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Sample records for phosphorylated histone h2ax

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

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    Serena Barral

    2014-01-01

    Full Text Available 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.

  2. Histone H2AX in DNA repair

    International Nuclear Information System (INIS)

    Lewandowska, H.; Szumiel, I.

    2002-01-01

    The paper reviews the recent reports on the role of the phosphorylated histone H2AX (γ-H2AX). The modification of this histone is an important part of the cellular response to the induction of DNA double strand brakes (DSB) by ionising radiation and other DSB-generating factors. In irradiated cells the modification is carried out mainly by ATM (ataxia-telangiectasia mutated) kinase, the enzyme that starts the alarm signalling upon induction of DSB.γ-H2AX molecules are formed within 1-3 min after irradiation and form foci at the sites of DSB. This seems to be necessary for the recruitment of repair factors that are later present in foci of damaged nuclei. Modification of a constant percentage of H2AX molecules per DSB takes place, corresponding to chromatin domains of megabase of DNA. (author)

  3. Phosphorylation of histone H2AX as an indicator of received dose of gamma radiation after whole-body irradiation of rats

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    Radim Havelek

    2011-01-01

    Full Text Available The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats. Wistar rats were irradiated by 1-10 Gy of gamma radiation by 60Co source. Value LD50/60 was 7.37 (4.68-8.05 Gy. Histone H2AX is phosphorylated by ATM kinase on serine 139 (γH2AX quickly after the irradiation. It forms microscopically visible foci in the site of double strand breaks of DNA. Flow-cytometric method was used for quantitative detection. This study is the first one that evaluated dose-dependency of H2AX phosphorylation in peripheral lymphocytes of rats irradiated by whole-body dose 1-10 Gy. Our data show a dose-dependent increase in γH2AX in rat peripheral blood lymphocytes 1 h after whole-body irradiation by the dose of 1-10 Gy. We proved that phosphorylation of histone H2AX is a prompt and reliable indicator of the received radiation dose suitable for rapid measurement before the number of lymphocytes in peripheral blood starts to decrease. It can be used already 1 h after the irradiation for an estimation of the received dose of radiation. Blood samples can be stored in 4 °C for 23 h without significantly affecting the result.

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

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    Kobayashi, Junya; Tauchi, Hiroshi; Chen, Benjamin; Burma, Sandeep; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J; Komatsu, Kenshi

    2009-03-20

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

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

    International Nuclear Information System (INIS)

    Kobayashi, Junya; Tauchi, Hiroshi; Chen, Benjamin; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J.; Komatsu, Kenshi

    2009-01-01

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

  6. Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

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    So Ra Yoon

    2017-01-01

    Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

  7. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    International Nuclear Information System (INIS)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-01-01

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications

  8. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

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    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  9. NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain

    International Nuclear Information System (INIS)

    Kobayashi, J.; Chen, D.J.; Sakamoto, S.; Matsuura, S.; Tanimoto, K.; Komatsu, K.

    2003-01-01

    Full text: DNA double-strand breaks (DSBs) represent the most potentially serious damage to a genome, and hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Histone H2AX, one of histone H2A family, is phosphorylated within a few minutes in response to ionizing radiation (IR) and the phosphorylated H2AX (gamma-H2AX) forms foci at the region of DSBs. Moreover, Histone H2AX is essential for the IR-induced focus formation of DNA repair proteins such as BRCA1, NBS1 and 53BP1. Hence, we investigated that the function of histone H2AX for the recruitment of NBS1/hMRE11/ hRAD50 complex to DSBs sites. We clarify that NBS1 physically interacts with histone H2AX independent of DNA. We also show that the NBS1-binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells. We also demonstrate that the FHA/BRCT domain of NBS1 is essential for this physical interaction by the immunoprecipitation studies and a pull-down assay with recombinant FHA/BRCT domain. These findings suggest that the FHA/BRCT domain have a crucial role for both binding to histone and for re-localization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage

  10. Histone H2AX is a critical factor for cellular protection against DNA alkylating agents.

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    Meador, J A; Zhao, M; Su, Y; Narayan, G; Geard, C R; Balajee, A S

    2008-09-25

    Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G(2)/M phase. Lack of G(2)/M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.

  11. Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX

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    Szilard, Rachel K.; Jacques, Pierre-Étienne; Laramée, Louise; Cheng, Benjamin; Galicia, Sarah; Bataille, Alain R.; Yeung, ManTek; Mendez, Megan; Bergeron, Maxime; Robert, François; Durocher, Daniel

    2011-01-01

    Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, γ-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes. PMID:20139982

  12. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

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    Duensing Stefan

    2008-07-01

    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

  13. Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

    International Nuclear Information System (INIS)

    Tian Mei; Pan Yan; Liu Jianxiang; Ruan Jianlei; Su Xu

    2011-01-01

    Objective: To investigate 60 Co γ-ray induced damage in lymphocytes and the relationship between doses of 60 Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM. Methods: Cells were irradiated with 60 Co γ-rays in the range of 0-8 Gy. The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively. The micronucleus(MN)was analyzed by CB method to evaluate DNA damage. Results: FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses, and the fitting curve was shown as Y=3.96+11.29D-0.45D 2 . The level of phosphorylated ATM (p-ATM) was not changed significantly by using the same method. Western blot showed that p-ATM protein expression was significantly increased after irradiation compared with sham, irradiated group. The MN assay which represented DNA damage was sensitive to different doses. Conclusions: γ-ray irradiation could induce the phosphorylation of H2AX and ATM, which may play an important role in indicating DNA damage. Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage. (authors)

  14. Histone H2A subfractions and their phosphorylation in cultured Peromyscus cells

    International Nuclear Information System (INIS)

    Halleck, M.S.; Gurley, L.R.

    1980-01-01

    Patterns of histone phosphorylation and histone H2A subfractionation have been compared in cultured cell lines from two species of deer mice, Peromyscus eremicus and Peromyscus boylii, which differ considerably in their content of heterochromatin but which contain essentially the same euchromatin content. DNA measurements by flow microfluorometry indicated that P. eremicus cells contained 34.2% more DNA than P. boylii cells, and C-band chromosome analysis indicated that the extra DNA in P. eremicus was present as constitutive heterochromatin. Subfraction of histone H2A by acid-urea polyacrylamide preparative gel electrophoresis in the presence of non-ionic detergent showed that each cell line contained two H2A subfractions. Incorporation of 32 PO 4 into these histones indicated that the steady state phosphorylation of the two H2A subfractions was not the same, the more hydrophobic H2A being greater than two times more phosphorylated than the less hydrophobic H2A in both cell lines. A comparison of the two cell lines indicated that the cell line with 34.2% greater constitutive heterochromatin contained a similar excess (29%) in its ratio of the more highly phosphorylated, more hydrophobic H2A subfraction to the less hydrophobic H2A subfraction. It is suggested that this enrichment of the more highly phosphorylated, more hydrophobic H2A subfraction may be related to the amount of constitutive heterochromatin present in the genome

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

    OpenAIRE

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

    2012-01-01

    Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (cH2AX) 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 cH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional respo...

  16. A preliminary study to detect CT radiation doses by using the γH2AX focus formation assay

    International Nuclear Information System (INIS)

    Zhang Bo; Gong Jianping; Zhang Wei; Yu Zeyang; Zhou Liying; Zhang Hong; Fu Jinxiang

    2010-01-01

    To prospectively determine if γH2AX (phosphorylated form of H2AX histone variant)-based visualization and quantification of DNA damage induced in peripheral blood mononuclear cells (PBMCs) can be used to estimate the radiation dose received after multi-detector computed tomography (CT) by the in vitro study, comprehend the correlation between the dose and the induced γH2AX foci, and explore its prospect. The result showed that, DNA damage first presented as γH2AX foci after CT, which can be detected by fluorescence microscope. The γH2AX focus yields linearly depend on the radiation dose after CT. γH2AX focus yield in blood cells may be a useful quantitative biomarker of human radiation exposure by CT scans. (authors)

  17. ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation

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    Yuko Atsumi

    2015-12-01

    Full Text Available In response to DNA double-strand breaks (DSBs, H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.

  18. Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation▿

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    Sanders, Steven L.; Arida, Ahmad R.; Phan, Funita P.

    2010-01-01

    Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability. PMID:20679488

  19. Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation.

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    Sanders, Steven L; Arida, Ahmad R; Phan, Funita P

    2010-10-01

    Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

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

    International Nuclear Information System (INIS)

    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

    2009-01-01

    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 H2AXH2AX) foci at 30 min and 6 h post-irradiation. Results: Survival curves showed the increasing effectiveness of radiation as a function of LET. γH2AX 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 γH2AX 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 γH2AX foci. We conclude that γH2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.

  1. Assessment of γ-H2AX levels in circulating tumor cells from patients receiving chemotherapy

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    Garcia-Villa, Alejandra; Balasubramanian, Priya; Miller, Brandon L. [William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH (United States); Lustberg, Maryam B.; Ramaswamy, Bhuvaneswari [Department of Internal Medicine, Breast Medical Oncology, James Cancer Hospital and Ohio State University Comprehensive Cancer Center, Columbus, OH (United States); Chalmers, Jeffrey J., E-mail: chalmers.1@osu.edu [William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH (United States)

    2012-10-25

    Circulating tumor cells (CTCs) are prognostic markers in a variety of solid tumor malignancies. The potential of CTCs to be used as a “liquid biopsy” to monitor a patient’s condition and predict drug response and resistance is currently under investigation. Using a negative depletion, enrichment methodology, CTCs isolated from the peripheral blood of breast cancer patients with stage IV breast cancer undergoing DNA damaging therapy with platinum-based therapy were enriched. The enriched cell suspensions were stained with an optimized labeling protocol targeting: nuclei, cytokeratins 8, 18, and 19, the surface marker CD45, and the presence of the protein γ-H2AX. As a direct or indirect result of platinum therapy, double-strand break of DNA initiates phosphorylation of the histone H2AX, at serine 139; this phosphorylated form is referred to as γ-H2AX. In addition to γ-H2AX staining in specific locations with the cell nuclei, consistent with previous reports and referred to as foci, more general staining in the cell cytoplasm was also observed in some cells suggesting the potential of cell apoptosis. Our study underscores the utility and the complexity of investigating CTCs as predictive markers of response to various therapies. Additional studies are ongoing to evaluate the diverse γ-H2AX staining patterns we report here which needs to be further correlated with patient outcomes.

  2. Age-related disease association of endogenous γ-H2AX foci in mononuclear cells derived from leukapheresis.

    Directory of Open Access Journals (Sweden)

    Shepherd H Schurman

    Full Text Available The phosphorylated form of histone H2AX (γ-H2AX forms immunohistochemically detectable foci at DNA double strand breaks. In peripheral blood mononuclear cells (PBMCs derived from leukapheresis from patients enrolled in the Baltimore Longitudinal Study of Aging, γ-H2AX foci increased in a linear fashion with regards to age, peaking at ~57 years. The relationship between the frequency of γ-H2AX foci and age-related pathologies was assessed. We found a statistically significant (p = 0.023 50% increase in foci in PBMCs derived from patients with a known history of vitamin D deficiency. In addition, there were trends toward increased γ-H2AX foci in patients with cataracts (34% increase, p<0.10 and in sleep apnea patients (44%, p<0.10. Among patients ≥57 y/o, we found a significant (p = 0.037 36% increase in the number of γ-H2AX foci/cell for patients with hypertension compared to non-hypertensive patients. Our results support a role for increased DNA damage in the morbidity of age-related diseases. γ -H2AX may be a biomarker for human morbidity in age-related diseases.

  3. Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci

    International Nuclear Information System (INIS)

    Djuzenova, Cholpon S; Elsner, Ines; Katzer, Astrid; Worschech, Eike; Distel, Luitpold V; Flentje, Michael; Polat, Bülent

    2013-01-01

    High expression of constitutive histone γ-H2AX, a sensitive marker of DNA damage, might be indicative of defective DNA repair pathway or genomic instability. 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. This study explores the relationship between the clinical radiosensitivity of tumor patients and the expression/induction of γ-H2AX and 53BP1 in vitro. Using immunostaining, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53 BP1 in peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=57) undergoing radiotherapy (RT). Cells from apparently healthy donors (n=12) served as references. Non-irradiated cells from controls and unselected BC patients exhibited similar baseline levels of DNA damage assessed by γ-H2AX and 53BP1 foci. At the same time, the γ-H2AX assay of in vitro irradiated cells revealed significant differences between the control group and the group of unselected BC patients with respect to the initial (0.5 Gy, 30 min) and residual (2 Gy, 24 h post-radiation) DNA damage. The numbers of 53BP1 foci analyzed in 35 BC patients were significantly higher than in controls only in case of residual DNA damage. A weak correlation was found between residual foci of both proteins tested. In addition, cells from cancer patients with an adverse acute skin reaction (grade 3) to RT showed significantly increased radiation-induced γ-H2AX foci and their protracted disappearance compared to the group of BC patients with normal skin reaction (grade 0–1). The mean number of γ-H2AX foci after 5 clinical fractions was significantly higher than that before RT, especially in clinically radiosensitive patients. The γ-H2AX assay may have potential for screening individual radiosensitivity of breast cancer patients.

  4. Use of γ-H2AX Foci Assay on Human Peripheral Blood Lymphocytes as Sensitive Biomarker of Exposure

    International Nuclear Information System (INIS)

    Gajski, G.; Garaj-Vrhovac, V.; Geric, M.; Filipic, M.; Nunic, J.; Straser, A.; Zegura, B.

    2013-01-01

    In modern medicine, it is impossible to imagine diagnostics and treatments without equipment that emit radiation (X-ray, CT, PET, etc.). At the same time there is a need to minimize the amount of radiation that the patient will gain during such medical examination. In that manner ALARA (As Low As Reasonably Achievable) principle and dosimetry are the bases of assuring patients safety. The induction of gamma phosphorylated H2AX histone is newly developed tool in biodosimetry, which is more sensitive for the detection of radiation caused DNA damage than currently used micronucleus and comet assay. Gamma phosphorylation of H2AX histone is a consequence of DNA double strand breaks and its role is to trigger the DNA repair mechanisms. In this study, we tested the effect of 2 and 4 Gy X-rays on human peripheral blood lymphocytes from two healthy volunteers using γ-H2AX foci assay. The FITC signal from labelled antibodies was monitored using flow cytometry and clearly demonstrated the difference in control samples and irradiated samples. There was also the difference between the exposed blood samples from the two volunteers. The results of present study reveal new sensitive method that is capable of detecting changes in DNA when exposed to different doses of radiation, and thus potentially optimizing the ALARA principle.(author)

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

    Science.gov (United States)

    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

  6. The quiescent and mitogen stimulated peripheral blood mononuclear cells after gamma irradiation and their P53, P21 and H2AX expression

    International Nuclear Information System (INIS)

    Vilasova, Z.; Vavrova, J.; Sinkorova, Z.; Tichy, A.; Oesterreicher, J.; Rezacova, M.; Zoelzer, F.

