[Show abstract][Hide abstract] ABSTRACT: B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation.
The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports
reactions such as class switch recombination of the antibody genes. We found that histone H3 and H4 around the Prdm1 intron
5 MARE were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its
expression. Conversely, methylation of H3 lysine 9 was lower in X63/0 cells than BAL17 cells. Purification of Bach2 complex
in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin
beta-like 1X-linked (Tbl1x), and RAP1 interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding
of Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased
Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate
the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.
Preview · Article · Jan 2016 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is
crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase
complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the
acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation
of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells
disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required
for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged
chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the
acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation
of NBS1 at DNA damage sites.
No preview · Article · Oct 2015 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an
acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA
required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2
subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a
transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target
genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate
kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we
propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for
histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively
proliferating cells such as cancer and fetal cells.
Preview · Article · Sep 2015 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.
[Show abstract][Hide abstract] ABSTRACT: The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER.
Full-text · Article · Jun 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Ser139-phosphorylated histone H2AX (γH2AX) is a useful biomarker of DNA double strand breaks. γH2AX has been conventionally detected by immunology-based methods using anti-γH2AX antibody, but quantitative analysis is difficult to perform with such methods. Here, we describe an absolute quantification method using liquid chromatography-triple quadrupole tandem mass spectrometry that is applicable to peptides derived from γH2AX (ATQA(pS)QEY) and unphosphorylated H2AX (ATQASQEY). Our method was successfully applied to histones extracted from human cervix adenocarcinoma HeLa S3 cells. The estimated number of molecules of γH2AX (ATQA(pS)QEY) per vehicle-treated HeLa S3 cell was 9.4 × 10(4) and increased to 6.2 × 10(5) molecules/cell after exposure to the DNA-damaging agent camptothecin (10 μM) for 1 h. The estimated total amount of H2AX (ATQA(pS)QEY + ATQASQEY) was 3.3-3.6 × 10(6) molecules/cell. Due to its broad adaptability and throughput performance, we believe that our method is a powerful tool for mechanistic studies of the DNA-damage response as well as for genotoxicity testing, cancer drug screening, clinical studies, and other fields.
No preview · Article · May 2015 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs).
Methods and materials:
To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation.
FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells.
We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation.
No preview · Article · Jul 2014 · International journal of radiation oncology, biology, physics
[Show abstract][Hide abstract] ABSTRACT: Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.
Full-text · Article · May 2014 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.
[Show abstract][Hide abstract] ABSTRACT: Histone H2AX phosphorylated at Ser139 (γ-H2AX) is a useful biomarker for DNA double-strand breaks. However, γ-H2AX detection has methodological disadvantages such as the requirement of expensive anti-γ-H2AX antibody and time-consuming handling for its staining. Mediator of DNA damage checkpoint 1 (MDC1) is a central adaptor protein which recruits various DNA damage response proteins to γ-H2AX and thus forms nuclear foci in the same location as γ-H2AX in response to DNA damage. Here, we describe an easy-to-use genotoxicity assay which combines enhanced green fluorescence protein (EGFP)-fused MDC1-expressing cells with a free R program for image-processing and quantification of foci area/nucleus. The workflow of this assay is simple: mutagen treatment, imaging, and R-processing. This assay does not need antibodies or staining handling and it detected the genotoxicity of a range of mutagens, including camptothecin (topoisomerase I inhibitior), cisplatin (crosslinker), and 4-nitroquinoline 1-oxide and benzo[a]pyrene (bulky DNA-adduct forming compounds), as increased fluorescence of EGFP-MDC1 foci. Furthermore, cotreatment with arabinofuranosyl cytosine/hydroxyurea and mutagens sensitized EGFP-MDC1 foci formation to bulky DNA adduct-type mutagens. Additionally, the established cells can be monitored in real-time using live cell imaging to obtain detailed dynamics of MDC1 in response to mutagens. The simple handling of this assay is expected to enable its full automation, thus making it useful for high-throughput genotoxicity screening of chemicals and monitoring of environmental mutagens.
No preview · Article · Jan 2014 · Genes and Environment
[Show abstract][Hide abstract] ABSTRACT: The E3 ubiquitin ligase RNF20 regulates chromatin structure through ubiquitination of histone H2B, so that early homologous recombination repair (HRR) proteins can access the DNA in eukaryotes during repair. However, it remains unresolved how RNF20 itself approaches the DNA in the presence of chromatin structure. Here, we identified the histone chaperone FACT as a key protein in the early step of HRR. Depletion of SUPT16H, a component of FACT, caused pronounced defects in accumulations of repair proteins, consequently decreased HRR activity. This led to enhanced sensitivity to ionizing radiation (IR) and mitomycin-C in a fashion similar to RNF20-deficient cells, indicating that SUPT16H is essential for RNF20-mediated pathway. Indeed, SUPT16H directly bound to RNF20 in vivo, and mutation at the RNF20's RING-finger domain abolished its interaction and accumulation, as well as RAD51 and BRCA1 at sites of DSBs, whereas the localization of SUPT16H remained intact. Interestingly, PAF1, implicated in transcription as a mediator of FACT and RNF20 association, was dispensable for DNA damage-induced interaction of RNF20 with SUPT16H. Furthermore, depletion of SUPT16H caused pronounced defects in RNF20-mediated H2B ubiquitination and thereby, impaired accumulation of the chromatin remodeling factor SNF2h. Consistent with this observation, the defective phenotypes of SUPT16H were effectively counteracted by enforced nucleosome relaxation. Taken together, present results indicate a primary role of FACT in RNF20 recruitment and the resulting chromatin remodeling for initiation of HRR.
