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Sample records for phosphorylates histone h2ax

  1. Cigarette sidestream smoke induces phosphorylated histone H2AX.

    PubMed

    Toyooka, Tatsushi; Ibuki, Yuko

    2009-05-31

    Cigarette sidestream smoke (CSS) is a widespread environmental pollutant having highly genotoxic potency. In spite of the overwhelming evidence that CSS induces a wide range of DNA damage such as oxidative base damage and DNA adducts, evidence that CSS can result in DNA double strand breaks (DSBs) is little. In this study, we showed that CSS generated phosphorylated histone H2AX (gamma-H2AX), recently considered as a sensitive marker of the generation of DSBs, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of gamma-H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of gamma-H2AX, suggesting that reactive oxygen species produced from CSS partially contributed to the phosphorylation. The generation of gamma-H2AX is considered to be accompanied the induction of DSBs. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis. Our results would increase the evidence of the strong genotoxicity of passive smoking.

  2. ATM is the predominant kinase involved in the phosphorylation of histone H2AX after heating.

    PubMed

    Takahashi, Akihisa; Mori, Eiichiro; Su, Xiaoming; Nakagawa, Yosuke; Okamoto, Noritomo; Uemura, Hirokazu; Kondo, Natsuko; Noda, Taichi; Toki, Atsushi; Ejima, Yosuke; Chen, David J; Ohnishi, Ken; Ohnishi, Takeo

    2010-01-01

    Heating induces histone H2AX phosphorylation at serine 139 (gammaH2AX). Phosphorylated H2AX subsequently forms foci in numerous mammalian cell lines. The aim of this study was to clarify details in the mechanisms involved in the phosphorylation of H2AX after heating. The cell lines used were DNA-PKcs knockout cells, ATM knockout cells, and their parental cell lines. To elucidate mechanisms of induction of phosphorylation of H2AX after heating, ATM/ATR inhibitor (CGK733) and DNA-PK inhibitor (NU7026) were used. The intensity of gammaH2AX signals was assayed with flow cytometry. The thermal dose-response curve for the fluorescence intensity of gammaH2AX appearance in DNA-PKcs-/- cells during the heating period was similar to that observed in DNA-PKcs+/+ cells. On the other hand, the slope of thermal dose-response curve for them in ATM-/- cells was lower than that in ATM+/+ cells. Phosphorylation of H2AX after heating was suppressed by a combination of CGK733 and NU7026 in the culture medium in DNA-PKcs-/- cells, ATM-/- cells and in their parental cells. Although the phosphorylation of H2AX after heating was not suppressed by NU7026 in their parental cells, such phosphorylation was suppressed by CGK733 in their parental cells. These results indicate that ATM is the predominant protein which is active in the phosphorylation of histone H2AX after heating.

  3. Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

    PubMed Central

    Barral, Serena; Beltramo, Riccardo; Salio, Chiara; Aimar, Patrizia; Lossi, Laura; Merighi, Adalberto

    2014-01-01

    Phosphorylation of the histone H2AXH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases. PMID:24451138

  4. Phosphorylation of histone H2AX in the mouse brain from development to senescence.

    PubMed

    Barral, Serena; Beltramo, Riccardo; Salio, Chiara; Aimar, Patrizia; Lossi, Laura; Merighi, Adalberto

    2014-01-21

    Phosphorylation of the histone H2AXH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

  5. Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks

    PubMed Central

    Rybak, Paulina; Hoang, Agnieszka; Bujnowicz, Lukasz; Bernas, Tytus; Berniak, Krzysztof; Zarębski, Mirosław; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy

    2016-01-01

    Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities. PMID:27391338

  6. Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

    PubMed

    Han, Jianxun; Hendzel, Michael J; Allalunis-Turner, Joan

    2006-03-01

    Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

  7. BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation.

    PubMed

    Turner, James M A; Aprelikova, Olga; Xu, Xiaoling; Wang, Ruihong; Kim, Sangsoo; Chandramouli, Gadisetti V R; Barrett, J Carl; Burgoyne, Paul S; Deng, Chu-Xia

    2004-12-14

    In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

  8. Slow elimination of phosphorylated histone {gamma}-H2AX from DNA of terminally differentiated mouse heart cells in situ

    SciTech Connect

    Gavrilov, Boris; Vezhenkova, Irina; Firsanov, Denis; Solovjeva, Liudmila; Svetlova, Maria; Mikhailov, Vyacheslav; Tomilin, Nikolai . E-mail: nvtom@hotmail.com

    2006-09-08

    Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called {gamma}-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of {gamma}-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% {gamma}-H2AX-positive cells and in tissues fixed 1 h after X-irradiation {gamma}-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of {gamma}-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the {gamma}-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of {gamma}-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.

  9. Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

    SciTech Connect

    Koike, Manabu Mashino, Minako; Sugasawa, Jun; Koike, Aki

    2007-11-30

    Histone H2AX undergoes phosphorylation on Ser 139 ({gamma}-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, {gamma}-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, {gamma}-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, {gamma}-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, {gamma}-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.

  10. AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

    PubMed Central

    Baritaud, M; Cabon, L; Delavallée, L; Galán-Malo, P; Gilles, M-E; Brunelle-Navas, M-N; Susin, S A

    2012-01-01

    The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AXH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis. PMID:22972376

  11. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome

    PubMed Central

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency. PMID:27907109

  12. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

  13. Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

    PubMed Central

    Sone, Kenbun; Piao, Lianhua; Nakakido, Makoto; Ueda, Koji; Jenuwein, Thomas; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    The presence of phosphorylated histone H2AX (γ-H2AX) is associated with the local activation of DNA-damage repair pathways. Although γ-H2AX deregulation in cancer has previously been reported, the molecular mechanism involved and its relationship with other histone modifications remain largely unknown. Here we find that the histone methyltransferase SUV39H2 methylates histone H2AX on lysine 134. When H2AX was mutated to abolish K134 methylation, the level of γ-H2AX became significantly reduced. We also found lower γ-H2AX activity following the introduction of double-strand breaks in Suv39h2 knockout cells or on SUV39H2 knockdown. Tissue microarray analyses of clinical lung and bladder tissues also revealed a positive correlation between H2AX K134 methylation and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemosensitivity of cancer cells. Overall, our results suggest that H2AX methylation plays a role in the regulation of γ-H2AX abundance in cancer. PMID:25487737

  14. Tissue-specific DNA-PK-dependent H2AX phosphorylation and {gamma}-H2AX elimination after X-irradiation in vivo

    SciTech Connect

    Koike, Manabu Sugasawa, Jun; Yasuda, Mariko; Koike, Aki

    2008-11-07

    Histone H2AX rapidly undergoes phosphorylation at Ser139 ({gamma}-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of {gamma}-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of {gamma}-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of {gamma}-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of {gamma}-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of {gamma}-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of {gamma}-H2AX level is tissue-specific.

  15. Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in Poly (ADP-ribose) polymerase (Parp-1) null oocytes

    PubMed Central

    Yang, Feikun; Baumann, Claudia; De La Fuente, Rabindranath

    2009-01-01

    In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1 (−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1 (−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains. PMID:19463809

  16. Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability.

    PubMed

    Bogliolo, Massimo; Lyakhovich, Alex; Callén, Elsa; Castellà, Maria; Cappelli, Enrico; Ramírez, María J; Creus, Amadeu; Marcos, Ricard; Kalb, Reinhard; Neveling, Kornelia; Schindler, Detlev; Surrallés, Jordi

    2007-03-07

    Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.

  17. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX

    PubMed Central

    Artus, Cédric; Boujrad, Hanan; Bouharrour, Aïda; Brunelle, Marie-Noëlle; Hoos, Sylviane; Yuste, Victor J; Lenormand, Pascal; Rousselle, Jean-Claude; Namane, Abdelkader; England, Patrick; Lorenzo, Hans K; Susin, Santos A

    2010-01-01

    Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif. PMID:20360685

  18. H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

    PubMed

    Dong, Yaqiong; Xiong, Min; Duan, Lianning; Liu, Ze; Niu, Tianhui; Luo, Yuan; Wu, Xinpin; Xu, Chengshan; Lu, Chengrong

    2014-08-01

    Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

  19. Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens.

    PubMed

    Albino, A P; Huang, X; Jorgensen, E; Yang, J; Gietl, D; Traganos, F; Darzynkiewicz, Z

    2004-08-01

    DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 microg/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G(1) vs S vs G(2)/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.

  20. Sumoylation precedes accumulation of phosphorylated H2AX on sex chromosomes during their meiotic inactivation.

    PubMed

    Vigodner, Margarita

    2009-01-01

    During meiosis in male mammals, X and Y chromosomes undergo the process of meiotic sex chromosome inactivation (MSCI). A crucial role in MSCI has recently been reported for BRCA1, ATR kinase, and phosphorylated histone H2AX, but the exact mechanism remains to be determined. Small ubiquitin-like modifier (SUMO) proteins have recently been shown to localize to the sex body in mouse meiotic spermatocytes, but the role they play during MSCI is unknown. In this study, in order to better understand the molecular events of MSCI, we followed dynamic changes in gammaH2AX and SUMO localization patterns during MSCI. Using confocal laser scanning microscopy (CLSM) as an analytical tool for visualizing numerous spermatocytes from the same development stage and for consecutively following the meiotic progression, we were able to demonstrate a very early appearance of SUMO-1, which preceded gammaH2AX accumulation on the sex chromosomes during their meiotic inactivation. In contrast to SUMO-1, SUMO-2/3 was undetectable in zygotene spermatocytes, suggesting a possible specific role for SUMO-1 in the initiation of MSCI.

  1. Control of radiosensitivity of F9 mouse teratocarcinoma cells by regulation of histone H2AX gene expression using a tetracycline turn-off system.

    PubMed

    Yoshida, Kayo; Morita, Takashi

    2004-06-15

    The mouse histone H2AX has unique COOH-terminal serine residues that are phosphorylated in response to double-strand DNA breaks introduced by ionizing radiation. This suggests that H2AX acts to maintain genomic stability. We constructed a tetracycline (tet)-directed turn-off vector and integrated it into F9 mouse teratocarcinoma cells by homologous recombination. In homozygously recombined cells, expression of the histone H2AX gene was repressed to 0.02% of the expression observed in wild-type cells by the addition of doxycycline, an analog of tet. Sensitivity of cells with repressed H2AX expression to X-irradiation was increased 1.95x, indicating that DNA repair was impaired by repression of H2AX. When we s.c. injected tet-regulated F9 cells into the flanks of mice, tumor growth was slightly suppressed by X-irradiation in H2AX-repressed tumors, whereas without X-irradiation, tumor growth did not differ by H2AX status. Thus, H2AX might be a potential molecular target for sensitizing cancer cells to radiotherapy to minimize required irradiation doses.

  2. γH2AX and cancer

    PubMed Central

    Bonner, William M.; Redon, Christophe E.; Dickey, Jennifer S.; Nakamura, Asako J.; Sedelnikova, Olga A.; Solier, Stéphanie; Pommier, Yves

    2011-01-01

    Histone H2AX phosphorylation on a serine four residues from the carboxyl terminus (producing γH2AX) is a sensitive marker for DNA double-strand breaks (DSBs). DSBs may lead to cancer but, paradoxically, are also used to kill cancer cells. Using γH2AX detection to determine the extent of DSB induction may help to detect precancerous cells, to stage cancers, to monitor the effectiveness of cancer therapies and to develop novel anticancer drugs. PMID:19005492

  3. Induction and Rejoining of DNA Double Strand Breaks Assessed by H2AX Phosphorylation in Melanoma Cells Irradiated with Proton and Lithium Beams

    SciTech Connect

    Ibanez, Irene L.; Bracalente, Candelaria; Molinari, Beatriz L.; Palmieri, Monica A.; Policastro, Lucia; Kreiner, Andres J.; Burlon, Alejandro A.; Valda, Alejandro; Navalesi, Daniela; Davidson, Jorge; Davidson, Miguel; Vazquez, Monica; Ozafran, Mabel; Duran, Hebe

    2009-07-15

    Purpose: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. Methods and Materials: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX ({gamma}H2AX) foci at 30 min and 6 h post-irradiation. Results: Survival curves showed the increasing effectiveness of radiation as a function of LET. {gamma}H2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of {gamma}H2AX foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. Conclusions: Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of {gamma}H2AX foci. We conclude that {gamma}H2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.

  4. Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM.

    PubMed

    Horn, Simon; Brady, Darren; Prise, Kevin

    2015-10-01

    The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

  5. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast

    PubMed Central

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-01-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain–phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  6. Phosphorylation of gH2AX as a novel prognostic biomarker for laryngoesophageal dysfunction-free survival

    PubMed Central

    de Miguel-Luken, María José; Chaves-Conde, Manuel; Quintana, Begoña; Menoyo, Alicia; Tirado, Isabel; de Miguel-Luken, Verónica; Pachón, Jerónimo; Chinchón, David; Suarez, Vladimir; Carnero, Amancio

    2016-01-01

    Current larynx preservation treatments have achieved an improvement of laryngoesophageal dysfunction-free survival (LDS) but lead to significant toxicities and recurrences. At present, there is no evidence to select the group of patients that may benefit from preservation approaches instead of surgery. Therefore, laryngeal biomarkers could facilitate pretreatment identification of patients who could respond to chemoradiation-based therapy. In this study, we evaluated retrospectively 53 patients with larynx cancer to determine whether gH2AX phosphorylation (pH2AX) alone or in combination with the membrane protein MAP17 (PDZK1IP1) could be used as prognostic biomarkers. We also evaluated whether the completion of cisplatin treatment and radiotherapy could predict survival in combination with pH2AX. We found that the dose of cisplatin received but not the length of the radiotherapy influenced LDS. High-pH2AX expression was associated with prolonged LDS (HR 0.26, p = 0.02) while MAP17 correlated with overall survival (OS) (HR 0.98, p = 0.05). High-MAP17 and high-pH2AX combined analysis showed improved LDS (with 61.35 months vs 32.2 months, p = 0.05) and OS (with 66.6 months vs 39.8 months, p = 0.01). Furthermore, the subgroup of high-pH2AX and optimal dose of cisplatin was also associated with OS (72 months vs 38.6 months, p = 0.03) and LDS (66.9 months vs 27 months, p = 0.017). These findings suggest that pH2AX alone or better in combination with MAP17 may become a novel and valuable prognostic biomarker for patients with laryngeal carcinoma treated with preservation approaches. PMID:27166270

  7. Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay

    PubMed Central

    Ji, Jiuping; Zhang, Yiping; Redon, Christophe E.; Reinhold, William C.; Chen, Alice P.; Fogli, Laura K.; Holbeck, Susan L.; Parchment, Ralph E.; Hollingshead, Melinda; Tomaszewski, Joseph E.; Dudon, Quentin; Pommier, Yves; Doroshow, James H.; Bonner, William M.

    2017-01-01

    Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents. PMID:28158293

  8. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    PubMed

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  9. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    SciTech Connect

    Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa; Komatsu, Kenshi; Oda, Shoji; Mitani, Hiroshi

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

  10. Intense THz pulses cause H2AX phosphorylation and activate DNA damage response in human skin tissue

    PubMed Central

    Titova, Lyubov V.; Ayesheshim, Ayesheshim K.; Golubov, Andrey; Fogen, Dawson; Rodriguez-Juarez, Rocio; Hegmann, Frank A.; Kovalchuk, Olga

    2013-01-01

    Recent emergence and growing use of terahertz (THz) radiation for medical imaging and public security screening raise questions on reasonable levels of exposure and health consequences of this form of electromagnetic radiation. In particular, picosecond-duration THz pulses have shown promise for novel diagnostic imaging techniques. However, the effects of THz pulses on human cells and tissues thus far remain largely unknown. We report on the investigation of the biological effects of pulsed THz radiation on artificial human skin tissues. We observe that exposure to intense THz pulses for ten minutes leads to a significant induction of H2AX phosphorylation, indicating that THz pulse irradiation may cause DNA damage in exposed skin tissue. At the same time, we find a THz-pulse-induced increase in the levels of several proteins responsible for cell-cycle regulation and tumor suppression, suggesting that DNA damage repair mechanisms are quickly activated. Furthermore, we find that the cellular response to pulsed THz radiation is significantly different from that induced by exposure to UVA (400 nm). PMID:23577291

  11. Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species.

    PubMed

    Hagen, Jodi; Schwartz, David; Kalyuzhny, Alexander E

    2017-01-01

    Multiplex staining of cell and tissue sections with antibodies raised in the same host species is a serious challenge because of unwanted but inevitable cross-reactivity of secondary antibodies with irrelevant primary antibodies. Several techniques can be used to overcome this obstacle including direct labeling of primary antibodies with fluorescent tags and using tyramide signal amplification. Unfortunately these techniques either lack sensitivity, or require a long multistep protocol which can cause physical damage of specimens. As an alternative, we have developed a protocol based on conjugation of primary antibodies to small-size hapten molecules which can be detected with hapten-specific fluorescent secondary antibodies. This technique has been used for two-color labeling of Y845 phosphorylated Epidermal Growth Factor Receptor (EGFR) and S139 phosphorylated histone H2AX protein in A431 human epidermoid carcinoma cells. Our novel hapten-anti-hapten detection chemistry allows for generating a stronger fluorescent signal and completely avoid cross-interactions of secondary antibodies with irrelevant primary antibodies.

  12. Genome-wide profiles of H2AX and γ-H2AX differentiate endogenous and exogenous DNA damage hotspots in human cells.

    PubMed

    Seo, Jungmin; Kim, Sang Cheol; Lee, Heun-Sik; Kim, Jung Kyu; Shon, Hye Jin; Salleh, Nur Lina Mohd; Desai, Kartiki Vasant; Lee, Jae Ho; Kang, Eun-Suk; Kim, Jin Sung; Choi, Jung Kyoon

    2012-07-01

    Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

  13. H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown.

    PubMed

    Dickey, Jennifer S; Zemp, Franz J; Altamirano, Alvin; Sedelnikova, Olga A; Bonner, William M; Kovalchuk, Olga

    2011-02-01

    Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs.

  14. Thermal response of proteins (histone H2AX, H3.1) by a coarse-grained Monte Carlo simulation with a knowledge-based phenomenological potential

    NASA Astrophysics Data System (ADS)

    Fritsche, Miriam; Heermann, Dieter; Pandey, Ras; Farmer, Barry

    2012-02-01

    Using a coarse-grained bond fluctuating model, we investigate structure and dynamics of two histones, H2AX (143 residues) and H3.1 (136 residues) as a function of temperature (T). A knowledged based contact matrix is used as an input for a phenomenological residue-residue interaction in a generalized Lennard-Jones potential. Metropolis algorithm is used to execute stochastic movement of each residue. A number of local and global physical quantities are analyzed. Despite unique energy and mobility profiles of its residues in a specific sequence, the histone H3.1 appears to undergo a structural transformation from a random coil to a globular conformation on reducing the temperature. The radius of gyration of the histone H2AX, in contrast, exhibits a non-monotonic dependence on temperature with a maximum at a characteristic temperature (Tc) where crossover occurs from a positive (stretching below Tc) to negative (contraction above Tc) thermal response on increasing T. Multi-scale structures of the proteins are examined by a detailed analysis of their structure functions.

  15. Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

    PubMed

    Sonoda, Eiichiro; Zhao, Guang Yu; Kohzaki, Masaoki; Dhar, Pawan Kumar; Kikuchi, Koji; Redon, Christophe; Pilch, Duane R; Bonner, William M; Nakano, Atsushi; Watanabe, Masami; Nakayama, Tatsuo; Takeda, Shunichi; Takami, Yasunari

    2007-03-01

    One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.

  16. A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair.

    PubMed

    Lee, Han-Sae; Park, Ji-Hye; Kim, So-Jung; Kwon, Su-Jung; Kwon, Jongbum

    2010-04-21

    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 gamma-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to gamma-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with gamma-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 gamma-H2AX nucleosomes. S139ph stimulates the H3 acetylation on gamma-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on gamma-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, gamma-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

  17. Conformational temperature-dependent behavior of a histone H2AX: a coarse-grained Monte Carlo approach via knowledge-based interaction potentials.

    PubMed

    Fritsche, Miriam; Pandey, Ras B; Farmer, Barry L; Heermann, Dieter W

    2012-01-01

    Histone proteins are not only important due to their vital role in cellular processes such as DNA compaction, replication and repair but also show intriguing structural properties that might be exploited for bioengineering purposes such as the development of nano-materials. Based on their biological and technological implications, it is interesting to investigate the structural properties of proteins as a function of temperature. In this work, we study the spatial response dynamics of the histone H2AX, consisting of 143 residues, by a coarse-grained bond fluctuating model for a broad range of normalized temperatures. A knowledge-based interaction matrix is used as input for the residue-residue Lennard-Jones potential.We find a variety of equilibrium structures including global globular configurations at low normalized temperature (T* = 0.014), combination of segmental globules and elongated chains (T* = 0.016,0.017), predominantly elongated chains (T* = 0.019,0.020), as well as universal SAW conformations at high normalized temperature (T* ≥ 0.023). The radius of gyration of the protein exhibits a non-monotonic temperature dependence with a maximum at a characteristic temperature (T(c)* = 0.019) where a crossover occurs from a positive (stretching at T* ≤ T(c)*) to negative (contraction at T* ≥ T(c)*) thermal response on increasing T*.

  18. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells.

    PubMed

    Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A; Tawbi, Hussein; Beumer, Jan H; Schmitz, John C

    2015-01-01

    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

  19. Chemical proteomics reveals a γH2AX-53BP1 interaction in the DNA damage response

    PubMed Central

    Kleiner, Ralph E.; Verma, Priyanka; Molloy, Kelly R.; Chait, Brian T.; Kapoor, Tarun M.

    2015-01-01

    DNA double-strand break repair involves phosphorylation of histone variant H2AX (‘γH2AX’), which accumulates in foci at sites of damage. In current models, the recruitment of multiple DNA repair proteins to γH2AX foci depends mainly on recognition of this ‘mark’ by a single protein, MDC1. However, DNA repair proteins accumulate at γH2AX sites without MDC1, suggesting that other ‘readers’ exist. Here, we use a quantitative chemical proteomics approach to profile direct, phospho-selective γH2AX binders in native proteomes. We identify γH2AX binders, including the DNA repair mediator, 53BP1, which we show recognizes γH2AX through its BRCT domains. Furthermore, we investigate targeting of wild-type 53BP1 or a mutant form deficient in γH2AX binding, to chromosomal breaks resulting from endogenous and exogenous DNA damage. Our results show how direct recognition of γH2AX modulates protein localization at DNA damage sites, and suggest how specific chromatin ‘mark’-‘reader’ interactions contribute to essential mechanisms ensuring genome stability. PMID:26344695

  20. Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation, p53 phosphorylation, Bcl-2 production, and cell death

    PubMed Central

    Kiang, Juliann G.; Garrison, Bradley R.; Smith, Joan T.; Fukumoto, Risaku

    2014-01-01

    Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53−/− of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy 60Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2, apoptotic proteins, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53+/+) within 1 hr in a radiation dose-dependent manner. CIP pretreatment and post-treatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 hr postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy. PMID:24802382

  1. Immunofluorescence detection of clustered gamma-H2AX foci induced by HZE-particle radiation.

    PubMed

    Desai, N; Davis, E; O'Neill, P; Durante, M; Cucinotta, F A; Wu, H

    2005-10-01

    We studied the spatial and temporal distributions of foci of the phosphorylated form of the histone protein H2AX (gamma-H2AX), which is known to be activated by double-strand breaks after irradiation of human fibroblast cells with high-energy silicon (54 keV/microm) and iron (176 keV/microm) ions. Here we present data obtained with the ion path parallel to a monolayer of human fibroblast cells that leads to gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane, thus enabling the analysis of the fluorescence distributions along the ion trajectories. Qualitative analyses of these distributions provide insights into DNA damage processing kinetics for high charge and energy (HZE) ions, including evidence of increased clustering of DNA damage and slower processing with increasing LET.

  2. Nitric oxide induces ataxia telangiectasia mutated (ATM) protein-dependent γH2AX protein formation in pancreatic β cells.

    PubMed

    Oleson, Bryndon J; Broniowska, Katarzyna A; Schreiber, Katherine H; Tarakanova, Vera L; Corbett, John A

    2014-04-18

    In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylationH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

  3. New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks.

    PubMed

    Zhao, Xiaoxu; Yang, Gang; Toyooka, Tatsushi; Ibuki, Yuko

    2015-12-01

    We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage.

  4. HSP72 depletion suppresses gammaH2AX activation by genotoxic stresses via p53/p21 signaling.

    PubMed

    Gabai, V L; Sherman, M Y; Yaglom, J A

    2010-04-01

    Knockout of heat shock protein Hsp72 was shown to promote chromosomal instability and increase radiation sensitivity of mouse fibroblasts. Here, we report that downregulation of Hsp72 in human tumor cells leads to suppression of a specific branch of the DNA damage response (DDR) that facilitates DNA repair following genotoxic insults, that is, reduced accumulation of the phosphorylated form of histone H2AX (gammaH2AX). This inhibition was due to decreased expression of H2AX as well as higher rate of gammaH2AX dephosphorylation. Formation of gammaH2AX and MDC1 radiation-induced foci was impaired in Hsp72-depleted cells, which in turn enhanced DNA damage, resulting in sensitization of cells to gamma-radiation and doxorubicin. These effects of Hsp72 knockdown were dependent on activation of the p53/p21-signaling pathway. Overall, permanent activation of the p53/p21 signaling in Hsp72-depleted cells specifically impaired the gammaH2AX pathway of the DDR, enhanced DNA damage following genotoxic insults, and led to further stimulation of the p53/p21 pathway, thus creating a positive feedback loop. The resulting strong induction of p21 precipitated senescence following exposure to DNA-damaging agents, thus accounting for higher sensitivity of cells to genotoxic stresses.

  5. Essential role of autoactivation circuitry on Aurora B-mediated H2AX-pS121 in mitosis.

    PubMed

    Shimada, Midori; Goshima, Takahiro; Matsuo, Hiromi; Johmura, Yoshikazu; Haruta, Mayumi; Murata, Kazuhiro; Tanaka, Hiromitsu; Ikawa, Masahito; Nakanishi, Keiko; Nakanishi, Makoto

    2016-07-08

    Proper deposition and activation of Aurora B at the centromere is critical for faithful chromosome segregation in mammals. However, the mechanistic basis for abrupt Aurora B kinase activation at the centromere has not yet been fully understood. We demonstrate here that Aurora B-mediated phosphorylation of histone H2AX at serine 121 (H2AX-pS121) promotes Aurora B autophosphorylation and is essential for proper chromosome segregation. Aurora B-mediated H2AX-pS121 is specifically detected at the centromere during mitosis. H2AX depletion results in a severe defect in activation and deposition of Aurora B at this locus. A phosphomimic mutant of H2AX at S121 interacts with activated Aurora B more efficiently than wild-type in vitro. Taken together, these results propose a model in which Aurora B-mediated H2AX-pS121 probably provide a platform for Aurora B autoactivation circuitry at centromeres and thus play a pivotal role in proper chromosome segregation.

  6. The γH2AX DNA damage assay from a drop of blood

    PubMed Central

    Heylmann, Daniel; Kaina, Bernd

    2016-01-01

    DNA double-strand breaks (DSB) and blocked replication forks activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of damage and can be detected as foci by immunocytochemistry. Therefore, γH2AX is a sensitive and robust biomarker of DNA damage, notably DSB. Cells from peripheral blood are often used for studies on genotoxic exposure of humans. They are limited, however, by the amount of blood required and the costly blood purification method. Here, we present a method that enables the detection of DNA damage by the analysis of γH2AX foci in a drop of blood. The blood drop method (BDM) is simple, fast, inexpensive and allows large series of blood sampling and storage over time. It can be combined with genotoxic treatment of cells in the collected blood sample for experimental purposes on DNA damage induction and repair. The BDM is suitable for rapid and large-scale screenings of genetic damage in human and animal populations. PMID:26940638

  7. Induction and quantification of gammma-H2AX foci following cx- and gamma-irradiaton

    NASA Technical Reports Server (NTRS)

    Leatherbarrow, E. L.; Cucinotta, F. A.; O'Neill, Peter

    2004-01-01

    Following DNA damage the histone H2AX becomes phosphorylated and can be visualised by immunofluorescence as an indicator of DSBs in individual cells. Using a wild type hamster cell line (V79-4) exposed to either a-particles or to Co-60 gamma-rays to induce DNA DSBs at different doses (20-200OmGy), the dose dependent induction of gamma-H2AX foci were scored both manually (by eye) and using image analysis. A linearly dependence on dose was found for both radiations. The number of DSBs determined by image analysis after a post-irradiation period of 30 minutes at 37 C, is 16.6 foci/cell/Gy for alpha-irradiation and 12.2 foci/cell/Gy for gamma-irradiation; the latter being 3-4 times the levels observed by eye and comparable to gamma-radiation-induced levels of prompt DSBs more recently reported using pulse field gel electrophoresis (approx. 16 DSBs/Gy). The average size of the gamma-H2AX foci induced by alpha-irradiation (0.30 square micrometers) is approximately 1.5 times larger than those induced by gamma-irradiation (0.19 square micrometers). The timescale of induction and removal of DSBs up to 24 hours post-irradiation, was investigated with gamma-H2AX foci levels found to remain significantly higher than controls for 4 or 6 hours in gamma-irradiated samples or alpha irradiated samples, respectively. These results demonstrate that not only gamma radiation but also alpha-radiation induce phosphorylation of the H2AX histone in response to DSBs even at low doses (20mGy for gamma-rays, 1 track/cell on average for alpha-particles) and the variation in size and dephosphorylation of the induced foci is dependent on radiation quality (LET).

  8. gammaH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products.

    PubMed

    Albino, Anthony P; Jorgensen, Ellen D; Rainey, Patrick; Gillman, Gene; Clark, T Jeffrey; Gietl, Diana; Zhao, Hong; Traganos, Frank; Darzynkiewicz, Zbigniew

    2009-08-01

    Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as gammaH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that gammaH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring gammaH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that gammaH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that gammaH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.

  9. DNA DSB measurements and modelling approaches based on gamma-H2AX foci time evolution

    NASA Astrophysics Data System (ADS)

    Esposito, Giuseppe; Campa, Alessandro; Antonelli, Francesca; Mariotti, Luca; Belli, Mauro; Giardullo, Paola; Simone, Giustina; Antonella Tabocchini, Maria; Ottolenghi, Andrea

    DNA double strand breaks (DSBs) induced by ionising radiation are considered the main dam-age related to the deleterious consequences in the cells. Unrepaired or mis-repaired DSBs can cause mutations or loss of chromosome regions which can eventually lead to cell death or neo-plastic transformation. Quantification of the number and complexity of DSBs induced by low doses of radiation remains a complex problem. About ten years ago Rogakou et al. proposed an immunofluorescent technique able to detect even a single DSB per cell. This approach is based on the serine 139 phosphorylation of many molecules (up to 2000) of histone H2AX (γg-H2AX) following the induction of a DSB in the DNA. DSB can be visualized as foci by immunofluores-cence by using phospho-specific antibodies, so that enumeration of foci can be used to measure DSB induction and processing. It is still not completely clear how γ-H2AX dephosphorylation takes place; however it has been related with DSB repair, in particular with the efficiency of DSB repair. In this work we analyse the H2AX phosphorylation-dephosphorylation kinetics after irradiation of primary human fibroblasts (AG1522 cell line) with radiation of differing quality, that is γ-rays and α-particles (125 keV/µm), with the aim of comparing the time evolution of γ-H2AX foci. Our results show that, after a dose of 0.5 Gy, both γ-rays and α-particles induce the maximum number of γ-H2AX foci within 30 minutes from irradiation, that this number depends on the radiation type and is consistent with the number of track traversal in α-irradiated nuclei, that the dephosphorylation kinetics are very different, being the α-induced foci rate of disappearence slower than that of γ-induced foci. In this work a modellistic approach to estimate the number of DSB induced by γ-rays detectable by using the γ-H2AX assay is presented. The competing processes of appearance and disappearance of visible foci will be modeled taking into account the

  10. Positive immunohistochemical staining of gammaH2AX is associated with tumor progression in gastric cancers from radiation-exposed patients.