    2008-01-01

    , phosphorylation at serine-15 was dose-dependent, while phosphorylation at serine-392 was not. We have shown that irradiation of stimulated lymphocytes causes an increase in phosphorylation of histone H2A.X, an increase in p53 and its phosphorylation, and an increase in p21. ELISA measurement of p53 phosphorylated on serine-15 and p21 can be used as indicator of received dose of IR. (authors)

  7. Histones H10a and H10b are the same as CHO histones H1(III) and H1(IV):new features of H10 phosphorylation during the cell cycle

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.

    1981-01-01

    Two histone H1 fractions [H1(I) and H1(II) and two histone H1 0 fractions (H1 0 a and H1 0 b) have been isolated from butyrate-treated Chinese hamster (line CHO) cells by guanidine hydrochloride gradient chromatography on Bio-Rex 70 ion-exchange resin. The fractions have been identified by electrophoresis and amino acid analyses. Electrophoretic analysis of cyanogen bromide treated H1 0 in long acid-urea-polyacrylamide gels suggests that H1 0 a and H1 0 b differ, at least, within the 20-30 residue fragment(s) removed by the cyanogen bromide clevage. Shallow-gradient Bio-Rex 70 chromatography indicates that histones H1 0 a and H1 0 b are the same as the respective CHO histones, H1(III) and H1(IV). This identification and the phosphate incorporation data of Gurley et al. (1975) reveal new features about H1 0 phosphorylation: (1) following release from G 1 arrest, H1 0 a and H1 0 b become phosphorylated in late G 1 prior to DNA synthesis; (2) H1 0 a and H1 0 b are phosphorylated at similar rates throughout the cell cycle. These and other data demonstrate that histone H1 0 is phosphorylated in a cell cycle dependent fashion which mimics that of histone H1

  8. Comparison of the induction and disappearance of DNA double strand breaks and gamma-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells.

    Science.gov (United States)

    Kato, Takamitsu A; Okayasu, Ryuichi; Bedford, Joel S

    2008-03-01

    The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the gamma-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the gamma-H2AX assay which depends on extensive phosphorylation and dephosphorylation of the H2AX histone variant surrounding DSBs. The disappearance of gamma-H2AX foci after irradiation was much slower for mitotic than for G1 cells. On the other hand, no difference was seen for the gel electrophoresis assay. Our data may suggest the limited accessibility of dephosphorylation enzyme in irradiated metaphase cells or trapped gamma-H2AX in condensed chromatin.

  9. Comparison of the induction and disappearance of DNA double strand breaks and γ-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells

    International Nuclear Information System (INIS)

    Kato, Takamitsu A.; Okayasu, Ryuichi; Bedford, Joel S.

    2008-01-01

    The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the γ-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the γ-H2AX assay which depends on extensive phosphorylation and dephosphorylation of the H2AX histone variant surrounding DSBs. The disappearance of γ-H2AX foci after irradiation was much slower for mitotic than for G1 cells. On the other hand, no difference was seen for the gel electrophoresis assay. Our data may suggest the limited accessibility of dephosphorylation enzyme in irradiated metaphase cells or trapped γ-H2AX in condensed chromatin

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. ► A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. ► DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. ► DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. ► 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 γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-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 γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation

  12. γH2AX foci as a marker for DNA double-strand breaks

    International Nuclear Information System (INIS)

    Deckbar, Dorothee

    2009-01-01

    Full text: The DNA double-strand break (DSB) is the most deleterious lesion of all DNA damages. Left unrepaired or being mis-rejoined it can lead to chromosome aberrations which compromise the genomic stability and carry the potential to initiate carcinogenesis. So DSB repair mechanisms are under intensive investigation for many years. As older techniques had to utilize non-physiological doses to monitor DSB repair, they did not allow repair studies on the cellular level or after in vivo irradiation. But during the last years, an upcoming method allows the detection of a single DSB after physiologically relevant doses. To maintain the genomic integrity after the occurrence of a DSB, cellular mechanisms have evolved that detect and repair DSBs and even halt cell cycle progression to provide time for repair. In these processes, one of the first steps is the phosphorylation of the histone H2AX at serine 139 (γH2AX). Within minutes after DSB induction, large numbers of H2AX molecules are phosphorylated around the break site leading to the accumulation of proteins involved in chromatin remodelling, to damage signal amplification, and eventually to checkpoint activation and DSB repair. The finding that DSB-surrounding proteins can be visualized as foci in immunofluorescence microscopy opened up new opportunities in cancer biology and radiation biology. It was now for the first time possible to measure DSB repair after physiologically relevant doses of ionizing radiation, i.e. after doses used for therapeutic as well as for diagnostic purposes. First reports even describe the measurement of DSB repair after in vivo irradiation in mice and humans. This did not only improve the basic research investigating the mechanisms of DSB repair but also the research on low-dose effects and radiation protection. So the potential of γH2AX foci analysis as a predictive marker for radiosensitivity or radiation induced side effects is actually discussed. (author)

  13. NoRC Recruitment by H2A.X Deposition at rRNA Gene Promoter Limits Embryonic Stem Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Boris Eleuteri

    2018-05-01

    Full Text Available Summary: Embryonic stem cells (ESCs display an abbreviated cell cycle, resulting in a short doubling time and rapid proliferation. The histone variant H2A.X is critical for proliferation of stem cells, although mechanistic insights have remained obscure. Here, we show that H2A.X defines the rate of mouse ESC proliferation independently of the DNA damage response pathway, and it associates with three major chromatin-modifying complexes. Our functional and biochemical analyses demonstrate that H2A.X-associated factors mediate the H2A.X-dependent effect on ESC proliferation and involve the nucleolar remodeling complex (NoRC. A specific H2A.X deposition at rDNA promoters determines the chromatin recruitment of the NoRC, histone modifications, the rRNA transcription, and the rate of proliferation. Collectively, our results suggest that NoRC assembly by H2A.X deposition at rRNA promoters silences transcription, and this represents an important regulatory component for ESC proliferation. : Histone variant H2A.X defines the rate of embryonic stem cell proliferation. Eleuteri et al. identify H2A.X-interacting proteins, and they show that H2A.X deposition at rDNA promoters assembles the NoRC, which represses rRNA transcription and determines the rate of self-renewal. Keywords: ribosomal biogenesis, rRNA, rDNA, stem cells, TIP5, SNF2H, SPT16, BRG1, H2A.X, G1, cell cycle, cell cycle arrest, proliferation

  14. Post-Translational Modifications of H2A Histone Variants and Their Role in Cancer

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    David Corujo

    2018-02-01

    Full Text Available Histone variants are chromatin components that replace replication-coupled histones in a fraction of nucleosomes and confer particular characteristics to chromatin. H2A variants represent the most numerous and diverse group among histone protein families. In the nucleosomal structure, H2A-H2B dimers can be removed and exchanged more easily than the stable H3-H4 core. The unstructured N-terminal histone tails of all histones, but also the C-terminal tails of H2A histones protrude out of the compact structure of the nucleosome core. These accessible tails are the preferential target sites for a large number of post-translational modifications (PTMs. While some PTMs are shared between replication-coupled H2A and H2A variants, many modifications are limited to a specific histone variant. The present review focuses on the H2A variants H2A.Z, H2A.X, and macroH2A, and summarizes their functions in chromatin and how these are linked to cancer development and progression. H2A.Z primarily acts as an oncogene and macroH2A and H2A.X as tumour suppressors. We further focus on the regulation by PTMs, which helps to understand a degree of context dependency.

  15. Super-Resolution Localization Microscopy of γ-H2AX and Heterochromatin after Folate Deficiency.

    Science.gov (United States)

    Bach, Margund; Savini, Claudia; Krufczik, Matthias; Cremer, Christoph; Rösl, Frank; Hausmann, Michael

    2017-08-08

    Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with "high-risk" human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access

  16. Microscopic evaluation of nuclear foci (gamma H2AX) in cells irradiated with protons and lithium ions

    International Nuclear Information System (INIS)

    Bracalente, C.; Molinari, Beatriz L.; Duran, Hebe; Ibanez, I.; Palmieri, M.; Kreiner, Andres J.; Burlon, Alejandro; Valda, Alejandro; Davidson, J.; Davidson, M.; Vazquez, Monica; Ozafran, Mabel J.

    2007-01-01

    Full text: The special properties of both physical and biological radiation particles with high-LET (Linear Transfer of Energy) have led to its increased use in cancer therapy. In this work, the effect of high and low LET radiation on cell lines with different radiosensitivity (Irs-20 and CHO-10B2) quantifying the number and size of nuclear foci obtained from histone H2AXH2AX) phosphorylation which plays an important role in DNA damage reparation is compared. Foci detection was performed by immunocytochemical methods and fluorescence microscopy. The cells cultures were irradiated with plateau-phase protons (14 MeV, LET: 3 keV/μ), on Bragg peak (3 MeV. LET: 14 KeV/μ) and with Lithium ions (7 MeV, LET: 250 KeV//μ) on the Tandar accelerator. A clonogenic analysis of the two cell lines was made. Irradiation with protons (low LET) showed a significant difference (p [es

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

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    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.

  18. The foci of DNA double strand break-recognition proteins localize with γH2AX after heat treatment

    International Nuclear Information System (INIS)

    Takahashi, Akihisa; Mori, Eiichiro; Ohnishi, Takeo

    2010-01-01

    Recently, there have been many reports concerning proteins which can recognize DNA double strand break (DSBs), and such proteins include histone H2AX phosphorylated at serine 139 (γH2AX), ataxia telangiectasia mutated (ATM) phospho-serine 1981, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phospho-threonine 2609, Nijmegen breakage syndrome 1 (NBS1) phospho-serine 343, checkpoint kinase 2 (CHK2), phospho-threonine 68, and structural maintenance of chromosomes 1 (SMC1) phospho-serine 966. Thus, it should be possible to follow the formation of DSBs and their repair using immunohistochemical methods with multiple antibodies to detect these proteins. When normal human fibroblasts (AG1522 cells) were exposed to 3 Gy of X-rays as a control, clearly discernable foci for these proteins were detected, and these foci localized with γH2AX foci. After heat treatment at 45.5 deg C for 20 min, these proteins are partially localized with γH2AX foci. Here we show that there were slight differences in the localization pattern among these proteins, such as a disappearance from the nucleus (phospho-ATM) and translocation to the cytoplasm (phospho-NBS1) at 30 min after heat treatment, and some foci (phospho-DNA-PKcs and phospho-CHK2) appeared at 8 h after heat treatment. These results are discussed from perspectives of heat-induced denaturation of proteins and formation of DSBs. (author)

  19. γ-H2AX foci are increased in lymphocytes in vivo in young children 1 h after very low-dose X-irradiation: a pilot study

    International Nuclear Information System (INIS)

    Halm, Brunhild M.; Franke, Adrian A.; Lai, Jennifer F.; Turner, Helen C.; Brenner, David J.; Zohrabian, Vatche M.; DiMauro, Robert

    2014-01-01

    Computed tomography (CT) is an imaging modality involving ionizing radiation. The presence of γ-H2AX foci after low to moderate ionizing radiation exposure has been demonstrated; however it is unknown whether very low ionizing radiation exposure doses from CT exams can induce γ-H2AX formation in vivo in young children. To test whether very low ionizing radiation doses from CT exams can induce lymphocytic γ-H2AX foci (phosphorylated histones used as a marker of DNA damage) formation in vivo in young children. Parents of participating children signed a consent form. Blood samples from three children (ages 3-21 months) undergoing CT exams involving very low blood ionizing radiation exposure doses (blood doses of 0.22-1.22 mGy) were collected immediately before and 1 h post CT exams. Isolated lymphocytes were quantified for γ-H2AX foci by a technician blinded to the radiation status and dose of the patients. Paired t-tests and regression analyses were performed with significance levels set at P < 0.05. We observed a dose-dependent increase in γ-H2AX foci post-CT exams (P = 0.046) among the three children. Ionizing radiation exposure doses led to a linear increase of foci per cell in post-CT samples (102% between lowest and highest dose). We found a significant induction of γ-H2AX foci in lymphocytes from post-CT samples of three very young children. When possible, CT exams should be limited or avoided by possibly applying non-ionizing radiation exposure techniques such as US or MRI. (orig.)

  20. γ-H2AX foci are increased in lymphocytes in vivo in young children 1 h after very low-dose X-irradiation: a pilot study

    Energy Technology Data Exchange (ETDEWEB)

    Halm, Brunhild M.; Franke, Adrian A.; Lai, Jennifer F. [University of Hawaii Cancer Center, Honolulu, HI (United States); Turner, Helen C.; Brenner, David J.; Zohrabian, Vatche M. [Columbia University Medical Center, Center for Radiological Research, New York, NY (United States); DiMauro, Robert [Kapi' olani Medical Center for Women and Children, Honolulu, HI (United States)

    2014-10-15

    Computed tomography (CT) is an imaging modality involving ionizing radiation. The presence of γ-H2AX foci after low to moderate ionizing radiation exposure has been demonstrated; however it is unknown whether very low ionizing radiation exposure doses from CT exams can induce γ-H2AX formation in vivo in young children. To test whether very low ionizing radiation doses from CT exams can induce lymphocytic γ-H2AX foci (phosphorylated histones used as a marker of DNA damage) formation in vivo in young children. Parents of participating children signed a consent form. Blood samples from three children (ages 3-21 months) undergoing CT exams involving very low blood ionizing radiation exposure doses (blood doses of 0.22-1.22 mGy) were collected immediately before and 1 h post CT exams. Isolated lymphocytes were quantified for γ-H2AX foci by a technician blinded to the radiation status and dose of the patients. Paired t-tests and regression analyses were performed with significance levels set at P < 0.05. We observed a dose-dependent increase in γ-H2AX foci post-CT exams (P = 0.046) among the three children. Ionizing radiation exposure doses led to a linear increase of foci per cell in post-CT samples (102% between lowest and highest dose). We found a significant induction of γ-H2AX foci in lymphocytes from post-CT samples of three very young children. When possible, CT exams should be limited or avoided by possibly applying non-ionizing radiation exposure techniques such as US or MRI. (orig.)

  1. Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

    International Nuclear Information System (INIS)

    Blattmann, C.; Oertel, S.; Thiemann, M.; Weber, K.J.; Schmezer, P.; Zelezny, O.; Lopez Perez, R.; Kulozik, A.E.; Debus, J.; Ehemann, V.

    2012-01-01

    Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors. (orig.)

  2. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  3. Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study.

    Directory of Open Access Journals (Sweden)

    Michael Brand

    Full Text Available Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes.Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC and Q 10. Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX.For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%, selenium (14%, vitamin E (12%, vitamin C (25%, NAC (43% and Q 10 (18% led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect.Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes.

  4. Histones and their phosphorylation during germination of rice seeds

    International Nuclear Information System (INIS)

    Iqbal Ahmed, C.M.; Padayatti, J.D.

