies performed with wild‐type H2AX and the ADP‐ribosylation‐deficient E141A mutant suggest that H2AX ADP‐ribosylation plays a critical role in base excision repair (BER). Mechanistically, ADP‐ribosylation on E141 mediates the recruitment of Neil3 glycosylase to the sites of DNA damage for BER. Moreover, loss of this ADP‐ribosylation enhances serine‐139 phosphorylation of H2AX (γH2AX) upon oxidative DNA damage and erroneously causes the accumulation of DNA double‐strand break (DSB) response factors. Taken together, these results reveal that H2AX ADP‐ribosylation not only facilitates BER repair, but also suppresses the γH2AX‐mediated DSB response.