    PubMed

    Sentani, Kazuhiro; Oue, Naohide; Sakamoto, Naoya; Nishisaka, Takashi; Fukuhara, Toshiyuki; Matsuura, Hiroo; Yasui, Wataru

    2008-11-01

    To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic bomb (A-bomb) exposure is important. DNA double-strand breaks (DSBs) are thought to be caused by the deleterious effects of ionizing radiation, and gammaH2AX (serine 139 phosphorylated form of histone H2AX) is reported to be a significant marker for DSBs. In the present study, we performed immunohistochemical analysis of gammaH2AX in gastric cancers (GCs) from 66 exposed and 47 non-exposed patients who developed GC after the bombing. Of the 47 GCs from non-exposed patients, 6 (13%) cases showed nuclear positive staining for gammaH2AX, whereas of the 66 GCs from exposed patients, 20 (30%) cases were positive (P=0.0405). However, among stage I GC, there was no significant difference in gammaH2AX expression frequency between exposed patients and non-exposed patients. Among exposed patients, stage II-IV cases were more frequently positive for gammaH2AX than stage I cases (P=0.0197). Among GCs from non-exposed patients, gammaH2AX staining showed no significant association with Lauren's classification, depth of invasion, lymph node metastasis or TNM stage. These results suggest that the characteristics of tumor cells differ between GCs from exposed and non-exposed patients. DSBs may be involved in progression of GC in exposed patients.

  11. γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin

    NASA Astrophysics Data System (ADS)

    Redon, Christophe E.; Dickey, Jennifer S.; Bonner, William M.; Sedelnikova, Olga A.

    2009-04-01

    Ionizing radiation (IR) exposure is inevitable in our modern society and can lead to a variety of deleterious effects including cancer and birth defects. A reliable, reproducible and sensitive assessment of exposure to IR and the individual response to that exposure would provide much needed information for the optimal treatment of each donor examined. We have developed a diagnostic test for IR exposure based on detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The cell responds to a nascent DSB through the phosphorylation of thousands of H2AX molecules flanking the damaged site. This highly amplified response can be visualized as a γ-H2AX focus in the chromatin that can be detected in situ with the appropriate antibody. Here we assess the usability of γ-H2AX focus formation as a possible biodosimeter for human exposure to IR using peripheral blood lymphocytes irradiated ex vivo and three-dimensional artificial models of human skin biopsies. In both systems, the tissues were exposed to 0.2-5 Gy, doses of IR that might be realistically encountered in various scenarios such as cancer radiotherapies or accidental exposure to radiation. Since the γ-H2AX response is maximal 30 min after exposure and declines over a period of hours as the cells repair the damage, we examined the time limitations of the useful detectability of γ-H2AX foci. We report that a linear response proportional to the initial radiation dose was obtained 48 and 24 h after exposure in blood samples and skin cells respectively. Thus, detection of γ-H2AX formation to monitor DNA damage in minimally invasive blood and skin tests could be useful tools to determine radiation dose exposure and analyze its effects on humans.

  12. Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break.

    PubMed

    Lee, Cheng-Sheng; Lee, Kihoon; Legube, Gaëlle; Haber, James E

    2014-01-01

    In budding yeast, a single double-strand break (DSB) triggers extensive Tel1 (ATM)- and Mec1 (ATR)-dependent phosphorylation of histone H2A around the DSB, to form γ-H2AX. We describe Mec1- and Tel1-dependent phosphorylation of histone H2B at T129. γ-H2B formation is impaired by γ-H2AX and its binding partner Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL7, GAL10 and GAL1 genes is turned off, γ-H2AX is restored within 5 min, in a Mec1-dependent manner; after reinduction of these genes, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other pericentromeric regions, again depending on Mec1. Our data provide new insights in the function and establishment of phosphorylation events occurring on chromatin after DSB induction.

  13. Plant γH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors.

    PubMed

    Lang, Julien; Smetana, Ondrej; Sanchez-Calderon, Lenin; Lincker, Frédéric; Genestier, Julie; Schmit, Anne-Catherine; Houlné, Guy; Chabouté, Marie-Edith

    2012-04-01

    Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AXH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of γH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses. We further establish the existence, restrictive to the G1/S transition, of specific DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

  14. Kinetics of gamma-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation.

    PubMed

    Paris, Lorena; Cordelli, Eugenia; Eleuteri, Patrizia; Grollino, Maria Giuseppa; Pasquali, Emanuela; Ranaldi, Roberto; Meschini, Roberta; Pacchierotti, Francesca

    2011-07-01

    Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.

  15. Low doses of X-rays induce prolonged and ATM-independent persistence of γH2AX foci in human gingival mesenchymal stem cells.

    PubMed

    Osipov, Andreyan N; Pustovalova, Margarita; Grekhova, Anna; Eremin, Petr; Vorobyova, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry Y; Eremin, Ilya

    2015-09-29

    Diagnostic imaging delivering low doses of radiation often accompany human mesenchymal stem cells (MSCs)-based therapies. However, effects of low dose radiation on MSCs are poorly characterized. Here we examine patterns of phosphorylated histone H2AXH2AX) and phospho-S1981 ATM (pATM) foci formation in human gingiva-derived MSCs exposed to X-rays in time-course and dose-response experiments. Both γH2AX and pATM foci accumulated linearly with dose early after irradiation (5-60 min), with a maximum induction observed at 30-60 min (37 ± 3 and 32 ± 3 foci/cell/Gy for γH2AX and pATM, respectively). The number of γH2AX foci produced by intermediate doses (160 and 250 mGy) significantly decreased (40-60%) between 60 and 240 min post-irradiation, indicating rejoining of DNA double-strand breaks. In contrast, γH2AX foci produced by low doses (20-80 mGy) did not change after 60 min. The number of pATM foci between 60 and 240 min decreased down to control values in a dose-independent manner. Similar kinetics was observed for pATM foci co-localized with γH2AX foci. Collectively, our results suggest differential DNA double-strand break signaling and processing in response to low vs. intermediate doses of X-rays in human MSCs. Furthermore, mechanisms governing the prolonged persistence of γH2AX foci in these cells appear to be ATM-independent.

  16. Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity.

    PubMed

    Vandersickel, Veerle; Depuydt, Julie; Van Bockstaele, Bram; Perletti, Gianpaolo; Philippe, Jan; Thierens, Hubert; Vral, Anne

    2010-01-01

    A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

  17. Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity.

    PubMed

    Tsamou, Maria; Jennen, Danyel G J; Claessen, Sandra M H; Magkoufopoulou, Christina; Kleinjans, Jos C S; van Delft, Joost H M

    2012-11-01

    The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery-Ames test, mouse lymphoma assay (MLA) and CA/MN test-resulted in low accuracy (55-65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62-70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell-based tool for early screening of compounds for in vivo genotoxicity.

  18. The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC inhibitors in ovarian cancer cells.

    PubMed

    Wilson, Andrew J; Holson, Edward; Wagner, Florence; Zhang, Yan-Ling; Fass, Daniel M; Haggarty, Stephen J; Bhaskara, Srividya; Hiebert, Scott W; Schreiber, Stuart L; Khabele, Dineo

    2011-09-15

    High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.

  19. Dub3 controls DNA damage signalling by direct deubiquitination of H2AX.

    PubMed

    Delgado-Díaz, M Rocío; Martín, Yusé; Berg, Anna; Freire, Raimundo; Smits, Veronique A J

    2014-07-01

    A crucial event in the DNA damage response is the phosphorylation and subsequent ubiquitination of H2AX, required for the recruitment of proteins involved in DNA repair. Here we identify a novel regulator of this process, the ubiquitin hydrolase Dub3. Overexpression of wild type, but not catalytic inactive, Dub3 decreases the DNA damage-induced mono-ubiquitination of H2A(X) whereas downregulation of Dub3 has the opposite effect. Dub3 overexpression abrogates focus formation of 53BP1 and BRCA1 in response to genotoxic stress. However, focus formation of MDC1 and γH2AX, earlier events in this response, are unaffected by Dub3 overexpression. We show that Dub3 counteracts H2AX E3 ligases RNF8 and RNF168. Moreover, Dub3 and H2AX interact and Dub3 deubiquitinates H2AX in vitro. Importantly, overexpression of Dub3 delays H2AX dephosphorylation and recovery of MDC1 focus formation at later time points after DNA damage, whereas H2AX dephosphorylation at later time points is faster after Dub3 depletion. Altogether these results show that Dub3 regulates a correct DNA damage response by controlling H2AX ubiquitination.

  20. Global quantification of γH2AX as a triage tool for the rapid estimation of received dose in the event of accidental radiation exposure.

    PubMed

    Viau, Muriel; Testard, Isabelle; Shim, Grace; Morat, Luc; Normil, Marie D; Hempel, William M; Sabatier, Laure

    2015-11-01

    The phosphorylation of the H2AX histone to form γH2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation between the number of γH2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of γH2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying γH2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global γH2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established γH2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global γH2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25-6Gy) using flow cytometry measurements showed that inter-individual variability in global γH2AX fluorescence is statistically insignificant at 4h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that

  1. Assessment of DNA double-strand breaks and γH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone

    PubMed Central

    Smart, Daniel J.; Halicka, H. Dorota; Schmuck, Gabriele; Traganos, Frank; Darzynkiewicz, Zbigniew; Williams, Gary M.

    2008-01-01

    Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as γH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of γH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells. DSBs were measured by the neutral comet assay, while γH2AX was quantified using immunocytochemistry and flow cytometry. Stabilized cleavage complexes (SCCs), lesions thought to be responsible for TOPO II poison-induced genotoxicity, were measured using a complex of enzyme–DNA assay. In the case of ETOP, a no observed adverse effect level (NOAEL) and lowest observed effect level (LOEL) for genotoxicity was determined; γH2AX levels paralleled DSBs at all concentrations but significant DNA damage was not detected below 0.5 μg/ml. Furthermore, DNA damage was dependent on the formation of SCCs. In contrast, at low MXT concentrations (0.0001–0.001 μg/ml), induction of γH2AX was not accompanied by increases in DSBs. Rather, DSBs were only significantly increased when SCCs were detected. These findings suggest MXT-induced genotoxicity occurred via at least two mechanisms, possibly related to DNA intercalation and/or redox cycling as well as TOPO II inhibition. Our findings also indicate that γH2AX can be induced by DNA lesions other than DSBs. In conclusion, γH2AX, when measured using immunocytochemical and flow cytometric methods, is a sensitive indicator of DNA damage and may be a useful tool in genetic toxicology screens. ETOP data are consistent with the threshold concept for TOPO II poison-induced genotoxicity and this should be considered in the safety assessment of chemicals displaying an affinity for TOPO II and genotoxic/clastogenic effects. PMID:18423498

  2. Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse.

    PubMed

    Chicheportiche, Alexandra; Bernardino-Sgherri, Jacqueline; de Massy, Bernard; Dutrillaux, Bernard

    2007-05-15

    Meiotic DNA double strand breaks (DSBs) are indicated at leptotene by the phosphorylated form of histone H2AX (gamma-H2AX). In contrast to previous studies, we identified on both zygotene and pachytene chromosomes two distinct types of gamma-H2AX foci: multiple small (S) foci located along autosomal synaptonemal complexes (SCs) and larger signals on chromatin loops (L-foci). The S-foci number gradually declined throughout pachytene, in parallel with the repair of DSBs monitored by repair proteins suggesting that S-foci mark DSB repair events. We validated this interpretation by showing the absence of S-foci in Spo11(-/-) spermatocytes. By contrast, the L-foci number was very low through pachytene. Based on the analysis of gamma-H2AX labeling after irradiation of spermatocytes, the formation of DSBs clearly induced L-foci formation. Upon DSB repair, these foci appear to be processed and lead to the above mentioned S-foci. The presence of L-foci in wild-type pachytene and diplotene could therefore reflect delayed or unregulated DSB repair events. Interestingly, their distribution was different in Spo11(+/-) spermatocytes compared with Spo11(+/+) spermatocytes, where DSB repair might be differently regulated as a response to homeostatic control of crossing-over. The presence of these L-foci in Spo11(-/-) spermatocytes raises the interesting possibility of yet uncharacterized alterations in DNA or chromosome structure in Spo11(-/-) cells.

  3. INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

    PubMed

    Morrison, Ashby J; Highland, Jessica; Krogan, Nevan J; Arbel-Eden, Ayelet; Greenblatt, Jack F; Haber, James E; Shen, Xuetong

    2004-12-17

    While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

  4. γ-H2AX/53BP1/pKAP-1 foci and their linear tracks induced by in vitro exposure to radon and its progeny in human peripheral blood lymphocytes

    PubMed Central

    Ding, Defang; Zhang, Yaping; Wang, Jing; Wang, Xufei; Fan, Dunhuang; He, Linfeng; Zhang, Xuxia; Gao, Yun; Li, Qiang; Chen, Honghong

    2016-01-01

    The biodosimetric information is critical for evaluating the human health hazards caused by radon and its progeny. Here, we demonstrated that the formation of phosphorylated histone variant H2AX (γ-H2AX), p53-binding protein 1 (53BP1) and phosphorylated KRAB-associated protein 1 (pKAP-1) foci and their linear tracks in human peripheral blood lymphocytes (HPBLs) in vitro exposed to radon and its progeny were dependent on the cumulative absorbed dose of radon exposure but was unrelated to the concentration of radon. Among them, γ-H2AX foci and its linear tracks were the most sensitive indicators with the lowest estimable cumulative absorbed dose of 1.74 mGy from their linear dose-response curves and sustained for 12 h after termination of radon exposure. In addition, three types of foci showed an overdispersed non-Poisson distribution in HPBLs. The ratios of pKAP-1/γ-H2AX foci co-localization, 53BP1/γ-H2AX foci co-localization and 53BP1/pKAP-1 foci co-localization were significantly increased in HPBLs exposed to radon while they were unrelated to the cumulative dose of radon exposure, suggesting that γ-H2AX, pKAP-1 and 53BP1 play an important role in the repair of heterochromatic double-strand breaks. Altogether, our findings provide an experimental basis for estimating the biological dose of internal α-particle irradiation from radon and its progeny exposure in humans. PMID:27922110

  5. Gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in targeted and bystander human artificial skin models and peripheral blood lymphocytes

    NASA Astrophysics Data System (ADS)

    Redon, Christophe; Dickey, Jennifer; Bonner, William; Sedelnikova, Olga

    Ionizing radiation (IR) exposure is inevitable. In addition to exposure from cosmic rays, the sun and radioactive substances, modern society has created new sources of radiation exposure such as space and high altitude journeys, X-ray diagnostics, radiological treatments and the increasing threat of radiobiological terrorism. For these reasons, a reliable, reproducible and sensitive assessment of dose and time exposure to IR is essential. We developed a minimally invasive diagnostic test for IR exposure based on detection of a phosphorylated variant of histone H2AX (gamma-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The phosphorylation of thousands of H2AX molecules forms a gamma-H2AX focus in the chromatin flanking the DSB site that can be detected in situ. We analyzed gamma- H2AX focus formation in both directly irradiated cells as well as in un-irradiated "bystanders" in close contact with irradiated cells. In order to insure minimal invasiveness, we examined commercially available artificial skin models as a surrogate for human skin biopsies as well as peripheral blood lymphocytes. In human skin models, cells in a thin plane were microbeamirradiated and gamma-H2AX formation was measured both in irradiated and in distal bystander cells over time. In irradiated cells DSB formation reached a maximum at 15-30 minutes post- IR and then declined within several hours; all cells were affected. In marked contrast, the incidence of DSBs in bystander cells reached a maximum by 12-48 hours post-irradiation, gradually decreasing over the 7 day time course. At the maxima, 40-60% of bystander cells were affected. Similarly, we analyzed blood samples exposed to IR ex vivo at doses ranging from 0.02 to 3 Gy. The amount of DNA damage was linear in respect to radiation dose and independent of the age or sex of the blood donor. The method is highly reproducible and highly sensitive. In directly irradiated cells, the number of gamma-H2AX foci peaked

  6. Persistence of gamma-H2AX and 53BP1 foci in proliferating and nonproliferating human mammary epithelial cells after exposure to gamma-rays or iron ions

    SciTech Connect

    Groesser, Torsten; Chang, Hang; Fontenay, Gerald; Chen, James; Costes, Sylvain V.; Barcellos-Hoff, Mary Helen; Parvin, Bahram; Rydberg, Bjorn

    2010-12-22

    To investigate {gamma}-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionizing radiation under different cell culture conditions. HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced {gamma}-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both {gamma}-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after {gamma}-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. Conclusions: The disappearance of radiation induced {gamma}-H2AX and 53BP1 foci in HMEC have different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent {gamma}-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodeling.

  7. Microwaves from GSM Mobile Telephones Affect 53BP1 and γ-H2AX Foci in Human Lymphocytes from Hypersensitive and Healthy Persons

    PubMed Central

    Markovà, Eva; Hillert, Lena; Malmgren, Lars; Persson, Bertil R. R.; Belyaev, Igor Y.

    2005-01-01

    The data on biologic effects of nonthermal microwaves (MWs) from mobile telephones are diverse, and these effects are presently ignored by safety standards of the International Commission for Non-Ionizing Radiation Protection (ICNIRP). In the present study, we investigated effects of MWs of Global System for Mobile Communication (GSM) at different carrier frequencies on human lymphocytes from healthy persons and from persons reporting hypersensitivity to electromagnetic fields (EMFs). We measured the changes in chromatin conformation, which are indicative of stress response and genotoxic effects, by the method of anomalous viscosity time dependence, and we analyzed tumor suppressor p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γ-H2AX), which have been shown to colocalize in distinct foci with DNA double-strand breaks (DSBs), using immunofluorescence confocal laser microscopy. We found that MWs from GSM mobile telephones affect chromatin conformation and 53BP1/γ-H2AX foci similar to heat shock. For the first time, we report here that effects of MWs from mobile telephones on human lymphocytes are dependent on carrier frequency. On average, the same response was observed in lymphocytes from hypersensitive and healthy subjects. PMID:16140623

  8. Genome-wide transcriptional analysis of apoptosis-related genes and pathways regulated by H2AX in lung cancer A549 cells.

    PubMed

    Lu, Chengrong; Xiong, Min; Luo, Yuan; Li, Jing; Zhang, Yanjun; Dong, Yaqiong; Zhu, Yanjun; Niu, Tianhui; Wang, Zhe; Duan, Lianning

    2013-09-01

    Histone H2AX is a novel tumor suppressor protein and plays an important role in apoptosis of cancer cells. However, the role of H2AX in lung cancer cells is unclear. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. We showed that H2AX was involved in apoptosis of lung cancer A549 cells as in other tumor cells. Knockdown of H2AX strongly suppressed apoptosis of A549 cells. We clarified the molecular mechanisms of apoptosis regulated by H2AX based on genome-wide transcriptional analysis. Microarray data analysis demonstrated that H2AX knockdown in A549 cells affected expression of 3,461 genes, including upregulation of 1,435 and downregulation of 2,026. These differentially expressed genes were subjected to bioinformatic analysis for exploring biological processes regulated by H2AX in lung cancer cells. Gene ontology analysis showed that H2AX affected expression of many genes, through which, many important functions including response to stimuli, gene expression, and apoptosis were involved in apoptotic regulation of lung cancer cells. Pathway analysis identified the mitogen-activated protein kinase signaling pathway and apoptosis as the most important pathways targeted by H2AX. Signal transduction pathway networks analysis and chromatin immunoprecipitation assay showed that two core genes, NFKB1 and JUN, were involved in apoptosis regulated by H2AX in lung cancer cells. Taken together, these data provide compelling clues for further exploration of H2AX function in cancer cells.

  9. Adapting the γ-H2AX Assay for Automated Processing in Human Lymphocytes. 1. Technological Aspects

    PubMed Central

    Turner, Helen C.; Brenner, David J.; Chen, Youhua; Bertucci, Antonella; Zhang, Jian; Wang, Hongliang; Lyulko, Oleksandra V.; Xu, Yanping; Shuryak, Igor; Schaefer, Julia; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y. Lawrence; Amundson, Sally A.; Garty, Guy

    2011-01-01

    The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes. PMID:21388271

  10. Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

    PubMed

    Quesnot, Nicolas; Rondel, Karine; Audebert, Marc; Martinais, Sophie; Glaise, Denise; Morel, Fabrice; Loyer, Pascal; Robin, Marie-Anne

    2016-01-01

    The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.

  11. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    SciTech Connect

    Yamagata, Kazutsune; Kitabayashi, Issay

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  12. Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

    PubMed

    Yamamoto, Takenobu; Ali, Mir A; Liu, XueQiao; Cohen, Jeffrey I

    2014-03-01

    Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

  13. Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells

    PubMed Central

    Pustovalova, Margarita; Grekhova, Anna; Astrelina, Tatiana; Nikitina, Viktoria; Dobrovolskaya, Ekaterina; Suchkova, Yulia; Kobzeva, Irina; Usupzhanova, Darya; Vorobyeva, Natalia; Samoylov, Aleksandr; Bushmanov, Andrey; Ozerov, Ivan V.; Zhavoronkov, Alex; Leonov, Sergey; Klokov, Dmitry; Osipov, Andreyan N.

    2016-01-01

    Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion. PMID:27959319

  14. Reduction of phosphorylated histone H3 serine 10 and serine 28 cell cycle marker intensities after DNA damage.

    PubMed

    Ozawa, Kazuo

    2008-06-01

    Phosphorylated histone H3 at serine 10 and serine 28 (H3Ser10 and H3Ser28) have been recognized as cell cycle markers to evaluate the late-G(2)/M status of cell and tissue samples. I report about the reduction phenomena of H3Ser10 and Ser28 phosphorylation (H3Ser10P and 28P) at the late-G(2) through M cell cycle phases in association with DNA damage caused by hydrogen peroxide (H(2)O(2)). The levels of H3Ser10P and Ser28P decreased between 15 and 60 min after H(2)O(2) addition in an inverse correlation manner with H2AX Ser139 phosphorylation (gammaH2AX). Experiments using wortmannin suggested that the reduction events of H3Ser10P/28P are under the control of phosphatidylinositol 3-kinase-like kinases. Fluorescence microscopic observation showed that H3Ser10 and Ser28 on telomeric portions of condensed M-phase chromosomes retained their strongly phosphorylated status even after 60 min of H(2)O(2) treatment. In addition, these chromosome parts were poorly gammaH2AX positive showing mutually exclusive distribution patterns between H3Ser10P/28P and gammaH2AX. Considering these data, I hypothesize that the reduction of the H3Ser10P/28P is a part of the DNA damage response processes. It is advisable to pay careful attention to these phenomena at the time of designing cell cycle assay protocols with H3Ser10P or Ser28P mitosis markers when DNA damaging process is expected to occur.

  15. HDAC inhibitor sodium butyrate sensitizes E1A+Ras-transformed cells to DNA damaging agents by facilitating formation and persistence of γH2AX foci.

    PubMed

    Abramova, Maria V; Svetlikova, Svetlana B; Kukushkin, Alexander N; Aksenov, Nikolai D; Pospelova, Tatiana V; Pospelov, Valery A

    2011-12-15

    HDAC inhibitors (HDACi) suppress the growth of tumor cells due to induction of cell cycle arrest, senescence or apoptosis. Recent data demonstrate that HDACi can interfere with DNA Damage Response (DDR) thereby sensitizing the cells to DNA damaging agents. Here, we show that HDACi sodium butyrate (NaBut) potentiates the formation of γH2AX foci predominantly in S-phase E1A+Ras cells. Accumulation of γH2AX foci sensitizes the cells toward such DNA damaging agents as irradiation (IR) and adriamycin. In fact, NaBut potentiates the persistence of γH2AX foci induced by genotoxic agents. The synergizing effects depend on DNA damaging factors and on the order of NaBut treatment. Indeed, NaBut treatment for 24 h leads to an accumulation of G 1-phase cells and a lack of S-phase cells, therefore, adriamycin, a powerful S-phase-specific inhibitor, when added to NaBut-treated cells, is unable to substantially add γH2AX foci. In contrast, IR produces both single- and double-strand DNA breaks at any stage of the cell cycle and was shown to increase γH2AX foci in NaBut-treated cells. Further, a lifetime of IR-induced γH2AX foci depends on the subsequent presence of HDACi. Correspondingly, NaBut withdrawal leads to the extinction of IR-induced γH2AX foci. This necessitates HDACi to hold the IR-induced γH2AX foci unrepaired. However, the IR-induced γH2AX foci persist after long-term NaBut treatment (72 h) even after washing the drug. Thus, although signaling pathways regulating H2AX phosphorylation in NaBut-treated cells remain to be investigated, the obtained results show that NaBut potentiates effects of DNA damaging agents by facilitating formation and persistence of γH2AX foci.

  16. X-Ray Induced Formation of γ-H2AX Foci after Full-Field Digital Mammography and Digital Breast-Tomosynthesis

    PubMed Central

    Schwab, Siegfried A.; Brand, Michael; Schlude, Ina-Kristin; Wuest, Wolfgang; Meier-Meitinger, Martina; Distel, Luitpold; Schulz-Wendtland, Ruediger; Uder, Michael; Kuefner, Michael A.

    2013-01-01

    Purpose To determine in-vivo formation of x-ray induced γ-H2AX foci in systemic blood lymphocytes of patients undergoing full-field digital mammography (FFDM) and to estimate foci after FFDM and digital breast-tomosynthesis (DBT) using a biological phantom model. Materials and Methods The study complies with the Declaration of Helsinki and was performed following approval by the ethic committee of the University of Erlangen-Nuremberg. Written informed consent was obtained from every patient. For in-vivo tests, systemic blood lymphocytes were obtained from 20 patients before and after FFDM. In order to compare in-vivo post-exposure with pre-exposure foci levels, the Wilcoxon matched pairs test was used. For in-vitro experiments, isolated blood lymphocytes from healthy volunteers were irradiated at skin and glandular level of a porcine breast using FFDM and DBT. Cells were stained against the phosphorylated histone variant γ-H2AX, and foci representing distinct DNA damages were quantified. Results Median in-vivo foci level/cell was 0.086 (range 0.067–0.116) before and 0.094 (0.076–0.126) after FFDM (p = 0.0004). In the in-vitro model, the median x-ray induced foci level/cell after FFDM was 0.120 (range 0.086–0.140) at skin level and 0.035 (range 0.030–0.050) at glandular level. After DBT, the median x-ray induced foci level/cell was 0.061 (range 0.040–0.081) at skin level and 0.015 (range 0.006–0.020) at glandular level. Conclusion In patients, mammography induces a slight but significant increase of γ-H2AX foci in systemic blood lymphocytes. The introduced biological phantom model is suitable for the estimation of x-ray induced DNA damages in breast tissue in different breast imaging techniques. PMID:23936236

  17. Activation of p38 mitogen-activated protein kinase by celecoxib oppositely regulates survivin and gamma-H2AX in human colorectal cancer cells

    SciTech Connect

    Hsiao, P.-W.; Chang, C.-C.; Liu, H.-F.; Tsai, C.-M.; Chiu, Ted H.; Chao, J.-I . E-mail: chaoji@mail.tcu.edu.tw

    2007-07-01

    Cancer cells express survivin that facilitates tumorigenesis. Celecoxib has been shown to reduce human colorectal cancers. However, the role and regulation of survivin by celecoxib in colorectal carcinoma cells remain unclear. Treatment with 40-80 {mu}M celecoxib for 24 h induced cytotoxicity and proliferation inhibition via a concentration-dependent manner in RKO colorectal carcinoma cells. Celecoxib blocked the survivin protein expression and increased the phosphorylation of H2AX at serine-193 ({gamma}-H2AX). The survivin gene knockdown by transfection with a survivin siRNA revealed that the loss of survivin correlated with the expression of {gamma}-H2AX. Meanwhile, celecoxib increased caspase-3 activation and apoptosis. Celecoxib activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The phosphorylated proteins of p38 MAP kinase and {gamma}-H2AX were observed in the apoptotic cells. SB203580, a specific p38 MAP kinase inhibitor, protected the survivin protein expression and decreased the levels of {gamma}-H2AX and apoptosis in the celecoxib-exposed cells. The blockade of survivin expression increased the celecoxib-induced cytotoxicity; conversely, overexpression of survivin by transfection with a survivin-expressing vector raised the cancer cell proliferation and resisted the celecoxib-induced cell death. Our results provide for the first time that p38 MAP kinase participates in the down-regulation of survivin and subsequently induces the activation of {gamma}-H2AX for mediating apoptosis following treatment with celecoxib in human colorectal cancer cells.

  18. Epstein-Barr Virus Essential Antigen EBNA3C Attenuates H2AX Expression

    PubMed Central

    Jha, Hem C.; AJ, Mahadesh Prasad; Saha, Abhik; Banerjee, Shuvomoy; Lu, Jie

    2014-01-01

    Epstein-Barr virus (EBV) latent antigen EBNA3C is implicated in B-cell immortalization and linked to several B-cell malignancies. Deregulation of H2AX can induce genomic instability with increased chromosomal aberrations, which ultimately leads to tumorigenesis. Here we demonstrated that EBNA3C can attenuate H2AX expression at the transcript and protein levels. A reduction of total H2AX levels was clearly observed upon infection of primary B cells with wild-type EBV but not with EBNA3C knockout recombinant EBV. H2AX also interacted with EBNA3C through its N-terminal domain (residues 1 to 100). Furthermore, H2AX mutated at Ser139 failed to interact with EBNA3C. Luciferase-based reporter assays also revealed that the binding domain of EBNA3C is sufficient for transcriptional inhibition of the H2AX promoter. EBNA3C also facilitated H2AX degradation through recruitment of components of the ubiquitin proteasome pathway. We further demonstrated that knockdown of H2AX in lymphoblastoid cell lines (LCLs) led to the upregulation of the Bub1 oncoprotein and downregulated expression of p53. Overall, our study provides additional insights into EBV-associated B-cell lymphomas, which are linked to the regulation of the DNA damage response system in infected cells. The importance of these insights are as follows: (i) EBNA3C downregulates H2AX expression at the protein and transcript levels in epithelial cells, B cells, and EBV-transformed LCLs, (ii) EBNA3C binds with wild-type H2AX but not with the Ser139 mutant of H2AX, (iii) the N terminus (residues 1 to 100) of EBNA3C is critical for binding to H2AX, (iv) localization of H2AX is predominantly nuclear in the presence of EBNA3C, and (v) H2AX knocked down in LCLs led to enhanced expression of Bub1 and downregulation of the tumor suppressor p53, which are both important for driving the oncogenic process. PMID:24429368

  19. Induction and inhibition of the pan-nuclear gamma-H2AX response in resting human peripheral blood lymphocytes after X-ray irradiation.

    PubMed

    Ding, D; Zhang, Y; Wang, J; Zhang, X; Gao, Y; Yin, L; Li, Q; Li, J; Chen, H

    2016-01-01

    Human peripheral blood lymphocytes (HPBLs) are one of the most sensitive cells to ionizing radiation (IR) in the human body, and IR-induced DNA damage and functional impairment of HPBLs are the adverse consequences of IR accidents and major side effects of radiotherapy. Phosphorylated H2AXH2AX) is a sensitive marker for DNA double-strand breaks, but the role and regulation of the pan-nuclear γH2AX response in HPBLs after IR remain unclear. We herein demonstrated that the pan-nuclear γH2AX signals were increased in a time- and dose-dependent manner, colocalized with >94% of TUNEL apoptotic staining, and displayed a typical apoptotic pattern in resting HPBLs after low LET X-ray IR. In addition, the X-irradiation-induced pan-nuclear p-ATM and p-DNA-PKcs responses also occurred in resting HPBLs, and were colocalized with 92-95% of TUNEL staining and 97-98% of the pan-nuclear γH2AX signals, respectively, with a maximum at 6 h post irradiation, but disappeared at 24 h post irradiation. Moreover, ATM/DNA-PKcs inhibitor KU55933, p53 inhibitor PFT-μ and pan-caspase inhibitor ZVAD-fmk significantly decreased X-irradiation-induced pan-nuclear γH2AX signals and TUNEL staining, protected HPBLs from apoptosis, but decreased the proliferative response to mitogen in X-irradiated HPBLs. Notably, whereas both KU55933 and PFT-μ increased the IR-induced chromosome breaks and mis-repair events through inhibiting the formation of p-ATM, p-DNA-PKcs and γH2AX foci in X-irradiated HPBLs, the ZVAD-fmk did not increase the IR-induced chromosomal instability. Taken together, our data indicate that pan-nuclear γH2AX response represents an apoptotic signal that is triggered by the transient pan-nuclear ATM and DNA-PKcs activation, and mediated by p53 and pan-caspases in X-irradiated HPBLs, and that caspase inhibitors are better than ATM/DNA-PKcs inhibitors and p53 inhibitors to block pan-nuclear γH2AX response/apoptosis and protect HPBLs from IR.