    1980-01-01

    Histones from nuclei of rice embryos were identified by their mobilities on 15% acid-urea polyacrylamide gel electrophoreogram, incorporation of ( 3 H)lysine and ( 14 C) arginine and lack of incorporation of ( 3 H)tryptophan. The ratio of histone to DNA in ungerminated embryos was 2.7 which decreased during germination reaching unity by 48 hr. There was preferential phosphorylation of lysine-rich histones, which paralleled the synthesis of DNA. In the presence of cytosine arabinoside and mitomycin-C, which inhibited the synthesis of DNA to the extend of 75-80% the phosphorylation of lysine-rich histone was reduced by 50-60% suggesting the dependence of phosphorylation on the ongoing synthesis of DNA. (auth.)

  5. Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

    Science.gov (United States)

    Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

    2010-01-01

    Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  7. Microwaves from GSM mobile telephones affect 53BP1 and gamma-H2AX foci in human lymphocytes from hypersensitive and healthy persons.

    Science.gov (United States)

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

    2005-09-01

    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 (gamma-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/gamma-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.

  8. A non-linear detection of phospho-histone H2AX in EA.hy926 endothelial cells following low-dose X-irradiation is modulated by reactive oxygen species

    International Nuclear Information System (INIS)

    Large, Martin; Reichert, Sebastian; Hehlgans, Stephanie; Fournier, Claudia; Rödel, Claus; Rödel, Franz

    2014-01-01

    A discontinuous dose response relationship is a major characteristic of the anti-inflammatory effects of low-dose X-irradiation therapy. Although recent data indicate an involvement of a variety of molecular mechanisms in these characteristics, the impact of reactive oxygen species (ROS) production to give rise or contribute to these phenomena in endothelial cells (EC) remains elusive. HUVEC derived immortalized EA.hy926 cells were stimulated by tumor necrosis factor-α (TNF-α, 20 ng/ml) 4 h before irradiation with doses ranging from 0.3 to 1 Gy. To analyse DNA repair capacity, phospho-histone H2AX foci were assayed at 1 h, 4 h and 24 h after irradiation. ROS production and superoxide dismutase (SOD) activity were analysed by fluorometric 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCFDA) and colorimetric assays. A functional impact of ROS on γH2AX production was analysed by treatment with the scavenger N-acetyl-L-cysteine (NAC). Irrespective of stimulation by TNF-α, EA.hy926 cells revealed a linear dose response characteristic of γH2AX foci detection at 1 h and 4 h after irradiation. By contrast, we observed a discontinuity in residual γH2AX foci detection at 24 h after irradiation with locally elevated values following a 0.5 Gy exposure that was abolished by inhibition of ROS by NAC. Moreover, SOD protein expression was significantly decreased at doses of 0.5 Gy and 0.7 Gy concomitant with a reduced SOD activity. These data implicate a non-linear regulation of ROS production and SOD activity in EA.hy926 EC following irradiation with doses < 1 Gy that may contribute to a discontinuous dose-response relationship of residual γH2AX foci detection

  9. Histone phosphorylation during radiation-induced mitotic delay in synchronous plasmodia of Physarum polycephalum

    International Nuclear Information System (INIS)

    Brewer, E.N.; Oleinick, N.L.

    1980-01-01

    Using the nearly perfect synchrony of the mitotic stages in Physarum plasmodia, and making use of 32 P as a tracer, studies were made to define the time course of histone phosphorylation during the late G2 and prophase and the alterations in that time course accompanying radiation-induced mitotic delay. Histone H1 was phosphorylated throughout the last 2-3 hours of the mitotic cycle coincident with the early stages of chromosome condensation. H1 phosphorylation appeared to be reduced in irradiated plasmodia. It is postulated that a longer time period, i.e. the mitotic delay, may be required to obtain the same eventual level of H1-phosphate. In normal cultures, nucleosome core histones were phosphorylated late in G2 and prophase, the peak corresponding closely with the γ-transition point. In irradiated plasmodia, phosphorylation of the core histones had an extended time course similar to H1. (U.K.)

  10. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

    Directory of Open Access Journals (Sweden)

    Justin W Leung

    2014-03-01

    Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

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

    Directory of Open Access Journals (Sweden)

    Ly-Thuy-Tram Le

    2013-02-01

    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.

  12. Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation

    OpenAIRE

    Metzger, Eric; Yin, Na; Wissmann, Melanie; Kunowska, Natalia; Fischer, Kristin; Friedrichs, Nicolaus; Patnaik, Debasis; Higgins, Jonathan M.G.; Potier, Noelle; Scheidtmann, Karl-Heinz; Buettner, Reinhard; Schüle, Roland

    2007-01-01

    Posttranslational modifications of histones such as methylation, acetylation, and phosphorylation regulate chromatin structure and gene expression. Here we show that protein kinase C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor (AR) target genes. PRK1 is pivotal to AR function since PRK1 knockdown or inhibition impedes AR-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphor...

  13. Ultrasound-induced DNA damage and signal transductions indicated by gammaH2AX

    Science.gov (United States)

    Furusawa, Yukihiro; Fujiwara, Yoshisada; Zhao, Qing-Li; Hassan, Mariame Ali; Ogawa, Ryohei; Tabuchi, Yoshiaki; Takasaki, Ichiro; Takahashi, Akihisa; Ohnishi, Takeo; Kondo, Takashi

    2011-09-01

    Ultrasound (US) has been shown to induce cancer cell death via different forms including apoptosis. Here, we report the potential of low-intensity pulsed US (LIPUS) to induce genomic DNA damage and subsequent DNA damage response. Using the ionizing radiation-induced DNA double-strand breaks (DSBs) as the positive control, we were able to observe the induction of DSBs (as neutral comet tails) and the subsequent formation of gammaH2AX-positive foci (by immunofluorescence detection) in human leukemia cells following exposure to LIPUS. The LIPUS-induced DNA damage arose most likely from the mechanical, but not sonochemical, effect of cavitation, based on our observation that the suppression of inertial cavitation abrogated the gammH2AX foci formation, whereas scavenging of free radical formation (e.g., hydroxyl radical) had no protective effect on it. Treatment with the specific kinase inhibitor of ATM or DNA-PKcs, which can phosphorylate H2AX Ser139, revealed that US-induced gammaH2AX was inhibited more effectively by the DNA-PK inhibitor than ATM kinase inhibitor. Notably, these inhibitor effects were opposite to those with radiation-induced gammH2AX. In conclusion, we report, for the first time that US can induce DNA damage and the DNA damage response as indicated by gammaH2AX was triggered by the cavitational mechanical effects. Thus, it is expected that the data shown here may provide a better understanding of the cellular responses to US.

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

    Science.gov (United States)

    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.

  15. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    International Nuclear Information System (INIS)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay

    2015-01-01

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

  16. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  17. Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Li, Binbin; Huang, Guoliang; Zhang, Xiangning; Li, Rong; Wang, Jian; Dong, Ziming; He, Zhiwei

    2013-01-01

    Increased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC). The expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed. Immunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (χ 2 =6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1-induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation. EBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells.

    Science.gov (United States)

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M; Garcia, Benjamin A

    2016-07-15

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells*

    Science.gov (United States)

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M.; Garcia, Benjamin A.

    2016-01-01

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. PMID:27226594

  1. Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.

    Science.gov (United States)

    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

    2011-03-01

    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.

  2. miR-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated blood cells

    Science.gov (United States)

    Lal, Ashish; Pan, Yunfeng; Navarro, Francisco; Dykxhoorn, Derek M.; Moreau, Lisa; Meire, Eti; Bentwich, Zvi; Lieberman, Judy; Chowdhury, Dipanjan

    2010-01-01

    Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3’UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damage-induced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. PMID:19377482

  3. Q{sub {gamma}-H2AX}, an analysis method for partial-body radiation exposure using {gamma}-H2AX in non-human primate lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Redon, Christophe E., E-mail: redonc@mail.nih.gov [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Nakamura, Asako J.; Gouliaeva, Ksenia [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Rahman, Arifur; Blakely, William F. [Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603 (United States); Bonner, William M. [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States)

    2011-09-15

    We previously used the {gamma}-H2AX assay as a biodosimeter for total-body irradiation (TBI) exposure ({gamma}-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant {gamma}-H2AX responses days after total-body exposure to 1-8.5 Gy ({sup 60}Co {gamma}-rays at 55 cGy min{sup -1}). Here, we introduce a partial-body exposure analysis method, Q{sub {gamma}-H2AX}, which is based on the number of {gamma}-H2AX foci per damaged cells as evident by having one or more {gamma}-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q{sub {gamma}-H2AX} dose-response calibration curves to assess acute partial-body exposures. {gamma}-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of {gamma}-H2AX may provide a robust biodosimeter for analyzing partial-body exposures to ionizing radiation in humans.

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

    Science.gov (United States)

    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

  5. Staining Against Phospho-H2AX (gamma-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells

    NARCIS (Netherlands)

    Nagelkerke, A.P.; Span, P.N.

    2016-01-01

    Phospho-H2AX or gamma-H2AX- is a marker of DNA double-stranded breaks and can therefore be used to monitor DNA repair after, for example, irradiation. In addition, positive staining for phospho-H2AX may indicate genomic instability and telomere dysfunction in tumour cells and tissues. Here, we

  6. Induction and disappearance of γH2AX foci and formation of micronuclei after exposure of human lymphocytes to ⁶⁰Co γ-rays and p(66)+ Be(40) neutrons.

    Science.gov (United States)

    Vandersickel, Veerle; Beukes, Philip; Van Bockstaele, Bram; Depuydt, Julie; Vral, Anne; Slabbert, Jacobus

    2014-02-01

    To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (γH2AX foci) after in vitro exposure of human lymphocytes to either (60)Co γ-rays or p(66)+ Be(40) neutrons. MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0-4 Gy γ-rays or 0-2 Gy neutrons. Also, γH2AX foci DR curves were obtained following exposure to doses ranging from 0-0.5 Gy of either γ-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy γ-rays or neutrons were studied up to 24 hours post-irradiation. Micronuclei yields following neutron exposure were consistently higher compared to that from (60)Co γ-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced γH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from γ-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from γ-rays. Foci formations were more likely to be over-dispersed for neutron irradiations. Although neutrons are more effective to induce MN, the absolute number of induced γH2AX foci are less at first compared to γ-rays. With time neutron-induced foci are more persistent. These findings are helpful for using γH2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

  7. X-ray induced formation of γ-H2AX foci after full-field digital mammography and digital breast-tomosynthesis.

    Directory of Open Access Journals (Sweden)

    Siegfried A Schwab

    Full Text Available 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.

  8. Phosphorylation of rat thymus histones, its control and the effects thereon of γ-irradiation

    International Nuclear Information System (INIS)

    Fonagy, A.; Ord, M.G.; Stocken, L.A.

    1977-01-01

    The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32 P uptakes than did histones from resting liver nuclei; the other histones all showed 32 P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [γ- 32 P]ATP was incorporated into non-histone proteins, including Pl, and into the ADP-ribosylated form of histone 1; negligible 32 P was incorporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein Pl was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. γ-irradiation decreased 32 P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed. (author)

  9. High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Giovannini, Caterina [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy); Piaggi, Simona [Sezione di Patologia Sperimentale, Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Pisa University, Pisa (Italy); Federico, Giovanni [Unità di Endocrinologia Pediatrica e Diabete, Dipartimento di Medicina Clinica e Sperimentale Pisa University, Pisa (Italy); Scarpato, Roberto, E-mail: roberto.scarpato@unipi.it [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy)

    2014-12-15

    Highlights: • We aimed to detect signs of very early damage in peripheral cells of T1DM adolescents. • T1DM patients had high levels of oxidized cells and reduced expression of iNOS and NO. • Highly mutagenic lesions were markedly increased in the diabetic group, mainly in females. • The observed damage might increase the risk of cancer in the patients later in life. - Abstract: Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H{sub 2}O{sub 2}-induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O{sub 2}{sup −}; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H{sub 2}O{sub 2}-induced (47.13 ± 0.02) were significantly higher than in controls (35.90 ± 0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15 ± 1.08% of γ-H2AX{sup +} cells; 8.72 ± 2.14 γ-H2AXF/n; 9.26 ± 2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents

  10. High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes

    International Nuclear Information System (INIS)

    Giovannini, Caterina; Piaggi, Simona; Federico, Giovanni; Scarpato, Roberto

    2014-01-01

    Highlights: • We aimed to detect signs of very early damage in peripheral cells of T1DM adolescents. • T1DM patients had high levels of oxidized cells and reduced expression of iNOS and NO. • Highly mutagenic lesions were markedly increased in the diabetic group, mainly in females. • The observed damage might increase the risk of cancer in the patients later in life. - Abstract: Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H 2 O 2 -induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O 2 − ; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H 2 O 2 -induced (47.13 ± 0.02) were significantly higher than in controls (35.90 ± 0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15 ± 1.08% of γ-H2AX + cells; 8.72 ± 2.14 γ-H2AXF/n; 9.26 ± 2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents. This also indicates that

  11. Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

    Science.gov (United States)

    Thiriet, Christophe; Hayes, Jeffrey J.

    2009-01-01

    Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

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

    Energy Technology Data Exchange (ETDEWEB)

    Bracalente, Candelaria; Ibañez, Irene L. [Departamento de Micro y Nanotecnología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Molinari, Beatriz [Departamento de Radiobiología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Palmieri, Mónica [Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina); Kreiner, Andrés [Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Gerencia de Investigación y Aplicaciones, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); Valda, Alejandro [Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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 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

  14. Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks.

    Science.gov (United States)

    Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; Vasireddy, Raja S; El-Osta, Assam; Karagiannis, Tom C

    2011-01-25

    Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

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

    Directory of Open Access Journals (Sweden)

    Miriam Fritsche

    Full Text Available 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*.

  16. Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons

    DEFF Research Database (Denmark)

    Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada

    2016-01-01

    Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol...... of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation...... of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K...

  17. Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Shisheng Li

    2012-09-01

    Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

  18. Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new Histones H3 and H4

    International Nuclear Information System (INIS)

    Jackson, V.

    1987-01-01

    The authors have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposits as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution of density gradients

  19. cAMP Signaling Regulates Histone H3 Phosphorylation and Mitotic Entry Through a Disruption of G2 Progression

    OpenAIRE

    Rodriguez-Collazo, Pedro; Snyder, Sara K.; Chiffer, Rebecca C.; Bressler, Erin A.; Voss, Ty C.; Anderson, Eric P.; Genieser, Hans-Gottfried; Smith, Catharine L.

    2008-01-01

    cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in ...

  20. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

    NARCIS (Netherlands)

    A. Lagarou (Anna); A.B. Mohd Sarip; Y.M. Moshkin (Yuri); G.E. Chalkley (Gillian); K. Bezstarosti (Karel); J.A.A. Demmers (Jeroen); C.P. Verrijzer (Peter)

    2008-01-01

    textabstractTranscription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was

  1. A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

    Directory of Open Access Journals (Sweden)

    Miriam Sansó

    Full Text Available Transcript elongation by RNA polymerase II (RNAPII is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1 plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb, in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell

  2. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

    Science.gov (United States)

    Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

    2013-01-01

    Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

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

    DEFF Research Database (Denmark)

    Klimovskaia, Ilnaz M; Young, Clifford; Strømme, Caroline B

    2014-01-01

    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...