No preview · Article · Dec 2013 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
Full-text · Article · Dec 2013 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous recombinational DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Posttranslational modifications of proteins, such as phosphorylation, acetylation and ubiquitination, play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been determined to play a role in the regulation of the DNA damage response. Here we show that the SUMOylation E2, UBC9, and E3, PIAS1 and PIAS4 enzymes are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for RAD51 accumulation at sites of DNA damage. These findings suggest that the SUMO-SIM system plays an important role in DNA repair, through the regulation of RAD51 dynamics.
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase (PARP) plays a critical role in responding to DNA damage, by activating DNA repair pathways responsible for cellular survival. PARP inhibition is used to treat certain solid cancers, such as breast and ovarian cancers. However, its effectiveness with other solid cancers, such as esophageal squamous cell carcinoma (ESCC), has not been clarified. We evaluated the effects of PARP inhibition on the survival of human esophageal cancer cells, with a special focus on the induction and repair of DNA double-strand breaks. The effects were monitored by colony formation assays and DNA damage responses, with immunofluorescence staining of γH2AX and RAD51. We found that PARP inhibition synergized with cisplatin, and the cells were highly sensitive, in a similar manner to the combination of cisplatin and 5-fluorouracil (5-FU). Comparable increases in RAD51 foci formation were observed after each combinational treatment with cisplatin and either 3-aminobenzamide (3-AB) or 5-FU in three human esophageal cancer cell lines, TE11, TE14 and TE15. In addition, decreasing the amount of RAD51 by RNA interference rendered the TE11 cells even more hypersensitive to these combinational treatments. Our findings suggested that the homologous recombinational repair pathway may be involved in the synergism between cisplatin and either 3-AB or 5-FU, and that 3-AB and 5-FU may similarly modify the cisplatin-induced DNA damage to types requiring the recruitment of RAD51 proteins for their repair. Understanding these mechanisms could be useful for improving the clinical outcome of ESCC patients, who suffer from aggressive diseases that presently lack effective treatment options. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle
progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its
partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also
activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little
is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking
damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core
complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence
that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation.
Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either
single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation
of the FA pathway.
Full-text · Article · May 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: 5-Fluorouracil (5-FU) is one of the most well established chemotherapeutic agents in the treatment of esophageal cancer. Ribonucleotide reductase M1 (RRM-1) is the rate‑limiting enzyme in de novo DNA synthesis, and has been considered to play an important role in the 5-FU metabolic pathway. However, the means by which RRM-1 participates in the anticancer effects of 5-FU and cisplatin (CDDP) have not been well studied. Here, we show that RRM-1 significantly contributes to the induction of DNA damage by 5-FU in esophageal cancer cell lines. An assay of γ-H2AX focus formation, a marker of DNA damage, after 5-FU treatment revealed good correlation with the levels of RRM-1 protein expression. Moreover, the increased sensitivity and RAD51 focus formation induced by the combination treatment of 5-FU and CDDP were significantly repressed by RRM-1 depletion. These results suggest that RRM-1 is involved not only in the induction of DNA damage by 5-FU but also in the synergistic cytotoxic effect in the combination therapy of 5-FU and CDDP.
Preview · Article · Apr 2013 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: Although nuclear actin and Arps (actin-related proteins) are often identified as components of multi-protein chromatin-modifying enzyme complexes, such as chromatin remodeling and histone acetyltransferase (HAT) complexes, their molecular functions still remain largely elusive. Here, we investigated the role of human Arp4 (BAF53, also known as actin-like protein 6A) in Brg1-containing chromatin remodeling complexes. Depletion of Arp4 by RNA interference impaired the integrity of these complexes and accelerated the degradation of Brg1, indicating a crucial role in their maintenance, at least in certain human cell lines. We further found that Arp4 can form a heterocomplex with β-actin. Based on structural similarities between conventional actin and Arp4, and the assumption that actin-Arp4 binding might mimic actin-actin binding, we introduced a series of mutations in Arp4 that might be expected to impair its interaction with β-actin. Some of them indeed caused reduced binding to β-actin. Interestingly, such mutant Arp4 proteins also showed reduced incorporation into Brg1 complexes, and their interaction with Myc-associated complexes as well as Tip60 HAT complexes were also impaired. Based on these findings, we propose that β-actin-Arp4 complex formation might be a crucial feature in some chromatin-modifying enzyme complexes, such as the Brg1 complex.
Full-text · Article · May 2012 · Journal of Cell Science