  20. Study on γH2AX Expression of Lymphocytes as a Biomarker In Radiation Biodosimetry

    PubMed Central

    Pan, Yan; Gao, Gang; Ruan, Jian Lei; Liu, Jian Xiang

    2016-01-01

    Flow cytometry analysis was used to detect the changes of γH2AX protein expression in human peripheral blood lymphocytes. In the dose-effect study, the expression of γH2AX was detected 1 h after irradiation with 60Co γ-rays at doses of 0, 0.5, 1, 2, 4, and 6 Gy. Blood was cultivated for 0, 1, 2, 4, 6, 12, and 24 h after 4 Gy 60Co γ-rays irradiation for the time-effect study. At the same time, the blood was divided into four treatment groups (ultraviolet [UV] irradiation, 60Co γ-rays irradiation, UV plus 60Co γ-rays irradiation, and control group) to detect the changes of protein expression of γH2AX. The results showed that the γH2AX protein expression was in dose-effect and time-effect relationship with 60Co γ-rays. The peak expression of γH2AX was at 1 h after 60Co γ-ray irradiation and began to decrease quickly. Compared to irradiation with 60Co γ-rays alone, the expression of γH2AX was not significantly changed after irradiation with 60Co γ-rays plus UV. Dose rate did not significantly change the expression of γH2AX. The expression of γH2AX induced by 60Co γ-rays was basically consistent with the mice in vivo and in vitro. The results revealed that the detection of γH2AX protein expression changes in peripheral blood lymphocyte by flow cytometry analysis is reasonable and may be useful for biodosimetry. PMID:28217286

  1. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

    PubMed

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-Ping; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-05-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AXH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.

  2. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors

    PubMed Central

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-ping; Young, Lauren F.; Shieh, Jae-Hung; Moore, Malcolm A.; van den Brink, Marcel R.M.; Kusunoki, Yoichiro

    2016-01-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AXH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34 + Lin−) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. PMID:27169377

  3. Early Detection of Genotoxic Urinary Bladder Carcinogens by Immunohistochemistry for γ-H2AX.

    PubMed

    Toyoda, Takeshi; Cho, Young-Man; Akagi, Jun-Ichi; Mizuta, Yasuko; Hirata, Tadashi; Nishikawa, Akiyoshi; Ogawa, Kumiko

    2015-12-01

    DNA double-strand breaks (DSBs) induced by exposure to genotoxic agents are known to cause genome instability and cancer development. To evaluate the applicability of γ-H2AX, a sensitive marker of DSBs, in the early detection of genotoxicity and carcinogenicity of chemicals using animal models, we examined γ-H2AX expression in urinary bladders of rats. Six-week-old male F344 rats were orally treated for 4 weeks with a total of 12 chemicals divided into 4 categories based on genotoxicity and carcinogenicity in the urinary bladder. Animals were sacrificed at the end of administration or after 2 weeks of recovery, and immunohistochemistry for γ-H2AX was performed. At week 4, γ-H2AX expression in bladder epithelial cells was significantly increased by all 4 genotoxic bladder carcinogens as compared with the controls, whereas the 3 chemicals that were genotoxic but not carcinogenic in the bladders did not cause upregulation of γ-H2AX. After the recovery period, γ-H2AX expression was markedly reduced in all groups but remained significantly elevated in rats treated with 3 of the 4 genotoxic bladder carcinogens. Although slight increases in γ-H2AX expression were induced by a weak bladder carcinogen with equivocal genotoxicity (phenethyl isothiocyanate) and 2 nongenotoxic bladder carcinogens (melamine and uracil) at week 4, these differences were not significant and were thought to be associated with activated proliferation by urothelial hyperplasia, as demonstrated by increased Ki67-positive cells. These results suggested that γ-H2AX may be a potential biomarker for the early detection of genotoxic bladder carcinogens.

  4. Evaluation of the gamma-H2AX assay for radiation biodosimetry in a swine model.

    PubMed

    Moroni, Maria; Maeda, Daisuke; Whitnall, Mark H; Bonner, William M; Redon, Christophe E

    2013-07-08

    There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo γ-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans.

  5. Evaluation of the Gamma-H2AX Assay for Radiation Biodosimetry in a Swine Model

    PubMed Central

    Moroni, Maria; Maeda, Daisuke; Whitnall, Mark H.; Bonner, William M.; Redon, Christophe E.

    2013-01-01

    There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo γ-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. PMID:23880859

  6. Induction and Persistence of Large γH2AX Foci by High Linear Energy Transfer Radiation in DNA-Dependent protein kinase–Deficient Cells

    SciTech Connect

    Bracalente, Candelaria; Ibañez, Irene L.; Molinari, Beatriz; Palmieri, Mónica; Kreiner, Andrés; Valda, Alejandro; and others

    2013-11-15

    Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AXH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm{sup 2}) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation.

  7. Study of γ-H2AX as DNA double strand break biomarker in resident living in high natural radiation area of Mamuju, West Sulawesi.

    PubMed

    Hasan Basri, Iin Kurnia; Yusuf, Darlina; Rahardjo, Tur; Nurhayati, Siti; Tetriana, Devita; Ramadhani, Dwi; Alatas, Zubaidah; Purnami, Sofiati; Kisnanto, Teja; Lusiyanti, Yanti; Syaifudin, Mukh

    2017-05-01

    High expression of phospho histone γ-H2AX, a sensitive marker of double stranded DNA damage, is believed to be an indication of defective DNA repair pathway or genomic instability that may cause mutations and ultimately cancer. DNA damage can be caused by ionizing radiation exposure. Beside in medical treatment/diagnosis or industry, ionizing radiation exposure can also be found in naturally in regions of high natural back ground radiation. In this study we collect the blood from 45 volunteers living in Mamuju, a region with highest natural radiation in Indonesia (dose of ∼7 mSv/year). Subjects were grouped as high natural background area (HNBA) (n = 37) and control area (n = 8). The expression γ-H2AX foci were evaluated by one of researcher fluorescence microscope examination. Our results show that the average foci numbers per cell were in the normal range. While not statistical different, the average of γ-H2AX foci in exposed area higher in the exposed compared to the control area, 0.31 versus 0.13 (p > 0.05), respectively. Moreover, there was also no statistical difference of average γ-H2AX foci between man and woman, old and young people in exposed and control area (p > 0.05). In this preliminary study we find that γ-H2AX foci (and thus DNA double strand break) frequency in residents living in the HNBA of Mamuju, West Sulawesi, show a trend towards higher (albeit not significant) average values relative to the control area. More research is needed to further scrutinize these observations.

  8. Involvement of ROS-p38-H2AX axis in novel curcumin analogues-induced apoptosis in breast cancer cells.

    PubMed

    Dong, Yinhui; Yin, Shutao; Song, Xinhua; Huo, Yazhen; Fan, Lihong; Ye, Min; Hu, Hongbo

    2016-04-01

    Curcumin-based structural modification for developing more effective curcumin analogues has been drawning increasing attention. As alternative approach, using LC/MS guided purification, we previously obtained a series of novel natural terpene-conjugated curcuminoids from turmeric, and some of them exhibited even more potent anti-cancer activity against multiple types of cancer cells than curcumin. The purpose of this follow-up study was designed to decipher the mechanisms involved in anti-cancer activity of these novel curcumin analogues. Apoptosis was evaluated using sub-G1 analysis by flow cytometry and Cell Death ELISA Kit. Changes of protein expression were analyzed by western blotting. RNA interference was employed to inhibit expression of specific protein. We found that bisabolocurcumin ether (T1) and demethoxybisabolocurcumin ether (T2) were able to trigger much stronger apoptosis induction in multiple types of cancer cells than curcumin, which was attributed to persistent and stronger ROS generation. ROS induction by T1 resulted in activation of p38/H2AX axis and p53. Inhibition of p38/H2AX led to a significant reduction of apoptosis, whereas inactivation of p53 caused a dramatically enhanced H2AX phosphorylation and apoptosis induction, suggesting activation of p38/H2AX contributed to apoptosis induction by T1, whereas p53 activation protected novel curcumins-induced apoptosis via suppression of H2AX activation. Our findings provide mechanistic support for the potential use of terpene-conjugated curcuminoids as a novel class of cancer chemopreventive agents. © 2015 Wiley Periodicals, Inc.

  9. Spatiotemporal kinetics of γ-H2AX protein on charged particles induced DNA damage

    NASA Astrophysics Data System (ADS)

    Niu, H.; Chang, H. C.; Cho, I. C.; Chen, C. H.; Liu, C. S.; Chou, W. T.

    2014-08-01

    In several researches, it has been demonstrated that charged particles can induce more complex DNA damages. These complex damages have higher ability to cause the cell death or cell carcinogenesis. For this reason, clarifying the DNA repair mechanism after charged particle irradiation plays an important role in the development of charged particle therapy and space exploration. Unfortunately, the detail spatiotemporal kinetic of DNA damage repair is still unclear. In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in alpha-particle irradiated HeLa cells. The result shows that the intensity of γ-H2AX foci increased gradually, and reached to its maximum at 30 min after irradiation. A good linear relationship can be observed between foci intensity and radiation dose. After 30 min, the γ-H2AX foci intensity was decreased with time passed, but remained a large portion (∼50%) at 48 h passed. The data show that the dissolution rate of γ-H2AX foci agreed with two components DNA repairing model. These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair.

  10. Generation of an alpaca-derived nanobody recognizing γ-H2AX

    PubMed Central

    Rajan, Malini; Mortusewicz, Oliver; Rothbauer, Ulrich; Hastert, Florian D.; Schmidthals, Katrin; Rapp, Alexander; Leonhardt, Heinrich; Cardoso, M. Cristina

    2015-01-01

    Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers. PMID:26500838

  11. USP11 Is a Negative Regulator to γH2AX Ubiquitylation by RNF8/RNF168*

    PubMed Central

    Yu, Miao; Liu, Kun; Mao, Zebin; Luo, Jianyuan; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Ubiquitin modification at double strand breaks (DSB) sites is an essential regulator of signaling and repair. γH2AX extends from DSB sites and provides a platform for subsequent recruitment and amplification of DNA repair proteins and signaling factors. Here, we found that RNF8/RNF168 ubiquitylates γH2AX. We identified that USP11 is a unique deubiquitylation enzyme for γH2AX. USP11 deubiquitylates γH2AX both in vivo and in vitro but not the canonical (ub)-K119-H2A and (ub)-K120-H2B in vitro, and USP11 ablation enhances the levels of γH2AX ubiquitylation. We also found that USP11 interacts with γH2AX both in vivo and in vitro. We found that 53BP1 and ubiquitin-conjugated proteins are misregulated to be retained longer and stronger at DSB sites after knockdown of USP11. We further found that cells are hypersensitive to γ-irradiation after ablation of USP11. Together, our findings elucidate deeply and extensively the mechanism of RNF8/RNF168 and USP11 to maintain the proper status of ubiquitylation γH2AX to repair DSB. PMID:26507658

  12. Association of γH2AX at Diagnosis with Chemotherapy Outcome in Patients with Breast Cancer

    PubMed Central

    Yang, Sherry X.; Polley, Eric C.; Nguyen, Dat

    2017-01-01

    γH2AX plays a role in DNA damage response signaling and facilitates the repair of DNA double strand breaks. However, it remains unknown whether constitutive tumor γH2AX expression is associated with treatment outcome in patients. γH2AX status was detected in primary tumors from 24% of 826 patients with stage I, II and III breast cancer by immunohistochemistry; overall survival was analyzed by Kaplan-Meier method. At median follow-up of 176 months (range 13 - 282 months), we found substantial survival heterogeneity in γH2AX-positive patients (P=0.002) among uniform treatment groups including radiation or endocrine therapy alone and no-treatment, as well as chemotherapy alone (being worst), in contrast to γH2AX-negative patients (P=0.2). In the chemotherapy group (n=118), median survival was 63 months (95% confidence interval [CI], 29 - 83) in patients with γH2AX-positive tumors compared with 170 months (95% CI 94 - 235) in those with γH2AX-negative tumors (P=0.0017). γH2AX remained a poor prognosis factor in the group by multivariable analysis (adjusted hazard ratio 2.12, P=0.009). Our data demonstrate that constitutive γH2AX positivity is significantly associated with survival heterogeneity in patients among uniform treatment groups, and its expression at diagnosis independently predicts poor chemotherapy outcome in breast cancer. PMID:28382166

  13. Calculation of Dose Deposition in Nanovolumes and Simulation of gamma-H2AX Experiments

    NASA Technical Reports Server (NTRS)

    Plante, Ianik

    2010-01-01

    Monte-Carlo track structure simulations can accurately simulate experimental data: a) Frequency of target hits. b) Dose per event. c) Dose per ion. d) Radial dose. The dose is uniform in micrometers sized voxels; at the nanometer scale, the difference in energy deposition between high and low-LET radiations appears. The calculated 3D distribution of dose voxels, combined with chromosomes simulated by random walk is very similar to the distribution of DSB observed with gamma-H2AX experiments. This is further evidenced by applying a visualization threshold on dose.

  14. ATM-mediated phosphorylation of the chromatin remodeling enzyme BRG1 modulates DNA double-strand break repair.

    PubMed

    Kwon, S-J; Park, J-H; Park, E-J; Lee, S-A; Lee, H-S; Kang, S W; Kwon, J

    2015-01-15

    ATP-dependent chromatin remodeling complexes such as SWI/SNF (SWItch/Sucrose NonFermentable) have been implicated in DNA double-strand break (DSB) repair and damage responses. However, the regulatory mechanisms that control the function of chromatin remodelers in DNA damage response are largely unknown. Here, we show that ataxia telangiectasia mutated (ATM) mediates the phosphorylation of BRG1, the catalytic ATPase of the SWI/SNF complex that contributes to DSB repair by binding γ-H2AX-containing nucleosomes via interaction with acetylated histone H3 and stimulating γ-H2AX formation, at Ser-721 in response to DNA damage. ATM-mediated phosphorylation of BRG1 occurs rapidly and transiently after DNA damage. Phosphorylated BRG1 binds γ-H2AX-containing nucleosomes to form the repair foci. The Ser-721 phosphorylation of BRG1 is critical for binding γ-H2AX-containing nucleosomes and stimulating γ-H2AX formation and DSB repair. BRG1 binds to acetylated H3 peptides much better after phosphorylation at Ser-721 by DNA damage. However, the phosphorylation of Ser-721 does not significantly affect the ATPase and transcriptional activities of BRG1. These results, establishing BRG1 as a novel and functional ATM substrate, suggest that the ATM-mediated phosphorylation of BRG1 facilitates DSB repair by stimulating the association of this remodeler with γ-H2AX nucleosomes via enhancing the affinity to acetylated H3. Our work also suggests that the mechanism of BRG1 stimulation of DNA repair is independent of the remodeler's enzymatic or transcriptional activities.

  15. Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

    PubMed

    Cheung, Jennifer R; Dickinson, Donna A; Moss, Jocelyn; Schuler, Maik J; Spellman, Richard A; Heard, Pamela L

    2015-01-01

    The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

  16. High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome.

    PubMed

    Iacovoni, Jason S; Caron, Pierre; Lassadi, Imen; Nicolas, Estelle; Massip, Laurent; Trouche, Didier; Legube, Gaëlle

    2010-04-21

    Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

  17. Quantification of gamma-H2AX foci in human lymphocytes: a method for biological dosimetry after ionizing radiation exposure.

    PubMed

    Roch-Lefèvre, Sandrine; Mandina, Tania; Voisin, Pascale; Gaëtan, Gruel; Mesa, Jorge Ernesto Gonzàlez; Valente, Marco; Bonnesoeur, Pierre; García, Omar; Voisin, Philippe; Roy, Laurence

    2010-08-01

    Recent studies have suggested that visualization of gamma-H2AX nuclear foci can be used to estimate exposure to very low doses of ionizing radiation. Although this approach is widely used for various purposes, its suitability for individual human biodosimetry has not yet been assessed. We therefore conducted such an assessment with the help of available software for observing and automatically scoring gamma-H2AX foci. The presence of gamma-H2AX foci was evaluated in human peripheral blood lymphocytes exposed ex vivo to gamma rays in a dose range of 0.02 to 2 Gy. We analyzed the response of gamma-H2AX to ionizing radiation in relation to dose, time after exposure, and individual variability. We constructed dose-effect calibration curves at 0.5, 8 and 16 h after exposure and evaluated the threshold of detection of the technique. The results show the promise of automatic gamma-H2AX scoring for a reliable assessment of radiation doses in a dose range of 0.6 Gy to 2 Gy up to 16 h after exposure. This gamma-H2AX-based assay may be useful for biodosimetry, especially for triage to distinguish promptly among individuals the ones who have received negligible doses from those with significantly exposures who are in need of immediate medical attention. However, additional in vivo experiments are needed for validation.

  18. High throughput measurement of γH2AX DSB repair kinetics in a healthy human population.

    PubMed

    Sharma, Preety M; Ponnaiya, Brian; Taveras, Maria; Shuryak, Igor; Turner, Helen; Brenner, David J

    2015-01-01

    The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

  19. Induction and Processing of the Radiation-Induced Gamma-H2AX Signal and Its Link to the Underlying Pattern of DSB: A Combined Experimental and Modelling Study.

    PubMed

    Tommasino, Francesco; Friedrich, Thomas; Jakob, Burkhard; Meyer, Barbara; Durante, Marco; Scholz, Michael

    2015-01-01

    We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed

  20. RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

    PubMed Central

    Mamouni, Kenza; Cristini, Agnese; Guirouilh-Barbat, Josée; Monferran, Sylvie; Lemarié, Anthony; Faye, Jean-Charles; Lopez, Bernard S.

    2014-01-01

    Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression. PMID:24912678

  1. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  2. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  3. γH2AX foci on apparently intact mitotic chromosomes: not signatures of misrejoining events but signals of unresolved DNA damage.

    PubMed

    Martín, Marta; Terradas, Mariona; Hernández, Laia; Genescà, Anna

    2014-01-01

    The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

  4. γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90.

    PubMed

    Turner, Helen C; Shuryak, Igor; Weber, Waylon; Doyle-Eisele, Melanie; Melo, Dunstana; Guilmette, Raymond; Amundson, Sally A; Brenner, David J

    2015-01-01

    In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as 137Cs and 90Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected 137CsCl and 90SrCl2 radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for 137Cs (0 to 10 Gy) and 90Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle

  5. γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90

    PubMed Central

    Turner, Helen C.; Shuryak, Igor; Weber, Waylon; Doyle-Eisele, Melanie; Melo, Dunstana; Guilmette, Raymond; Amundson, Sally A.; Brenner, David J.

    2015-01-01

    In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as 137Cs and 90Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected 137CsCl and 90SrCl2 radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for 137Cs (0 to 10 Gy) and 90Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle

  6. Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

    PubMed Central

    Reindl, Judith; Girst, Stefanie; Walsh, Dietrich W. M.; Greubel, Christoph; Schwarz, Benjamin; Siebenwirth, Christian; Drexler, Guido A.; Friedl, Anna A.; Dollinger, Günther

    2017-01-01

    The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened. PMID:28094292

  7. The first gamma-H2AX biodosimetry intercomparison exercise of the developing European biodosimetry network RENEB.

    PubMed

    Barnard, S; Ainsbury, E A; Al-hafidh, J; Hadjidekova, V; Hristova, R; Lindholm, C; Monteiro Gil, O; Moquet, J; Moreno, M; Rößler, U; Thierens, H; Vandevoorde, C; Vral, A; Wojewódzka, M; Rothkamm, K

    2015-04-01

    In the event of a mass casualty radiation incident, the gamma-H2AX foci assay could be a useful tool to estimate radiation doses received by individuals. The rapid processing time of blood samples of just a few hours and the potential for batch processing, enabling high throughput, make the assay ideal for early triage categorisation to separate the 'worried well' from the low and critically exposed by quantifying radiation-induced foci in peripheral blood lymphocytes. Within the RENEB framework, 8 European laboratories have taken part in the first European gamma-H2AX biodosimetry exercise, which consisted of a telescoring comparison of 200 circulated foci images taken from 8 samples, and a comparison of 10 fresh blood lymphocyte samples that were shipped overnight to participating labs 4 or 24 h post-exposure. Despite large variations between laboratories in the dose-response relationship for foci induction, the obtained results indicate that the network should be able to use the gamma-H2AX assay for rapidly identifying the most severely exposed individuals within a cohort who could then be prioritised for accurate chromosome dosimetry.

  8. γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis

    PubMed Central

    Ivashkevich, Alesia N.; Martin, Olga A.; Smith, Andrea J.; Redon, Christophe E.; Bonner, William M.; Martin, Roger F.; Lobachevsky, Pavel N.

    2011-01-01

    The γH2AX focus assay represents a fast and sensitive approach for detection of one of the critical types of DNA damage – double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable approach due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens. PMID:21216255

  9. γH2Ax Expression as a Potential Biomarker Differentiating between Low and High Grade Cervical Squamous Intraepithelial Lesions (SIL) and High Risk HPV Related SIL

    PubMed Central

    Kefala, Maria; Kottaridi, Christine; Spathis, Aris; Gouloumi, Alina-Roxani; Pouliakis, Abraham; Pappas, Asimakis; Sioulas, Vasileios; Chrelias, Charalambos; Karakitsos, Petros; Panayiotides, Ioannis

    2017-01-01

    Background γH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas. Methods Immunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed. Results Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. Conclusions γH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state. PMID:28118377

  10. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  11. Calculation of Dose Deposition in 3D Voxels by Heavy Ions and Simulation of gamma-H2AX Experiments

    NASA Technical Reports Server (NTRS)

    Plante, I.; Ponomarev, A. L.; Wang, M.; Cucinotta, F. A.

    2011-01-01

    The biological response to high-LET radiation is different from low-LET radiation due to several factors, notably difference in energy deposition and formation of radiolytic species. Of particular importance in radiobiology is the formation of double-strand breaks (DSB), which can be detected by -H2AX foci experiments. These experiments has revealed important differences in the spatial distribution of DSB induced by low- and high-LET radiations [1,2]. To simulate -H2AX experiments, models based on amorphous track with radial dose are often combined with random walk chromosome models [3,4]. In this work, a new approach using the Monte-Carlo track structure code RITRACKS [5] and chromosome models have been used to simulate DSB formation. At first, RITRACKS have been used to simulate the irradiation of a cubic volume of 5 m by 1) 450 1H+ ions of 300 MeV (LET 0.3 keV/ m) and 2) by 1 56Fe26+ ion of 1 GeV/amu (LET 150 keV/ m). All energy deposition events are recorded to calculate dose in voxels of 20 m. The dose voxels are distributed randomly and scattered uniformly within the volume irradiated by low-LET radiation. Many differences are found in the spatial distribution of dose voxels for the 56Fe26+ ion. The track structure can be distinguished, and voxels with very high dose are found in the region corresponding to the track "core". These high-dose voxels are not found in the low-LET irradiation simulation and indicate clustered energy deposition, which may be responsible for complex DSB. In the second step, assuming that DSB will be found only in voxels where energy is deposited by the radiation, the intersection points between voxels with dose > 0 and simulated chromosomes were obtained. The spatial distribution of the intersection points is similar to -H2AX foci experiments. These preliminary results suggest that combining stochastic track structure and chromosome models could be a good approach to understand radiation-induced DSB and chromosome aberrations.

  12. Dicentric chromosomes and gamma-H2AX foci formation in lymphocytes of human blood samples exposed to a CT scanner: a direct comparison of dose response relationships.

    PubMed

    Golfier, Sven; Jost, Gregor; Pietsch, Hubertus; Lengsfeld, Philipp; Eckardt-Schupp, Friederike; Schmid, Ernst; Voth, Matthias

    2009-02-01

    Experiments using the induction of dicentric chromosomes (dicentrics) as well as the gamma-H2AX foci formation in lymphocytes of blood samples from a healthy donor were performed to directly evaluate the radiation sensitivity of both biological endpoints. For computed tomography scans at dose levels from 0.025 to 1 Gy, a linear-quadratic dose-response relationship for dicentrics and a linear dose-response relationship for gamma-H2AX foci were obtained. The coefficients of the dose-response relationship for dicentrics are alpha = (3.76 +/- 0.29) x 10(-2) Gy(-1) and beta = (5.54 +/- 0.45) x 10(-2) Gy(-2), the linear coefficient for gamma-H2AX foci is (7.38 +/- 0.11) Gy(-1). The findings indicate that scoring of dicentrics as well as microscopic analysis of gamma-H2AX foci are sensitive methods to quantify a radiation-induced biological damage at low doses. However, since gamma-H2AX foci can be partially repaired within a few hours, biological damages present for days or even months, which constitute the clinically relevant endpoints, can only be quantified reliably by scoring of chromosome aberrations. Thus currently the quantification of dicentrics or reciprocal translocations remains the recommended method for estimating the effect of exposures to low dose levels of radiation ('biological dosimetry'). However, owing to the high radiation sensitivity of the gamma-H2AX foci assay observed in the present study, further investigations on the effectiveness of low-linear energy transfer radiation qualities in producing gamma-H2AX foci in lymphocytes from healthy donors should be performed.

  13. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    PubMed Central

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-01-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure. PMID:27445126

  14. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    NASA Astrophysics Data System (ADS)

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-07-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure.

  15. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.

  16. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    PubMed Central

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-01

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  17. Histone H3 lysine 9 acetylation pattern suggests that X and B chromosomes are silenced during entire male meiosis in a grasshopper.

    PubMed

    Cabrero, J; Teruel, M; Carmona, F D; Jiménez, R; Camacho, J P M

    2007-01-01

    The facultative heterochromatic X chromosome in leptotene spermatocytes of the grasshopper Eyprepocnemis plorans showed marked hypoacetylation for lysine 9 in the H3 histone (H3-K9) with no sign of histone H2AX phosphorylation. Since H3-K9 hypoacetylation precedes the meiotic appearance of phosphorylated H2AX (gamma-H2AX), which marks the beginning of recombinational DNA double-strand breaks (DSBs), it seems that meiotic sex-chromosome inactivation (MSCI) in this grasshopper occurs prior to the beginning of recombination and hence synapsis (which in this species begins later than recombination). In addition, all constitutively heterochromatic chromosome regions harbouring a 180-bp tandem-repeat DNA and rDNA (B chromosomes and pericentromeric regions of A chromosomes) were H3-K9 hypoacetylated at early leptotene even though they will synapse at subsequent stages. This also suggests that meiotic silencing in this grasshopper might be independent of synapsis. The H3-K9 hypoacetylated state of facultative and constitutive heterochromatin persisted during subsequent meiotic stages and was even apparent in round spermatids. Finally, the fact that B chromosomes are differentially hypoacetylated in testis and embryo interphase cells suggests that they might be silenced early in development and remain this way for most (or all) life-cycle stages.

  18. Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles

    PubMed Central

    2012-01-01

    Background Little is known about the cellular effects of exposure to mixed beams of high and low linear energy transfer radiation. So far, the effects of combined exposures have mainly been assessed with clonogenic survival or cytogenetic methods, and the results are contradictory. The gamma-H2AX assay has up to now not been applied in this context, and it is a promising tool for investigating the early cellular response to mixed beam irradiation. Purpose To determine the dose response and repair kinetics of gamma-H2AX ionizing radiation-induced foci in VH10 human fibroblasts exposed to mixed beams of 241Am alpha particles and X-rays. Results VH10 human fibroblasts were irradiated with each radiation type individually or both in combination at 37°C. Foci were scored for repair kinetics 0.5, 1, 3 and 24 h after irradiation (one dose per irradiation type), and for dose response at the 1 h time point. The dose response effect of mixed beam was additive, and the relative biological effectiveness for alpha particles (as compared to X-rays) was of 0.76 ± 0.52 for the total number of foci, and 2.54 ± 1.11 for large foci. The repair kinetics for total number of foci in cells exposed to mixed beam irradiation was intermediate to that of cells exposed to alpha particles and X-rays. However, for mixed beam-irradiated cells the frequency and area of large foci were initially lower than predicted and increased during the first 3 hours of repair (while the predicted number and area did not). Conclusions The repair kinetics of large foci after mixed beam exposure was significantly different from predicted based on the effect of the single dose components. The formation of large foci was delayed and they did not reach their maximum area until 1 h after irradiation. We hypothesize that the presence of low X-ray-induced damage engages the DNA repair machinery leading to a delayed DNA damage response to the more complex DNA damage induced by alpha particles. PMID:23121736

  19. Comparison of two methods for measuring γ-H2AX nuclear fluorescence as a marker of DNA damage in cultured human cells: applications for microbeam radiation therapy

    NASA Astrophysics Data System (ADS)

    Anderson, D.; Andrais, B.; Mirzayans, R.; Siegbahn, E. A.; Fallone, B. G.; Warkentin, B.

    2013-06-01

    Microbeam radiation therapy (MRT) delivers single fractions of very high doses of synchrotron x-rays using arrays of microbeams. In animal experiments, MRT has achieved higher tumour control and less normal tissue toxicity compared to single-fraction broad beam irradiations of much lower dose. The mechanism behind the normal tissue sparing of MRT has yet to be fully explained. An accurate method for evaluating DNA damage, such as the γ-H2AX immunofluorescence assay, will be important for understanding the role of cellular communication in the radiobiological response of normal and cancerous cell types to MRT. We compare two methods of quantifying γ-H2AX nuclear fluorescence for uniformly irradiated cell cultures: manual counting of γ-H2AX foci by eye, and an automated, MATLAB-based fluorescence intensity measurement. We also demonstrate the automated analysis of cell cultures irradiated with an array of microbeams. In addition to offering a relatively high dynamic range of γ-H2AX signal versus irradiation dose ( > 10 Gy), our automated method provides speed, robustness, and objectivity when examining a series of images. Our in-house analysis facilitates the automated extraction of the spatial distribution of the γ-H2AX intensity with respect to the microbeam array — for example, the intensities in the peak (high dose area) and valley (area between two microbeams) regions. The automated analysis is particularly beneficial when processing a large number of samples, as is needed to systematically study the relationship between the numerous dosimetric and geometric parameters involved with MRT (e.g., microbeam width, microbeam spacing, microbeam array dimensions, peak dose, valley dose, and geometric arrangement of multiple arrays) and the resulting DNA damage.

  20. Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

    SciTech Connect

    Audebert, M.; Zeman, F.; Beaudoin, R.; Péry, A.; Cravedi, J.-P.

    2012-04-01

    Polycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24 h treatment. The dose–response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose–response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment. -- Highlights: ► Examination of the genotoxic properties of 13 PAHs on two human cell lines. ► Modelization with a Hill model of the genotoxic dose–response. ► First investigation of the genotoxicity of benzo[c]fluorene on human cell lines. ► Establishment of relevant TEFs for PAHs to improve cancer risk assessment.

  1. Histone H3 phosphorylation in the rat pineal gland: adrenergic regulation and diurnal variation.

    PubMed

    Chik, C L; Arnason, T G; Dukewich, W G; Price, D M; Ranger, A; Ho, A K

    2007-04-01

    In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

  2. Synthesis, Acetylation, and Phosphorylation of Histone IV and Its Binding to DNA During Spermatogenesis in Trout*

    PubMed Central

    Louie, Andrew J.; Dixon, Gordon H.