  4. Identification of the elementary structural units of the DNA damage response.

    Science.gov (United States)

    Natale, Francesco; Rapp, Alexander; Yu, Wei; Maiser, Andreas; Harz, Hartmann; Scholl, Annina; Grulich, Stephan; Anton, Tobias; Hörl, David; Chen, Wei; Durante, Marco; Taucher-Scholz, Gisela; Leonhardt, Heinrich; Cardoso, M Cristina

    2017-06-12

    Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

  5. Increased histone H3 phosphorylation in neurons in specific brain structures after induction of status epilepticus in mice.

    Directory of Open Access Journals (Sweden)

    Tetsuji Mori

    Full Text Available 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.

  6. Phosphorylation-mediated control of histone chaperone ASF1 levels by Tousled-like kinases.

    Directory of Open Access Journals (Sweden)

    Maxim Pilyugin

    Full Text Available Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.

  7. Rapid divergence of histones in Hydrozoa (Cnidaria) and evolution of a novel histone involved in DNA damage response in hydra.

    Science.gov (United States)

    Reddy, Puli Chandramouli; Ubhe, Suyog; Sirwani, Neha; Lohokare, Rasika; Galande, Sanjeev

    2017-08-01

    Histones are fundamental components of chromatin in all eukaryotes. Hydra, an emerging model system belonging to the basal metazoan phylum Cnidaria, provides an ideal platform to understand the evolution of core histone components at the base of eumetazoan phyla. Hydra exhibits peculiar properties such as tremendous regenerative capacity, lack of organismal senescence and rarity of malignancy. In light of the role of histone modifications and histone variants in these processes it is important to understand the nature of histones themselves and their variants in hydra. Here, we report identification of the complete repertoire of histone-coding genes in the Hydra magnipapillata genome. Hydra histones were classified based on their copy numbers, gene structure and other characteristic features. Genomic organization of canonical histone genes revealed the presence of H2A-H2B and H3-H4 paired clusters in high frequency and also a cluster with all core histones along with H1. Phylogenetic analysis of identified members of H2A and H2B histones suggested rapid expansion of these groups in Hydrozoa resulting in the appearance of unique subtypes. Amino acid sequence level comparisons of H2A and H2B forms with bilaterian counterparts suggest the possibility of a highly mobile nature of nucleosomes in hydra. Absolute quantitation of transcripts confirmed the high copy number of histones and supported the canonical nature of H2A. Furthermore, functional characterization of H2A.X.1 and a unique variant H2A.X.2 in the gastric region suggest their role in the maintenance of genome integrity and differentiation processes. These findings provide insights into the evolution of histones and their variants in hydra. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Study of the dose-effect relationship of γ-H2AX radiation

    International Nuclear Information System (INIS)

    Cui Shuangshuang; Fan Yaguang; Gun Zhijuan; Wang Jixian; Zhao Yongcheng; Sun Yuping

    2010-01-01

    Objective: By using laser scanning confocal microscopy (LSCM) to test the dose-effects relationship between ionizing radiation intensity and quantity of the γ-H2AX in vivo and in vitro respectively, and the consistency relationship between the vivo and vitro retrial. Methods: To irradiate the peripheral blood from Wister female rats by 137 Cs at 7 with different doses (0, 0.5, 1, 2, 2.54, 4, 6, 8 Gy) extract the lymphocytes from the peripheral blood and detect the dose-effects relationship between irradiation intensity and number of γ-H2AX foci. Results: There are good dose-effects relationships between the irradiation and foci number both in vivo and in vitro, which are linear, Y vivo =0.096+0.13X; Y vitro =0.040+0.21X. And there is good consistency (R=0.98) between the γ-H2AX in vivo and in vitro. Conclusions: γ-H2AX has the possibility for clinical trial as an indicator, and we can use vitro trials in place of the vivo trails to evaluate the dose people received. (authors)

  9. Stress-induced brain histone H3 phosphorylation: contribution of the intensity of stressors and length of exposure.

    Science.gov (United States)

    Rotllant, David; Pastor-Ciurana, Jordi; Armario, Antonio

    2013-05-01

    Expression of c-fos is used for the characterization of brain areas activated by stressors. Recently, some epigenetic markers associated with enhanced transcription have been identified that may be also useful to detect neuronal populations important for the processing of stressors: phosphorylation of histone H3 in serine 10 or 28 (pH3S₁₀ or pH3S₂₈). Then, we compared in rats the response to stress of c-fos and these epigenetic changes. More specifically, we studied the influence of the type of stressor (novel environment vs. immobilization, IMO) and the dynamics of the response to IMO. Stress increased pH3S₁₀ positive neurons, with a more restricted pattern than that of c-fos, both in terms of brain areas activated and number of positive neurons. Changes in pH3S₁₀ showed a maximum at 30 min, then progressively declining in most areas in spite of the persistence of IMO. Moreover, the decline was in general more sensitive than c-fos to the termination of IMO. The pattern of pH3S₂₈ was even more restricted that of pH3S₁₀, but they showed co-localization. The present data demonstrate a more selective pattern of stress-induced histone H3 phosphorylation than c-fos. The factors determining such a selectivity and its biological meaning remain to be studied. © 2013 International Society for Neurochemistry.

  10. Distribution pattern of histone H3 phosphorylation at serine 10 ...

    Indian Academy of Sciences (India)

    2013-08-06

    Aug 6, 2013 ... tant consequences for chromatin packing due to change in histone load ... Minas Gerais, Brazil), in B. brizantha (cultivar Marandu, ... (2005), who state that the ..... Mitotic microtubule development and histone H3 phosphoryla-.

  11. Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons.

    Science.gov (United States)

    Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada; Hu, Xiaochen; Hellysaz, Arash; Falconi, Anastasia; Gomes, Ana-Luisa; Broberger, Christian; Hansen, Klaus; Fisone, Gilberto

    2016-08-01

    Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol, a typical antipsychotic drug, increase the phosphorylation of H3 at S28 and that this effect occurs in the context of H3K27me3. The increases in H3K27me3S28p occur in distinct populations of projection neurons located in the striatum, the major component of the basal ganglia. Genetic inactivation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K27me3S28p as a potential mediator of the effects exerted by amphetamine and haloperidol, and suggest that these drugs may act by re-activating PcG repressed target genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Preety M Sharma

    Full Text Available 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.

  13. Residual γH2AX foci as an indication of lethal DNA lesions

    Directory of Open Access Journals (Sweden)

    Banuelos C Adriana

    2010-01-01

    Full Text Available Abstract Background Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication. Methods To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. Results For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Conclusion Retention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci.

  14. Residual γH2AX foci as an indication of lethal DNA lesions

    International Nuclear Information System (INIS)

    Banáth, Judit P; Klokov, Dmitry; MacPhail, Susan H; Banuelos, C Adriana; Olive, Peggy L

    2010-01-01

    Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication. To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Retention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci

  15. Lestaurtinib inhibits histone phosphorylation and androgen-dependent gene expression in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Jens Köhler

    Full Text Available BACKGROUND: Epigenetics is defined as heritable changes in gene expression that are not based on changes in the DNA sequence. Posttranslational modification of histone proteins is a major mechanism of epigenetic regulation. The kinase PRK1 (protein kinase C related kinase 1, also known as PKN1 phosphorylates histone H3 at threonine 11 and is involved in the regulation of androgen receptor signalling. Thus, it has been identified as a novel drug target but little is known about PRK1 inhibitors and consequences of its inhibition. METHODOLOGY/PRINCIPAL FINDING: Using a focused library screening approach, we identified the clinical candidate lestaurtinib (also known as CEP-701 as a new inhibitor of PRK1. Based on a generated 3D model of the PRK1 kinase using the homolog PKC-theta (protein kinase c theta protein as a template, the key interaction of lestaurtinib with PRK1 was analyzed by means of molecular docking studies. Furthermore, the effects on histone H3 threonine phosphorylation and androgen-dependent gene expression was evaluated in prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: Lestaurtinib inhibits PRK1 very potently in vitro and in vivo. Applied to cell culture it inhibits histone H3 threonine phosphorylation and androgen-dependent gene expression, a feature that has not been known yet. Thus our findings have implication both for understanding of the clinical activity of lestaurtinib as well as for future PRK1 inhibitors.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated...

  18. Histone H2A mobility is regulated by its tails and acetylation of core histone tails

    International Nuclear Information System (INIS)

    Higashi, Tsunehito; Matsunaga, Sachihiro; Isobe, Keisuke; Morimoto, Akihiro; Shimada, Tomoko; Kataoka, Shogo; Watanabe, Wataru; Uchiyama, Susumu; Itoh, Kazuyoshi; Fukui, Kiichi

    2007-01-01

    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

  19. Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Qianzheng; Battu, Aruna; Ray, Alo; Wani, Gulzar; Qian, Jiang; He, Jinshan; Wang, Qi-en [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Wani, Altaf A., E-mail: wani.2@osu.edu [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210 (United States); James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 (United States)

    2015-06-15

    Highlights: • HDAC1 and HDAC2 co-localize with UV radiation-induced DNA damage sites. • HDAC1 translocation to chromatin is dependent on DDB2 function. • HDAC1 and HDAC2 are involved in H3K56Ac deacetylation. • H3K56Ac deacetylation requires DDB1 and DDB2 but not XPA or XPC functions. • HDAC1/2 depletion decreases XPC ubiquitination and local γH2AX accumulation. - Abstract: Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

  20. Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.

    Directory of Open Access Journals (Sweden)

    Mo-bin Cheng

    2014-12-01

    Full Text Available Histone lysine (K residues, which are modified by methyl- and acetyl-transferases, diversely regulate RNA synthesis. Unlike the ubiquitously activating effect of histone K acetylation, the effects of histone K methylation vary with the number of methyl groups added and with the position of these groups in the histone tails. Histone K demethylases (KDMs counteract the activity of methyl-transferases and remove methyl group(s from specific K residues in histones. KDM3A (also known as JHDM2A or JMJD1A is an H3K9me2/1 demethylase. KDM3A performs diverse functions via the regulation of its associated genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanism by which the activity of KDM3A is regulated is largely unknown. Here, we demonstrated that mitogen- and stress-activated protein kinase 1 (MSK1 specifically phosphorylates KDM3A at Ser264 (p-KDM3A, which is enriched in the regulatory regions of gene loci in the human genome. p-KDM3A directly interacts with and is recruited by the transcription factor Stat1 to activate p-KDM3A target genes under heat shock conditions. The demethylation of H3K9me2 at the Stat1 binding site specifically depends on the co-expression of p-KDM3A in the heat-shocked cells. In contrast to heat shock, IFN-γ treatment does not phosphorylate KDM3A via MSK1, thereby abrogating its downstream effects. To our knowledge, this is the first evidence that a KDM can be modified via phosphorylation to determine its specific binding to target genes in response to thermal stress.

  1. Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

    Science.gov (United States)

    Yang, Tao-Tao; Cao, Na; Zhang, Hai-Hui; Wei, Jian-Bo; Song, Xiao-Xia; Yi, Dong-Min; Chao, Shuai-Heng; Zhang, Li-Da; Kong, Ling-Fei; Han, Shuang-Yin; Yang, Yu-Xiu; Ding, Song-Ze

    2018-04-15

    Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased

  2. Effect of prolonging radiation delivery time on retention of gammaH2AX

    International Nuclear Information System (INIS)

    Moiseenko, Vitali; Banáth, Judit P; Duzenli, Cheryl; Olive, Peggy L

    2008-01-01

    Compared to conventional external beam radiotherapy, IMRT requires significantly more time to deliver the dose. Prolonging dose delivery potentially increases DNA repair which would reduce the biological effect. We questioned whether retention of γH2AX, a measure of lack of repair of DNA damage, would decrease when dose delivery was protracted. Exponentially growing SiHa cervical carinoma cells were irradiated with 6 MV photons in a water tank using a VarianEX linear accelerator. Cells held at 37°C received 2 Gy in 0.5 min and 4 Gy in 1 min. To evaluate effect of dose delivery prolongation, 2 and 4 Gy were delivered in 30 and 60 min. After 24 h recovery, cells were analyzed for clonogenic survival and for residual γH2AX as measured using flow cytometry. Increasing the dose delivery time from 0.5 or 1 min to 30 or 60 min produced a signficant increase in cell survival from 0.45 to 0.48 after 2 Gy, and from 0.17 to 0.20 after 4 Gy. Expression of residual γH2AX decreased from 1.27 to 1.22 relative to background after 2 Gy and 1.46 to 1.39 relative to background after 4 Gy, but differences were not statistically significant. The relative differences in the slopes of residual γH2AX versus dose for acute versus prolonged irradiation bordered on significant (p = 0.055), and the magnitude of the change was consistent with the observed increase in surviving fraction. These results support the concept that DNA repair underlies the increase in survival observed when dose delivery is prolonged. They also help to establish the limits of sensitivity of residual γH2AX, as measured using flow cytometry, for detecting differences in response to irradiation

  3. Characterization of the UV-crosslinked heterodimer of histones H2B and H4

    International Nuclear Information System (INIS)

    Johnson, E.R.; Brown, D.M.; DeLange, R.J.

    1986-01-01

    At relatively high salt concentrations (1.2 M), histone 2B (H2B) and histone 4 (H4) can be covalently crosslinked by irradiation with ultraviolet light to yield a mixture of the three possible dimers: H2B-H2B, H4-H4, and H2B-H4. The formation of the H2B-H4 heterodimer was found to be favored at lower histone concentrations (> 90% H2B-H4 at 0.1 mg/ml total histone protein). CNBr cleavage of the H2B-H4 dimer produced three fragments which were separated by reverse phase HPLC. These fragments were identified by amino acid compositional analysis to be H4(85-102), H2B(62-125), and the crosslinked N-terminal regions H2B(1-59)-H4(1-84). Amino acid sequence analysis of the crosslinked fragment indicated that tyrosine-40 of H2B is likely involved in the covalent crosslinkage which joins the histone monomers to form the heterodimer

  4. Histone H1 interphase phosphorylation becomes largely established in G1 or early S phase and differs in G1 between T-lymphoblastoid cells and normal T cells

    Directory of Open Access Journals (Sweden)

    Gréen Anna

    2011-08-01

    Full Text Available Abstract Background Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G1, S and G2/M populations. Results We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G1 or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. Conclusion Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G1 may be affecting the G1/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.

  5. Neutron scattering studies of the H2a-H2b and (H3-H4)2 histone complexes

    International Nuclear Information System (INIS)

    Carlson, R.D.

    1984-01-01

    Neutron scattering experiments have shown that both the (H3-H4)2 and H2a-H2b histone complexes are quite asymmetric in solution. The (H3-H4)2 tetramer is an oblate or flattened structure, with a radius of gyration almost as large as that of the core octamer. If the tetramer is primarily globular, it must have an axial ratio of about 1:5. It is more likely, however, that this asymmetry results in part from N-terminal arms that extend outward approximately within the major plane of the particle. If this is the case, less asymmetric models for the globular part of the tetramer, including a dislocated disk of the type proposed by Klug et al. (23), can be made consistent with the scattering data. The H2a-H2b dimer, on the other hand, is an elongated structure. The low resolution data are in good agreement with those calculated for a cylindrical model 64 X 27 A, but other elongated models fit those data almost as well, including one that approximates free N-terminal arms at each end. Free arms are not necessary, but they must extend from the ends if they exist. A contrast matching experiment done with 50% deuterated H2b and undeuterated H2a in the reconstituted dimer showed that these two histones must each be rather elongated within the complex and are not just confined to one end. The amount of scattering contrast between the undeuterated and 50% deuterated histones was sufficient to suggest further experiments using complexes reconstituted from mixtures of undeuterated and partially deuterated histones which will help elucidate their arrangement within the histone complexes and within the octamer core of the nucleosome core particle

  6. DNA damage and γH2AX expression in EJ cells induced by 60Co gamma-rays

    International Nuclear Information System (INIS)

    Pan Yan; Tian Mei; Liu Jianxiang; Ruan Jianlei; Su Xu

    2009-01-01

    Objective: To investigate 60 Co γ-rays induced DNA damage of human bladder cancer cell line EJ cells and the relationship between different doses of 60 Co γ-rays, γH2AX foci number and γH2AX expression level. Methods: EJ cells were exposed to different doses of 60 Co γ-rays and the oliver tail moment (TM) of EJ cells were analyzed with single cell gel electrophoresis (SCGE) . Immunofluorescent microscopy was used to analysis γH2AX foci formation in EJ cells after exposure to different doses of γ-ray irradiation and time-course after exposure to 2 Gy γ-ray irradiation. FACSAria was used to detect the changes of γH2AX protein expression in EJ cells. Results: The TMs of EJ cells were increased with the irradiation dose. The TM of 0 Gy group and 4 Gy group was 0.24 and 5.26, respectively. Immunofluorescent analysis demonstrated that γH2AX foci could be induced by γ-ray irradiation in dose-dependent and time-dependent manners. The foci number and size in nuclei of EJ cells was significantly increased after exposed to different doses of γ-ray irradiation and the foci remained detectable at 24 h after exposed to irradiation. The dose range in which foci could be clearly detected was from 0.1 to 4 Gy. FACSDiva showed that γH2AX protein expression was increased after exposure to different doses of γ-ray irradiation. γH2AX protein expression level of 0.1 Gy group and 4 Gy group was 7.4% and 29.2% , respectively. Conclusions: γH2AX foci could be the most sensitive indicator for DNA damage and repair in mammalian cells, and it might be a new biomarker for radiological emergency. (authors)

  7. Different reaction of core histones H2A and H2B to the red laser radiation

    Directory of Open Access Journals (Sweden)

    Brill G.E.

    2017-09-01

    Full Text Available Aim: to investigate the influence of red laser irradiation on the processes of self-assembly of core histones H2A and H2B. Material and Methods. Solutions of human histone proteins were used in the work. Self-assembly was studied by the method of wedge dehydration. Image facies analysis consisted in their qualitative characterization and calculation of quantitative indicators with subsequent statistical processing. Results. It was established that linearly polarized laser light of the red region of the spectrum (A=660 nm, 1 J/cm2 significantly modifies the process of self-assembly of core histone H2B, while the structure of the facies of H2A histone changing to a lesser extent. Conclusion. Red laser radiation influences on the on the processes of self-assembly of core histones H2A and H2B. There is a differential sensitivity of different classes of histones to laser action. Histone proteins used in the experiments are present in the form of aqueous salt solutions. Red light realizes the effect seems to be due to the formation of singlet oxygen by direct laser excitation of molecular oxygen.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Niu, H., E-mail: hniu@mx.nthu.edu.tw [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Chang, H.C. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China); Cho, I.C. [Institute for Radiological Research, Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, C.H. [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, C.S. [Cancer Center of Taipei Veterans General Hospital, Taipei, Taiwan (China); Chou, W.T. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China)

    2014-08-15

    Highlights: • Charged particles can induce more complex DNA damages, and these complex damages have higher ability to cause the cell death or cell carcinogenesis. • In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in particle irradiated HeLa cells. • The HeLa cells were irradiated by 400 keV alpha-particles in four different dosages. • The result shows that a good linear relationship can be observed between foci number and radiation dose. • The data shows that the dissolution rate of γ-H2AX foci agree with the two components DNA repairing model, and it was decreasing as the radiation dose increased. • These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair. - Abstract: 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

  10. Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

    Directory of Open Access Journals (Sweden)

    Lisa Trisciuoglio

    Full Text Available A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

  11. Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

    DEFF Research Database (Denmark)

    Mistrik, Martin; Oplustilova, Lenka; Lukas, Jiri

    2009-01-01

    sensitive, quantitative, rapid and simple fluorescence image analysis in thousands of adherent cells per day. Sensitive DNA breakage estimation through analysis of phosphorylated histone H2AX (gamma-H2AX), and homologous recombination (HR) assessed by a new RPA/Rad51 dual-marker approach illustrate...

  12. AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

    Science.gov (United States)

    Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

    2015-05-19

    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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Study on ?H2AX Expression of Lymphocytes as a Biomarker In Radiation Biodosimetry

    OpenAIRE

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

    2016-01-01

    Flow cytometry analysis was used to detect the changes of ?H2AX protein expression in human peripheral blood lymphocytes. In the dose-effect study, the expression of ?H2AX was detected 1 h after irradiation with 60Co ?-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 ?-rays irradiation for the time-effect study. At the same time, the blood was divided into four treatment groups (ultraviolet [UV] irradiation, 60Co ?-rays irradiati...

  14. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

    Science.gov (United States)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

  15. Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

    Science.gov (United States)

    Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

    2016-09-01

    In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Individual Impact of Distinct Polysialic Acid Chain Lengths on the Cytotoxicity of Histone H1, H2A, H2B, H3 and H4

    Directory of Open Access Journals (Sweden)

    Kristina Zlatina

    2017-12-01

    Full Text Available Neutrophils are able to neutralize pathogens by phagocytosis, by the release of antimicrobial components, as well as by the formation of neutrophil extracellular traps (NETs. The latter possibility is a DNA-meshwork mainly consisting of highly concentrated extracellular histones, which are not only toxic for pathogens, but also for endogenous cells triggering several diseases. To reduce the negative outcomes initiated by extracellular histones, different approaches like antibodies against histones, proteases, and the polysaccharide polysialic acid (polySia were discussed. We examined whether each of the individual histones is a binding partner of polySia, and analyzed their respective cytotoxicity in the presence of this linear homopolymer. Interestingly, all of the histones (H1, H2A, H2B, H3, and H4 seem to interact with α2,8-linked sialic acids. However, we observed strong differences regarding the required chain length of polySia to bind histone H1, H2A, H2B, H3, and H4. Moreover, distinct degrees of polymerization were necessary to act as a cytoprotective agent in the presence of the individual histones. In sum, the outlined results described polySia-based strategies to bind and/or to reduce the cytotoxicity of individual histones using distinct polySia chain length settings.

  17. Distribution pattern of histone H3 phosphorylation at serine 10

    Indian Academy of Sciences (India)

    We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated ...

  18. H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

    Science.gov (United States)

    Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

    2012-09-21

    Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Antibodies to H2a and H2b histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

    Science.gov (United States)

    Baranova, Svetlana V; Dmitrienok, Pavel S; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

    2017-06-01

    Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical enzymes were isolated from the sera of HIV-infected patients by chromatography on several affinity sorbents including anti-histone Sepharose. In contrast to canonical proteases (trypsin, chymotrypsin, proteinase K), IgGs from HIV-infected patients specifically hydrolyzed only histones but not many other tested globular proteins. Using MALDI mass spectrometry the sites of H2a and H2b histone cleavage by anti-histone IgGs were determined for the first time. One cluster of H2a hydrolysis contains two major (↕) and four moderate (↓) cleavage sites: 31-H↓R↓L↓L↓R↕K G↕N-38. One major and two moderate sites of cleavage were revealed in the second cluster: 14-A↕KSRS↓SRA↓G-22. The third cluster corresponding to the H2a C-terminal part contains only five minor (†) sites of cleavage: 82-H†LQLAIRNDEELN†KLLG†RV†T†I-102. It was shown that two major and four moderate sites of cleavage were present in the main cluster of H2b hydrolysis: 46-K↕QvhpD↓TgiS↓SkA↓M↕GiM↓N-63. Two moderate sites of cleavage correspond to a relatively short 6-mer cluster: 12-K↓GskK↓A-17. The third relatively long 9-mer cluster contains one major and two minor sites of H2b cleavage: 80-L↕AHYN†KRS†T-88. In the nucleosome core particle, most of the major and moderate cleavage sites are located at the H2a/H2b interaction interface. Minor cleavage sites of H2a are involved in binding with H3 in the nucleosome core. Two moderate cleavage sites of H2b and one

  20. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks

    DEFF Research Database (Denmark)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia

    2015-01-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase......, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required...... for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling...

  1. Different reaction of the core histones H2A and H2B to red laser irradiation

    Science.gov (United States)

    Brill, G. E.; Egorova, A. V.; Bugaeva, I. O.; Postnov, D. E.; Ushakova, O. V.

    2017-03-01

    Analysis of the influence of red laser irradiation on the processes of self-assembly of the core histones H2A and H2B was performed using a wedge dehydration method. Image-analysis of facies included their qualitative characteristics and calculation of quantitative parameters with subsequent statistical processing. It was established that linearly polarized red laser light (λ - 660 nm, 1 J/cm2) significantly modified the process of self-assembly of core histone H2B, whereas the structure of the facies of H2A histone changed to a lesser extent. Histones were used in the form of aqueous salt solutions. The effect of red light seems to result from the formation of singlet oxygen by direct laser excitation of molecular oxygen.

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

    Science.gov (United States)

    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

  3. Synthesis and phosphorylation of histones and nonhistone proteins in the cycloheximide-synchronized hepatocytes after the effect of radiation and serotonin

    International Nuclear Information System (INIS)

    Aslamova, L.I.; Blyum, Ya.B.; Tsudzevich, B.A.; Kucherenko, N.E.

    1984-01-01

    Phosphorylation and synthesis of histones and nonhistone proteins were studied after the inhibition of translation by sublethal cycloheximide doses. Activation of the chromatin protein phosphorylation was noted: (1) at the stage of recovery and stimulation of the protein synthesis (18-24 h), and (2) at the stage of activation of the replicative DNA synthesis (30-60 h). Phosphorylation and synthesis of the chromatin poteins depended upon the individual or combined effect of X-radiation and serotonin. The possible role of the chromatin protein phosphorylation in the response of the nuclear apparatus to the effect of radiation and serotonin the latter being used as a radioprotective agent is discussed

  4. Endogenous and radiation-induced expression of γH2AX in biopsies from patients treated for carcinoma of the uterine cervix

    International Nuclear Information System (INIS)

    Olive, Peggy L.; Banuelos, C. Adriana; Durand, Ralph E.; Kim, Joo-Young; Aquino-Parsons, Christina

    2010-01-01

    Background and purpose: The possibility of using γH2AX foci as a marker of DNA damage and as a potential predictor of tumour response to treatment was examined using biopsies from 3 sets of patients with advanced carcinoma of the cervix. The relation between endogenous γH2AX expression and hypoxia was also examined. Materials and methods: Set 1 consisted of 26 biopsies that included pre-treatment and 24 h post-radiation treatment samples. Pre-treatment biopsies from 12 patients in Set 2 were used to develop image analysis software while pre-treatment biopsies from 33 patients in Set 3 were examined for the relation between staining for the hypoxia marker pimonidazole and endogenous γH2AX expression. Formalin-fixed paraffin-embedded sections were analyzed after antigen retrieval and fluorescence antibody labeling for the hypoxia markers CAIX or pimonidazole in combination with γH2AX staining. Results: Before treatment, 24 ± 19% of cells contained γH2AX foci, with most positive cells containing fewer than 5 foci per nucleus. Twenty-four hours after exposure to the first fraction of 1.8-2.5 Gy, 38 ± 19% contained foci. CAIX positive cells were 1.4 times more likely to exhibit endogenous γH2AX foci, and pimonidazole-positive cells were 2.8 times more likely to contain γH2AX foci. For 18 patients for whom both pre-treatment and 24 h post-irradiation biopsies were available, local control was unrelated to the fraction of cells that retained γH2AX foci. However, 24 h after irradiation, tumours that had received 2.5 Gy showed a significantly higher fraction of cells with residual γH2AX foci than tumours given 1.8 Gy. Conclusions: Endogenous γH2AX foci are enriched in hypoxic tumour regions. Small differences in delivered dose can produce quantifiable differences in residual DNA damage that can overshadow inter-tumour differences in response.

  5. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  6. Core Histones H2B and H4 Are Mobilized during Infection with Herpes Simplex Virus 1 ▿

    Science.gov (United States)

    Conn, Kristen L.; Hendzel, Michael J.; Schang, Luis M.

    2011-01-01

    The infecting genomes of herpes simplex virus 1 (HSV-1) are assembled into unstable nucleosomes soon after nuclear entry. The source of the histones that bind to these genomes has yet to be addressed. However, infection inhibits histone synthesis. The histones that bind to HSV-1 genomes are therefore most likely those previously bound in cellular chromatin. In order for preexisting cellular histones to associate with HSV-1 genomes, however, they must first disassociate from cellular chromatin. Consistently, we have shown that linker histones are mobilized during HSV-1 infection. Chromatinization of HSV-1 genomes would also require the association of core histones. We therefore evaluated the mobility of the core histones H2B and H4 as measures of the mobilization of H2A-H2B dimers and the more stable H3-H4 core tetramer. H2B and H4 were mobilized during infection. Their mobilization increased the levels of H2B and H4 in the free pools and decreased the rate of H2B fast chromatin exchange. The histones in the free pools would then be available to bind to HSV-1 genomes. The mobilization of H2B occurred independently from HSV-1 protein expression or DNA replication although expression of HSV-1 immediate-early (IE) or early (E) proteins enhanced it. The mobilization of core histones H2B and H4 supports a model in which the histones that associate with HSV-1 genomes are those that were previously bound in cellular chromatin. Moreover, this mobilization is consistent with the assembly of H2A-H2B and H3-H4 dimers into unstable nucleosomes with HSV-1 genomes. PMID:21994445

  7. Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex.

    Science.gov (United States)

    van de Werken, C; Avo Santos, M; Laven, J S E; Eleveld, C; Fauser, B C J M; Lens, S M A; Baart, E B

    2015-10-01

    Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the

  8. Effects of Florida Red Tides on histone variant expression and DNA methylation in the Eastern oyster Crassostrea virginica.