    1972-01-01

    During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [3H]aminoacids and inorganic [32P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [3H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [3H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total 3H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an “obligatory” acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that ε-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH2-terminal region of histone IV to assume the correct conformation (in this case, an α-helix), and fit into the major groove of DNA. Deacetylation then “locks” histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known. Images PMID:4505675

  3. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    NASA Astrophysics Data System (ADS)

    Klimovskaia, Ilnaz M.; Young, Clifford; Strømme, Caroline B.; Menard, Patrice; Jasencakova, Zuzana; Mejlvang, Jakob; Ask, Katrine; Ploug, Michael; Nielsen, Michael L.; Jensen, Ole N.; Groth, Anja

    2014-03-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

  4. Manual versus automated γ-H2AX foci analysis across five European laboratories: can this assay be used for rapid biodosimetry in a large scale radiation accident?

    PubMed

    Rothkamm, Kai; Barnard, Stephen; Ainsbury, Elizabeth A; Al-Hafidh, Jenna; Barquinero, Joan-Francesc; Lindholm, Carita; Moquet, Jayne; Perälä, Marjo; Roch-Lefèvre, Sandrine; Scherthan, Harry; Thierens, Hubert; Vral, Anne; Vandersickel, Veerle

    2013-08-30

    The identification of severely exposed individuals and reassurance of the 'worried well' are of prime importance for initial triage following a large scale radiation accident. We aim to develop the γ-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo γ-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate γ-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2-4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the γ-H2AX assay. We conclude that the γ-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods.

  5. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline

    PubMed Central

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-01-01

    SUMMARY A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here we show that phosphorylation at Threonine 3 of H3 (H3T3P) distinguishes preexisting versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. Together these studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3, coordinating asymmetric segregation of “old” H3 into GSCs, and that a tight regulation of H3T3 phosphorylation is required for male germline activity. PMID:26522592

  6. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline.

    PubMed

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-11-05

    A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

  7. ERK/MAPK Regulates Hippocampal Histone Phosphorylation Following Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Levenson, Jonathan M.; Sweatt, J. David; Chwang, Wilson B.; O'Riordan, Kenneth J.

    2006-01-01

    Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the…

  8. Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4.

    PubMed

    Utley, Rhea T; Lacoste, Nicolas; Jobin-Robitaille, Olivier; Allard, Stéphane; Côté, Jacques

    2005-09-01

    The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

  9. Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila.

    PubMed

    Deng, Huai; Bao, Xiaomin; Cai, Weili; Blacketer, Melissa J; Belmont, Andrew S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-02-01

    Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

  10. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  11. Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells.

    PubMed

    Okamura, Hirohiko; Yoshida, Kaya; Amorim, Bruna Rabelo; Haneji, Tatsuji

    2008-04-01

    Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.

  12. Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly

    PubMed Central

    1994-01-01

    We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions. PMID:8045925

  13. Microwaves from UMTS/GSM mobile phones induce long-lasting inhibition of 53BP1/gamma-H2AX DNA repair foci in human lymphocytes.

    PubMed

    Belyaev, Igor Y; Markovà, Eva; Hillert, Lena; Malmgren, Lars O G; Persson, Bertil R R

    2009-02-01

    We have recently described frequency-dependent effects of mobile phone microwaves (MWs) of global system for mobile communication (GSM) on human lymphocytes from persons reporting hypersensitivity to electromagnetic fields and healthy persons. Contrary to GSM, universal global telecommunications system (UMTS) mobile phones emit wide-band MW signals. Hypothetically, UMTS MWs may result in higher biological effects compared to GSM signal because of eventual "effective" frequencies within the wideband. Here, we report for the first time that UMTS MWs affect chromatin and inhibit formation of DNA double-strand breaks co-localizing 53BP1/gamma-H2AX DNA repair foci in human lymphocytes from hypersensitive and healthy persons and confirm that effects of GSM MWs depend on carrier frequency. Remarkably, the effects of MWs on 53BP1/gamma-H2AX foci persisted up to 72 h following exposure of cells, even longer than the stress response following heat shock. The data are in line with the hypothesis that the type of signal, UMTS MWs, may have higher biological efficiency and possibly larger health risk effects compared to GSM radiation emissions. No significant differences in effects between groups of healthy and hypersensitive subjects were observed, except for the effects of UMTS MWs and GSM-915 MHz MWs on the formation of the DNA repair foci, which were different for hypersensitive (P < 0.02[53BP1]//0.01[gamma-H2AX]) but not for control subjects (P > 0.05). The non-parametric statistics used here did not indicate specificity of the differences revealed between the effects of GSM and UMTS MWs on cells from hypersensitive subjects and more data are needed to study the nature of these differences.

  14. Synchrotron-based imaging of chromium and  γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water

    DOE PAGES

    Thompson, Chad M.; Seiter, Jennifer; Chappell, Mark A.; ...

    2014-10-28

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodenamore » from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ~2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. Lastly, these findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.« less

  15. Synchrotron-based imaging of chromium and  γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water

    SciTech Connect

    Thompson, Chad M.; Seiter, Jennifer; Chappell, Mark A.; Tappero, Ryan V.; Proctor, Deborah M.; Suh, Mina; Wolf, Jeffrey C.; Haws, Laurie C.; Vitale, Rock; Mittal, Liz; Kirman, Christopher R.; Hays, Sean M.; Harris, Mark A.

    2014-10-28

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ~2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. Lastly, these findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.

  16. EGFR Modulates DNA Synthesis and Repair through Tyr Phosphorylation of Histone H4

    PubMed Central

    Chou, Ruey-Hwang; Wang, Ying-Nai; Hsieh, Yi-Hsien; Li, Long-Yuan; Xia, Weiya; Chang, Wei-Chao; Chang, Ling-Chu; Cheng, Chien-Chia; Lai, Chien-Chen; Hsu, Jennifer L.; Chang, Wei-Jung; Chiang, Shu-Ya; Lee, Hong-Jen; Liao, Hsin-Wei; Chuang, Pei-Huan; Chen, Hui-Yu; Wang, Hung-Ling; Kuo, Sheng-Chu; Chen, Chung-Hsuan; Yu, Yung-Luen; Hung, Mien-Chie

    2014-01-01

    Summary Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical in DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 mono-methylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair. PMID:25073158

  17. Genotoxicity evaluation of individual cigarette smoke toxicants using the in vitro γH2AX assay by high content screening.

    PubMed

    Garcia-Canton, Carolina; Anadon, Arturo; Meredith, Clive

    2013-10-23

    Cigarette smoke is a complex mixture consisting of more than 5600 identified chemical constituents of which approximately 150 have been identified so far as "tobacco smoke toxicants". Proposals made by the World Health Organisation Framework Convention on Tobacco Control mandate the lowering of nine tobacco smoke priority toxicants, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), and benzo[a]pyrene (B[a]P) and monitoring the levels of a further nine including cadmium. Here, we evaluated the genotoxic potential in human bronchial epithelial BEAS-2B cells of four cigarette smoke toxicants; NNK, NNN, B[a]P and cadmium using the novel in vitro γH2AX assay by High Content Screening (HCS). We also examined the genotoxicity of binary mixtures of NNK and NNN reporting their relative contribution to the genotoxic end-point. The results of this preliminary assessment showed that the in vitro γH2AX assay by HCS could be used as a pre-screening tool to detect and quantify the genotoxicity effect of cigarette smoke toxicants individually and in binary mixture. Moreover, the data produced could contribute to the prioritisation of toxicant reduction research in modified tobacco products.

  18. Survival Fraction at 2 Gy and γH2AX Expression Kinetics in Peripheral Blood Lymphocytes From Cancer Patients: Relationship With Acute Radiation-Induced Toxicities

    SciTech Connect

    Pouliliou, Stamatia E.; Dimitriou, Thespis; Giatromanolaki, Alexandra; Papazoglou, Dimitrios; Pappa, Aglaia; Pistevou, Kyriaki

    2015-07-01

    Purpose: Predictive assays for acute radiation toxicities would be clinically relevant in radiation oncology. We prospectively examined the predictive role of the survival fraction at 2 Gy (SF2) and of γH2AX (double-strand break [DSB] DNA marker) expression kinetics in peripheral blood mononuclear cells (PBMCs) from cancer patients before radiation therapy. Methods and Materials: SF2 was measured with Trypan Blue assay in the PBMCs from 89 cancer patients undergoing radiation therapy at 4 hours (SF2{sub [4h]}) and 24 hours (SF2{sub [24h]}) after ex vivo irradiation. Using Western blot analysis and band densitometry, we further assessed the expression of γH2AX in PBMC DNA at 0 hours, 30 minutes, and 4 hours (33 patients) and 0 hour, 4 hours, and 24 hours (56 patients), following ex vivo irradiation with 2 Gy. Appropriate ratios were used to characterize each patient, and these were retrospectively correlated with early radiation therapy toxicity grade. Results: The SF2{sub (4h)} was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio{sub (30min)} (band density of irradiated/non-irradiated cells at 30 minutes) revealed, similarly, a significant inverse association (P=.0001). The DSB DNA repair rate from 30 minutes to 4 hours, calculated as the relative RγH2AX-ratio (γH2AX-ratio{sub (4h)}/γH2AX-ratio{sub (30min)}) showed a significant direct association with high toxicity grade (P=.01). Conclusions: Our results suggest that SF2 is a significant radiation sensitivity index for patients undergoing radiation therapy. γH2AX Western blot densitometry analysis provided 2 important markers of normal tissue radiation sensitivity. Low γH2AX expression at 30 minutes was linked with high toxicity grade, suggesting that poor γH2AX repair activity within a time frame of 30 minutes after irradiation predicts for poor radiation tolerance. On the other hand, rapid γH2AX content restoration at 4 hours after irradiation, compatible with

  19. The profiles of gamma-H2AX along with ATM/DNA-PKcs activation in the lymphocytes and granulocytes of rat and human blood exposed to gamma rays.

    PubMed

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong

    2016-08-01

    Establishing a rat model suitable for γ-H2AX biodosimeter studies has important implications for dose assessment of internal radionuclide contamination in humans. In this study, γ-H2AX, p-ATM and p-DNA-PKcs foci were enumerated using immunocytofluorescence method, and their protein levels were measured by Western blot in rat blood lymphocytes and granulocytes exposed to γ-rays compared with human blood lymphocytes and granulocytes. It was found that DNA double-strand break repair kinetics and linear dose responses in rat lymphocytes were similar to those observed in the human counterparts. Moreover, radiation induced clear p-ATM and p-DNA-PKcs foci formation and an increase in ratio of co-localization of p-ATM or p-DNA-PKcs with γ-H2AX foci in rat lymphocytes similar to those of human lymphocytes. The level of γ-H2AX protein in irradiated rat and human lymphocytes was significantly reduced by inhibitors of ATM and DNA-PKcs. Surprisingly, unlike human granulocytes, rat granulocytes with DNA-PKcs deficiency displayed a rapid accumulation, but delayed disappearance of γ-H2AX foci with essentially no change from 10 h to 48 h post-irradiation. Furthermore, inhibition of ATM activity in rat granulocytes also decreased radiation-induced γ-H2AX foci formation. In comparison, human granulocytes showed no response to irradiation regarding γ-H2AX, p-ATM or p-DNA-PKcs foci. Importantly, incidence of γ-H2AX foci in lymphocytes after total-body radiation of rats was consistent with that of in vitro irradiation of rat lymphocytes. These findings show that rats are a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. ATM and DNA-PKcs participate together in DSB repair in rat lymphocytes similar to that of human lymphocytes. Further, rat granulocytes, which have the characteristic of delayed disappearance of γ-H2AX foci in response to radiation, may be a useful experimental system for biodosimetry studies.

  20. Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors.

    PubMed

    Lee, E; Nakatsuma, A; Hiraoka, R; Ishikawa, E; Enomoto, R; Yamauchi, A

    1999-07-01

    Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.

  1. Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure.

    PubMed

    North, Justin A; Šimon, Marek; Ferdinand, Michelle B; Shoffner, Matthew A; Picking, Jonathan W; Howard, Cecil J; Mooney, Alex M; van Noort, John; Poirier, Michael G; Ottesen, Jennifer J

    2014-04-01

    Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.

  2. The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

    PubMed Central

    de la Barre, Anne-Elisabeth; Angelov, Dimitri; Molla, Annie; Dimitrov, Stefan

    2001-01-01

    We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed ‘the ready production label’ was suggested to explain the role of histone H3 phosphorylation during cell division. PMID:11707409

  3. Calibration of the γ-H2AX DNA Double Strand Break Focus Assay for Internal Radiation Exposure of Blood Lymphocytes

    PubMed Central

    Eberlein, Uta; Peper, Michel; Fernández, Maria; Lassmann, Michael; Scherthan, Harry

    2015-01-01

    DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations. PMID:25853575

  4. Investigating chromosome damage and gammaH2AX response in human lymphocytes and lymphocyte subsets as potential biomarkers of radiation sensitivity

    NASA Astrophysics Data System (ADS)

    Beaton, Lindsay A.

    This thesis examines in vitro irradiated blood samples from prostate cancer patients exhibiting late normal tissue damage after receiving radiotherapy, for lymphocyte response. Chromosomal aberrations, translocations and proliferation rate are measured, as well as gammaH2AX response in lymphocytes and lymphocyte subsets. The goal of this thesis is to determine whether the lymphocyte response to in vitro radiation could be used as a marker for radiosensitivity. Patients were selected from a randomized clinical trial evaluating the optimal timing of Dose Escalated Radiation and short course Androgen Deprivation Therapy. Of 438 patients, 3% developed Grade 3 late radiation proctitis and were considered to be radiosensitive. Blood was drawn from 10 of these patients along with 20 matched samples from patients with grade 0 proctitis. The samples were irradiated and were analyzed for dicentric chromosomes, excess fragments and proliferation rates (at 6 Gy), translocations, stable and unstable damage (at 4 Gy), and dose response (up to 10 Gy), along with time response after 2 Gy (0 -- 24 h). Chromosome aberrations, excess fragments per cell, translocations per cell and proliferation rates were analyzed by brightfield and fluorescent microscopy, while the gammaH2AX response in lymphocytes and lymphocyte subsets was analyzed by flow cytometry. Both groups were statistically similar for all endpoints at 0 Gy. At 6 Gy, there were statistically significant differences between the radiosensitive and control cohorts for three endpoints; the mean number of dicentric chromosomes per cell, the mean number of excess fragments per cell and the proportion of cells in second metaphase. At 4 Gy, there were statistically significant differences between the two cohorts for three endpoints; the mean number of translocations per cell, the mean number of dicentric chromosomes per cell and the mean number of deletions per cell. There were no significant differences between the gammaH2AX

  5. Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein

    PubMed Central

    Kurat, Christoph F.; Lambert, Jean-Philippe; van Dyk, Dewald; Tsui, Kyle; van Bakel, Harm; Kaluarachchi, Supipi; Friesen, Helena; Kainth, Pinay; Nislow, Corey; Figeys, Daniel; Fillingham, Jeffrey; Andrews, Brenda J.

    2011-01-01

    The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach–release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters. PMID:22156209

  6. Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein.

    PubMed

    Kurat, Christoph F; Lambert, Jean-Philippe; van Dyk, Dewald; Tsui, Kyle; van Bakel, Harm; Kaluarachchi, Supipi; Friesen, Helena; Kainth, Pinay; Nislow, Corey; Figeys, Daniel; Fillingham, Jeffrey; Andrews, Brenda J

    2011-12-01

    The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.

  7. Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation

    PubMed Central

    Zhao, Hong; Traganos, Frank; Darzynkiewicz, Zbigniew

    2008-01-01

    The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15P) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT- induced p53-Ser15P with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15P had “patchy” localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15P cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15P appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15P was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15P expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4–6 h. This maximum expression of p53-Ser15P coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1–2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by

  8. Involvement of histone methylation and phosphorylation in regulation of transcription by thyroid hormone receptor.

    PubMed

    Li, Jiwen; Lin, Qiushi; Yoon, Ho-Geun; Huang, Zhi-Qing; Strahl, Brian D; Allis, C David; Wong, Jiemin

    2002-08-01

    Previous studies have established an important role of histone acetylation in transcriptional control by nuclear hormone receptors. With chromatin immunoprecipitation assays, we have now investigated whether histone methylation and phosphorylation are also involved in transcriptional regulation by thyroid hormone receptor (TR). We found that repression by unliganded TR is associated with a substantial increase in methylation of H3 lysine 9 (H3-K9) and a decrease in methylation of H3 lysine 4 (H3-K4), methylation of H3 arginine 17 (H3-R17), and a dual modification of phosphorylation of H3 serine 10 and acetylation of lysine 14 (pS10/acK14). On the other hand, transcriptional activation by liganded TR is coupled with a substantial decrease in both H3-K4 and H3-K9 methylation and a robust increase in H3-R17 methylation and the dual modification of pS10/acK14. Trichostatin A treatment results in not only histone hyperacetylation but also an increase in methylation of H3-K4, increase in dual modification of pS10/acK14, and reduction in methylation of H3-K9, revealing an extensive interplay between histone acetylation, methylation, and phosphorylation. In an effort to understand the underlying mechanism for an increase in H3-K9 methylation during repression by unliganded TR, we demonstrated that TR interacts in vitro with an H3-K9-specific histone methyltransferase (HMT), SUV39H1. Functional analysis indicates that SUV39H1 can facilitate repression by unliganded TR and in so doing requires its HMT activity. Together, our data uncover a novel role of H3-K9 methylation in repression by unliganded TR and provide strong evidence for the involvement of multiple distinct histone covalent modifications (acetylation, methylation, and phosphorylation) in transcriptional control by nuclear hormone receptors.

  9. Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy

    SciTech Connect

    Thompson, Chad M.; Wolf, Jeffrey C.; Elbekai, Reem H.; Paranjpe, Madhav G.; Seiter, Jennifer M.; Chappell, Mark A.; Tappero, Ryan V.; Suh, Mina; Proctor, Deborah M.; Bichteler, Anne; Haws, Laurie C.; Harris, Mark A.

    2015-08-01

    Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50 mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

  10. Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation

    NASA Astrophysics Data System (ADS)

    Jiang, Yan-Ke; Zou, Jian-Wei; Wu, Yu-Qian; Zhang, Na; Yu, Qing-Sen; Jiang, Yong-Jun

    Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3-HP1 trajectory. It is clear from the 10-ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications-a stable methylation and a dynamic phosphorylation mark-establish a regulatory mechanism of protein-protein interactions.

  11. Phosphorylation of histone H3 is functionally linked to retinoic acid receptor β promoter activation

    PubMed Central

    Lefebvre, Bruno; Ozato, Keiko; Lefebvre, Philippe

    2002-01-01

    Ligand-dependent transcriptional activation of retinoic acid receptors (RARs) is a multistep process culminating in the formation of a multimeric co-activator complex on regulated promoters. Several co-activator complexes harbor an acetyl transferase activity, which is required for retinoid-induced transcription of reporter genes. Using murine P19 embryonal carcinoma cells, we examined the relationship between histone post-translational modifications and activation of the endogenous RARβ2 promoter, which is under the control of a canonical retinoic acid response element and rapidly induced upon retinoid treatment. While histones H3 and H4 were constitutively acetylated at this promoter, retinoid agonists induced a rapid phosphorylation at Ser10 of histone H3. A retinoid antagonist, whose activity was independent of co-repressor binding to RAR, could oppose this agonist-induced H3 phosphorylation. Since such post-translational modifications were not observed at several other promoters, we conclude that histone H3 phosphorylation may be a molecular signature of the activated, retinoid-controlled mRARβ2 gene promoter. PMID:11897660

  12. Human linker histones: interplay between phosphorylation and O-β-GlcNAc to mediate chromatin structural modifications

    PubMed Central

    2011-01-01

    Eukaryotic chromatin is a combination of DNA and histone proteins. It is established fact that epigenetic mechanisms are associated with DNA and histones. Initial studies emphasize on core histones association with DNA, however later studies prove the importance of linker histone H1 epigenetic. There are many types of linker histone H1 found in mammals. These subtypes are cell specific and their amount in different types of cells varies as the cell functions. Many types of post-translational modifications which occur on different residues in each subtype of linker histone H1 induce conformational changes and allow the different subtypes of linker histone H1 to interact with chromatin at different stages during cell cycle which results in the regulation of transcription and gene expression. Proposed O-glycosylation of linker histone H1 promotes condensation of chromatin while phosphorylation of linker histone H1 is known to activate transcription and gene regulation by decondensation of chromatin. Interplay between phosphorylation and O-β-GlcNAc modification on Ser and Thr residues in each subtype of linker histone H1 in Homo sapiens during cell cycle may result in diverse functional regulation of proteins. This in silico study describes the potential phosphorylation, o-glycosylation and their possible interplay sites on conserved Ser/Thr residues in various subtypes of linker histone H1 in Homo sapiens. PMID:21749719

  13. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response

    PubMed Central

    Ben Yehuda, Adi; Risheq, Marwa; Novoplansky, Ofra; Bersuker, Kirill; Kopito, Ron R.; Goldberg, Michal; Brandeis, Michael

    2017-01-01

    Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington’s disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality. PMID:28052107

  14. SWI/SNF recruitment to a DNA double-strand break by the NuA4 and Gcn5 histone acetyltransferases.

    PubMed

    Bennett, Gwendolyn; Peterson, Craig L

    2015-06-01

    The DNA damage response to double-strand breaks (DSBs) is critical for cellular viability. Recent work has shown that a host of chromatin regulators are recruited to a DSB, and that they are important for the DNA damage response. However, the functional relationships between different chromatin regulators at DSBs remain unclear. Here we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.XH2AX). Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.

  15. Individual and Combined Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predict Shorter Survival of Soft Tissue Sarcoma Patients

    PubMed Central

    Park, See-Hyoung; Park, Hye Jeong; Wang, Sung Il; Park, Ho Sung; Lee, Ho; Kwon, Keun Sang; Moon, Woo Sung; Lee, Dong Geun; Kim, Jung Ryul; Jang, Kyu Yun

    2016-01-01

    DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, γH2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1, γH2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and γH2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, γH2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P < 0.001) and EFS (P = 0.016). The ten-year DSS rate of the CSddrm-low, CSddrm-intermediate, and CSddrm-high subgroups were 81%, 26%, and 0%, respectively. In conclusion, this study demonstrates that the individual and combined expression patterns of the DDR molecules PARP1, γH2AX, BRCA1, and BRCA2 could be predictive of the prognosis of STS patients and suggests that controlling the activity of these DDR molecules could be employed in new therapeutic stratagems for the treatment of STS. PMID:27643881

  16. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

    PubMed Central

    Hait, Nitai C; Wise, Laura E; Allegood, Jeremy C; O’Brien, Megan; Avni, Dorit; Reeves, Thomas M; Knapp, Pamela E; Lu, Junyan; Luo, Cheng; Miles, Michael F; Milstien, Sheldon; Lichtman, Aron H; Spiegel, Sarah

    2014-01-01

    FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories. PMID:24859201

  17. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory.

    PubMed

    Hait, Nitai C; Wise, Laura E; Allegood, Jeremy C; O'Brien, Megan; Avni, Dorit; Reeves, Thomas M; Knapp, Pamela E; Lu, Junyan; Luo, Cheng; Miles, Michael F; Milstien, Sheldon; Lichtman, Aron H; Spiegel, Sarah

    2014-07-01

    FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

  18. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase.

    PubMed

    Le, Ly-Thuy-Tram; Vu, Hong-Lien; Nguyen, Chi-Hung; Molla, Annie

    2013-04-15

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  19. Histone H1 Phosphorylation by Cdk2 Selectively Modulates Mouse Mammary Tumor Virus Transcription through Chromatin Remodeling

    PubMed Central

    Bhattacharjee, Rabindra N.; Banks, Geoffrey C.; Trotter, Kevin W.; Lee, Huay-Leng; Archer, Trevor K.

    2001-01-01

    Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo. PMID:11463824

  20. Histone deacetylase 2 is phosphorylated, ubiquitinated, and degraded by cigarette smoke.

    PubMed

    Adenuga, David; Yao, Hongwei; March, Thomas H; Seagrave, Jeanclare; Rahman, Irfan

    2009-04-01

    Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke.

  1. Quantitative analysis of γ-H2AX and p53 nuclear expression levels in ovarian and fallopian tube epithelium from risk-reducing salpingo-oophorectomies in BRCA1 and BRCA2 mutation carriers.

    PubMed

    Staff, Synnöve; Tolonen, Teemu; Laasanen, Satu-Leena; Mecklin, Jukka-Pekka; Isola, Jorma; Mäenpää, Johanna

    2014-05-01

    Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Increased lifetime ovarian cancer risk among BRCA1/BRCA2 mutation carriers can be substantially decreased by risk-reducing salpingo-oophorectomy (RRSO), which also provides material for molecular research on early pathogenesis of serous ovarian cancer. RRSO studies have suggested fallopian tube as a primary site of serous high-grade ovarian cancer. In this study, the nuclear expression levels of γ-H2AX and p53 using immunohistochemical (IHC) study was quantitatively assessed in ovarian and fallopian tube epithelium derived from RRSOs in 29 BRCA1 and BRCA2 mutation carriers and in 1 patient with a strong family history of breast and ovarian cancer but showing an unknown BRCA status. Both p53 and γ-H2AX nuclear staining levels were significantly higher in BRCA1/2 mutation-positive fallopian tube epithelium compared with the control fallopian tube epithelium (P<0.006 and P=0.011, respectively). Nuclear expression levels of p53 and γ-H2AX were similar between the BRCA1/2 mutation-positive ovarian epithelium and controls. Both γ-H2AX and p53 showed significantly higher nuclear expression levels in BRCA1/2 mutation-positive fallopian tube epithelium compared with BRCA1/2 mutation-positive ovarian epithelium (P<0.0001 and P<0.0001, respectively). BRCA1/2 mutation-positive fallopian tube epithelium showed a positive correlation between the γ-H2AX and p53 nuclear expression levels (Pearson r=0.508, P=0.003). Our results of quantitative nuclear p53 and γ-H2AX expression levels in ovarian and fallopian tube epithelium derived from RRSO in high-risk patients support the previously suggested role of fallopian tube epithelium serving as a possible site of initial serous ovarian carcinogenesis.

  2. Histone H3 phosphorylation and elimination of paternal X chromosomes at early cleavages in sciarid flies.

    PubMed

    Escribá, M Carmen; Goday, Clara

    2013-07-15

    In sciarid flies (Diptera, Sciaridae), one or two paternally derived X chromosomes are discarded from the soma at early cleavages to determine the sex of the embryo (XX, females; X0, males). X chromosome(s) elimination is achieved by an abnormal anaphase segregation so that X sister chromatids do not reach the poles and are not included in the daughter nuclei. A cis-acting locus (CE) within the heterochromatin proximal to the centromere is known to regulate X chromosome elimination. By immunofluorescence analysis in early embryos from Sciara ocellaris and Sciara coprophila, we investigated histone H3 phosphorylation at Ser10, Ser28 and Thr3 prior to, and during, the X elimination process. We found that the regular syncytial nuclear divisions are characterized by a gradual loss of H3S10 phosphorylation along the chromosome arms at anaphase. Importantly, the eliminating X chromosomes show a retardation in anaphase chromatid segregation and high levels of H3S10 phosphorylation in the chromosome arms. In the present study, we provide the first evidence linking the hyper-phosphorylated H3 status of the X chromosome with a delay in sister chromatid separation at anaphase. Our findings support the idea that the CE induces a deficiency in H3 dephosphorylation in the paternal X chromosomes to be eliminated.

  3. The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements.

    PubMed

    Thorne, David; Larard, Sophie; Baxter, Andrew; Meredith, Clive; Gaҫa, Marianna

    2017-01-04

    DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated. E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a "cigalike" and a closed "modular" system) and cigarette smoke. A Vitrocell(®) VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120min exposure (diluting airflow, 0.25-1L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% γH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform. The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM

  4. Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae.

    PubMed

    Govin, Jérôme; Schug, Jonathan; Krishnamoorthy, Thanuja; Dorsey, Jean; Khochbin, Saadi; Berger, Shelley L

    2010-08-01

    We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair.

  5. The influence of x-ray contrast agents in computed tomography on the induction of dicentrics and γ-H2AX foci in lymphocytes of human blood samples

    NASA Astrophysics Data System (ADS)

    Jost, G.; Golfier, S.; Pietsch, H.; Lengsfeld, P.; Voth, M.; Schmid, T. E.; Eckardt-Schupp, F.; Schmid, E.

    2009-10-01

    The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and γ-H2AX foci) in human lymphocytes after CT scans in the presence of an iodinated contrast agent. Blood samples from a healthy donor were exposed to CT scans in the absence or presence of iotrolan 300 at iodine concentrations of 5 or 50 mg ml-1 blood. The samples were exposed to 0.025, 0.05, 0.1 and 1 Gy in a tissue equivalent body phantom. Chromosome aberration scoring and automated microscopic analysis of γ-H2AX foci were performed in parts of the same samples. The theoretical physical dose enhancement factor (DEF) was calculated on the basis of the mass energy-absorption coefficients of iodine and blood and the photon energy spectrum of the CT tube. No significant differences in the yields of dicentrics and γ-H2AX foci were observed in the absence or presence of 5 mg iodine ml-1 blood up to 0.1 Gy, whereas at 1 Gy the yields were elevated for both biomarkers. At an iodine concentration of 50 mg ml-1 serving as a positive control, a biological DEF of 9.5 ± 1.4 and 2.3 ± 0.5 was determined for dicentrics and γ-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml-1, respectively. Thus, it can be concluded that in the diagnostic dose range (radiation and contrast dose), no relevant biological dose-enhancing effect could be detected, whereas a clear biological dose-enhancing effect could be found for a contrast dose well outside the diagnostic CT range for the complete radiation dose range with both methods.

  6. DNA Double-Strand Break Analysis by {gamma}-H2AX Foci: A Useful Method for Determining the Overreactors to Radiation-Induced Acute Reactions Among Head-and-Neck Cancer Patients

    SciTech Connect

    Goutham, Hassan Venkatesh; Mumbrekar, Kamalesh Dattaram; Vadhiraja, Bejadi Manjunath; Fernandes, Donald Jerard; Sharan, Krishna; Kanive Parashiva, Guruprasad; Kapaettu, Satyamoorthy; Bola Sadashiva, Satish Rao

    2012-12-01

    Purpose: Interindividual variability in normal tissue toxicity during radiation therapy is a limiting factor for successful treatment. Predicting the risk of developing acute reactions before initiation of radiation therapy may have the benefit of opting for altered radiation therapy regimens to achieve minimal adverse effects with improved tumor cure. Methods and Materials: DNA double-strand break (DSB) induction and its repair kinetics in lymphocytes of head-and-neck cancer patients undergoing chemoradiation therapy was analyzed by counting {gamma}-H2AX foci, neutral comet assay, and a modified version of neutral filter elution assay. Acute normal tissue reactions were assessed by Radiation Therapy Oncology Group criteria. Results: The correlation between residual DSBs and the severity of acute reactions demonstrated that residual {gamma}-H2AX foci in head-and-neck cancer patients increased with the severity of oral mucositis and skin reaction. Conclusions: Our results suggest that {gamma}-H2AX analysis may have predictive implications for identifying the overreactors to mucositis and skin reactions among head-and-neck cancer patients prior to initiation of radiation therapy.

  7. Importance of protamine phosphorylation to histone displacement in spermatids: can the disruption of this process be used for male contraception

    SciTech Connect

    Balhorn, R.; Hud, N.V.; Corzett, M.; Mazrimas, J.

    1995-06-01

    Protamine is a small protein that packages DNA in the sperm of most vertebrates. Shortly after its synthesis, the serine and threonine residues in each protamine are phosphorylated and the modified proteins are deposited onto DNA, displacing the histones and other chromatin proteins. We have hypothesized that the phosphorylation of protamine 1 induces protamine dimerization and these dimers are required for efficient histone displacement. Histone displacement by protamines in late-step spermatids appears to be essential for the production of fertile sperm in man and other mammals, and the disruption of this process could provide a new approach for male contraception. As a first step towards testing this theory, we have initiated a set of in vitro experiments to determine whether of not protamine phosphorylation is essential for histone displacement. Thee results of these experiments, although incomplete, confirm that unphosphorylated protamine cannot effectively displace histone from DNA. Polyarginine molecules twice the size of a protamine molecule and salmine dimer were found to be more effective. These results are consistent with the theory that the disruption of protamine phosphorylation may prove to be a useful new approach for male contraception if it can be shown to facilitate or induce protamine dimerization.

  8. Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids.

    PubMed

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2017-01-01

    Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique "meiosis-mitosis shift" at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.

  9. P-TEFb Kinase Complex Phosphorylates Histone H1 to Regulate Expression of Cellular and HIV-1 Genes*

    PubMed Central

    O'Brien, Siobhan K.; Cao, Hong; Nathans, Robin; Ali, Akbar; Rana, Tariq M.

    2010-01-01

    Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription. PMID:20551309

  10. Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo*

    PubMed Central

    Li, Yeran; Cai, Weili; Wang, Chao; Yao, Changfu; Bao, Xiaomin; Deng, Huai; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH2-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH2-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein. PMID:23723094

  11. Analysis of the interplay between all-trans retinoic acid and histone deacetylase inhibitors in leukemic cells.

    PubMed

    Noack, Katrin; Mahendrarajah, Nisintha; Hennig, Dorle; Schmidt, Luisa; Grebien, Florian; Hildebrand, Dagmar; Christmann, Markus; Kaina, Bernd; Sellmer, Andreas; Mahboobi, Siavosh; Kubatzky, Katharina; Heinzel, Thorsten; Krämer, Oliver H

    2016-11-02

    The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.

  12. Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition.

    PubMed

    Liu, Zhan-Guo; Ni, Shu-Yuan; Chen, Gui-Ming; Cai, Jing; Guo, Zhen-Hui; Chang, Ping; Li, Yu-Sheng

    2013-01-01

    Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte

  13. Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis

    PubMed Central

    Kikani, Chintan K; Wu, Xiaoying; Paul, Litty; Sabic, Hana; Shen, Zuolian; Shakya, Arvind; Keefe, Alexandra; Villanueva, Claudio; Kardon, Gabrielle; Graves, Barbara; Tantin, Dean; Rutter, Jared

    2016-01-01

    PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells. DOI: http://dx.doi.org/10.7554/eLife.17985.001 PMID:27661449

  14. Increased Histone H3 Phosphorylation in Neurons in Specific Brain Structures after Induction of Status Epilepticus in Mice

    PubMed Central

    Mori, Tetsuji; Wakabayashi, Taketoshi; Ogawa, Haruyuki; Hirahara, Yukie; Koike, Taro; Yamada, Hisao

    2013-01-01

    Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3+ (PH3+) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation. PMID:24147063

  15. Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata.

    PubMed

    Vacquier, V D; Porter, D C; Keller, S H; Aukerman, M

    1989-05-01

    When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.

  16. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    PubMed

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.

  17. Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro.

    PubMed

    Prusov, A N; Smirnova, T A; Kolomijtseva, G Ya

    2013-02-01

    In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100-200 nm; N-1) and partly decondensed (diameter of fibrils ~30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4-7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin

  18. Association Between Phosphorylated Histone H3 and Oncotype DX Recurrence Scores in Breast Cancer

    PubMed Central

    Lee, Lik Hang; Swanson, Paul E.; Tang, Patricia A.; Bigras, Gilbert

    2017-01-01

    We investigate the association between phosphorylated histone H3 (PhH3) and Oncotype DX recurrence score (RS). All invasive breast carcinoma with RS results from our city between 2007 and 2010 (n=47) were reviewed. Whole-tumor sections were stained for PhH3. Mitotic and PhH3 counts were performed and clinical charts reviewed. PhH3 correlated well with RS (r=0.69, P<0.001). Other correlations were: PhH3 versus mitotic count (r=0.87, P<0.001), PhH3 versus mitotic score (r=0.71, P<0.001), PhH3 versus modified Bloom-Richardson-Elston (MBR) grade (r=0.65, P<0.001), RS versus mitotic count (r=0.62, P<0.001), RS versus mitotic score (r=0.44, P=0.002), and RS versus MBR grade (r=0.49, P=0.001). Significant correlation between PhH3 and RS remained after controlling for mitotic count (r=0.39, P=0.007), mitotic score (r=0.60, P<0.001), MBR grade (r=0.56, P<0.001), and all 3 (r=0.37, P=0.014) by partial correlation. Two patients died of metastasis at 12 and 38 months after diagnosis. One had intermediate RS, and 1 high RS; both were in the top-third of PhH3 count. All other patients are alive and recurrence free. Correlation between PhH3 and RS was statistically significant in our cohort, and remained significant after controlling for traditional measures of proliferation. Given that RS has an established strong relationship with prognosis and therapy responsiveness, PhH3 may thus also be an important prognostic/predictive marker in breast cancer. PMID:26371428

  19. Histone H3 Phosphorylation in Human Skin Histoculture as a Tool to Evaluate Patient’s Response to Antiproliferative Drugs

    PubMed Central

    Ugarte, Fernando; Porth, Katherine; Sadekova, Svetlana

    2015-01-01

    Evaluation of patient’s response to chemotherapeutic drugs is often difficult and time consuming. Skin punch biopsies are easily accessible material that can be used for the evaluation of surrogate biomarkers of a patient’s response to a drug. In this study, we hypothesized that assessment of phosphorylated histone H3 in human skin punch biopsies could be used as a pharmacodynamics biomarker of patient’s response to the kinesin spindle protein inhibitor SCH2047069. To test this hypothesis, we used a human skin histoculture technique that allows culturing intact human skin in the presence of the drug. Human melanoma and skin histocultures were treated with SCH2047069, and the effect of the drug was assessed by increasing histone H3 phosphorylation using immunohistochemistry. Our results demonstrate that SCH2047069 has a significant effect on cell proliferation in human melanoma and skin histoculture and justify using human skin punch biopsies for evaluation of the pharmacodynamic changes induced by SCH2047069. ACRONYMS Histone subunit H3 (H3), Kinesin spindle protein (KSP), 5-ethynyl-2′-deoxyuridine (EDU), Dimethyl sulfoxide (DMSO), Formalin-fixed paraffin embedded (FFPE). PMID:26917945

  20. Drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin

    PubMed Central

    Pang, Baoxu; Qiao, Xiaohang; Janssen, Lennert; Velds, Arno; Groothuis, Tom; Kerkhoven, Ron; Nieuwland, Marja; Ovaa, Huib; Rottenberg, Sven; van Tellingen, Olaf; Janssen, Jeroen; Huijgens, Peter; Zwart, Wilbert; Neefjes, Jacques

    2013-01-01

    DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy. PMID:23715267

  1. Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

    SciTech Connect

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M.

    2014-08-06

    Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

  2. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  3. ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation

    PubMed Central

    Elaimy, Ameer L.; Ahsan, Aarif; Marsh, Katherine; Pratt, William B.; Ray, Dipankar; Lawrence, Theodore S.; Nyati, Mukesh K.

    2016-01-01

    Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylationH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies. PMID:27738310

  4. Utilisation de l'essai comete et du biomarqueur gamma-H2AX pour detecter les dommages induits a l'ADN cellulaire par le 5-bromodeoxyuridine post-irradiation

    NASA Astrophysics Data System (ADS)

    La Madeleine, Carole

    Ce memoire est presente a la Faculte de medecine et des sciences de la sante de l'Universite de Sherbrooke en vue de l'obtention du grade de maitre es sciences (M.Sc.) en radiobiologie (2009). Un jury a revise les informations contenues dans ce memoire. Il etait compose de professeurs de la Faculte de medecine et des sciences de la sante soit : Darel Hunting PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Leon Sanche PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Richard Wagner PhD, membre du programme (departement de medecine nucleaire et radiobiologie) et Guylain Boissonneault PhD, membre exterieur au programme (departement de biochimie). Le 5-bromodeoxyuridine (BrdU), un analogue halogene de la thymidine reconnu depuis les annees 60 comme etant un excellent radiosensibilisateur. L'hypothese la plus repandue au sujet de l'effet radio sensibilisant du BrdU est qu'il augmente le nombre de cassures simple et double brin lorsqu'il est incorpore dans l'ADN de la cellule et expose aux radiations ionisantes. Toutefois, de nouvelles recherches semblent remettre en question les observations precedentes. Ces dernieres etudes ont confirme que le BrdU est un bon radiosensibilisateur, car il augmente les dommages radio-induits dans l'ADN. Mais, c'est en etant incorpore dans une region simple brin que le BrdU radiosensibilise l'ADN. Ces recherches ont egalement revele pour la premiere fois un nouveau type de dommages produits lors de l'irradiation de l'ADN contenant du BrdU : les dimeres interbrins. Le but de ces travaux de recherche est de determiner si la presence de bromodeoxyuridine dans l'ADN augmente l'induction de bris simple et / ou double brin chez les cellules irradiees en utilisant de nouvelles techniques plus sensibles et specifiques que celles utilisees auparavant. Pour ce faire, les essais cometes et la detection des foci H2AX phosphorylee pourraient permettre d'etablir les effets engendres par

  5. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    SciTech Connect

    Cooper, E.; Spaulding, S.W.

    1983-05-01

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with /sup 32/Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of /sup 32/P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of /sup 32/P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the /sup 32/P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, (/sup 32/P) HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

  6. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

    PubMed Central

    Yoshida, Makoto M.; Ting, Lily; Gygi, Steven P.

    2016-01-01

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  7. Nocturnal activation of aurora C in rat pineal gland: its role in the norepinephrine-induced phosphorylation of histone H3 and gene expression.

    PubMed

    Price, D M; Kanyo, R; Steinberg, N; Chik, C L; Ho, A K

    2009-05-01

    We have shown previously that Ser10 phosphorylation of histone H3 occurs in rat pinealocytes after stimulation with norepinephrine (NE) and that histone modifications such as acetylation appear to play an important role in pineal gene transcription. Here we report the nocturnal phosphorylation of a Ser10 histone H3 kinase, Aurora C, in the rat pineal gland. The time profile of this phosphorylation parallels the increase in the level of phospho-Ser10 histone H3. Studies with cultured pinealocytes indicate that Aurora C phosphorylation is induced by NE and this induction can be blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor. Moreover, only treatment with dibutyryl cAMP, but not other kinase activators, mimics the effect of NE on Aurora C phosphorylation. These results indicate that Aurora C is phosphorylated primarily by a beta-adrenergic/protein kinase A-mediated mechanism. Treatment with an Aurora C inhibitor reduces the NE-induced histone H3 phosphorylation and suppresses the NE-stimulated induction of arylalkylamine N-acetyltransferase (AA-NAT), the rhythm-controlling enzyme of melatonin synthesis, and melatonin production. The effects of Aurora C inhibitors on adrenergic-induced genes in rat pinealocytes are gene specific: inhibitory for Aa-nat and inducible cAMP repressor but stimulatory for c-fos. Together our results support a role for the NE-stimulated phosphorylation of Aurora C and the subsequent remodeling of chromatin in NE-stimulated Aa-nat transcription. This phenomenon suggests that activation of this mitotic kinase can be induced by extracellular signals to participate in the transcriptional induction of a subset of genes in the rat pineal gland.

  8. Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

    PubMed Central

    Sur, Swastika; Swier, Vicki J.; Radwan, Mohamed M.; Agrawal, Devendra K.

    2016-01-01

    Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia. PMID:26820885

  9. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

    PubMed

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-06-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

  10. Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts.

    PubMed

    Herrera, R E; Chen, F; Weinberg, R A

    1996-10-15

    Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity. Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts. This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1. H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation. In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts. Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function.

  11. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

    PubMed Central

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.11402.001 PMID:26878753

  12. Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

    SciTech Connect

    Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

    2009-12-15

    Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

  13. Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

    PubMed Central

    Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

    2015-01-01

    Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

  14. Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase.

    PubMed

    Attwood, Paul V; Ludwig, Katrin; Bergander, Klaus; Besant, Paul G; Adina-Zada, Abdussalam; Krieglstein, Josef; Klumpp, Susanne

    2010-01-01

    Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

  15. Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

    SciTech Connect

    Aoyama, Kazumasa; Fukumoto, Yasunori; Ishibashi, Kenichi; Kubota, Sho; Morinaga, Takao; Horiike, Yasuyoshi; Yuki, Ryuzaburo; Takahashi, Akinori; Nakayama, Yuji; Yamaguchi, Naoto

    2011-12-10

    c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

  16. Vitamin K3 (menadione)-induced oncosis associated with keratin 8 phosphorylation and histone H3 arylation.

    PubMed

    Scott, Gary K; Atsriku, Christian; Kaminker, Patrick; Held, Jason; Gibson, Brad; Baldwin, Michael A; Benz, Christopher C

    2005-09-01

    The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 microM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.

  17. Cell cycle–regulated phosphorylation of p220NPAT by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription

    PubMed Central

    Ma, Tianlin; Van Tine, Brian A.; Wei, Yue; Garrett, Michelle D.; Nelson, David; Adams, Peter D.; Wang, Jin; Qin, Jun; Chow, Louise T.; Harper, J. Wade

    2000-01-01

    Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220NPAT and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle–specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220NPAT links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription. PMID:10995387

  18. Histone Deacetylation Critically Determines T-cell Subset Radiosensitivity1

    PubMed Central

    Pugh, Jason L.; Sukhina, Alona S.; Seed, Thomas M.; Manley, Nancy R.; Sempowski, Gregory A.; van den Brink, Marcel R.M.; Smithey, Megan J.; Nikolich-Zugich, Janko

    2014-01-01

    Lymphocytes are sensitive to ionizing radiation and naïve lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naïve (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice, and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and : (i) levels of pro- and anti-apoptotic Bcl-2 family members; or (ii) the H2-AX content and maximal γH2-AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2-AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment, improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells, but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences. PMID:24990082

  19. Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos.

    PubMed

    Bohrer, Rodrigo Camponogara; Duggavathi, Raj; Bordignon, Vilceu

    2014-01-01

    Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos.

  20. Phosphorylated Histone 3 at Serine 10 Identifies Activated Spinal Neurons and Contributes to the Development of Tissue Injury-Associated Pain

    PubMed Central

    Torres-Pérez, Jose Vicente; Sántha, Péter; Varga, Angelika; Szucs, Peter; Sousa-Valente, Joao; Gaal, Botond; Sivadó, Miklós; Andreou, Anna P; Beattie, Sara; Nagy, Bence; Matesz, Klara; C. Arthur, J. Simon; Jancsó, Gábor; Nagy, Istvan

    2017-01-01

    Transcriptional changes in superficial spinal dorsal horn neurons (SSDHN) are essential in the development and maintenance of prolonged pain. Epigenetic mechanisms including post-translational modifications in histones are pivotal in regulating transcription. Here, we report that phosphorylation of serine 10 (S10) in histone 3 (H3) specifically occurs in a group of rat SSDHN following the activation of nociceptive primary sensory neurons by burn injury, capsaicin application or sustained electrical activation of nociceptive primary sensory nerve fibres. In contrast, brief thermal or mechanical nociceptive stimuli, which fail to induce tissue injury or inflammation, do not produce the same effect. Blocking N-methyl-D-aspartate receptors or activation of extracellular signal-regulated kinases 1 and 2, or blocking or deleting the mitogen- and stress-activated kinases 1 and 2 (MSK1/2), which phosphorylate S10 in H3, inhibit up-regulation in phosphorylated S10 in H3 (p-S10H3) as well as fos transcription, a down-stream effect of p-S10H3. Deleting MSK1/2 also inhibits the development of carrageenan-induced inflammatory heat hyperalgesia in mice. We propose that p-S10H3 is a novel marker for nociceptive processing in SSDHN with high relevance to transcriptional changes and the development of prolonged pain. PMID:28120884

  1. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    SciTech Connect

    Eto, Masumi; Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  2. Effect of SPL (Spent Pot Liner) and its main components on root growth, mitotic activity and phosphorylation of Histone H3 in Lactuca sativa L.

    PubMed

    Freitas, Aline Silva; Fontes Cunha, Isabela Martinez; Andrade-Vieira, Larissa Fonseca; Techio, Vânia Helena

    2016-02-01

    Spent Pot Liner (SPL) is a solid waste from the aluminum industry frequently disposed of in industrial landfills; it can be leached and contaminate the soil, sources of drinking water and plantations, and thus may pose a risk to human health and to ecosystems. Its composition is high variable, including cyanide, fluoride and aluminum salts, which are highly toxic and environmental pollutants. This study evaluated the effect of SPL and its main components on root growth and the mitosis of Lactuca sativa, by investigating the mechanisms of cellular and chromosomal alterations with the aid of immunolocalization. To this end, newly emerged roots of L. sativa were exposed to SPL and its main components (solutions of cyanide, fluoride and aluminum) and to calcium chloride (control) for 48h. After this, root length was measured and cell cycle was examined by means of conventional cytogenetics and immunolocalization. Root growth was inhibited in the treatments with SPL and aluminum; chromosomal and nuclear alterations were observed in all treatments. The immunolocalization evidenced normal dividing cells with regular temporal and spatial distribution of histone H3 phosphorylation at serine 10 (H3S10ph). However, SPL and its main components inhibited the phosphorylation of histone H3 at serine 10, inactivated pericentromeric regions and affected the cohesion of sister chromatids, thus affecting the arrangement of chromosomes in the metaphase plate and separation of chromatids in anaphase. In addition, these substances induced breaks in pericentromeric regions, characterized as fragile sites.

  3. Loss of centromeric histone H2AT120 phosphorylation accompanies somatic chromosomes inactivation in the aberrant spermatocytes of Acricotopus lucidus (Diptera, Chironomidae).

    PubMed

    Staiber, Wolfgang

    2016-01-01

    In the germ line of the chironomid Acricotopus lucidus, two cells with quite different chromosome constitutions result from the last unequal gonial mitosis. In the male, the future primary spermatocyte receives all the germ line-limited chromosomes (=Ks) together with somatic chromosomes (=Ss), and later on undergoes meiotic divisions, while the connected aberrant spermatocyte gets only Ss and remains undivided with chromosomes inactivated in a metaphase-like condensed state. This raises the question whether the centromeres of the permanently condensed Ss of the aberrant spermatocyte remain active during meiosis of the connected regular spermatocyte. Active centromeres exhibit an epigenetic phosphorylation mark at threonine 120 of histone H2A. To visualise the centromeric H2A phosphorylation of the Ss in the aberrant spermatocyte, meiotic stages were immunostained with different anti-phospho histone H2AT120 antibodies. Clear H2AT120ph signals appear at the centromeres of the Ss during prophase, persist on the metaphase-like condensed Ss during meiosis I of the connected primary spermatocyte and disappear during transition to meiosis II. The centromeres of the Ss and Ks of the regular spermatocytes display H2AT120ph signals from prophase I to anaphase II. The loss of the H2AT120 phosphorylation detected on the centromeres of the Ss of the aberrant spermatocyte indicating their deactivation supports the idea of a programmed inactivation of the Ss to block the entry of the germ line-derived aberrant spermatocyte, lacking Ks, into meiosis, and thus to prevent the generation of sperms possessing only Ss. This mechanism would ensure the presence of the Ks in the germ line.

  4. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells.

    PubMed

    Eto, Masumi; Kirkbride, Jason A; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In

    2013-04-26

    CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  5. Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

    PubMed Central

    Ho, Catherine M. K.; Donovan-Banfield, I’ah Z.; Tan, Li; Zhang, Tinghu; Gray, Nathanael S.; Strang, Blair L.

    2016-01-01

    Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. PMID:26930276

  6. Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

    PubMed Central

    Barlev, Nickolai A.; Poltoratsky, Vladimir; Owen-Hughes, Tom; Ying, Carol; Liu, Lin; Workman, Jerry L.; Berger, Shelley L.

    1998-01-01

    GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity. PMID:9488450

  7. The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer.

    PubMed

    Hait, N C; Avni, D; Yamada, A; Nagahashi, M; Aoyagi, T; Aoki, H; Dumur, C I; Zelenko, Z; Gallagher, E J; Leroith, D; Milstien, S; Takabe, K; Spiegel, S

    2015-06-08

    Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

  8. The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer

    PubMed Central

    Hait, N C; Avni, D; Yamada, A; Nagahashi, M; Aoyagi, T; Aoki, H; Dumur, C I; Zelenko, Z; Gallagher, E J; Leroith, D; Milstien, S; Takabe, K; Spiegel, S

    2015-01-01

    Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer. PMID:26053034

  9. Heterochromatin protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II

    PubMed Central

    Kwon, So Hee; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2010-01-01

    Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II. PMID:20889714

  10. Structure of C-terminal Tandem BRCT Repeats of Rtt107 Protein Reveals Critical Role in Interaction with Phosphorylated Histone H2A during DNA Damage Repair*

    PubMed Central

    Li, Xinxin; Liu, Kaixian; Li, Fudong; Wang, Juncheng; Huang, Hongda; Wu, Jihui; Shi, Yunyu

    2012-01-01

    Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT5-BRCT6) of Rtt107. The crystal structures of BRCT5-BRCT6 alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT5-BRCT6 and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks. PMID:22262834

  11. Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

    PubMed

    Chen, Changchao; Zhang, Zixiao; Cui, Panpan; Liao, Yaya; Zhang, Yue; Yao, Lingyun; Rui, Rong; Ju, Shiqiang

    2017-02-20

    Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.

  12. Induction of ERK1/2 and histone H3 phosphorylation within the outer stripe of the outer medulla of the Eker rat by 2,3,5-tris-(glutathion-S-yl)hydroquinone.

    PubMed

    Dong, Jing; Everitt, Jeffrey I; Lau, Serrine S; Monks, Terrence J

    2004-08-01

    2,3,5-tris-(glutathion-S-yl)-hydroquinone (TGHQ), a metabolite of hydroquinone (HQ), generates reactive oxygen species (ROS) in cultured renal epithelial cells and binds to tissue macromolecules within the rat kidney. The potential mechanisms by which TGHQ induces nephrotoxicity and nephrocarcinogenesis have been examined in cell culture models, but less is known concerning the molecular mechanisms of TGHQ-induced nephrotoxicity in vivo. In LLC-PK1 cells, TGHQ induces phosphorylation of both mitogen-activated protein kinase and histone H3, which likely promotes inappropriate chromatin condensation and mitotic catastrophe. Using the Eker (Tsc-2 mutant) rat as a model, we show by immunohistochemistry that TGHQ (7.5 micromol/kg) selectively induces ERK1/2 phosphorylation within the outer stripe of the outer medulla (OSOM) of the kidney. ERK1/2 phosphorylation is time-dependant, occurring as early as 1 h following treatment, and reaching maximal levels by 4 h. Subsequently, ERK1/2 phosphorylation returns to baseline levels by 24 h post treatment. ERK1/2 phosphorylation was confirmed by western blot analysis of OSOM tissue. Increases in histone H3 phosphorylation occurred subsequent to ERK1/2 phosphorylation (8 h), and reached a peak by 24 h, coincident with histological evidence of tissue necrosis. In contrast to studies in cell culture, neither JNK/SAPK nor p38 MAPK phosphorylation were significantly altered after TGHQ administration in vivo, as evidenced by western blot and immunohistochemical analyses. These data indicate that activation of the ERK1/2 pathway precedes overt cytotoxicity and that the signaling pathways activated by TGHQ in vivo and in vitro differ.

  13. Effects of Forced Swimming Stress on ERK and Histone H3 Phosphorylation in Limbic Areas of Roman High- and Low-Avoidance Rats

    PubMed Central

    Piludu, Maria Antonietta; Poddighe, Laura; Serra, Maria Pina; Quartu, Marina; Corda, Maria Giuseppa; Giorgi, Osvaldo

    2017-01-01

    Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent. PMID:28107383

  14. Role of phosphorylated histone H3 serine 10 in DEN-induced deregulation of Pol III genes and cell proliferation and transformation

    PubMed Central

    Zhong, Shuping

    2013-01-01

    The products of Pol III genes (RNA polymerase III-dependent genes), such as tRNAs and 5S rRNA, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. An increase in Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB, augments Pol III gene transcription and is sufficient for cell transformation and tumor formation. We have demonstrated that enhancement of Brf1 and Pol III gene expression is associated with the occurrences of hepatocellular carcinoma (HCC) in mice. This suggests that Brf1 may be a key molecule during HCC development. Diethylnitrosamine (DEN), a chemical carcinogen, has been used to induce HCC in rodents. To determine the role of Brf1 and the epigenetic-regulating events in cell proliferation and transformation, hepatocytes were treated with DEN. The results indicate that DEN increases proliferation and transformation of AML-12 cells. DEN enhanced Brf1 expression and tRNALeu and 5S rRNA transcription, as well as H3S10ph (phosphorylation of histone H3 serine 10). Interestingly, DEN-induced Pol III gene transcription and H3S10ph in tumor cells of liver are significantly higher than in non-tumor cells. Inhibition of H3S10ph by H3S10A attenuates the induction of Brf1 and Pol III genes. Further analysis indicates that H3S10ph occupies the promoters of Brf1 and Pol III genes to modulate their expression. Blocking H3S10ph represses cell proliferation and transformation. These results demonstrate that DEN induces H3S10ph, which mediate Brf1 expression, including but not limited Brf1-dependent genes, to upregulate Pol III gene transcription, resulting in an increase in cell proliferation and transformation. PMID:23774401

  15. Purification and characterization of protein phosphatase 2C in rat parotid acinar cells: two forms of Mg(2+)-activated histone phosphatase and phosphorylation by cAMP-dependent protein kinase.

    PubMed

    Yokoyama, N; Kobayashi, T; Tamura, S; Sugiya, H

    1996-07-01

    Two forms of Mg(2+)-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography. Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA. The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak. These two peaks eluted at distinct positions on the gel filtration column. The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS-gels. The first 46-kDa peak immunoreacted with anti-PP2Calpha phosphatase antibody and like PP2Calpha phosphatase could be phosphorylated by cAMP-dependent protein kinase. The second 55-kDa peak showed neither reactivity with anti-PP2Calpha phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+ or Mn2+ dependence for its histone phosphatase activity. Ca2+ showed a strong inhibition on this activity. On the basis of these observations, we have identified the first peak enzyme as PP2Calpha phosphatase and the second peak as a novel PP2C-like phosphatase.

  16. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair.

    PubMed

    Kato, Akihiro; Komatsu, Kenshi

    2015-07-14

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair.

  17. Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4+ T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb

    PubMed Central

    Jamaluddin, Md Saha; Hu, Pei-Wen; Jan, Yih; Siwak, Edward B.

    2016-01-01

    Abstract Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4+ T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4+ cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4+ T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4+ T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation. PMID:26727990

  18. Analysis of histones and histone variants in plants.

    PubMed

    Trivedi, Ila; Rai, Krishan Mohan; Singh, Sunil Kumar; Kumar, Verandra; Singh, Mala; Ranjan, Amol; Lodhi, Niraj; Sawant, Samir V

    2012-01-01

    Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

  19. Phosphorylation of CDK2 on threonine 160 influences silencing of sex chromosome during male meiosis.

    PubMed

    Wang, Lu; Liu, Wenjing; Zhao, Weidong; Song, Gendi; Wang, Guishuan; Wang, Xiaorong; Sun, Fei

    2014-06-01

    In mammalian meiosis, the X and Y chromosomes are largely unsynapsed and transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation), forming a specialized nuclear territory called sex or XY body. An increasing number of proteins and noncoding RNAs were found to localize to the sex body and take part in influencing expression of sex chromosome genes. Cyclin-dependent kinase 2 (Cdk2 (-/-)) spermatocytes show incomplete sex chromosome pairing. Here, we further showed that phosphorylation of CDK2 isoform 1 (p-CDK2(39) [39 kDa]) on threonine 160 localizes to the sites of asynapsis and the sex body, interacting with phosphorylated gamma-H2AX. Meanwhile, p-CDK2(39) is frequently mislocalized throughout the sex body, and meiotic sex chromosome inactivation is disrupted in PWK×C57BL/6J hybrid mice. Furthermore, pachytene spermatocytes treated with mevastatin (an inhibitor of p-CDK2) showed overexpression of sex chromosome-linked genes. Our results highlight an important role for p-CDK2(39) in influencing silencing of the sex chromosomes during male meiosis by interacting with gamma-H2AX.

  20. Histone modifying enzymes: novel disease biomarkers and assay development.

    PubMed

    Ma, Fei; Zhang, Chun-yang

    2016-01-01

    Histones are the chief components of chromatin. When being catalyzed by a series of histone modifying enzymes, histones may undergo various post-translational modifications such as acetylation, methylation, phosphorylation, ubiquitylation and SUMOylation. The dysregulation of histone modifying enzymes will alter the histone post-modification patterns and cause diverse diseases including cancers. Consequently, the histone modifying enzymes have emerged as the promising biomarkers for disease diagnosis and prognosis. In this review, we summarize the recent researches about the histone modifying enzymes as the disease biomarkers, and highlight the development of methods for histone modifying enzyme assays.

  1. A mouse speciation gene encodes a meiotic histone H3 methyltransferase.

    PubMed

    Mihola, Ondrej; Trachtulec, Zdenek; Vlcek, Cestmir; Schimenti, John C; Forejt, Jiri

    2009-01-16

    Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

  2. In vivo efficacy of the histone deacetylase inhibitor suberoylanilide hydroxamic acid in combination with radiotherapy in a malignant rhabdoid tumor mouse model

    PubMed Central

    2012-01-01

    Purpose Histone deacetylase inhibitors are promising new substances in cancer therapy and have also been shown to sensitize different tumor cells to irradiation (XRT). We explored the effect as well as the radiosensitizing properties of suberoylanilide hydroxamic acid (SAHA) in vivo in a malignant rhabdoid tumor (MRT) mouse model. Methods and material Potential radiosensitization by SAHA was assessed in MRT xenografts by analysis of tumor growth delay, necrosis (HE), apoptosis (TUNEL), proliferation (ki-67) and γH2AX expression as well as dynamic 18F-Fluorodeoxyglucose Positron Emission Tomography (18F-FDG -PET) after treatment with either SAHA alone, single-dose (10 Gy) or fractionated XRT (3 × 3Gy) solely as well as in combination with SAHA compared to controls. Results SAHA only had no significant effect on tumor growth. Combination of SAHA for 8 days with single-dose XRT resulted in a higher number of complete remissions, but failed to prove a significant growth delay compared to XRT only. In contrast fractionated XRT plus SAHA for 3 weeks did induce significant tumor growth delay in MRT-xenografts. The histological examination showed a significant effect of XRT in tumor necrosis, expression of Ki-67, γH2AX and apoptosis. SAHA only had no significant effect in the histological examination. Comparison of xenografts treated with XRT and XRT plus SAHA revealed a significantly increased γH2AX expression and apoptosis induction in the mice tumors after combination treatment with single-dose as well as fractionated XRT. The combination of SAHA with XRT showed a tendency to increased necrosis and decrease of proliferation compared to XRT only, which, however, was not significant. The 18F-FDG-PET results showed no significant differences in the standard uptake value or glucose transport kinetics after either treatment. Conclusion SAHA did not have a significant effect alone, but proved to enhance the effect of XRT in our MRT in vivo model. PMID:22458853

  3. Immunocytochemical and immunogold analyses of histone H4 acetylation during Chara vulgaris spermiogenesis.

    PubMed

    Wojtczak, Agnieszka

    2016-03-01

    Histone acetylation is one of the epigenetic modifications which play a significant role in chromatin remodeling during spermiogenesis. Acetylation of the histone H4 makes the exchange of nucleoproteins easy. Research on mouse spermatogenesis showed that H4 histone acetylated at Lys 12 (H4K12ac) was specific only to spermatids. Immunocytochemical studies of Chara vulgaris spermatids with the use of antibodies against the histone H4K12ac revealed positive reactions in spermatid nuclei at stages I-VII. This reaction, connected with nuclear condensation, was much stronger at the early stages of spermiogenesis than later on. Moreover, it showed that at the stages V-VII in spermatid nuclei the presence of the histone H4K12ac corresponded with DNA double-strand breaks. Electron microscopy studies with the use of immunogold technique revealed an almost twofold difference between the mean total numbers of gold grains in the examined chromatin in both stages. This study showed nearly equal distribution of gold grains on condensed and non-condensed chromatin of spermatids at the stage III/IV (48.11% and 51.89%, respectively). In the later stage-VI, when chromatin condensation proceeded, labeling of condensed chromatin reached 57.27%, while in the case of non-condensed chromatin it dropped to 42.73%. The percentage analysis also revealed an increase (above 9%) in condensed chromatin labeling in relation to the stage III/IV. Intensive acetylation of histone H4 at the early stages is correlated with DNA DSBs and transcriptional activity. It facilitates chromatin loosening, which enables the correct course of chromatin remodeling at a later stage. Histone γH2AX also influences chromatin structure in many biological processes in different cell types. Current studies reveal other similarities regarding histone H4 acetylation, not only between Chara and mammals but between invertebrates (molluscs) and vertebrates (bony fishes) as well.