    Science.gov (United States)

    Gonzalez-Romero, Rodrigo; Suarez-Ulloa, Victoria; Rodriguez-Casariego, Javier; Garcia-Souto, Daniel; Diaz, Gabriel; Smith, Abraham; Pasantes, Juan Jose; Rand, Gary; Eirin-Lopez, Jose M

    2017-05-01

    Massive algal proliferations known as Harmful Algal Blooms (HABs) represent one of the most important threats to coastal areas. Among them, the so-called Florida Red Tides (FRTs, caused by blooms of the dinoflagellate Karenia brevis and associated brevetoxins) are particularly detrimental in the southeastern U.S., causing high mortality rates and annual losses in excess of $40 million. The ability of marine organisms to cope with environmental stressors (including those produced during HABs) is influenced by genetic and epigenetic mechanisms, the latter resulting in phenotypic changes caused by heritable modifications in gene expression, without involving changes in the genetic (DNA) sequence. Yet, studies examining cause-effect relationships between environmental stressors, specific epigenetic mechanisms and subsequent responses are still lacking. The present work contributes to increase this knowledge by investigating the effects of Florida Red Tides on two types of mechanisms participating in the epigenetic memory of Eastern oysters: histone variants and DNA methylation. For that purpose, a HAB simulation was conducted in laboratory conditions, exposing oysters to increasing concentrations of K. brevis. The obtained results revealed, for the first time, the existence of H2A.X, H2A.Z and macroH2A genes in this organism, encoding histone variants potentially involved in the maintenance of genome integrity during responses to the genotoxic effect of brevetoxins. Additionally, an increase in H2A.X phosphorylationH2A.X, a marker of DNA damage) and a decrease in global DNA methylation were observed as the HAB simulation progressed. Overall, the present work provides a basis to better understand how epigenetic mechanisms participate in responses to environmental stress in marine invertebrates, opening new avenues to incorporate environmental epigenetics approaches into management and conservation programs. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Effects of Florida Red Tides on histone variant expression and DNA methylation in the Eastern oyster Crassostrea virginica

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez-Romero, Rodrigo; Suarez-Ulloa, Victoria [Environmental Epigenetics Group, Department of Biological Sciences, Florida International University, North Miami, FL 33181 (United States); Rodriguez-Casariego, Javier [Environmental Epigenetics Group, Department of Biological Sciences, Florida International University, North Miami, FL 33181 (United States); Ecotoxicology and Risk Assessment Laboratory, Southeast Environmental Research Center, Florida International University, North Miami, FL 33181 (United States); Garcia-Souto, Daniel [Departamento de Bioquimica, Xenetica e Inmunoloxia, Universidade de Vigo, E-36310 Vigo (Spain); Diaz, Gabriel [Environmental Epigenetics Group, Department of Biological Sciences, Florida International University, North Miami, FL 33181 (United States); Smith, Abraham [Ecotoxicology and Risk Assessment Laboratory, Southeast Environmental Research Center, Florida International University, North Miami, FL 33181 (United States); Pasantes, Juan Jose [Departamento de Bioquimica, Xenetica e Inmunoloxia, Universidade de Vigo, E-36310 Vigo (Spain); Rand, Gary [Ecotoxicology and Risk Assessment Laboratory, Southeast Environmental Research Center, Florida International University, North Miami, FL 33181 (United States); Eirin-Lopez, Jose M., E-mail: jeirinlo@fiu.edu [Environmental Epigenetics Group, Department of Biological Sciences, Florida International University, North Miami, FL 33181 (United States)

    2017-05-15

    Massive algal proliferations known as Harmful Algal Blooms (HABs) represent one of the most important threats to coastal areas. Among them, the so-called Florida Red Tides (FRTs, caused by blooms of the dinoflagellate Karenia brevis and associated brevetoxins) are particularly detrimental in the southeastern U.S., causing high mortality rates and annual losses in excess of $40 million. The ability of marine organisms to cope with environmental stressors (including those produced during HABs) is influenced by genetic and epigenetic mechanisms, the latter resulting in phenotypic changes caused by heritable modifications in gene expression, without involving changes in the genetic (DNA) sequence. Yet, studies examining cause-effect relationships between environmental stressors, specific epigenetic mechanisms and subsequent responses are still lacking. The present work contributes to increase this knowledge by investigating the effects of Florida Red Tides on two types of mechanisms participating in the epigenetic memory of Eastern oysters: histone variants and DNA methylation. For that purpose, a HAB simulation was conducted in laboratory conditions, exposing oysters to increasing concentrations of K. brevis. The obtained results revealed, for the first time, the existence of H2A.X, H2A.Z and macroH2A genes in this organism, encoding histone variants potentially involved in the maintenance of genome integrity during responses to the genotoxic effect of brevetoxins. Additionally, an increase in H2A.X phosphorylationH2A.X, a marker of DNA damage) and a decrease in global DNA methylation were observed as the HAB simulation progressed. Overall, the present work provides a basis to better understand how epigenetic mechanisms participate in responses to environmental stress in marine invertebrates, opening new avenues to incorporate environmental epigenetics approaches into management and conservation programs.

  10. Effects of Florida Red Tides on histone variant expression and DNA methylation in the Eastern oyster Crassostrea virginica

    International Nuclear Information System (INIS)

    Gonzalez-Romero, Rodrigo; Suarez-Ulloa, Victoria; Rodriguez-Casariego, Javier; Garcia-Souto, Daniel; Diaz, Gabriel; Smith, Abraham; Pasantes, Juan Jose; Rand, Gary; Eirin-Lopez, Jose M.

    2017-01-01

    Massive algal proliferations known as Harmful Algal Blooms (HABs) represent one of the most important threats to coastal areas. Among them, the so-called Florida Red Tides (FRTs, caused by blooms of the dinoflagellate Karenia brevis and associated brevetoxins) are particularly detrimental in the southeastern U.S., causing high mortality rates and annual losses in excess of $40 million. The ability of marine organisms to cope with environmental stressors (including those produced during HABs) is influenced by genetic and epigenetic mechanisms, the latter resulting in phenotypic changes caused by heritable modifications in gene expression, without involving changes in the genetic (DNA) sequence. Yet, studies examining cause-effect relationships between environmental stressors, specific epigenetic mechanisms and subsequent responses are still lacking. The present work contributes to increase this knowledge by investigating the effects of Florida Red Tides on two types of mechanisms participating in the epigenetic memory of Eastern oysters: histone variants and DNA methylation. For that purpose, a HAB simulation was conducted in laboratory conditions, exposing oysters to increasing concentrations of K. brevis. The obtained results revealed, for the first time, the existence of H2A.X, H2A.Z and macroH2A genes in this organism, encoding histone variants potentially involved in the maintenance of genome integrity during responses to the genotoxic effect of brevetoxins. Additionally, an increase in H2A.X phosphorylationH2A.X, a marker of DNA damage) and a decrease in global DNA methylation were observed as the HAB simulation progressed. Overall, the present work provides a basis to better understand how epigenetic mechanisms participate in responses to environmental stress in marine invertebrates, opening new avenues to incorporate environmental epigenetics approaches into management and conservation programs.

  11. Histone peptide AKRHRK enhances H2O2-induced DNA damage and alters its site specificity

    International Nuclear Information System (INIS)

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke

    2005-01-01

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition

  12. Structural Insights into the Association of Hif1 with Histones H2A-H2B Dimer and H3-H4 Tetramer.

    Science.gov (United States)

    Zhang, Mengying; Liu, Hejun; Gao, Yongxiang; Zhu, Zhongliang; Chen, Zijun; Zheng, Peiyi; Xue, Lu; Li, Jixi; Teng, Maikun; Niu, Liwen

    2016-10-04

    Histone chaperones are critical for guiding specific post-transcriptional modifications of histones, safeguarding the histone deposition (or disassociation) of nucleosome (dis)assembly, and regulating chromatin structures to change gene activities. HAT1-interacting factor 1 (Hif1) has been reported to be an H3-H4 chaperone and to be involved in telomeric silencing and nucleosome (dis)assembly. However, the structural basis for the interaction of Hif1 with histones remains unknown. Here, we report the complex structure of Hif1 binding to H2A-H2B for uncovering the chaperone specificities of Hif1 on binding to both the H2A-H2B dimer and the H3-H4 tetramer. Our findings reveal that Hif1 interacts with the H2A-H2B dimer and the H3-H4 tetramer via distinct mechanisms, suggesting that Hif1 is a pivotal scaffold on alternate binding of H2A-H2B and H3-H4. These specificities are conserved features of the Sim3-Hif1-NASP interrupted tetratricopeptide repeat proteins, which provide clues for investigating their potential roles in nucleosome (dis)assembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

    OpenAIRE

    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 ...

  14. Dose-response relationship of γ-H2AX foci induction in human lymphocytes after X-rays exposure

    International Nuclear Information System (INIS)

    Mandina, Tania; Roch-Lefevre, Sandrine H.; Voisin, Pascale; Gonzalez, Jorge E.; Lamadrid, Ana I.; Romero, Ivonne; Garcia, Omar; Voisin, Philippe; Roy, Laurence

    2011-01-01

    Biological dosimeters are recommended for dose estimation in case of human overexposure to ionising radiation. Rapid measurement of γ-H2AX foci as a marker of DNA double-strand breaks (DSB) induction has been recently tested with this purpose. Here we reported a dose-response relationship after X-ray irradiation at different times post-exposure. Blood samples were obtained from several healthy donors and exposed to doses between 0 and 2 Gy. After irradiation, blood samples were incubated at 37 deg. C during 0.5 h, 5 h, and 8 h. Scoring of cells and γ-H2AX foci was performed by software. The dose-response curves for different incubation times were as follows: Y (0.5h) = 11.66D + 0.15 (R 2 = 0.99), Y (5h) = 2.44D + 0.15 (R 2 = 0.99), Y (8h) = 1.57D + 0.22 (R 2 = 0.99). At 0.5 h post-exposure, the dose-response relationship for X-irradiated lymphocytes was similar to the one obtained after gamma-irradiation using the same protocol. On the other hand, the results were not similar after 8 h due to different kinetics after gamma- and X-irradiation. Our results confirm the possibilities of using γ-H2AX foci method for dose estimation in a period from 0.5 h up to 8 h post X-irradiation and support the hypothesis of differences in γ-H2AX foci kinetics after gamma- and X-irradiation in vitro.

  15. Dose-response relationship of {gamma}-H2AX foci induction in human lymphocytes after X-rays exposure

    Energy Technology Data Exchange (ETDEWEB)

    Mandina, Tania [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Roch-Lefevre, Sandrine H.; Voisin, Pascale [Institut de Radioprotection et de Surete Nucleaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses (France); Gonzalez, Jorge E.; Lamadrid, Ana I.; Romero, Ivonne [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Garcia, Omar, E-mail: omar@cphr.edu.cu [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Voisin, Philippe; Roy, Laurence [Institut de Radioprotection et de Surete Nucleaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses (France)

    2011-09-15

    Biological dosimeters are recommended for dose estimation in case of human overexposure to ionising radiation. Rapid measurement of {gamma}-H2AX foci as a marker of DNA double-strand breaks (DSB) induction has been recently tested with this purpose. Here we reported a dose-response relationship after X-ray irradiation at different times post-exposure. Blood samples were obtained from several healthy donors and exposed to doses between 0 and 2 Gy. After irradiation, blood samples were incubated at 37 deg. C during 0.5 h, 5 h, and 8 h. Scoring of cells and {gamma}-H2AX foci was performed by software. The dose-response curves for different incubation times were as follows: Y{sub (0.5h)} = 11.66D + 0.15 (R{sup 2} = 0.99), Y{sub (5h)} = 2.44D + 0.15 (R{sup 2} = 0.99), Y{sub (8h)} = 1.57D + 0.22 (R{sup 2} = 0.99). At 0.5 h post-exposure, the dose-response relationship for X-irradiated lymphocytes was similar to the one obtained after gamma-irradiation using the same protocol. On the other hand, the results were not similar after 8 h due to different kinetics after gamma- and X-irradiation. Our results confirm the possibilities of using {gamma}-H2AX foci method for dose estimation in a period from 0.5 h up to 8 h post X-irradiation and support the hypothesis of differences in {gamma}-H2AX foci kinetics after gamma- and X-irradiation in vitro.

  16. Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus.

    Science.gov (United States)

    Rivera-Casas, Ciro; González-Romero, Rodrigo; Vizoso-Vazquez, Ángel; Cheema, Manjinder S; Cerdán, M Esperanza; Méndez, Josefina; Ausió, Juan; Eirin-Lopez, Jose M

    2016-10-01

    Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.

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

    International Nuclear Information System (INIS)

    Pouliliou, Stamatia E.; Lialiaris, Theodoros S.; Dimitriou, Thespis; Giatromanolaki, Alexandra; Papazoglou, Dimitrios; Pappa, Aglaia; Pistevou, Kyriaki; Kalamida, Dimitra; Koukourakis, Michael I.

    2015-01-01

    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 [4h] ) and 24 hours (SF2 [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 (4h) was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio (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 (4h) /γH2AX-ratio (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 efficient DSB repair ability

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

    Energy Technology Data Exchange (ETDEWEB)

    Pouliliou, Stamatia E. [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Lialiaris, Theodoros S. [Department of Medical Genetics, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Dimitriou, Thespis [Department of Anatomy, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Giatromanolaki, Alexandra [Department of Pathology, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Papazoglou, Dimitrios [Department of Internal Medicine, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Pappa, Aglaia [Department of Molecular Biology and Genetics, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Pistevou, Kyriaki [Department of Radiotherapy/Oncology, Aristotle University of Thessalonica, Thessalonica (Greece); Kalamida, Dimitra [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Koukourakis, Michael I., E-mail: targ@her.forthnet.gr [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece)

    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. Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

    Science.gov (United States)

    Achel, Daniel G; Serafin, Antonio M; Akudugu, John M

    2016-08-01

    Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5 Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6 h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors.

  20. Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Balajee, Adayabalam S.; Geard, Charles R.

    2004-01-01

    Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, γ-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and γ-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and γ-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and γ-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and γ-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with γ-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and γ-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with γ-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells

  1. H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

    Science.gov (United States)

    Belle, Jad I; Nijnik, Anastasia

    2014-05-01

    Posttranslational modifications of histone H2A through the attachment of ubiquitin or poly-ubiquitin conjugates are common in mammalian genomes and play an important role in the regulation of chromatin structure, gene expression, and DNA repair. Histone H2A deubiquitinases (H2A-DUBs) are a group of structurally diverse enzymes that catalyze the removal ubiquitin from histone H2A. In this review we provide a concise summary of the mechanisms that mediate histone H2A ubiquitination in mammalian cells, and review our current knowledge of mammalian H2A-DUBs, their biochemical activities, and recent developments in our understanding of their functions in mammalian physiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Investigation of early DNA damage after radioiodine therapy in patients with thyroid cancer using the gamma-H2AX focus assay

    International Nuclear Information System (INIS)

    Eberlein, U.; Lassmann, M.; Bluemel, C.; Buck, A.K.; Nowak, C.; Meineke, V.; Scherthan, H.