  4. Chatting histone modifications in mammals

    PubMed Central

    Izzo, Annalisa

    2010-01-01

    Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks. PMID:21266346

  5. Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide.

    PubMed

    Sato, Yuichi; Kurose, Akira; Ogawa, Akira; Ogasawara, Kuniaki; Traganos, Frank; Darzynkiewicz, Zbigniew; Sawai, Takashi

    2009-03-01

    Phosphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (gammaH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced gammaH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G(2)/M. NHA cells were arrested in G(1) with no evidence of gammaH2AX induction. A172 responded by rise in gammaH2AX throughout all phases of the cycle, arrest at the late S- to G(2)/M-phase, and appearance of senescence features: induction of p53, p21(WAF1/CIP1), p16(INK4A) and beta-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of gammaH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G(1) was seen in YKG-1 cells with no rise in gammaH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs. mutated, or inhibited by pifithrin-alpha) nor the expression of O(6)-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.

  6. Transcription-coupled replacement of histones: degradation or recycling?

    PubMed

    Chen, Yu-Shan; Qiu, Xiao-Bo

    2012-11-20

    Histone modifications are proposed to constitute a "histone code" for epigenetic regulation of gene expression. However, recent studies demonstrate that histones have to be disassembled from chromatin during transcription. Recent evidence, though not conclusive, suggests that histones might be degradable after being removed from chromatin during transcription. Degradation of overexpressed excessive histones, instead of native histones, has been shown to be dependent on proteasomes and ubiquitination. Since the 26S proteasome usually recognizes polyubiquitinated substrates, it is critical to demonstrate whether degradation of histones is mediated by polyubiquitination. Unexpectedly, there is almost no evidence that any ubiquitin ligase can promote polyubiquitination-dependent degradation of constitutive histones. Meanwhile, acetylation and phosphorylation are also associated with histone degradation. This review attempts to summarize the current knowledge on the transcription-coupled degradation of histones and its regulation by posttranslational protein modifications.

  7. Activation of ATM by DNA Damaging Agents

    DTIC Science & Technology

    2004-09-01

    all cases, observe a dependence on ATM for phosphorylation. These phosphorylation events are attenuated by pretreatment of cells with N-acetyl cysteine...Chemistry requested that I evaluate the effect of N-acetyl cysteine pretreatment on doxorubicin-induced phosphorylation of histone H2AX at serine 139. In...S33/35, T68) in response to doxorubicin treatment * I detennined that phosphorylation at these sites can be partially attenuated by pretreatment of

  8. Numerical Analysis of Etoposide Induced DNA Breaks

    PubMed Central

    Muslimović, Aida; Nyström, Susanne; Gao, Yue; Hammarsten, Ola

    2009-01-01

    Background Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs. Methodology/Principal Findings To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity. Conclusions/Significance These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity. PMID:19516899

  9. Histone Deacetylases

    PubMed Central

    Parbin, Sabnam; Kar, Swayamsiddha; Shilpi, Arunima; Sengupta, Dipta; Deb, Moonmoon; Rath, Sandip Kumar

    2014-01-01

    In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer. PMID:24051359

  10. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    found on the tubulin-binding domain of tau, leading us to the idea that the interaction of tau with tubulin could induce conformational changes on tau making it accessible to effects of the kinase. We detected -SP- as a sequence common to three major phosphorylation sites on K1, K2 and K3 fragments. Neurofilament-specific kinase and growth-associated histone H1 kinase are known to recognize the consensus sequence including -SP-. These enzymes exhibit certain properties similar to tau protein kinase and seem to play a crucial role in the regulation of neurite outgrowth or cell growth, through the phosphorylation of a specific substrate, neurofilaments or histone H1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

  11. HISTONE H3S10 PHOSPHORYLATION BY THE JIL-1 KINASE IN PERICENTRIC HETEROCHROMATIN AND ON THE 4TH CHROMOSOME CREATES A COMPOSITE H3S10phK9me2 EPIGENETIC MARK

    PubMed Central

    Wang, Chao; Li, Yeran; Cai, Weili; Bao, Xiaomin; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2014-01-01

    The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, we demonstrate that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the 4th chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus our findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and 4th chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase. PMID:24429699

  12. Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis.

    PubMed

    Huang, Xuan; Halicka, H Dorota; Traganos, Frank; Tanaka, Toshiki; Kurose, Akira; Darzynkiewicz, Zbigniew

    2005-08-01

    Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

  13. Targeting post-translational modifications of histones for cancer therapy.

    PubMed

    Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H

    2015-10-30

    Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.

  14. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  15. Histone Octamer

    NASA Technical Reports Server (NTRS)

    1997-01-01

    1 mm histone octamer crystal grown on STS-81. A very dynamic structure which functions in many aspects of gene regulation from control of gene activity to the more subtle mechanisms of genetic imprinting. Principle Investigator is Dan Carter of New Century Pharmaceuticals.

  16. Histone Octamer

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This is a large 2 mm crystal of histone octamer, grown on STS-81. A very dynamic structure which functions in many aspects of gene regulation from control of gene activity to the more subtle mechanisms of genetic imprinting. Principle Investigator is Dan Carter of New Century Pharmaceuticals.

  17. Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

    SciTech Connect

    Petrillo, Stephanie K.; Desmeules, Patrice; Truong, To-Quyen; Devine, Patrick J.

    2011-06-01

    Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM ({>=} 3 {mu}M) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation ({gamma}H2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 {mu}M PM induced significant losses of primordial and small primary follicles, respectively. PM-induced {gamma}H2AX was observed predominantly in oocytes, in which foci of {gamma}H2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 {mu}M PM. Numbers of oocytes with {>=} 5 {gamma}H2AX foci were significantly increased both 1 and 8 days after exposure to {>=} 1 {mu}M PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers ({>=} 30 {mu}M) and increased {gamma}H2AX foci ({>=} 3 {mu}M) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

  18. Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

    PubMed

    Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

    2013-10-01

    A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry.

  19. Interaction of calmodulin with histones. Alteration of histone dephosphorylation.

    PubMed

    Wolff, D J; Ross, J M; Thompson, P N; Brostrom, M A; Brostrom, C O

    1981-02-25

    The Ca2+-dependent regulator protein (CDR), also frequently termed "calmodulin" was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1, 2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increase the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities of CDR. Dephosphorylation of casein and phosphorylase alpha by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1, 2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 microM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent Km of the phosphatase for the various histones 2- to 6-fold, while maximal velocities were 4- to 12-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by chelation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(L-aspartate) or poly(L-glutamate). The phosphorylated fragment from the NH2-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of

  20. Dynamic monitoring of oxidative DNA double-strand break and repair in cardiomyocytes.

    PubMed

    Ye, Bo; Hou, Ning; Xiao, Lu; Xu, Yifan; Xu, Haodong; Li, Faqian

    2016-01-01

    DNA double-strand breaks (DSBs) are most dangerous lesions. To determine whether oxidative stress can induce DSBs and how they are repaired in cardiomyocytes (CMs), cultured neonatal rat CMs were treated with different doses of H2O2 and followed for up to 72 h for monitoring the spatiotemporal dynamics of DNA repair protein assembly/disassembly at DSB foci. The protein levels and foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) increased proportionally to 50, 100, and 200 μmol/L H2O2 after 30 min treatment. When H2O2 was at or above 400 μmol/L, γ-H2AX became predominantly pannuclear. After 30 min, 200 μmol/L of H2O2 treatment, γ-H2AX levels were highest within the first hour and then gradually declined during the recovery and returned to basal levels at 48 h. Among DNA damage transducer kinases, ataxia telangiectasia mutated (ATM) was significantly activated by H2O2 in contrast to mild activation of ATR (ATM and Rad3-related). A DSB binding protein, p53 binding protein 1, formed distinct nuclear foci that colocalized with γ-H2AX foci and phosphorylated ATM. Our findings indicate that DSBs can be induced by H2O2 and ATM is the main kinase to mediate DSB repair in CMs. Therefore, monitoring DSB repair can assess oxidative injury and response in CMs.

  1. Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin.

    PubMed

    Soubeyrand, Sébastien; Pope, Louise; De Chasseval, Régina; Gosselin, Dominique; Dong, Fumin; de Villartay, Jean-Pierre; Haché, Robert J G

    2006-05-19

    Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in naïve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both naïve and damaged cells.

  2. The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity.

    PubMed

    Cui, Fengmei; Fan, Rong; Chen, Qiu; He, Yongming; Song, Man; Shang, Zengfu; Zhang, Shimeng; Zhu, Wei; Cao, Jianping; Guan, Hua; Zhou, Ping-Kun

    2015-08-01

    Deregulation of c-Myc often occurs in various human cancers, which not only contributes to the genesis and progression of cancers but also affects the outcomes of cancer radio- or chemotherapy. In this study, we have investigated the function of c-Myc in the repair of DNA double-strand break (DSB) induced by γ-ray irradiation. A c-Myc-silenced Hela-630 cell line was generated from HeLa cells using RNA interference technology. The DNA DSBs were detected by γ-H2AX foci, neutral comet assay and pulsed-field gel electrophoresis. We found that the capability of DNA DSB repair in Hela-630 cells was significantly reduced, and the repair kinetics of DSB was delayed as compared to the control Hela-NC cells. Silence of c-myc sensitized the cellular sensitivity to ionizing radiation. The phosphorylated c-Myc (Thr58/pSer62) formed the consistent co-localisation foci with γ-H2AX as well as the phosphorylated DNA-PKcs/S2056 in the irradiated cells. Moreover, depression of c-Myc largely attenuated the ionizing radiation-induced phosphorylation of the ataxia telangiectasia mutated (ATM) and decreased the in vitro kinase activity of DNA-PKcs. Taken together, our results demonstrated that c-Myc protein functions in the process of DNA double-strand break repair, at least partially, through affecting the ATM phosphorylation and DNA-PKcs kinase activity. The overexpression of c-Myc in tumours can account for the radioresistance of some tumour cell types.

  3. Histone Modifications and Nuclear Architecture: A Review

    PubMed Central

    Bártová, Eva; Krejčí, Jana; Harničarová, Andrea; Galiová, Gabriela; Kozubek, Stanislav

    2008-01-01

    Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei. (J Histochem Cytochem 56:711–721, 2008) PMID:18474937

  4. S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

    SciTech Connect

    Lee, Haemi; Kwak, Hee-Jin; Cho, Il-taeg; Park, Seok Hee; Lee, Chang-Hun

    2009-01-02

    53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and {gamma}H2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.

  5. S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response.

    PubMed

    Lee, Haemi; Kwak, Hee-Jin; Cho, Il-taeg; Park, Seok Hee; Lee, Chang-Hun

    2009-01-02

    53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and gammaH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.

  6. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

    PubMed

    Elsen, Sylvie; Collin-Faure, Véronique; Gidrol, Xavier; Lemercier, Claudie

    2013-11-01

    Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AXH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

  7. Linker histones in hormonal gene regulation.

    PubMed

    Vicent, G P; Wright, R H G; Beato, M

    2016-03-01

    In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms.

  8. A Novel Approach for Studying Histone H1 Function in Vivo

    PubMed Central

    Siriaco, Giorgia; Deuring, Renate; Mawla, Gina D.; Tamkun, John W.

    2015-01-01

    In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1. PMID:25805849

  9. Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

    2004-01-01

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  10. Activation of p53 Transcriptional Activity by SMRT: a Histone Deacetylase 3-Independent Function of a Transcriptional Corepressor

    PubMed Central

    Adikesavan, Anbu Karani; Karmakar, Sudipan; Pardo, Patricia; Wang, Liguo; Liu, Shuang; Li, Wei

    2014-01-01

    The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression. PMID:24449765

  11. Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

    PubMed

    Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-11-09

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AXH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

  12. Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

    PubMed

    Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

    2014-10-10

    Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.

  13. A brief histone in time: understanding the combinatorial functions of histone PTMs in the nucleosome context.

    PubMed

    Ng, Marlee K; Cheung, Peter

    2016-02-01

    It has been over 50 years since Allfrey et al. proposed that histone acetylation regulates RNA synthesis, and the study of histone modifications has progressed at an extraordinary pace for the past two decades. In this review, we provide a perspective on some key events and advances in our understanding of histone modifications. We also highlight reagents and tools from past to present that facilitated progress in this research field. Using histone H3 phosphorylation as an underlying thread, we review the rationale that led to the proposal of the histone code hypothesis, as well as examples that illustrate the concepts of combinatorial histone modifications and cross-talk pathways. We further highlight the importance of investigating these mechanisms in the context of nucleosomes rather than just at the histone level and present current and developing approaches for such studies. Overall, research on histone modifications has yielded great mechanistic insights into the regulation of genomic functions, and extending these studies using nucleosomes will further elucidate the complexity of these pathways in a more physiologically relevant context.

  14. Histone deacetylases: Targets for antifungal drug development

    PubMed Central

    Kmetzsch, Livia

    2015-01-01

    The interaction of pathogens and its hosts causes a drastic change in the transcriptional landscape in both cells. Among the several mechanisms of gene regulation, transcriptional initiation is probably the main point. In such scenario, the access of transcriptional machinery to promoter is highly regulated by post-translational modification of histones, such as acetylation, phosphorylation and others. Inhibition of histone deacetylases is able to reduce fungal pathogens fitness during infection and, therefore, is currently being considered for the development of new antifungal therapy strategies. PMID:26151486

  15. Tetrandrine Exerts a Radiosensitization Effect on Human Glioma through Inhibiting Proliferation by Attenuating ERK Phosphorylation

    PubMed Central

    Ma, Ji-wei; Zhang, Yong; Ye, Ji-cheng; Li, Ru; Wen, Yu-Lin; Huang, Jian-xian; Zhong, Xue-yun

    2017-01-01

    Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, has been reported to have a radiosensitization effect on tumors. However, its effects on human glioma and the specific molecular mechanisms of these effects remain unknown. In this study, we demonstrated that Tet has a radiosensitization effect on human glioma cells. It has been hypothesized that Tet has a radiosensitization effect on glioma cells by affecting the glioma cell cycle and DNA repair mechanism and that ERK mediates these activities. Therefore, we conducted detailed analyses of the effects of Tet on the cell cycle by performing flow cytometric analysis and on DNA repair by detecting the expression of phosphorylated H2AX by immunofluorescence. We used western blot analysis to investigate the role of ERK in the effect of Tet on the cell cycle and DNA repair. The results revealed that Tet exerts its radiosensitization effect on glioma cells by inhibiting proliferation and decreasing the expression of phosphorylated ERK and its downstream proteins. In summary, our data indicate that ERK is involved in Tet-induced radiosensitization of glioma cells via inhibition of glioma cell proliferation or of the cell cycle at G0/G1 phase. PMID:27829269

  16. Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation

    PubMed Central

    2011-01-01

    Introduction Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation. Methods IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated. Results The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked

  17. An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts

    PubMed Central

    Pinazza, M; Borga, C; Agnusdei, V; Minuzzo, S; Fossati, G; Paganin, M; Michielotto, B; De Paoli, A; Basso, G; Amadori, A; te Kronnie, G; Indraccolo, S

    2016-01-01

    Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia. PMID:26764573

  18. Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Halicka, H. Dorota; Zhao, Hong

    2010-01-01

    Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G2 and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase. PMID:21755443

  19. Extracellular histones inhibit efferocytosis.

    PubMed

    Friggeri, Arnaud; Banerjee, Sami; Xie, Na; Cui, Huachun; De Freitas, Andressa; Zerfaoui, Mourad; Dupont, Hervé; Abraham, Edward; Liu, Gang

    2012-07-18

    The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest-specific gene 6 (Gas6) and milk fat globule-epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble α(v)β₅ integrin and Mer, but not with α(v)β₃, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.

  20. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila

    PubMed Central

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S.; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2014-01-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing. PMID:24598257

  1. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila.

    PubMed

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2014-05-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing.

  2. Histone chaperones: assisting histone traffic and nucleosome dynamics.

    PubMed

    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  3. Detection of histone modifications in plant leaves.

    PubMed

    Jaskiewicz, Michal; Peterhansel, Christoph; Conrath, Uwe

    2011-09-23

    Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles(1-2). H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues(1-2). These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)(3-7). Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde(8,9), extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies(9,10), de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C(4;) photosynthesis in maize(5,11) and systemic immunity in Arabidopsis(3).

  4. The Role of Co-transcriptional Histone Methylations

    PubMed Central

    Buratowski, Stephen; Kim, TaeSoo

    2011-01-01

    The C-terminal domain (CTD) of the RNA polymerase II subunit Rpb1 undergoes dynamic phosphorylation, with different phosphorylation sites predominating at different stages of transcription. Our lab studies how various mRNA processing and chromatin-modifying enzymes interact with the phosphorylated CTD to efficiently produce mRNAs. The H3K36 methyltransferase Set2 interacts with CTD carrying phosphorylations characteristic of downstream elongation complexes, and the resulting co-transcriptional H3K36 methylation targets the Rpd3S histone deacetylase to downstream transcribed regions. Although positively correlated with gene activity, this pathway actually inhibits transcription elongation as well as initiation from cryptic promoters within genes. During early elongation, CTD serine 5 phosphorylation helps recruit the H3K4 methyltransferase complex containing Set1. Within 5' transcribed regions, co-transcriptional H3K4 dimethylation (H3K4me2) by Set1 recruits the deacetylase complex Set3C. Finally, H3K4 trimethylation at the most promoter-proximal nucleosomes is thought to stimulate transcription by promoting histone acetylation by complexes containing the ING/Yng PHD finger proteins. Surprisingly, the Rpd3L histone deacetylase complex, normally a transcription repressor, may also recognize H3K4me3. Together, the cotranscriptional histone methylations appear to primarily function to distinguish active promoter regions, which are marked by high levels of acetylation and nucleosome turnover, from the deacetylated, downstream transcribed regions of genes. PMID:21447819

  5. Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly.

    PubMed

    Sampath, Srihari C; Marazzi, Ivan; Yap, Kyoko L; Sampath, Srinath C; Krutchinsky, Andrew N; Mecklenbräuker, Ingrid; Viale, Agnes; Rudensky, Eugene; Zhou, Ming-Ming; Chait, Brian T; Tarakhovsky, Alexander

    2007-08-17

    Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.

  6. Inhibition of mitotic-specific histone phophorylation by sodium arsenite

    SciTech Connect

    Cobo, J.M.; Valdez, J.G.; Gurley, L.R.

    1994-10-01

    Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

  7. Readers of histone modifications

    PubMed Central

    Yun, Miyong; Wu, Jun; Workman, Jerry L; Li, Bing

    2011-01-01

    Histone modifications not only play important roles in regulating chromatin structure and nuclear processes but also can be passed to daughter cells as epigenetic marks. Accumulating evidence suggests that the key function of histone modifications is to signal for recruitment or activity of downstream effectors. Here, we discuss the latest discovery of histone-modification readers and how the modification language is interpreted. PMID:21423274

  8. Histone Arginine Methylation

    PubMed Central

    Lorenzo, Alessandra Di; Bedford, Mark T.

    2012-01-01

    Arginine methylation is a common posttranslational modification (PTM). This type of PTM occurs on both nuclear and cytoplasmic proteins, and is particularly abundant on shuttling proteins. In this review, we will focus on one aspect of this PTM: the diverse roles that arginine methylation of the core histone tails play in regulating chromatin function. A family of nine protein arginine methyltransferases (PRMTs) catalyze methylation reactions, and a subset target histones. Importantly, arginine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, thus playing a central role in the orchestration of the histone code. PMID:21074527

  9. Histone Variants and Epigenetics

    PubMed Central

    Henikoff, Steven; Smith, M. Mitchell

    2015-01-01

    Histones package and compact DNA by assembling into nucleosome core particles. Most histones are synthesized at S phase for rapid deposition behind replication forks. In addition, the replacement of histones deposited during S phase by variants that can be deposited independently of replication provide the most fundamental level of chromatin differentiation. Alternative mechanisms for depositing different variants can potentially establish and maintain epigenetic states. Variants have also evolved crucial roles in chromosome segregation, transcriptional regulation, DNA repair, and other processes. Investigations into the evolution, structure, and metabolism of histone variants provide a foundation for understanding the participation of chromatin in important cellular processes and in epigenetic memory. PMID:25561719

  10. Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response

    PubMed Central

    Yang, Seungchan; Quaresma, Alexandre J. C.; Nickerson, Jeffrey A.; Green, Karin M.; Shaffer, Scott A.; Imbalzano, Anthony N.; Martin-Buley, Lori A.; Lian, Jane B.; Stein, Janet L.; van Wijnen, Andre J.; Stein, Gary S.

    2015-01-01

    ABSTRACT Cancer cells exhibit modifications in nuclear architecture and transcriptional control. Tumor growth and metastasis are supported by RUNX family transcriptional scaffolding proteins, which mediate the assembly of nuclear-matrix-associated gene-regulatory hubs. We used proteomic analysis to identify RUNX2-dependent protein–protein interactions associated with the nuclear matrix in bone, breast and prostate tumor cell types and found that RUNX2 interacts with three distinct proteins that respond to DNA damage – RUVBL2, INTS3 and BAZ1B. Subnuclear foci containing these proteins change in intensity or number following UV irradiation. Furthermore, RUNX2, INTS3 and BAZ1B form UV-responsive complexes with the serine-139-phosphorylated isoform of H2AXH2AX). UV irradiation increases the interaction of BAZ1B with γH2AX and decreases histone H3 lysine 9 acetylation levels, which mark accessible chromatin. RUNX2 depletion prevents the BAZ1B–γH2AX interaction and attenuates loss of H3K9 and H3K56 acetylation. Our data are consistent with a model in which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in cancer cells might clarify its expression in chemotherapy-resistant and/or metastatic tumors. PMID:25609707

  11. MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.

    PubMed

    Ichijima, Yosuke; Ichijima, Misako; Lou, Zhenkun; Nussenzweig, André; Camerini-Otero, R Daniel; Chen, Junjie; Andreassen, Paul R; Namekawa, Satoshi H

    2011-05-01

    Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AXH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and γH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the γH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.

  12. Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

    PubMed

    Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

    2017-02-15

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1-10. ©2017 AACR.

  13. The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair.

    PubMed

    Kari, Vijayalakshmi; Shchebet, Andrei; Neumann, Heinz; Johnsen, Steven A

    2011-10-15

    Many anticancer therapies function largely by inducing DNA double-strand breaks (DSBs) or altering the ability of cancer cells to repair them. Proper and timely DNA repair requires dynamic changes in chromatin assembly and disassembly characterized by histone H3 lysine 56 acetylation (H3K56ac) and phosphorylation of the variant histone H2AXH2AX). Similarly, histone H2B monoubiquitination (H2Bub1) functions in DNA repair, but its role in controlling dynamic changes in chromatin structure following DSBs and the histone chaperone complexes involved remain unknown. Therefore, we investigated the role of the H2B ubiquitin ligase RNF40 in the DSB response. We show that RNF40 depletion results in sustained H2AX phosphorylation and a decrease in rapid cell cycle checkpoint activation. Furthermore, RNF40 knockdown resulted in decreased H3K56ac and decreased recruitment of the facilitates chromatin transcription (FACT) complex to chromatin following DSB. Knockdown of the FACT component suppressor of Ty homolog-16 (SUPT16H) phenocopied the effects of RNF40 knockdown on both γH2AX and H3K56ac following DSB induction. Consistently, both RNF40 and SUPT16H were required for proper DNA end resection and timely DNA repair, suggesting that H2Bub1 and FACT cooperate to increase chromatin dynamics, which facilitates proper checkpoint activation and timely DNA repair. These results provide important mechanistic insights into the tumor suppressor function of H2Bub1 and provide a rational basis for pursuing H2Bub1-based therapies in conjunction with traditional chemo- and radiotherapy.

  14. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  15. Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin

    PubMed Central

    Raghuram, Nikhil; Strickfaden, Hilmar; McDonald, Darin; Williams, Kylie; Fang, He; Mizzen, Craig; Hayes, Jeffrey J.; Th’ng, John

    2013-01-01

    Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin. PMID:24100296

  16. Telomeres, histone code, and DNA damage response.

    PubMed

    Misri, S; Pandita, S; Kumar, R; Pandita, T K

    2008-01-01

    Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability.

  17. The Histone Database: an integrated resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Levine, Kevin M; Morales, Mario; Zhang, Suiyuan; Moreland, R Travis; Baxevanis, Andreas D; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

  18. Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells.

    PubMed

    Muthna, D; Vavrova, J; Lukasova, E; Tichy, A; Knizek, J; Kohlerova, R; Mazankova, N; Rezacova, M

    2012-01-01

    The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.

  19. Histone acetylation in neurodevelopment.

    PubMed

    Contestabile, Antonio; Sintoni, Silvia

    2013-01-01

    Post-translational modification of histones is a primary mechanism through which epigenetic regulation of DNA transcription does occur. Among these modifications, regulation of histone acetylation state is an important tool to influence gene expression. Epigenetic regulation of neurodevelopment contributes to the structural and functional shaping of the brain during neurogenesis and continues to impact on neural plasticity lifelong. Alterations of these mechanisms during neurodevelopment may result in later occurrence of neuropsychatric disorders. The present paper reviews and discusses available data on histone modifications, in particular histone acetylation, in neurogenesis considering results obtained in culture systems of neural progenitors as well as in in vivo studies. Possible teratogenic effects of altered histone acetylation state during development are also considered. The use during pregnancy of drugs such as valproic acid, which acts as a histone deacetylase inhibitor, may result during postnatal development in autistic-like symptoms. The effect of gestational administration of the drug has been, therefore, tested on adult hippocampal neurogenesis in animals showing behavioral impairment as a consequence of the drug administration at a specific stage of pregnancy. These experimental results show that adult neurogenesis in the hippocampal dentate gyrus is not quantitatively altered by gestational valproic acid administration. Future steps and goals of research on the role and mechanisms of histone acetylation in neurodevelopment are briefly discussed.

  20. Brownian dynamics simulation of the effect of histone modification on nucleosome structure

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2007-05-01

    Using Brownian dynamics we simulate the effect of histone modification, such as phosphorylation, acetylation, and methylation, on nucleosome structure by varying the interaction force between DNA and the histone octamer. The simulation shows that the structural stability of nucleosome is very sensitive to the interaction force, and the DNA unwrapping from the modified histone octamer usually occurs turn by turn. Furthermore, the effects of temperature and DNA break as well as the competition between modified and normal histone octamers are investigated, with the simulation results being in agreement with the experimental observation that phosphorylated nucleosomes near DNA breaks are more easily depleted. Though the simulation study may only give a coarse grained view of the DNA unwrapping process for the modified histone octamer, it may provide insight into the mechanism of DNA repair.

  1. K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase.

    PubMed

    Kobza, Keyna; Camporeale, Gabriela; Rueckert, Brian; Kueh, Alice; Griffin, Jacob B; Sarath, Gautam; Zempleni, Janos

    2005-08-01

    Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

  2. Characterization of antimicrobial histone sequences and posttranslational modifications by mass spectrometry.

    PubMed

    Ouvry-Patat, Séverine A; Schey, Kevin L

    2007-05-01

    Histones typically play a role in DNA packaging and transcription regulation. These proteins are heavily modified by acetylation, methylation, phosphorylation and/or ubiquitination, and various combinations of these modifications alter histone functions and form the basis of the histone code. Furthermore, histones, including those found in shrimp, have recently been found to possess antimicrobial properties; however, the sequences and posttranslational modifications of shrimp histones are largely unknown. In this study mass spectrometry was used to characterize the primary structure of the shrimp antimicrobial histone. A combination of in-solution digestion and in-gel propionylation/digestion followed by LC-MS-MS and MALDI-TOF-TOF analysis was used. Over 80% of each histone sequence was obtained by in-solution digestion; however, none of the N-terminal domains was sequenced with this method. An in-gel propionylation method was optimized to recover and sequence the extremely hydrophilic histone N-termini. This method was then applied to shrimp hemocyte lysates separated on a 1-D SDS-PAGE gel. Overall, 95% coverage was obtained for the histone sequences as well as the identification of posttranslational sites such as acetylation, methylation and phosphorylation.

  3. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-03

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

  4. Histone chaperones FACT and Spt6 prevent histone variants from turning into histone deviants.

    PubMed

    Jeronimo, Célia; Robert, François

    2016-05-01

    Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones.

  5. Modification of histones by sugar β-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

    PubMed

    Zhang, Suisheng; Roche, Kevin; Nasheuer, Heinz-Peter; Lowndes, Noel Francis

    2011-10-28

    The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

  6. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.

    PubMed

    Mitchell, Jonathan K; Friesen, Paul D

    2012-12-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.

  7. Induction of DNA Damage Response by the Supravital Probes of Nucleic Acids

    PubMed Central

    Zhao, Hong; Traganos, Frank; Dobrucki, Jurek; Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2009-01-01

    The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV) and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981) and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2 or p53 phosphorylation. However, pronounced ATM, Chk2 and p53 phosphorylation and perturbed G2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2 and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2 and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G2M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5 and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. PMID:19373929

  8. Synthesis, Biological Evaluation, and Structure–Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks

    PubMed Central

    2012-01-01

    Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AXH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents. PMID:22694057

  9. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways.

    PubMed

    Ichijima, Yosuke; Sin, Ho-Su; Namekawa, Satoshi H

    2012-08-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AXH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.

  10. Adamantanyl-Histone Deacetylase Inhibitor H6CAHA Exhibits Favorable Pharmacokinetics and Augments Prostate Cancer Radiation Sensitivity

    SciTech Connect

    Konsoula, Zacharoula; Cao Hong; Velena, Alfredo; Jung, Mira

    2011-04-01

    Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to {gamma}-radiation in vitro and in vivo. Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. Results: H6CAHA demonstrated good solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 {+-} 0.91 {mu}M x h, and its half-life was 11.17 {+-} 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of {gamma}H2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with {gamma}-radiation (10 x 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.

  11. Trichostatin-A induces differential changes in histone protein dynamics and expression in HeLa cells

    SciTech Connect

    Rao, Jyothsna; Bhattacharya, Dipanjan; Banerjee, Bidisha; Sarin, Apurva; Shivashankar, G.V.

    2007-11-16

    Trichostatin-A (TSA), a histone deacetylase (HDAC) inhibitor, results in enhanced acetylation of core histones thereby disrupting chromatin organization within living cells. We report on changes in chromatin organization and the resultant alteration in nuclear architecture following treatment with TSA using fluorescence imaging. TSA triggers an expected increase in the euchromatin fraction which is accompanied by a significant increase in nuclear volume and alterations in chromatin compaction mapped using fluorescence anisotropy imaging. We observe differential changes in the mobility of core and linker histones as measured by fluorescence recovery after photo-bleaching (FRAP) and fluorescence correlation spectroscopy (FCS) methods. Further TSA induces a differential increase in linker histone transcription and increased phosphorylation of linker histone proteins accompanying an expected increase in core histone acetylation patterns. Thus subtle feedback responses triggered by changes in chromatin configurations impinge selectively on linker histone mobility and its expression. These observations have implications for understanding the role of HDAC in the dynamic maintenance of chromatin organization.

  12. A Phosphotyrosine Switch Controls the Association of Histone Mark Readers with Methylated Proteins.

    PubMed

    Irving-Hooper, Bronwyn Kate; Binda, Olivier

    2016-03-22

    Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information.

  13. The human actin-related protein hArp5: nucleo-cytoplasmic shuttling and involvement in DNA repair.

    PubMed

    Kitayama, Kumiko; Kamo, Mariko; Oma, Yukako; Matsuda, Ryo; Uchida, Takafumi; Ikura, Tsuyoshi; Tashiro, Satoshi; Ohyama, Takashi; Winsor, Barbara; Harata, Masahiko

    2009-01-15

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

  14. Rapid purification of recombinant histones.

    PubMed

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  15. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients

    PubMed Central

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing. PMID:26237679

  16. Assaying Pharmacodynamic Endpoints with Targeted Therapy: Flavopiridol and 17AAG Induced Dephosphorylation of Histone H1.5 in Acute Myeloid Leukemia

    PubMed Central

    Wang, Liwen; Harshman, Sean W.; Liu, Shujun; Ren, Chen; Xu, Hua; Sallans, Larry; Grever, Michael; Byrd, John C.; Marcucci, Guido; Freitas, Michael A.

    2011-01-01

    Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this report we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 Acute Myeloid Leukemia (AML) cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. Liquid chromatography mass spectrometry profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin dependent kinase inhibitor, flavopiridol, and the Hsp90 inhibitor, 17AAG (17-(Allylamino)-17-demethoxygeldanamycin). In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation. PMID:21110323

  17. Assaying pharmacodynamic endpoints with targeted therapy: flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia.

    PubMed

    Wang, Liwen; Harshman, Sean W; Liu, Shujun; Ren, Chen; Xu, Hua; Sallans, Larry; Grever, Michael; Byrd, John C; Marcucci, Guido; Freitas, Michael A

    2010-12-01

    Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin-dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

  18. Phase I Study of Single-Agent AZD1775 (MK-1775), a Wee1 Kinase Inhibitor, in Patients With Refractory Solid Tumors

    PubMed Central

    Do, Khanh; Wilsker, Deborah; Ji, Jiuping; Zlott, Jennifer; Freshwater, Tomoko; Kinders, Robert J.; Collins, Jerry; Chen, Alice P.; Doroshow, James H.; Kummar, Shivaani

    2015-01-01

    Purpose Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AXH2AX) levels in paired tumor biopsies. Patients and Methods AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. Results Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Confirmed partial responses were observed in two patients carrying BRCA mutations: one with head and neck cancer and one with ovarian cancer. Common toxicities were myelosuppression and diarrhea. Dose-limiting toxicities were supraventricular tachyarrhythmia and myelosuppression. Accumulation of drug (t1/2 approximately 11 hours) was observed. Reduction in pY15-Cdk levels (two of five paired biopsies) and increases in γH2AX levels (three of five paired biopsies) were demonstrated. Conclusion This is the first report of AZD1775 single-agent activity in patients carrying BRCA mutations. Proof-of-mechanism was demonstrated by target modulation and DNA damage response in paired tumor biopsies. PMID:25964244

  19. Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

    PubMed

    Zhao, Hong; Li, Jiangwei; Traganos, Frank; Halicka, H Dorota; Zarebski, Mirosław; Dobrucki, Jurek; Darzynkiewicz, Zbigniew

    2011-06-01

    By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

  20. Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

    PubMed

    Pietrzak, Monika; Halicka, H Dorota; Wieczorek, Zbigniew; Wieczorek, Jolanta; Darzynkiewicz, Zbigniew

    2008-06-01

    We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

  1. Attenuation of acridine mutagen ICR-191 — DNA interactions and DNA damage by the mutagen interceptor chlorophyllin

    PubMed Central

    Pietrzak, Monika; Halicka, H. Dorota; Wieczorek, Zbigniew; Wieczorek, Jolanta; Darzynkiewicz, Zbigniew

    2009-01-01

    We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR–DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AXH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL. PMID:18423964

  2. A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

    PubMed

    Bouley, Julien; Saad, Lina; Grall, Romain; Schellenbauer, Amelie; Biard, Denis; Paget, Vincent; Morel-Altmeyer, Sandrine; Guipaud, Olivier; Chambon, Christophe; Salles, Bernard; Maloum, Karim; Merle-Béral, Hélène; Chevillard, Sylvie; Delic, Jozo

    2015-09-29

    Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

  3. A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    PubMed Central

    Schellenbauer, Amelie; Biard, Denis; Paget, Vincent; Morel-Altmeyer, Sandrine; Guipaud, Olivier; Chambon, Christophe; Salles, Bernard; Maloum, Karim; Merle-Béral, Hélène; Chevillard, Sylvie; Delic, Jozo

    2015-01-01

    Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells. PMID:26337656

  4. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.

    PubMed

    Rodriguez-Collazo, Pedro; Leuba, Sanford H; Zlatanova, Jordanka

    2009-06-01

    Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.

  5. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  6. The Role of Histone Protein Modifications and Mutations in Histone Modifiers in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia

    PubMed Central

    Janczar, Szymon; Janczar, Karolina; Pastorczak, Agata; Harb, Hani; Paige, Adam J. W.; Zalewska-Szewczyk, Beata; Danilewicz, Marian; Mlynarski, Wojciech

    2017-01-01

    While cancer has been long recognized as a disease of the genome, the importance of epigenetic mechanisms in neoplasia was acknowledged more recently. The most active epigenetic marks are DNA methylation and histone protein modifications and they are involved in basic biological phenomena in every cell. Their role in tumorigenesis is stressed by recent unbiased large-scale studies providing evidence that several epigenetic modifiers are recurrently mutated or frequently dysregulated in multiple cancers. The interest in epigenetic marks is especially due to the fact that they are potentially reversible and thus druggable. In B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) there is a relative paucity of reports on the role of histone protein modifications (acetylation, methylation, phosphorylation) as compared to acute myeloid leukemia, T-cell ALL, or other hematologic cancers, and in this setting chromatin modifications are relatively less well studied and reviewed than DNA methylation. In this paper, we discuss the biomarker associations and evidence for a driver role of dysregulated global and loci-specific histone marks, as well as mutations in epigenetic modifiers in BCP-ALL. Examples of chromatin modifiers recurrently mutated/disrupted in BCP-ALL and associated with disease outcomes include MLL1, CREBBP, NSD2, and SETD2. Altered histone marks and histone modifiers and readers may play a particular role in disease chemoresistance and relapse. We also suggest that epigenetic regulation of B-cell differentiation may have parallel roles in leukemogenesis. PMID:28054944

  7. Histone acetylation in insect chromosomes.

    PubMed

    Allfrey, V G; Pogo, B G; Littau, V C; Gershey, E L; Mirsky, A E

    1968-01-19

    Acetylation of histones takes place along the salivary gland chromosomes of Chironomus thummi when RNA synthesis is active. It can be observed but not measured quantitatively by autoradiography of chromosome squashes. The "fixatives" commonly used in preparing squashes of insect chromosomes preferentially extract the highly acetylated "arginine-rich" histone fractions; the use of such fixatives may explain the reported absence of histone acetylation in Drosophila melanogaster.

  8. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    SciTech Connect

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  9. Gamma irradiation induces DNA double-strand breaks in fibroblasts: a model study for the development of biodosimetry

    NASA Astrophysics Data System (ADS)

    Uttayarat, P.; Tangtong, T.; Sukapirom, K.; Boonsirichai, K.

    2015-05-01

    Double-strand breaks (DSBs) of DNAs induced by ionizing radiation can pose detrimental damages on organisms which include genetic instability and cell death. It is necessary to be able to assess health risks associated with irradiation from both accidental and therapeutic exposures in a timely manner for proper medical treatments. This present study showed the first attempt to develop a biodosimetric measure in Thailand based on the quantification of phosphorylated histone H2AX (γ-H2AX) formed at DSB sites with an aim to establish a dose response curve using a two-dimensional (2D) cell culture model. Human dermal fibroblasts were grown into monolayers before irradiated by gamma rays from a Co-60 source in a custom-made lead chamber at doses 0, 0.2, 1, 2 and 4 Gy and a dose rate of 0.21 Gy/min. After 30 min post exposure, γ-H2AX proteins were immunofluorescently labelled for evaluation by confocal microscopy and flow cytometry. The accumulation of phosphorylated γ-H2AX proteins at DSBs appeared as nuclear foci with the most prominent intensity at 4 Gy. Linear regression analysis of flow cytometric data showed a linear response (R2 = 0.9862) of foci intensity in proportion to irradiation dose. In addition, the fraction of cell viability was shown to decrease at higher doses. This technique can be further developed as a quick assessment tool to identify individuals subjected to accidental radiation in parallel to other established biodosimetric measures.

  10. Assessment of Cr(VI)-Induced Cytotoxicity and Genotoxicity Using High Content Analysis

    PubMed Central

    Thompson, Chad M.; Fedorov, Yuriy; Brown, Daniel D.; Suh, Mina; Proctor, Deborah M.; Kuriakose, Liz; Haws, Laurie C.; Harris, Mark A.

    2012-01-01

    Oral exposure to high concentrations of hexavalent chromium [Cr(VI)] induces intestinal redox changes, villus cytotoxicity, crypt hyperplasia, and intestinal tumors in mice. To assess the effects of Cr(VI) in a cell model relevant to the intestine, undifferentiated (proliferating) and differentiated (confluent) Caco-2 cells were treated with Cr(VI), hydrogen peroxide or rotenone for 2–24 hours. DNA damage was then assessed by nuclear staining intensity of 8-hydroxydeoxyguanosine (8-OHdG) and phosphorylated histone variant H2AX (γ-H2AX) measured by high content analysis methods. In undifferentiated Caco-2, all three chemicals increased 8-OHdG and γ-H2AX staining at cytotoxic concentrations, whereas only 8-OHdG was elevated at non-cytotoxic concentrations at 24 hr. Differentiated Caco-2 were more resistant to cytotoxicity and DNA damage than undifferentiated cells, and there were no changes in apoptotic markers p53 or annexin-V. However, Cr(VI) induced a dose-dependent translocation of the unfolded protein response transcription factor ATF6 into the nucleus. Micronucleus (MN) formation was assessed in CHO-K1 and A549 cell lines. Cr(VI) increased MN frequency in CHO-K1 only at highly cytotoxic concentrations. Relative to the positive control Mitomycin-C, Cr(VI) only slightly increased MN frequency in A549 at mildly cytotoxic concentrations. The results demonstrate that Cr(VI) genotoxicity correlates with cytotoxic concentrations, and that H2AX phosphorylation occurs at higher concentrations than oxidative DNA damage in proliferating Caco-2 cells. The findings suggest that in vitro genotoxicity of Cr(VI) is primarily oxidative in nature at low concentrations. Implications for in vivo intestinal toxicity of Cr(VI) will be discussed. PMID:22905163

  11. Histone deacetylases and atherosclerosis.

    PubMed

    Zheng, Xia-xia; Zhou, Tian; Wang, Xin-An; Tong, Xiao-hong; Ding, Jia-wang

    2015-06-01

    Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC inhibitors(HDACi) on the progress of atherosclerosis.

  12. LINE-1 retrotransposition events regulate gene expression after X-ray irradiation.

    PubMed

    Banaz-Yaşar, Ferya; Gedik, Nilgün; Karahan, Selda; Diaz-Carballo, David; Bongartz, Birthe M; Ergün, Süleyman

    2012-09-01

    Long interspersed nuclear element-1 (LINE-1) retrotransposons are mobile elements that insert into new genomic locations via reverse transcription of an RNA intermediate. The mechanism of retrotransposition is not entirely understood. The integration of these elements occurs by target-primed reverse transcription (TPRT), which initiates double-strand breaks (DSBs) during the LINE-1 integration. Also, X-ray is known to induce DNA damage. The aim of this study was to evaluate the potential effects of LINE-1 de novo retrotransposition on the expression of different genes after X-ray irradiation in human endothelial cells. After stable transfection of the human hybrid endothelial cell line EA.hy926 with the human LINE-1 element, we analyzed the expression of different genes after irradiation with 5 Gy X-rays by reverse transcription-polymerase chain reaction (RT-PCR). We determine the expression level of phosphorylated p53 and γ-histone H2AX protein levels upon X-ray irradiation with 5 Gy for 24 h. Our results showed that EA.hy926 LINE-1 cell clones react with a strong upregulation of phosphorylated p53 protein, already 15 min after irradiation compared to the wild type (WT) cells. Also, the expression of γ-histone H2AX protein was elevated in the cell clones with retrotransposition events 15 min after irradiation, whereas the WT cells have a delayed expression of phosphorylated histone H2AX protein. Taken together, our findings provide that LINE-1 retrotransposition events regulate different gene expression after irradiation in the EA.hy926 cell line.

  13. Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

    PubMed

    Zunder, Rachel M; Rine, Jasper

    2012-11-01

    In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

  14. The C terminus of the histone chaperone Asf1 cross-links to histone H3 in yeast and promotes interaction with histones H3 and H4.

    PubMed

    Dennehey, Briana K; Noone, Seth; Liu, Wallace H; Smith, Luke; Churchill, Mair E A; Tyler, Jessica K

    2013-02-01

    The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

  15. Insulin induced alteration in post-translational modifications of histone H3 under a hyperglycemic condition in L6 skeletal muscle myoblasts.

    PubMed

    Kabra, Dhiraj G; Gupta, Jeena; Tikoo, Kulbhushan

    2009-06-01

    Chromatin remodelling events, especially histone modifications are proposed to form the mainstay for most of the biological processes. However, the role of these histone modifications in the progression of diabetes is still unknown. Hyperglycemia plays a major role in diabetes and its complications. The present study was undertaken to check the effect of insulin on alterations in post-translational modifications of histone H3 in L6 myoblasts under a hyperglycemic condition. We provide first evidence that insulin under hyperglycemic condition alters multiple histone modifications by enhanced production of reactive oxygen species. Insulin induces dose dependent changes in Lysine 4 and 9 methylation, Ser 10 phosphorylation and acetylation of histone H3. Interestingly, insulin induced generation of reactive oxygen species induces dephosphorylation and deacetylation of histone H3. Preincubation with catalase and DPI prevents these changes in post-translational modifications of histone H3. Furthermore, changes in histone H3 phosphorylation was found to be independent of ERK, p38, RSK2 and MSK1. Moreover, serine/threonine phosphatase inhibitor, okadaic acid attenuates insulin induced dephosphorylation and deacetylation of histone H3, suggesting a role of serine/threonine phosphatases in altering modifications of histone H3. These changes in epigenetic modifications can provide new insights into pathogenesis of diabetes.

  16. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  17. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

    PubMed Central

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A.; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  18. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    PubMed

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

  19. Generation of DNA single-strand displacement by compromised nucleotide excision repair

    PubMed Central

    Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

    2012-01-01

    Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylationH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

  20. Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

    PubMed Central

    Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

    2011-01-01

    Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AXH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

  1. Intermediate frequency magnetic field generated by a wireless power transmission device does not cause genotoxicity in vitro.

    PubMed

    Shi, Dejing; Zhu, Chunbo; Lu, Rengui; Mao, Shitong; Qi, Yanhua

    2014-10-01

    The aim of this study was to evaluate effects of intermediate frequency magnetic fields (IFMF) generated by a wireless power transmission (WPT) based on magnetic resonance from the perspective of cellular genotoxicity on cultured human lens epithelial cells (HLECs). We evaluated the effects of exposure to 90 kHz magnetic fields at 93.36 µT on cellular genotoxicity in vitro for 2 and 4 h. The magnetic flux density is approximately 3.5 times higher than the reference level recommended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cellular proliferation, apoptosis and DNA damage by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, alkaline comet assay and phosphorylated histone H2AXH2AX) foci formation test. We did not detect any effect of a 90 kHz IFMF generated by WPT based on magnetic resonance on cell proliferation, apoptosis, comet assay, and γH2AX foci formation test. Our results indicated that exposure to 90 kHz IFMF generated by WPT based on magnetic resonance at 93.36 µT for 2 and 4 h does not cause detectable cellular genotoxicity.

  2. The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers.

    PubMed

    Cabrero, Josefa; Palomino-Morales, Rogelio J; Camacho, Juan Pedro M

    2007-01-01

    Fluorescence immunostaining for the phosphorylated H2AX histone (gammaH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of gammaH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.

  3. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

    PubMed Central

    Seto, Edward; Yoshida, Minoru

    2014-01-01

    Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

  4. A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor

    PubMed Central

    Sawatsubashi, Shun; Murata, Takuya; Lim, Jinseon; Fujiki, Ryoji; Ito, Saya; Suzuki, Eriko; Tanabe, Masahiko; Zhao, Yue; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Umetsu, Daiki; Ito, Takashi; Takeyama, Ken-ichi; Kato, Shigeaki

    2010-01-01

    Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors. PMID:20040570

  5. Structure and mechanism of non-histone protein acetyltransferase enzymes

    PubMed Central

    Friedmann, David R.

    2014-01-01

    Post translational modification (PTM) of proteins is ubiquitous and mediates many cellular processes including intracellular localization, protein-protein interactions, enzyme activity, transcriptional regulation and protein stability. While the role of phosphorylation as a key PTM has been well studied, the more evolutionarily conserved acetylation PTM has only recently attracted attention as a key regulator of cellular events. Protein acetylation has been largely studied in the context of its role in histone modification and gene regulation, where histones are modified by histone acetyltransferases (HATs) to promote transcription. However, more recent acetylomic and biochemical studies have revealed that acetylation is mediated by a broader family of protein acetyltransferases (PATs). The recent structure determination of several PATs has provided a wealth of molecular information regarding structural features of PATs, their enzymatic mechanisms, their mode of substrate-specific recognition and their regulatory elements. In this minireview, we will briefly describe what is known about non-histone protein substrates, but mainly focus on a few recent structures of PATs to compare and contrast them with HATs to better understand the molecular basis for protein recognition and modification by this burgeoning family of protein modification enzymes. PMID:23742047

  6. Histone deacetylases: salesmen and customers in the post-translational modification market.

    PubMed

    Brandl, André; Heinzel, Thorsten; Krämer, Oliver H

    2009-04-01

    HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N-epsilon-acetylated lysine residues in histones and non-histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post-translational modification. Such structural alterations can determine the stability, localization, activity and protein-protein interactions of HDACs. This subsequently affects the modification of their substrates and the co-ordination of cellular signalling networks. Intriguingly, physiologically relevant non-histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post-translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post-translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.

  7. Histone deacetylases: unique players in shaping the epigenetic histone code.

    PubMed

    Thiagalingam, Sam; Cheng, Kuang-Hung; Lee, Hyunjoo J; Mineva, Nora; Thiagalingam, Arunthathi; Ponte, Jose F

    2003-03-01

    The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones. This histone code determines the expression status of individual genes dependent upon their localization on the chromatin. The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3. In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3. The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin. Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD-dependent enzymes that are similar to the yeast SIR2 proteins. Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer. Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy. Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized. Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made. With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

  8. Histone chaperone networks shaping chromatin function.

    PubMed

    Hammond, Colin M; Strømme, Caroline B; Huang, Hongda; Patel, Dinshaw J; Groth, Anja

    2017-03-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

  9. The Role of Dietary Histone Deacetylases (HDACs) Inhibitors in Health and Disease

    PubMed Central

    Bassett, Shalome A.; Barnett, Matthew P. G.

    2014-01-01

    Modification of the histone proteins associated with DNA is an important process in the epigenetic regulation of DNA structure and function. There are several known modifications to histones, including methylation, acetylation, and phosphorylation, and a range of factors influence each of these. Histone deacetylases (HDACs) remove the acetyl group from lysine residues within a range of proteins, including transcription factors and histones. Whilst this means that their influence on cellular processes is more complex and far-reaching than histone modifications alone, their predominant function appears to relate to histones; through deacetylation of lysine residues they can influence expression of genes encoded by DNA linked to the histone molecule. HDAC inhibitors in turn regulate the activity of HDACs, and have been widely used as therapeutics in psychiatry and neurology, in which a number of adverse outcomes are associated with aberrant HDAC function. More recently, dietary HDAC inhibitors have been shown to have a regulatory effect similar to that of pharmacological HDAC inhibitors without the possible side-effects. Here, we discuss a number of dietary HDAC inhibitors, and how they may have therapeutic potential in the context of a whole food. PMID:25322459

  10. Enhanced top-down characterization of histone post-translational modifications

    SciTech Connect

    Tian, Zhixin; Tolić, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Paša-Tolić, Ljiljana

    2012-01-01

    Background: Multiple post-translational modifications (PTMs) on core histones often work synergistically to fine tune chromatin structure and functions, generating a “histone code” that can be interpreted by a variety of chromatin interacting proteins. Although previous bottom-up and middle-down proteomic approaches have been developed for limited characterization of PTMs on histone N-terminal tails, high-throughput methods for comprehensive identification of PTMs distributed along the entire primary amino acid sequence are yet to be implemented. Results: Here we report a novel online two-dimensional liquid chromatography - tandem mass spectrometry (2D LC–MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The metal-free LC system with reverse phase separation followed by weak cation exchange – hydrophilic interaction chromatography (WCX-HILIC) and online Orbitrap Velos tandem mass spectrometry allowed for unambiguous identification of over 700 histone isoforms from a single 2D LC–MS/MS analysis of 7.5 µg of purified core histones. In comparison with previous offline top-down analysis of H4, this online study identified 100 additional isoforms from 100-fold less sample. This platform enabled comprehensive characterization of histone modifications, including those beyond tail regions, with dramatically improved throughput and sensitivity compared to more traditional platforms. Isoforms identified included those with combinatorial PTMs extending well beyond the N-terminal tail regions as well as a large number of phosphorylated isoforms.

  11. Transcriptional alterations of ET-1 axis and DNA damage in lung tissue of a rat obesity model.

    PubMed

    Del Ry, Silvia; Cabiati, Manuela; Salvadori, Costanza; Guiducci, Letizia; Caselli, Chiara; Prescimone, Tommaso; Facioni, Maria Sole; Azzarà, Alessia; Chiaramonte, Anna; Mazzoni, Stefano; Bruschi, Fabrizio; Giannessi, Daniela; Scarpato, Roberto

    2015-03-01

    Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.

  12. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    PubMed Central

    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F.

    2014-01-01

    7,12-dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1mg/kg; intraperitoneal injection) at 18 wks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in chinese hamster cells 6 (Xrcc6), Breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), Poly [ADP-ribose] polymerase 1 (Parp1) and Protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AXH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. PMID:25448685

  13. Spatio-temporal radiation biology with conventionally or laser-accelerated particles for ELIMED

    NASA Astrophysics Data System (ADS)

    Ristić-Fira, A.; Bulat, T.; Keta, O.; Romano, F.; Cirrone, P.; Cuttone, G.; Petrović, I.

    2013-07-01

    The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumor cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (γ-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (γ-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of γ-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between γ-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the data base that might promote pulsed sources for medical treatments of malignant growths.

  14. Spatio-temporal radiation biology with conventionally or laser-accelerated particles for ELIMED

    SciTech Connect

    Ristić-Fira, A.; Bulat, T.; Keta, O.; Petrović, I.; Romano, F.; Cirrone, P.; Cuttone, G.

    2013-07-26

    The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumor cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (γ-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (γ-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of γ-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between γ-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the data base that might promote pulsed sources for medical treatments of malignant growths.

  15. Extremely Low Frequency Magnetic Fields Do Not Induce DNA Damage in Human Lens Epithelial Cells In Vitro.

    PubMed

    Zhu, Kan; Lv, Ye; Cheng, Qian; Hua, Jianing; Zeng, Qunli

    2016-05-01

    Non-ionizing radiations, e.g., radiofrequency electromagnetic fields, could induce DNA damage and oxidative stress in human lens epithelial cells (LECs) which can be early events in cataractogenesis. Extremely low frequency magnetic fields (ELF MF) as another common form of man-made electromagnetic fields has been considered as suspected human carcinogen by International Agency for Research on Cancer (IARC) and become a focus that people play more and more attentions to. This study aimed to determine whether ELF MF can induce DNA damage in cultured human LECs at a relatively low intensity. Human LECs were exposed or sham-exposed to a 50 Hz ELF MF which produced by a well-designed exposure system at the intensity of 0.4 mT. DNA damage in human LECs was examined by the phosphorylated form of histone variant H2AXH2AX) foci formation assay and further explored with western blot, flow cytometry, and alkaline comet assay. Immunofluorescence analysis showed that 0.4 mT ELF MF did not significantly increase γH2AX foci formation in human LECs after 2, 6, 12, 24, or 48 hr exposure. No significant differences had been detected in γH2AX expression level between the ELF MF- and sham-exposure groups, while no obvious chromosomal DNA fragmentation was detected by alkaline comet assay after ELF MF exposure. The results indicate an absence of genotoxicity in ELF MF-exposed human epithelial cells and do not support the hypothesis that environmental ELF MF might be causally led to genomic instability via chromosomal damage response processes. Neither short nor long term continuous exposure to 50 Hz ELF MF at 0.4 mT could induce DNA damage in human lens epithelial cells in vitro.

  16. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    SciTech Connect

    Ganesan, Shanthi Nteeba, Jackson Keating, Aileen F.

    2014-12-01

    7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AXH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity.

  17. Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

    PubMed Central

    Liu, Juan; Zhou, Xiang; Li, Qing; Zhou, Shu-Min; Hu, Bin; Hu, Guo-Wen; Niu, Xin; Guo, Shang-Chun

    2017-01-01

    Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke. PMID:28127553

  18. Butyrate Histone Deacetylase Inhibitors

    PubMed Central

    Boosalis, Michael S.; Perrine, Susan P.; Sangerman, José

    2012-01-01

    Abstract In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition. PMID:23514803

  19. Exercise-induced histone modifications in human skeletal muscle.

    PubMed

    McGee, Sean L; Fairlie, Erin; Garnham, Andrew P; Hargreaves, Mark

    2009-12-15

    Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP-activated protein kinase (AMPK) and the calcium-calmodulin-dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation-dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

  20. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  1. Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation.

    PubMed

    Mikami, Suzuka; Kanaba, Teppei; Takizawa, Naoki; Kobayashi, Ayaho; Maesaki, Ryoko; Fujiwara, Toshinobu; Ito, Yutaka; Mishima, Masaki

    2014-01-07

    The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.

  2. Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression.

    PubMed

    Borgal, Lori; Rinschen, Markus M; Dafinger, Claudia; Liebrecht, Valérie I; Abken, Hinrich; Benzing, Thomas; Schermer, Bernhard

    2016-01-01

    The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair.

  3. Histone code or not? Combinatorial pattern analyses of histone modifications

    NASA Astrophysics Data System (ADS)

    Zang, Chongzhi; Peng, Weiqun; Wang, Zhibin; Schones, Dustin E.; Barski, Artem; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Zhao, Keji; Rosenfeld, Jeffrey; Zhang, Michael

    2008-03-01

    Eukaryotic genomes are organized into chromatin, the structure of which plays critical role in the program of gene expression. Chromatin structure and function is regulated by a myriad of posttranslational modifications on histone tails of the nucleosomes, the fundamental unit of chromatin. It remains unclear how different modifications interact. Based on high- resolution genomic maps of close to 40 histone methylations and acetylations in human T-cells obtained experimentally by ChIP- Seq technique, we investigated the combinatorial patterns of histone modifications at gene promoter regions. We found that a very limited number of patterns dominate. Modifications within a pattern are strongly correlated and each pattern is associated with a distinct gene expression distribution. Our results suggest that it is the patterns rather than the individual modifications that affect the downstream readout.

  4. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes

    PubMed Central

    Govind, Chhabi K.; Qiu, Hongfang; Ginsburg, Daniel S.; Ruan, Chun; Hofmeyer, Kimberly; Hu, Cuihua; Swaminathan, Venkatesh; Workman, Jerry L.; Li, Bing; Hinnebusch, Alan G.

    2010-01-01

    Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are co-transcriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing co-transcriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is a key determinant of nucleosome eviction. PMID:20670892

  5. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  6. HHMD: the human histone modification database.

    PubMed

    Zhang, Yan; Lv, Jie; Liu, Hongbo; Zhu, Jiang; Su, Jianzhong; Wu, Qiong; Qi, Yunfeng; Wang, Fang; Li, Xia

    2010-01-01

    Histone modifications play important roles in chromatin remodeling, gene transcriptional regulation, stem cell maintenance and differentiation. Alterations in histone modifications may be linked to human diseases especially cancer. Histone modifications including methylation, acetylation and ubiquitylation probed by ChIP-seq, ChIP-chip and qChIP have become widely available. Mining and integration of histone modification data can be beneficial to novel biological discoveries. There has been no comprehensive data repository that is exclusive for human histone modifications. Therefore, we developed a relatively comprehensive database for human histone modifications. Human Histone Modification Database (HHMD, http://bioinfo.hrbmu.edu.cn/hhmd) focuses on the storage and integration of histone modification datasets that were obtained from laboratory experiments. The latest release of HHMD incorporates 43 location-specific histone modifications in human. To facilitate data extraction, flexible search options are built in HHMD. It can be searched by histone modification, gene ID, functional categories, chromosome location and cancer name. HHMD also includes a user-friendly visualization tool named HisModView, by which genome-wide histone modification map can be shown. HisModView facilitates the acquisition and visualization of histone modifications. The database also has manually curated information of histone modification dysregulation in nine human cancers.

  7. Charge State of the Globular Histone Core Controls Stability of the Nucleosome

    PubMed Central

    Fenley, Andrew T.; Adams, David A.; Onufriev, Alexey V.

    2010-01-01

    Presented here is a quantitative model of the wrapping and unwrapping of the DNA around the histone core of the nucleosome that suggests a mechanism by which this transition can be controlled: alteration of the charge state of the globular histone core. The mechanism is relevant to several classes of posttranslational modifications such as histone acetylation and phosphorylation; several specific scenarios consistent with recent in vivo experiments are considered. The model integrates a description based on an idealized geometry with one based on the atomistic structure of the nucleosome, and the model consistently accounts for both the electrostatic and nonelectrostatic contributions to the nucleosome free energy. Under physiological conditions, isolated nucleosomes are predicted to be very stable (38 ± 7 kcal/mol). However, a decrease in the charge of the globular histone core by one unit charge, for example due to acetylation of a single lysine residue, can lead to a significant decrease in the strength of association with its DNA. In contrast to the globular histone core, comparable changes in the charge state of the histone tail regions have relatively little effect on the nucleosome's stability. The combination of high stability and sensitivity explains how the nucleosome is able to satisfy the seemingly contradictory requirements for thermodynamic stability while allowing quick access to its DNA informational content when needed by specific cellular processes such as transcription. PMID:20816070

  8. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

    PubMed Central

    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David

    2015-01-01

    Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. PMID:25680960

  9. Histone Deacetylases and Cardiometabolic Diseases

    PubMed Central

    Yiew, Kan Hui; Chatterjee, Tapan K.; Hui, David Y.; Weintraub, Neal L.

    2015-01-01

    Cardiometabolic disease, emerging as a worldwide epidemic, is a combination of metabolic derangements leading to type 2 diabetes and cardiovascular disease. Genetic and environmental factors are linked through epigenetic mechanisms to the pathogenesis of cardiometabolic disease. Post-translational modifications of histone tails, including acetylation and deacetylation, epigenetically alter chromatin structure and dictate cell-specific gene expression patterns. The histone deacetylase (HDAC) family is comprised of 18 members that regulate gene expression by altering the acetylation status of nucleosomal histones and by functioning as nuclear transcriptional co-repressors. HDACs regulate key aspects of metabolism, inflammation, and vascular function pertinent to cardiometabolic disease in a cell- and tissue-specific manner. HDACs also likely play a role in the “metabolic memory” of diabetes, an important clinical aspect of the disease. Understanding the molecular, cellular, and physiological functions of HDACs in cardiometabolic disease is expected to provide insight into disease pathogenesis, risk factor control, and therapeutic development. PMID:26183616

  10. Non-thermal DNA damage of cancer cells using near-infrared irradiation.

    PubMed

    Tanaka, Yohei; Tatewaki, Naoto; Nishida, Hiroshi; Eitsuka, Takahiro; Ikekawa, Nobuo; Nakayama, Jun

    2012-08-01

    Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J/cm(2). Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser(1981), H2AX at Ser(139), Chk1 at Ser(317), structural maintenance of chromosome (SMC) 1 at Ser(966), and p53 at Ser(15) in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of γH2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J/cm(2). Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser(15), Chk1 at Ser(317), SMC1 at Ser(966), and H2AX at Ser(139) compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer.