    2015-01-01

    Full text of publication follows. Objectives: the Aim of the study is to investigate the DNA damage formation in blood lymphocytes and the correlation to the absorbed dose to the blood in patients with differentiated thyroid carcinoma (DTC) after their first radionuclide therapy with I-131 as measured by the induction, persistence and decay behaviour of γ-H2AX and 53BP1 DNA damage-induced foci. Radiation-induced DNA double strand breaks (DSBs) cause in their vicinity the formation of microscopically visible foci of the phospho-histone H2AX (γ-H2AX) and the 53BP1 protein that binds to and signals damaged chromatin at the DSB site. Nuclear foci containing both markers thus represent radiation-induced DSBs. Methods: we investigated 19 patients with DTC during the first treatment with 3.5±0.3 GBq I-131. Between 7 and 10 sequential peripheral blood samples (at least four within the first 5 hours) were taken before and between 0.5 h and 144 h post administration. The physical dosimetry procedures were performed according to the EANM DTC SOP. White blood cells were recovered by density centrifugation in CPT tubes (BD Biosciences) and subjected to two-colour immunofluorescence staining. The average frequencies of the radiation-induced γ-H2AX foci/nucleus that co localized with 53BP1 foci were derived from immuno-stained mononuclear peripheral blood lymphocyte samples. The number of foci was counted manually using a red/green double band pass filter (Chroma) in a Zeiss microscope by an experienced observer. Results: The mean I-131 absorbed dose to the blood was (0.04±0.01) Gy at t=2 h, (0.07±0.02) Gy at t=4 h, and (0.21±0.05) Gy at t=24 h, respectively. The mean value of the total absorbed dose to the blood was (0.36±0.08) Gy. The highest number of radiation-induced foci per nucleus (RIF) and per absorbed dose (median: 8.8 RIF/Gy, range 3.1-10.9 RIF/Gy) was observed in the first three hours post administration. Four hours after radioiodine administration the number

  3. Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

    Directory of Open Access Journals (Sweden)

    Catherine A Hazzalin

    2005-12-01

    Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

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

    Directory of Open Access Journals (Sweden)

    Adi Ben Yehuda

    Full Text Available 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.

  5. Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

    1980-01-01

    A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

  6. Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

    Science.gov (United States)

    Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

    2016-08-17

    The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

  7. Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

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    Jean-Luc Perfettini

    Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

  8. Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

    Directory of Open Access Journals (Sweden)

    Moreira J.C.F.

    2000-01-01

    Full Text Available Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

  9. γ-H2AX as a marker for dose deposition in the brain of wistar rats after synchrotron microbeam radiation.

    Directory of Open Access Journals (Sweden)

    Cristian Fernandez-Palomo

    Full Text Available Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT.Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.

  10. Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy.

    Science.gov (United States)

    Wang, Wenjun; Wang, Qing; Wan, Danyang; Sun, Yue; Wang, Lin; Chen, Hong; Liu, Chengyu; Petersen, Robert B; Li, Jianshuang; Xue, Weili; Zheng, Ling; Huang, Kun

    2017-05-04

    Autophagy plays critical and complex roles in many human diseases, including diabetes and its complications. However, the role of autophagy in the development of diabetic retinopathy remains uncertain. Core histone modifications have been reported involved in the development of diabetic retinopathy, but little is known about the histone variants. Here, we observed increased autophagy and histone HIST1H1C/H1.2, an important variant of the linker histone H1, in the retinas of type 1 diabetic rodents. Overexpression of histone HIST1H1C upregulates SIRT1 and HDAC1 to maintain the deacetylation status of H4K16, leads to upregulation of ATG proteins, then promotes autophagy in cultured retinal cell line. Histone HIST1H1C overexpression also promotes inflammation and cell toxicity in vitro. Knockdown of histone HIST1H1C reduces both the basal and stresses (including high glucose)-induced autophagy, and inhibits high glucose induced inflammation and cell toxicity. Importantly, AAV-mediated histone HIST1H1C overexpression in the retinas leads to increased autophagy, inflammation, glial activation and neuron loss, similar to the pathological changes identified in the early stage of diabetic retinopathy. Furthermore, knockdown of histone Hist1h1c by siRNA in the retinas of diabetic mice significantly attenuated the diabetes-induced autophagy, inflammation, glial activation and neuron loss. These results indicate that histone HIST1H1C may offer a novel therapeutic target for preventing diabetic retinopathy.

  11. Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription

    International Nuclear Information System (INIS)

    Davie, J.R.; Murphy, L.C.

    1990-01-01

    The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extend, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, the authors show that treatment of T-47D-5 human breast cancer cells with actinomycin D or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription

  12. Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predicts Poor Survival of Breast Carcinoma Patients

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    See-Hyoung Park

    2015-08-01

    Full Text Available BACKGROUND: Poly(ADP-ribose polymerase 1 (PARP1, γH2AX, BRCA1, and BRCA2 are conventional molecular indicators of DNA damage in cells and are often overexpressed in various cancers. In this study, we aimed, using immunohistochemical detection, whether the co-expression of PARP1, γH2AX, BRCA1, and BRCA2 in breast carcinoma (BCA tissue can provide more reliable prediction of survival of BCA patients. MATERIALS AND METHODS: We investigated immunohistochemical expression and prognostic significance of the expression of PARP1, γH2AX, BRCA1, and BRCA2 in 192 cases of BCAs. RESULTS: The expression of these four molecules predicted earlier distant metastatic relapse, shorter overall survival (OS, and relapse-free survival (RFS by univariate analysis. Multivariate analysis revealed the expression of PARP1, γH2AX, and BRCA2 as independent poor prognostic indicators of OS and RFS. In addition, the combined expressional pattern of BRCA1, BRCA2, PARP1, and γH2AX (CSbbph was an additional independent prognostic predictor for OS (P < .001 and RFS (P < .001. The 10-year OS rate was 95% in the CSbbph-low (CSbbph scores 0 and 1 subgroup, but that was only 35% in the CSbbph-high (CSbbph score 4 subgroup. CONCLUSION: This study has demonstrated that the individual and combined expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 could be helpful in determining an accurate prognosis for BCA patients and for the selection of BCA patients who could potentially benefit from anti-PARP1 therapy with a combination of genotoxic chemotherapeutic agents.

  13. Histone H3 is absent from organelle nucleoids in BY-2 cultured tobacco cells.

    Science.gov (United States)

    Takusagawa, Mari; Tamotsu, Satoshi; Sakai, Atsushi

    2013-07-01

    The core histone proteins (H2A, H2B, H3 and H4) are nuclear-localised proteins that play a central role in the formation of nucleosome structure. They have long been considered to be absent from extra-nuclear, DNA-containing organelles; that is plastids and mitochondria. Recently, however, the targeting of core histone H3 to mitochondria, and the presence of nucleosome-like structures in mitochondrial nucleoids, were proposed in cauliflower and tobacco respectively. Thus, we examined whether histone H3 was present in plant organelles and participated in the organisation of nucleoid structure, using highly purified organelles and organelle nucleoids isolated from BY-2 cultured tobacco cells. Immunofluorescence microscopic observations and Western blotting analyses demonstrated that histone H3 was absent from organelles and organelle nucleoids, consistent with the historical hypothesis. Thus, the organisation of organelle nucleoids, including putative nucleosome-like repetitive structures, should be constructed and maintained without participation of histone H3. © 2013 International Federation for Cell Biology.

  14. Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Pasini, Diego; Helin, Kristian

    2010-01-01

    distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides....... The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27......me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone...

  15. Some physico-chemical characteristics of a modified histone H2b on acute radiation affection

    International Nuclear Information System (INIS)

    Khrapunov, S.N.; Mel'nik, G.G.; Blyum, Ya.B.; Tsudzevich, B.A.; Kucherenko, N.E.

    1980-01-01

    A study was made of optical characteristics of histone H2b isolated from liver nuclei 12 h following irradiation in a dose of 0.21 C/kg. It was demonstrated that under similar conditions, the control and exposed histones H2b have different steric organization which correlates with radiation-induced modifications of lateral radicals in H2b histone molecules

  16. Histone H4 Lysine 20 methylation

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Schotta, Gunnar; Sørensen, Claus Storgaard

    2013-01-01

    of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin...... compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic...

  17. Ionizing radiation-induced DNA double-strand break and repair assessed by γ-H2AX foci analysis in neurons in mice

    International Nuclear Information System (INIS)

    Dong Xiaorong; Wu Gang; Ruebe Claudia; Ruebe Christian

    2009-01-01

    Objective: To investigate if the γ-H2AX foci is a precise index for the DSB formation and repair in mature neurons of brain in vivo after clinically relevant doses irradiation. Methods: For the DSB formation experiment, the mature neurons in the neocortex of brain tissue of C57BL/6 mice were analyzed at 10 rain after whole-body irradiation with 0.1, 0.5 and 1.0 Gy. For the DSB repair kinetics experiment, the mature neurons in the neocortex of brain tissue of repair-proficient (C57BL/6 mice) and repair-deficient mouse strains (BALB/c, A-T and SCID mice) were analyzed at 0.5, 2.5, 5, 24 and 48 h after whole-body irradiation with 2 Gy. The mature neurons in the neocortex of brain tissue of sham-irradiated mice of each strain served as controls. γ-H2AX immunohistochemistry and γ-H2AX and NeuN double immunofluorescence analysis was used to measure DSBs formation and repair in the mature neurons in the neocortex of brain tissue of the different mouse strains. Results: For the DSB formation experiment, γ-H2AX foci levels with a clear linear close correlation and very low backgrounds in the nuclei in the neocortex of brain tissue were observed. Scoring the loss of γ-H2AX foci allowed us to verify the different, genetically determined DSB repair deficiencies, including the minor impairment of BALB/c mice. Repair-proficient C57BL/6 mice exhibited the fastest decrease in foci number with time, and displayed low levels of residual damage at 24 h and 48 h post-irradiation. In contrast, SCID mice showed highly increased γ-H2AX foci levels at all repair times (0.5 h to 48 h) while A-T mice exhibited a lesser defect which was most significant at later repair times (≥ 5 h). Radiosensitive BALB/c mice exhibited slightly elevated foci numbers compared with C57BL/6 mice at 5 h and 24 h but not at 48 h post-irradiation. Conclusion: Quantifying the γ-H2AX foci in normal tissue represents a sensitivie tool for the detection of induction and repair of radiation-induced DSBs at

  18. 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

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

    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.

  19. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamashita, Sachiko [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Tanaka, Masakazu [Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010 (Japan); Sato, Teruaki [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Ida, Chieri [Department of Applied Life Studies, College of Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610 (Japan); Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Moss, Joel [Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590 (United States); Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)

    2016-08-05

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  20. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    International Nuclear Information System (INIS)

    Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki; Ida, Chieri; Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke; Moss, Joel; Miwa, Masanao

    2016-01-01

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  1. Antifungal properties of wheat histones (H1-H4) and purified wheat histone H1

    Science.gov (United States)

    Wheat (Triticum sp.) histones H1, H2, H3, and H4 were extracted. H1 was further purified. Their activities against fungi with varying degrees of wheat pathogenicity were determined. They included Aspergillus flavus, A. fumigatus, A. niger, F. oxysporum, F. verticillioides, F. solani, F. graminearu...

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

    Science.gov (United States)

    Morello, Noemi; Plicato, Ornella; Piludu, Maria Antonietta; Poddighe, Laura; Serra, Maria Pina; Quartu, Marina; Corda, Maria Giuseppa; Giorgi, Osvaldo; Giustetto, Maurizio

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cobo, J.M. [Universidad de Alcala de Henares, Madrid (Spain); Valdez, J.G.; Gurley, L.R. [Los Alamos National Lab., NM (United States)

    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.

  4. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

    Directory of Open Access Journals (Sweden)

    Andrea J Hartlerode

    Full Text Available Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me. Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

  5. 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.

    Science.gov (United States)

    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.

  6. 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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong [Fudan University, Department of Radiation Biology, Institute of Radiation Medicine, Shanghai (China)

    2016-08-15

    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. (orig.)

  7. 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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. (orig.)

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster.

    Science.gov (United States)

    Seo, Jung-Kil; Stephenson, Jeana; Noga, Edward J

    2011-03-01

    We have previously identified a histone H2B isomer (cvH2B-1) from tissue extracts of the bivalve mollusk, the American oyster (Crassostrea virginica). In this paper, we isolate an additional three antibacterial proteins from acidified gill extract by preparative acid-urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Extraction of these proteins from tissue was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Addition of protease inhibitors while boiling resulted in somewhat lower yields, with one protein being totally absent with this method. Via mass spectrometry, the masses of one of these purified proteins was 13607.0Da (peak 2), which is consistent with the molecular weight of histone H2B. In addition, via western-blotting using anti-calf histone H2B antibody, all three proteins were positive and were thus named cvH2B-2, cvH2B-3 and cvH2B-4. The antibacterial activity of cvH2B-2 was similar to that of cvH2B-1, with activity against a Gram-positive bacterium (Lactococcus lactis subsp. lactis; minimum effective concentration [MEC] 52-57μg/mL) but inactive against Staphylococcus aureus (MEC>250μg/mL). However, both proteins had relatively potent activity against the Gram-negative oyster pathogen Vibrio parahemolyticus (MEC 11.5-14μg/mL) as well as the human pathogen Vibrio vulnificus (MEC 21.3-25.3μg/mL). cvH2B-3 and cvH2B-4 also had similarly strong activity against Vibrio vulnificus. These data provide further evidence for the antimicrobial function of histone H2B isomers in modulating bacterial populations in oyster tissues. The combined estimated concentrations of these histone H2B isomers were far above the inhibitory concentrations for the tested vibrios, including human pathogens. Our results indicate that the highly conserved histone proteins might be important components not only of immune defenses in oysters but have the potential to influence the abundance of a

  10. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    Science.gov (United States)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  11. Dual function of Swc5 in SWR remodeling ATPase activation and histone H2A eviction.

    Science.gov (United States)

    Sun, Lu; Luk, Ed

    2017-09-29

    The chromatin remodeler SWR deposits histone H2A.Z at promoters and other regulatory sites via an ATP-driven histone exchange reaction that replaces nucleosomal H2A with H2A.Z. Simultaneous binding of SWR to both H2A nucleosome and free H2A.Z induces SWR ATPase activity and engages the histone exchange mechanism. Swc5 is a conserved subunit of the 14-polypeptide SWR complex that is required for the histone exchange reaction, but its molecular role is unknown. We found that Swc5, although not required for substrate binding, is required for SWR ATPase stimulation, suggesting that Swc5 is required to couple substrate recognition to ATPase activation. A biochemical complementation assay was developed to show that a unique, conserved domain at the C-terminus of Swc5, called Bucentaur (BCNT), is essential for the histone exchange activity of SWR, whereas an acidic region at the N-terminus is required for optimal SWR function. In vitro studies showed the acidic N-terminus of Swc5 preferentially binds to the H2A-H2B dimer and exhibits histone chaperone activity. We propose that an auxiliary function of Swc5 in SWR is to assist H2A ejection as H2A.Z is inserted into the nucleosome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

    Science.gov (United States)

    Nakajima, Nakako Izumi; Brunton, Holly; Watanabe, Ritsuko; Shrikhande, Amruta; Hirayama, Ryoichi; Matsufuji, Naruhiro; Fujimori, Akira; Murakami, Takeshi; Okayasu, Ryuichi; Jeggo, Penny; Shibata, Atsushi

    2013-01-01

    Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle. PMID:23967070

  13. Neutron scattering studies of the H2a-H2b and (H3-H4)2 histone complexes

    International Nuclear Information System (INIS)

    Carlson, R.D.