  11. Histone deacetylases (HDACs): characterization of the classical HDAC family.

    PubMed Central

    de Ruijter, Annemieke J M; van Gennip, Albert H; Caron, Huib N; Kemp, Stephan; van Kuilenburg, André B P

    2003-01-01

    Transcriptional regulation in eukaryotes occurs within a chromatin setting, and is strongly influenced by the post-translational modification of histones, the building blocks of chromatin, such as methylation, phosphorylation and acetylation. Acetylation is probably the best understood of these modifications: hyperacetylation leads to an increase in the expression of particular genes, and hypoacetylation has the opposite effect. Many studies have identified several large, multisubunit enzyme complexes that are responsible for the targeted deacetylation of histones. The aim of this review is to give a comprehensive overview of the structure, function and tissue distribution of members of the classical histone deacetylase (HDAC) family, in order to gain insight into the regulation of gene expression through HDAC activity. SAGE (serial analysis of gene expression) data show that HDACs are generally expressed in almost all tissues investigated. Surprisingly, no major differences were observed between the expression pattern in normal and malignant tissues. However, significant variation in HDAC expression was observed within tissue types. HDAC inhibitors have been shown to induce specific changes in gene expression and to influence a variety of other processes, including growth arrest, differentiation, cytotoxicity and induction of apoptosis. This challenging field has generated many fascinating results which will ultimately lead to a better understanding of the mechanism of gene transcription as a whole. PMID:12429021

  12. Histone variants: emerging players in cancer biology

    PubMed Central

    Vardabasso, Chiara; Hasson, Dan; Ratnakumar, Kajan; Chung, Chi-Yeh; Duarte, Luis F.

    2014-01-01

    Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology. PMID:23652611

  13. Histone acetylation in heterochromatin assembly

    PubMed Central

    Kim, Jeong-Hoon; Workman, Jerry L.

    2010-01-01

    Histone acetylation is generally considered a mark involved in activating gene expression by making chromatin structures less compact. In the April 1, 2010, issue of Genes & Development, Xhemalce and Kouzarides (pp. 647–652) demonstrate that the acetylation of histone H3 at Lys 4 (H3K4) plays a role in the formation of repressive heterochromatin in Schizosaccharomyces pombe. H3K4 acetylation mediates a switch of chromodomain proteins associated with methylated H3K9 during heterochromatin assembly. PMID:20395362

  14. Diversity and Divergence of Dinoflagellate Histone Proteins.

    PubMed

    Marinov, Georgi K; Lynch, Michael

    2015-12-08

    Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed.

  15. Chromium Cross-Links Histone Deacetylase 1-DNA Methyltransferase 1 Complexes to Chromatin, Inhibiting Histone-Remodeling Marks Critical for Transcriptional Activation▿

    PubMed Central

    Schnekenburger, Michael; Talaska, Glenn; Puga, Alvaro

    2007-01-01

    Transcriptional regulation of gene expression requires posttranslational modification of histone proteins, which, in concert with chromatin-remodeling factors, modulate chromatin structure. Exposure to environmental agents may interfere with specific histone modifications and derail normal patterns of gene expression. To test this hypothesis, we coexposed cells to binary mixtures of benzo[a]pyrene (B[a]P), an environmental procarcinogen that activates Cyp1a1 transcriptional responses mediated by the aryl hydrocarbon receptor (AHR), and chromium, a carcinogenic heavy metal that represses B[a]P-inducible AHR-mediated gene expression. We show that chromium cross-links histone deacetylase 1-DNA methyltransferase 1 (HDAC1-DNMT1) complexes to Cyp1a1 promoter chromatin and inhibits histone marks induced by AHR-mediated gene transactivation, including phosphorylation of histone H3 Ser-10, trimethylation of H3 Lys-4, and various acetylation marks in histones H3 and H4. These changes inhibit RNA polymerase II recruitment without affecting the kinetics of AHR DNA binding. HDAC1 and DNMT1 inhibitors or depletion of HDAC1 or DNMT1 with siRNAs blocks chromium-induced transcriptional repression by decreasing the interaction of these proteins with the Cyp1a1 promoter and allowing histone acetylation to proceed. By inhibiting Cyp1a1 expression, chromium stimulates the formation of B[a]P DNA adducts. Epigenetic modification of gene expression patterns may be a key element of the developmental and carcinogenic outcomes of exposure to chromium and to other environmental agents. PMID:17682057

  16. Epigenetic Modifications and Accumulation of DNA Double-Strand Breaks in Oral Lichen Planus Lesions Presenting Poor Response to Therapy

    PubMed Central

    Dillenburg, Caroline S.; Martins, Marco A.T.; Almeida, Luciana O.; Meurer, Luise; Squarize, Cristiane H.; Martins, Manoela D.; Castilho, Rogerio M.

    2015-01-01

    Abstract Epigenetics refers to changes in cell characteristics that occur independently of modifications to the deoxyribonucleic acid (DNA) sequence. Alterations mediated by epigenetic mechanisms are important factors in cancer progression. Although an exciting prospect, the identification of early epigenetic markers associated with clinical outcome in premalignant and malignant disorders remains elusive. We examined alterations in chromatin acetylation in oral lichen planus (OLP) with distinct clinical behavior and compared the alterations to the levels of DNA double-strand breaks (DSBs). We analyzed 42 OLP patients, who had different responses to therapy, for acetyl-histone H3 at lys9 (H3K9ac), which is associated with enhanced transcription and nuclear decondensation, and the presence of DSBs, as determined by accumulation of phosphorylated γH2AX foci. Patients with high levels of H3K9ac acetylation failed to respond to therapy or experienced disease recurrence shortly after therapy. Similar to H3K9ac, patients who responded poorly to therapy had increased accumulation of DNA DSB, indicating genomic instability. These findings suggest that histone modifications occur in OLP, and H3K9ac and γH2AX histones may serve as epigenetic markers for OLP recurrence. PMID:26222871

  17. Phosphorylation: Implications in Cancer.

    PubMed

    Singh, Vishakha; Ram, Mahendra; Kumar, Rajesh; Prasad, Raju; Roy, Birendra Kumar; Singh, Kaushal Kumar

    2017-02-01

    Post translational modifications (PTMs) are involved in variety of cellular activities and phosphorylation is one of the most extensively studied PTM, which regulates a number of cellular functions like cell growth, differentiation, apoptosis and cell signaling in healthy condition. However, alterations in phosphorylation pathways result in serious outcomes in the form of diseases, especially cancer. Many signalling pathways including Tyrosine kinase, MAP kinase, Cadherin-catenin complex, Cyclin-dependent kinase etc. are major players of the cell cycle and deregulation in their phosphorylation-dephosphorylation cascade has been shown to be manifested in the form of various types of cancers. Tyrosine kinase family encompasses the greatest number of oncoproteins. MAPK cascade has an importance role in cancer growth and progression. Bcl-2 family proteins serve either proapoptotic or antiapoptotic function. Cadherin-catenin complex regulates cell adhesion properties and cyclins are the key regulators of cell cycle. Altered phosphorylations in any of the above pathways are strongly associated with cancer, at the same time they serve as the potential tergets for drug development against cancer. Drugs targeting tyrosine kinase are potent anticancer drugs. Inhibitors of MEK, PI3K and ERK signalling pathways are undergoing clinical trials. Thus, drugs targeting phosphorylation pathways represent a promising area for cancer therapy.

  18. Histone-modifying genes as biomarkers in hepatocellular carcinoma

    PubMed Central

    Hung, Shih-Ya; Lin, Hui-Hua; Yeh, Kun-Tu; Chang, Jan-Gowth

    2014-01-01

    Hepatocellular carcinoma (HCC) is the world’s fifth most common cancer and second leading cause of cancer-related death in Taiwan. Over 600,000 HCC patients die each year worldwide despite recent advances in surgical techniques and medical treatments. Epigenetic regulations including DNA methylation and histone modification control gene expressions and play important roles during tumorigenesis. This study evaluates association between histone-modifying genes and prognosis of HCC to ferret out new diagnostic markers. We collected 50 paired HCC and adjacent non-cancerous tissues from Taiwanese patients for survey by RT-qPCR and tissue microarray-based immunohistochemistry (TMA-based IHC) staining. RT-qPCR data showed four of twenty-four genes over eightfold up-regulated in tumor tissues: e.g., histone phosphorylation gene-ARK2, methylation genes-G9a, SUV39H2, and EZH2 (n = 50, all p < 0.0001). Results of TMA-based IHC staining showed proteins of ARK2, EZH2, G9a, and SUV39H2 also overexpressed in tumor tissues. Staining intensity of SUV39H2 correlated with HCV infection (p = 0.025). We further restricted the analysis only in tumor tissues, we found EZH2 staining intensity associated with tumor stage (p = 0.016) and survival (p = 0.007); SUV39H2 intensity associated with tumor stage (p = 0.044). Our findings indicate overexpression of histone-modifying genes EZH2 and SUV39H2 associated with prognosis of HCC cases. EZH2 expression can serve as a novel prognostic biomarker during HCC progression among Taiwanese. PMID:24966962

  19. The Use of Gamma-H2AX as a Biodosimeter for Total-Body Radiation Exposure in Non-Human Primates

    DTIC Science & Technology

    2010-11-23

    1986) Rapid isolation in large numbers of intact, viable, individual hair follicles from skin: biochemical and ultrastructural characterization. J...Radiol 65: 148–151. 23. Kyoizumi S, Suzuki T, Teraoka S, Seyama T (1998) Radiation sensitivity of human hair follicles in SCID-hu mice. Radiat Res...Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to

  20. H3S10 phosphorylation-mediated transcriptional regulation by Aurora kinase A.

    PubMed

    Kim, Se-Ryeon; Kim, Kee-Beom; Chae, Yun-Cheol; Park, Jin Woo; Seo, Sang-Beom

    2016-01-01

    Histone H3S10 phosphorylation has been known as a cell cycle-specific marker and has a role in transcriptional activation. Various kinases phosphorylate H3S10 in different species, however, the role of the mitotic serine/threonine protein kinase Aurora A (AURKA) is largely unknown. Here we present evidence that AURKA phosphorylates H3S10 and activates target gene transcription. We show that down-regulation of AURKA level during leukemia cell differentiation results in decreased H3S10 phosphorylation level. We further show that AURKA is recruited to target gene promoters and activates transcription via H3S10 phosphorylation. Furthermore, this recruitment can be disrupted by the AURKA inhibitor Alisertib and results in H3K9-me2 recruitment by G9a.

  1. Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

    SciTech Connect

    Westwood, J.T.; Church, R.B.; Wagenaar, E.B.

    1985-08-25

    The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with (TSP)orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.

  2. Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

    PubMed Central

    Ahmed, K; Wilson, M J; Goueli, S A; Williams-Ashman, H G

    1978-01-01

    Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves. PMID:747650

  3. Mass Spectrometric Analysis of Histone Proteoforms

    NASA Astrophysics Data System (ADS)

    Yuan, Zuo-Fei; Arnaudo, Anna M.; Garcia, Benjamin A.

    2014-06-01

    Histones play important roles in chromatin, in the forms of various posttranslational modifications (PTMs) and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an ongoing challenge. Previous methods are based on antibodies, and because they usually detect only one modification at a time, they are not suitable for studying the various combinations of modifications on histones. Fortunately, mass spectrometry (MS) has emerged as a high-throughput technology for histone analysis and does not require prior knowledge about any modifications. From the data generated by mass spectrometers, both identification and quantification of modifications, as well as variants, can be obtained easily. On the basis of this information, the functions of histones in various cellular contexts can be revealed. Therefore, MS continues to play an important role in the study of histone proteoforms. In this review, we discuss the analysis strategies of MS, their applications on histones, and some key remaining challenges.

  4. Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells.

    PubMed

    Stuhldreier, Fabian; Kassel, Stefanie; Schumacher, Lena; Wesselborg, Sebastian; Proksch, Peter; Fritz, Gerhard

    2015-05-28

    We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10-20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AXH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy.

  5. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    SciTech Connect

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B.; Su, Tsann-Long; Yih, Ling-Huei

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  6. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest.

    PubMed

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B; Su, Tsann-Long; Yih, Ling-Huei

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

  7. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  8. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in histone H3.

    PubMed

    Kobza, Keyna; Sarath, Gautam; Zempleni, Janos

    2008-04-30

    BirA ligase is a prokaryotic ortholog of holocarboxylase synthetase (HCS) that can biotinylate proteins. This study tested the hypothesis that BirA ligase catalyzes the biotinylation of eukaryotic histones. If so, this would mean that recombinant BirA ligase is a useful surrogate for HCS in studies of histone biotinylation. The biological activity of recombinant BirA ligase was confirmed by enzymatic biotinylation of p67. In particular, it was found that BirA ligase biotinylated both calf thymus histone H1 and human bulk histone extracts. Incubation of recombinant BirA ligase with H3-based synthetic peptides showed that lysines 4, 9, 18, and 23 in histone H3 are the targets for the biotinylation by BirA ligase. Modification of the peptides (e.g., serine phosphorylation) affected the subsequent biotinylation by BirA ligase, suggesting crosstalk between modifications. In conclusion, this study suggests that prokaryotic BirA ligase is a promiscuous enzyme and biotinylates eukaryotic histones. Moreover the biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin.

  9. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  10. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  11. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  12. Identification of a variant-specific phosphorylation of TH2A during spermiogenesis

    PubMed Central

    Hada, Masashi; Masuda, Koji; Yamaguchi, Kosuke; Shirahige, Katsuhiko; Okada, Yuki

    2017-01-01

    Tissue-specific histone variant incorporation into chromatin plays dynamic and important roles in tissue development. Testis is one such tissue, and a number of testis-specific histone variants are expressed that have unique roles. While it is expected that such variants acquire post-transcriptional modifications to be functional, identification of variant-specific histone modifications is challenging because of the high similarity of amino acid sequences between canonical and variant versions. Here we identified a novel phosphorylation on TH2A, a germ cell-specific histone H2A variant. TH2A-Thr127 is unique to the variant and phosphorylated concomitant with chromatin condensation including spermiogenesis and early embryonic mitosis. In sperm chromatin, phosphorylated TH2A-Thr127 (=pTH2A) is co-localized with H3.3 at transcriptional starting sites of the genome, and subsequently becomes absent from the paternal genome upon fertilization. Notably, pTH2A is recurrent and accumulated in the pericentromeric heterochromatin of both paternal and maternal chromosomes in the first mitosis of embryos, suggesting its unique regulation during spermiogenesis and early embryogenesis. PMID:28387373

  13. 14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

    PubMed Central

    Karam, Caline S.; Kellner, Wendy A.; Takenaka, Naomi; Clemmons, Alexa W.; Corces, Victor G.

    2010-01-01

    Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation. PMID:20532201

  14. Properties of the yeast nuclear histone deacetylase.

    PubMed Central

    Sanchez del Pino, M M; Lopez-Rodas, G; Sendra, R; Tordera, V

    1994-01-01

    A nuclear histone deacetylase from yeast was partially purified and some of its characteristics were studied. Histone deacetylase activity was stimulated in vitro by high-mobility-group nonhistone chromatin proteins 1 and 2 and ubiquitin and inhibited by spermine and spermidine, whereas n-butyrate had no significant inhibitory effect. Like the mammalian enzyme, partially purified histone deacetylase from yeast was strongly inhibited by trichostatin A. However, in crude extract preparations the yeast enzyme was not inhibited and treatment with trichostatin in vivo did not show any effect, either on the histone acetylation level or on cell viability. At low ionic strength, the enzyme can be isolated as a complex of high molecular mass that is much less inhibited by trichostatin A than is partially purified histone deacetylase activity. Furthermore, radiolabelled oligonucleosomes were more efficiently deacetylated by the complex than by the low-molecular-mass form of the enzyme. The histone deacetylase activity was separated from a polyamine deacetylase activity and its specificity studied. Using h.p.l.c.-purified core histone species as substrate, histone deacetylase from yeast is able to deacetylate all core histones with a slight preference for H3. Our results support the idea that the yeast histone deacetylase may act as a high-molecular-mass complex in vivo. Images Figure 3 PMID:7980438

  15. Mitotic Activation of a Novel Histone Deacetylase 3-Linker Histone H1.3 Protein Complex by Protein Kinase CK2.

    PubMed

    Patil, Hemangi; Wilks, Carrie; Gonzalez, Rhiannon W; Dhanireddy, Sudheer; Conrad-Webb, Heather; Bergel, Michael

    2016-02-12

    Histone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis and the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II, or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (N-CoR) that accumulated in synchronized HeLa cells in late G2 phase and mitosis. Nonetheless, the deacetylation activity by HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated with H1.3 was highly phosphorylated on Ser-424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late G2 phase cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2) activated the HDAC3 in vitro. In vivo, CK2α and CK2α' double knockdown cells demonstrated a significant decrease in HDAC3 Ser-424 phosphorylation during mitosis. HDAC3 and H1.3 co-localized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially co-localized with chromatin during prophase and interphase. H1 has been reported previously to associate with microtubules and, therefore, could potentially function in targeting HDAC3 to the microtubules. We suggest that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin and regulation of polar microtubules dynamic instability.

  16. Regulation of RNA polymerase II activation by histone acetylation in single living cells.

    PubMed

    Stasevich, Timothy J; Hayashi-Takanaka, Yoko; Sato, Yuko; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Nagase, Takahiro; Nozaki, Naohito; McNally, James G; Kimura, Hiroshi

    2014-12-11

    In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

  17. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  18. Histone chaperone specificity in Rtt109 activation

    PubMed Central

    Park, Young-Jun; Sudhoff, Keely B; Andrews, Andrew J; Stargell, Laurie A; Luger, Karolin

    2008-01-01

    Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone 3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. Here we show that both Rtt109 and Vps75 bind histones with high affinity, but only the complex is efficient for catalysis. The C-terminal acidic domain of Vps75 contributes to activation of Rtt109 and is necessary for in vivo functionality of Vps75, but it is not required for interaction with either Rtt109 or histones. We demonstrate that Vps75 is a structural homolog of yeast Nap1 by solving its crystal structure. Nap1 and Vps75 interact with histones and Rtt109 with comparable affinities. However, only Vps75 stimulates Rtt109 enzymatic activity. Our data highlight the functional specificity of Vps75 in Rtt109 activation. PMID:19172749

  19. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    SciTech Connect

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  20. Uncoupling histone turnover from transcription-associated histone H3 modifications.

    PubMed

    Ferrari, Paolo; Strubin, Michel

    2015-04-30

    Transcription in eukaryotes is associated with two major changes in chromatin organization. Firstly, nucleosomal histones are continuously replaced by new histones, an event that in yeast occurs predominantly at transcriptionally active promoters. Secondly, histones become modified post-translationally at specific lysine residues. Some modifications, including histone H3 trimethylation at lysine 4 (H3K4me3) and acetylation at lysines 9 (H3K9ac) and 14 (H3K14ac), are specifically enriched at active promoters where histones exchange, suggesting a possible causal relationship. Other modifications accumulate within transcribed regions and one of them, H3K36me3, is thought to prevent histone exchange. Here we explored the relationship between these four H3 modifications and histone turnover at a few selected genes. Using lysine-to-arginine mutants and a histone exchange assay, we found that none of these modifications plays a major role in either promoting or preventing histone turnover. Unexpectedly, mutation of H3K56, whose acetylation occurs prior to chromatin incorporation, had an effect only when introduced into the nucleosomal histone. Furthermore, we used various genetic approaches to show that histone turnover can be experimentally altered with no major consequence on the H3 modifications tested. Together, these results suggest that transcription-associated histone turnover and H3 modification are two correlating but largely independent events.

  1. Cyclin B targets p34cdc2 for tyrosine phosphorylation.

    PubMed

    Meijer, L; Azzi, L; Wang, J Y

    1991-06-01

    A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.

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

    PubMed Central

    van Attikum, Haico; Fritsch, Olivier; Gasser, Susan M

    2007-01-01

    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 (γH2AX). 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, γH2AX 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. PMID:17762868

  3. Zalypsis: a novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double-strand breaks.

    PubMed

    Ocio, Enrique M; Maiso, Patricia; Chen, Xi; Garayoa, Mercedes; Alvarez-Fernández, Stela; San-Segundo, Laura; Vilanova, David; López-Corral, Lucía; Montero, Juan C; Hernández-Iglesias, Teresa; de Alava, Enrique; Galmarini, Carlos; Avilés, Pablo; Cuevas, Carmen; San-Miguel, Jesús F; Pandiella, Atanasio

    2009-04-16

    Multiple myeloma (MM) remains incurable, and new drugs with novel mechanisms of action are still needed. In this report, we have analyzed the action of Zalypsis, an alkaloid analogous to certain natural marine compounds, in MM. Zalypsis turned out to be the most potent antimyeloma agent we have tested so far, with IC(50) values from picomolar to low nanomolar ranges. It also showed remarkable ex vivo potency in plasma cells from patients and in MM cells in vivo xenografted in mice. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double-strand breaks (DSBs), evidenced by an increase in phospho-histone-H2AX and phospho-CHK2, followed by a striking overexpression of p53 in p53 wild-type cell lines. In addition, in those cell lines in which p53 was mutated, Zalypsis also provoked DSBs and induced cell death, although higher concentrations were required. Immunohistochemical studies in tumors also demonstrated histone-H2AX phosphorylation and p53 overexpression. Gene expression profile studies were concordant with these results, revealing an important deregulation of genes involved in DNA damage response. The potent in vitro and in vivo antimyeloma activity of Zalypsis uncovers the high sensitivity of tumor plasma cells to DSBs and strongly supports the use of this compound in MM patients.

  4. The interplay of histone modifications – writers that read

    PubMed Central

    Zhang, Tianyi; Cooper, Sarah; Brockdorff, Neil

    2015-01-01

    Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. PMID:26474904

  5. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    PubMed

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  6. The histone deacetylase inhibitor valproic acid inhibits NKG2D expression in natural killer cells through suppression of STAT3 and HDAC3

    PubMed Central

    Ni, Lulu; Wang, Lixin; Yao, Chao; Ni, Zhongya; Liu, Fei; Gong, Chenyuan; Zhu, Xiaowen; Yan, Xuewei; Watowich, Stephanie S.; Lee, Dean A.; Zhu, Shiguo

    2017-01-01

    NKG2D is a major activating receptor of NK cells and plays a critical role in tumor immunosurveillance. NKG2D expression in NK cells is inhibited by the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and enhanced by the narrow-spectrum HDAC inhibitor entinostat. We previously demonstrated that entinostat enhanced NKG2D transcription by increasing acetylation of Histones H3 and H4. However, the mechanism by which VPA reduces NKG2D expression in NK cells is not known. We have also shown that NKG2D transcription is regulated by STAT3 phosphorylation. In this study, we investigated regulation of NKG2D expression in NK cells by VPA and entinostat by assessing protein expression, phosphorylation, and interaction of HDACs and STAT3. We find that VPA selectively inhibits STAT3 tyrosine705 phosphorylation, but entinostat does not. STAT3 complexes with HDAC3, and HDAC3 inhibition represses STAT3 phosphorylation and therefore NKG2D expression. NK cells from STAT3 wild-type mice downregulate NKG2D in response to VPA, but not NK cells from STAT3 knockout mice. These results show that VPA is a potent inhibitor of STAT3 phosphorylation and demonstrate that histone acetylation and STAT3 tyrosine705 phosphorylation cooperate in regulating NKG2D expression in NK cells. PMID:28338101

  7. Remodeling somatic nuclei in Xenopus laevis egg extracts: molecular mechanisms for the selective release of histones H1 and H1(0) from chromatin and the acquisition of transcriptional competence.

    PubMed Central

    Dimitrov, S; Wolffe, A P

    1996-01-01

    The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription. Images PMID:8918467

  8. Canonical and variant histones of protozoan parasites.

    PubMed

    Dalmasso, Maria Carolina; Sullivan, William Joseph; Angel, Sergio Oscar

    2011-06-01

    Protozoan parasites have tremendously diverse lifestyles that require adaptation to a remarkable assortment of different environmental conditions. In order to complete their life cycles, protozoan parasites rely on fine-tuning gene expression. In general, protozoa use novel regulatory elements, transcription factors, and epigenetic mechanisms to regulate their transcriptomes. One of the most surprising findings includes the nature of their histones--these primitive eukaryotes lack some histones yet harbor novel histone variants of unknown function. In this review, we describe the histone components of different protozoan parasites based on literature and database searching. We summarize the key discoveries regarding histones and histone variants and their impact on chromatin regulation in protozoan parasites. In addition, we list histone genes IDs, sequences, and genomic localization of several protozoan parasites and Microsporidia histones, obtained from a thorough search of genome databases. We then compare these findings with those observed in higher eukaryotes, allowing us to highlight some novel aspects of epigenetic regulation in protists and to propose questions to be addressed in the upcoming years.

  9. Assessment of estrogen receptor--histone interactions.

    PubMed Central

    Kallos, J; Fasy, T M; Hollander, V P

    1981-01-01

    Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules. PMID:6942408

  10. Targeting histone methylation for colorectal cancer

    PubMed Central

    Huang, Tao; Lin, Chengyuan; Zhong, Linda L. D.; Zhao, Ling; Zhang, Ge; Lu, Aiping; Wu, Jiang; Bian, Zhaoxiang

    2016-01-01

    As a leading cause of cancer deaths worldwide, colorectal cancer (CRC) results from accumulation of both genetic and epigenetic alterations. Disruption of epigenetic regulation in CRC, particularly aberrant histone methylation mediated by histone methyltransferases (HMTs) and demethylases (HDMs), have drawn increasing interest in recent years. In this paper, we aim to review the roles of histone methylation and associated enzymes in the pathogenesis of CRC, and the development of small-molecule modulators to regulate histone methylation for treating CRC. Multiple levels of evidence suggest that aberrant histone methylations play important roles in CRC. More than 20 histone-methylation enzymes are found to be clinically relevant to CRC, including 17 oncoproteins and 8 tumor suppressors. Inhibitors of EZH2 and DOT1L have demonstrated promising therapeutic effects in preclinical CRC treatment. Potent and selective chemical probes of histone-methylation enzymes are required for validation of their functional roles in carcinogenesis and clinical translations as CRC therapies. With EZH2 inhibitor EPZ-6438 entering into phase I/II trials for advanced solid tumors, histone methylation is emerging as a promising target for CRC. PMID:28286564

  11. Histone H2A mobility is regulated by its tails and acetylation of core histone tails

    SciTech Connect

    Higashi, Tsunehito; Matsunaga, Sachihiro; Isobe, Keisuke; Morimoto, Akihiro; Shimada, Tomoko; Kataoka, Shogo; Watanabe, Wataru; Uchiyama, Susumu; Itoh, Kazuyoshi; Fukui, Kiichi . E-mail: kfukui@bio.eng.osaka-u.ac.jp

    2007-06-08

    Histone tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions.

  12. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-06-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

  13. Histone variants in plant transcriptional regulation.

    PubMed

    Jiang, Danhua; Berger, Frédéric

    2017-01-01

    Chromatin based organization of eukaryotic genome plays a profound role in regulating gene transcription. Nucleosomes form the basic subunits of chromatin by packaging DNA with histone proteins, impeding the access of DNA to transcription factors and RNA polymerases. Exchange of histone variants in nucleosomes alters the properties of nucleosomes and thus modulates DNA exposure during transcriptional regulation. Growing evidence indicates the important function of histone variants in programming transcription during developmental transitions and stress response. Here we review how histone variants and their deposition machineries regulate the nucleosome stability and dynamics, and discuss the link between histone variants and transcriptional regulation in plants. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

  14. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  15. Structure and Functions of Linker Histones.

    PubMed

    Lyubitelev, A V; Nikitin, D V; Shaytan, A K; Studitsky, V M; Kirpichnikov, M P

    2016-03-01

    Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

  16. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  17. Microvesicles Contribute to the Bystander Effect of DNA Damage.

    PubMed

    Lin, Xiaozeng; Wei, Fengxiang; Major, Pierre; Al-Nedawi, Khalid; Al Saleh, Hassan A; Tang, Damu

    2017-04-07

    Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

  18. ATM and p53 are essential in the cell-cycle containment of DNA breaks during V(D)J recombination in vivo.

    PubMed

    Dujka, M E; Puebla-Osorio, N; Tavana, O; Sang, M; Zhu, C

    2010-02-18

    V(D)J recombination is essential for the maturation of lymphocytes. Because of the involvement of cutting and joining DNA double strands, this recombination activity is strictly contained within the noncycling phases of the cell cycle. Such containment is crucial for the maintenance of genomic integrity. The ataxia telangiectasia mutated (ATM) gene is known to have a central role in sensing general DNA damage and mediating cell-cycle checkpoint. In this study, we investigated the role of ATM and its downstream targets in the cell-cycle control of V(D)J recombination in vivo. Our results revealed the persistence of double-strand breaks (DSBs) throughout the cell cycle in ATM(-/-) and p53(-/-) thymocytes, but the cell-cycle regulation of a V(D)J recombinase, Rag-2, was normal. The histone variant H2AX, which is phosphorylated during normal V(D)J recombination, was dispensable for containing DSBs. H2AX was still phosphorylated at V(D)J loci in the absence of ATM. Therefore, V(D)J recombination, a physiological DNA rearrangement process, activates the ATM/p53 pathway to contain DNA breaks within the noncycling cells and surprisingly this pathway is not important for containing Rag-2 activity. This study shows the dynamic multiple functions of ATM in maintaining genomic stability and preventing tumorigenesis in developing lymphocytes.

  19. Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota

    2016-07-01

    Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression.

  20. AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

    PubMed Central

    Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

    2015-01-01

    Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3′ end of DNA damage-activated genes to facilitate transcriptional termination. PMID:25813038

  1. A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes.

    PubMed

    Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2010-10-01

    Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone-replacement system provides a well-defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism.

  2. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  3. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community.

  4. Protein tyrosine phosphorylation in streptomycetes.

    PubMed

    Waters, B; Vujaklija, D; Gold, M R; Davies, J

    1994-07-01

    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  5. Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics.

    PubMed

    Hadnagy, Annamaria; Beaulieu, Raymond; Balicki, Danuta

    2008-04-01

    Over the past few years, the histone deacetylase (HDAC) inhibitors have occupied an important place in the effort to develop novel, but less toxic, anticancer therapy. HDAC inhibitors block HDACs, which are the enzymes responsible for histone deacetylation, and therefore they modulate gene expression. The cellular effects of HDAC inhibitors include growth arrest and the induction of differentiation. Early successes in cancer therapeutics obtained using these drugs alone or in combination with other anticancer drugs emphasize the important place of posttranslational modifications of histones in cancer therapy. Histone tail modifications along with DNA methylation are the most studied epigenetic events related to cancer progression. Moreover, extranuclear functions of histones have also been described. Because HDAC inhibitors block HDACs and thereby increase histone acetylation, we propose a model wherein exogenous acetylated histones or other related acetylated proteins that are introduced into the nucleus become HDAC substrates and thereby compete with endogenous histones for HDACs. This competition may lead to the increased acetylation of the endogenous histones, as in the case of HDAC inhibitor therapy. Moreover, other mechanisms of action, such as binding to chromatin and modulating gene expression, are also possible for exogenously introduced histones.

  6. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms.

    PubMed

    Wang, Huanyu; Ge, Zhongqi; Walsh, Scott T R; Parthun, Mark R

    2012-01-01

    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

  7. DNA methylation and histone acetylation work in concert to regulate memory formation and synaptic plasticity.

    PubMed

    Miller, Courtney A; Campbell, Susan L; Sweatt, J David

    2008-05-01

    A clear understanding is developing concerning the importance of epigenetic-related molecular mechanisms in transcription-dependent long-term memory formation. Chromatin modification, in particular histone acetylation, is associated with transcriptional activation, and acetylation of histone 3 (H3) occurs in Area CA1 of the hippocampus following contextual fear conditioning training. Conversely, DNA methylation is associated with transcriptional repression, but is also dynamically regulated in Area CA1 following training. We recently reported that inhibition of the enzyme responsible for DNA methylation, DNA methyltransferase (DNMT), in the adult rat hippocampus blocks behavioral memory formation. Here, we report that DNMT inhibition also blocks the concomitant memory-associated H3 acetylation, without affecting phosphorylation of its upstream regulator, extracellular signal-regulated kinase (ERK). Interestingly, the DNMT inhibitor-induced deficit in memory consolidation, along with