    1982-01-01

    Neutron scattering experiments have shown that both the (H3-H4) 2 and H2a-H2b histone complexes are quite asymmetric in solution. The (H3-H4) 2 tetramer is an oblate or flattened structure, with a radius of gyration almost as large as that of the core octamer. If the tetramer is primarily globular, it must have an axial ratio of about 1:5. It is more likely, however, that this asymmetry results in part from N-terminal arms that extend outward approximately within the major plane of the particle. If this is the case, less asymmetric models for the globular part of the tetramer, including a dislocated disk, can be made consistent with the scattering data. The H2a-H2b dimer, on the other hand, is an elongated structure. 48 references, 12 figures, 1 table

  14. Production of DNA Double Strand Breaks in Human Cells due to Acute Exposure to Tritiated Water (HTO)

    International Nuclear Information System (INIS)

    Gonen, R.; German, U.; Alfassi, Z. B.; Priel, E.

    2014-01-01

    The average and maximum energies of the beta emission from 3H are 5.69 keV and 18.6 keV respectively. The average range in water (or soft tissues), around 0.5 1/4m (500 nm), is considerably less than the typical diameter of a cell (10-30 1/4m), and even of a cell nucleus (5-10 1/4m), thus the micro-location of the tritium atom may well be crucial in determining its biochemical consequences. Due to the high ionization density of the beta particles emitted by tritium (about 400 ion pairs/1/4m) possible interaction of tritium beta radiation with DNA may play a significant role. Tritiated water (HTO) is the main chemical form in which tritium is found in the environment. In the body it may be retained as organically bound tritium (OBT), binding to biological molecules or remaining as OBT with various degrees of solubility. OBT can be retained in the human body much longer than HTO and therefore the dose arising from OBT can reach 50% of the total tritium dose . Histones are major protein components of chromatin. They function as spools around which DNA winds and play an important role in the regulation of gene expression. In the absence of histones, the DNA in chromosomes would be unmanageably long, as human cells each have about 1.8 m of DNA. During mitosis, DNA is duplicated and condensed, resulting in about 120 1/4m of chromosomes. It was recently reported that the phosphorylation of histone H2AX on serine residue 139 (D 3 -H2AX) is associated with Double Strand Breaks (DSB) sites in DNA), which indicates the possibility of research based on the detection of DSBs in DNA. The phosphorylated megabase chromatin domain surrounding the DSB can be immunostained and visualized as discrete foci by fluorescence microscopy, as each DNA DSB formed produces a visible D 3 -H2AX focus. Since 1 Gy of radiation produces approximately 60 DSBs/cell, doses of a few mGy should be distinguishable from the background, and it was recently shown that the exposure to 1 mGy of X-rays induces

  15. Quantitative evaluation of radiation dose by γ-H2AX on a microfluidic chip in a miniature fluorescence cytometer

    International Nuclear Information System (INIS)

    Wang, Junsheng; Song, Wendong; Song, Yongxin; Xu, Dan; Zhang, Min; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2014-01-01

    Evaluation of radiation dose is very important for the detection of radiation damage. γ-H2AX is a popular biological dosimeter to evaluate the radiation effect. Typically, bulky and expensive commercial flow cytometers are used to detect γ-H2AX. This paper presents a miniaturized and high sensitive cytometer using a microfluidic chip for evaluating the radiation dose by detecting the mean immunofluorescence intensity of γ-H2AX. A compact optical focusing system and a shift-phase differential amplifier are designed to improve the detection sensitivity. Sample lymphocyte cells are stained by FITC fluorescent dye after being irradiated by UVC. Comparison experiments between the developed miniature cytometer and a commercial flow cytometer were conducted under different radiation doses. The developed microfluidic cytometer also demonstrates a good linear correlation between the measured fluorescence intensity and the irradiation dose with a detection limit similar to that of the commercial flow cytometer. The developed cytometer can evaluate quantitatively the radiation dose by the mean fluorescence intensity of γ-H2AX with a significantly smaller amount of blood samples than a commercial flow cytometer. - Highlights: • A new microfluidic cytometer for evaluating irradiation dose was developed. • The utility of this biosensor is verified by comparison experiments using FCM. • The developed cytometer is small size, high sensitivity, low cost, and simple. • The cytometer can dramatically reduce sample consumption and analysis time

  16. Reliability of a Fully Automated Interpretation of γ-H2AX Foci in Lymphocytes of Moderately Trained Subjects under Resting Conditions

    Directory of Open Access Journals (Sweden)

    Juliane Heydenreich

    2014-01-01

    Full Text Available Background. Analysis of γ-H2AX foci is a promising approach to evaluate exercise-induced DNA damage. However, baseline levels and day-to-day variability of γ-H2AX foci have not been investigated in healthy subjects at rest. Methods. Blood was taken from eight moderately trained healthy males (29 ± 3 yrs, 1.84 ± 0.03 m, and 85 ± 6 kg at two separate days (M1/M2 after 24-hour exercise cessation. Number of γ-H2AX foci per 100 lymphocytes (N, number of foci per affected lymphocyte (NAL, percentage of affected lymphocytes (PAL, and diameter (D of γ-H2AX foci were analyzed (mean ± SD. Differences between M1 and M2 were analyzed using paired t-tests (α = 0.05. Day-to-day variability was evaluated by calculating the coefficients of variation (CV%, bias, and limits of agreement (LoA. Results. There were no statistically significant differences between M1 (N: 7.6 ± 4.4, NAL: 1.2 ± 0.2, PAL: 5.9 ± 2.6%, and D: 0.63 ± 0.07 and M2 (N: 8.4 ± 4.6, NAL: 1.3 ± 0.1, PAL: 6.9 ± 4.2%, and D: 0.66 ± 0.06. CV was calculated to be 98.5% (N, 88.9% (PAL, 11.3% (NAL, and 8.0% (D. Bias (LoA was 0.75 (−15.2/13.7, −0.02 (−0.36/0.33, −1.0 (−11.9/9.9, and −0.04 (−0.16/0.09, respectively. Conclusions. Background level in healthy subjects is approximately 0.07 to 0.09 γ-H2AX foci/cell. NAL and D are reliable measures.

  17. Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

    KAUST Repository

    Chauhan, Sakshi

    2016-09-02

    Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

  18. Effect of dose rate on residual γ-H2AX levels and frequency of micronuclei in X-irradiated mouse lymphocytes.

    Science.gov (United States)

    Turner, H C; Shuryak, I; Taveras, M; Bertucci, A; Perrier, J R; Chen, C; Elliston, C D; Johnson, G W; Smilenov, L B; Amundson, S A; Brenner, D J

    2015-03-01

    The biological risks associated with low-dose-rate (LDR) radiation exposures are not yet well defined. To assess the risk related to DNA damage, we compared the yields of two established biodosimetry end points, γ-H2AX and micronuclei (MNi), in peripheral mouse blood lymphocytes after prolonged in vivo exposure to LDR X rays (0.31 cGy/min) vs. acute high-dose-rate (HDR) exposure (1.03 Gy/min). C57BL/6 mice were total-body irradiated with 320 kVP X rays with doses of 0, 1.1, 2.2 and 4.45 Gy. Residual levels of total γ-H2AX fluorescence in lymphocytes isolated 24 h after the start of irradiation were assessed using indirect immunofluorescence methods. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptotic cell frequency in lymphocytes sampled at 24 h. Curve fitting analysis suggested that the dose response for γ-H2AX yields after acute exposures could be described by a linear dependence. In contrast, a linear-quadratic dose-response shape was more appropriate for LDR exposure (perhaps reflecting differences in repair time after different LDR doses). Dose-rate sparing effects (P effect across the dose range 24 h or 7 days post exposure. In conclusion, the γ-H2AX biomarker showed higher sensitivity to measure dose-rate effects after low-dose LDR X rays compared to MNi formation; however, confounding factors such as variable repair times post exposure, increased cell killing and cell cycle block likely contributed to the yields of MNi with accumulating doses of ionizing radiation.

  19. Germline-specific H1 variants: the "sexy" linker histones.

    Science.gov (United States)

    Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

    2016-03-01

    The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

  20. Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

    International Nuclear Information System (INIS)

    Watanabe, Fumiaki; Fukazawa, Hidesuke; Masutani, Mitsuko; Suzuki, Hiroshi; Teraoka, Hirobumi; Mizutani, Shuki; Uehara, Yoshimasa

    2004-01-01

    DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1 -/- ) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1 +/+ ) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1 -/- embryonic stem cell (ES) clones was also higher than that in Parp-1 +/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32 P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB

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

    International Nuclear Information System (INIS)

    Elsen, S.; Collin-Faure, V.; Gidrol, X.; Lemercier, C.

    2013-01-01

    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. (authors)

  2. BRCA1 Is a Histone-H2A-Specific Ubiquitin Ligase

    Directory of Open Access Journals (Sweden)

    Reinhard Kalb

    2014-08-01

    Full Text Available The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3 ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.

  3. Neutron scattering studies of the H2a-H2b and (H3-H4)/sub 2/ histone complexes

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, R.D.

    1982-01-01

    Neutron scattering experiments have shown that both the (H3-H4)/sub 2/ and H2a-H2b histone complexes are quite asymmetric in solution. The (H3-H4)/sub 2/ tetramer is an oblate or flattened structure, with a radius of gyration almost as large as that of the core octamer. If the tetramer is primarily globular, it must have an axial ratio of about 1:5. It is more likely, however, that this asymmetry results in part from N-terminal arms that extend outward approximately within the major plane of the particle. If this is the case, less asymmetric models for the globular part of the tetramer, including a dislocated disk, can be made consistent with the scattering data. The H2a-H2b dimer, on the other hand, is an elongated structure. 48 references, 12 figures, 1 table.

  4. Impact of High Glucose and Proteasome Inhibitor MG132 on Histone H2A and H2B Ubiquitination in Rat Glomerular Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Chenlin Gao

    2013-01-01

    Full Text Available Background. Hyperglycemia plays a pivotal role in the development of diabetic nephropathy (DN and may be related to epigenetic metabolic memory. One of the most crucial epigenetic mechanisms is histone modification, which is associated with the expression of a fibrosis factor in vascular injury. Aim .In this study, we investigated the ubiquitination of histones H2A and H2B to explore the epigenetic mechanisms of DN. Materials and Methods. The GMCs were cultured as follows: normal group, high glucose group, mannitol group, and intervention group. After 12 hr, 24 hr, and 48 hr, histones ubiquitination, transforming growth factor-β (TGF-β, and fibronectin (FN were measured using WB, RT-PCR, and IF. Result. High glucose can induce the upregulation of FN. H2A ubiquitination in GMCs increased in high glucose group (P<0.01, whereas it decreased significantly in intervention group (P<0.05. In contrast, H2B ubiquitination decreased with an increasing concentration of glucose, but it was recovered in the intervention group (P<0.05. Expression of TGF-β changed in response to abnormal histone ubiquitination. Conclusions. The high glucose may induce H2A ubiquitination and reduce H2B ubiquitination in GMCs. The changes of histone ubiquitination may be due in part to DN by activating TGF-β signaling pathway.

  5. Defects in Histone H3.3 Phosphorylation and ATRX Recruitment to Misaligned Chromosomes during Mitosis Contribute to the Development of Pediatric Glioblastomas

    Science.gov (United States)

    2015-09-01

    aneuploidy. 2. Keywords: aneuploidy, ATRX, cell cycle, chromosome missegregation, CRISPR /Cas9, DAXX, glioblastoma, histone H3.3, microinjection, mitosis...histone H3.3 with mutant constructs. We have switched from shRNA hairpins to CRISPR /Cas9 gene editing to silence both alleles of H3.3 (and an H3.3...plasmids against H3F3B. Both plasmids had the Cas9 gene and a soluble GFP reporter. The CRISPR guide sequence in one of these plasmids was 100% match

  6. Development of a new rapid HPLC method for the fractionation of histones

    International Nuclear Information System (INIS)

    Gurley, L.R.; Valdez, J.G.; Prentice, D.A.; Spall, W.D.

    1983-01-01

    To study histone functions, it is necessary to fractionate the histones into their five classes (H1, H2A, H2B, H3 and H4) and then to subfractionate these classes into variants having slightly different primary structures and into different phosphorylated and acetylated forms. With the advent of high-performance liquid chromatography (HPLC), it was hoped that laborious and time-consuming conventional methods could be replaced by a simple, rapid, high-resolving HPLC method for fractionating histones. However, problems of irreversible adsorption of the histones to HPLC column packings discouraged this development. Our laboratory has now determined that the strong adsorption of histones to HPLC columns results from two different forces: (1) polar interactions between the histones and the silanol groups of silica-based HPLC column packing, and (2) hydrophobic interactions between the histones and the bound organic phase of the column packings. By minimizing these forces, we have succeeded in developing an HPLC method suitable for histone studies

  7. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

    Science.gov (United States)

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

    2016-01-21

    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Masatoshi Suzuki

    2012-01-01

    Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    Experiments using the induction of dicentric chromosomes (dicentrics) as well as the γ-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 γ-H2AX foci were obtained. The coefficients of the dose - response relationship for dicentrics are α = (3.76 ± 0.29) x 10 -2 Gy -1 and β = (5.54 ± 0.45) x 10 -2 Gy -2 , the linear coefficient for γ-H2AX foci is (7.38 ± 0.11) Gy -1 . The findings indicate that scoring of dicentrics as well as microscopic analysis of γ-H2AX foci are sensitive methods to quantify a radiation-induced biological damage at low doses. However, since γ-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 γ-H2AX foci assay observed in the present study, further investigations on the effectiveness of low-linear energy transfer radiation qualities in producing γ-H2AX foci in lymphocytes from healthy donors should be performed. (authors)

  10. The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.

    Directory of Open Access Journals (Sweden)

    Christophe E Redon

    2010-11-01

    Full Text Available There is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person.We evaluated immunocytofluorescence-based quantitation of γ-H2AX foci as a biodosimeter of total-body radiation exposure ((60Co γ-rays in a rhesus macaque (Macaca mulatta model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual γ-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs.These findings indicate that quantitation of γ-H2AX foci may make a robust biodosimeter for analyzing total-body exposure to ionizing radiation in humans. This tool would help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. These results also demonstrate that the use of a high throughput γ-H2AX biodosimeter would be useful for days post-exposure in applications like large-scale radiological events or radiation therapy. In addition, this study validates a possibility to use plucked hair in future clinical trials investigating genotoxic effects of drugs and radiation treatments.

  11. A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours.

    Science.gov (United States)

    Reddy, Divya; Khade, Bharat; Pandya, Riddhi; Gupta, Sanjay

    2017-01-01

    Dysregulation in post-translational modifications of histones and their modifiers are now well-recognized as a hallmark of cancer and can be used as biomarkers and potential therapeutic targets for disease progression and prognosis. In most solid tumours, a biopsy is challenging, costly, painful or potentially risky for the patient. Therefore, non-invasive methods like 'liquid biopsy' for analysis of histone modifications and their modifiers if possible will be helpful in the better clinical management of cancer patients. Here, we have developed a cost-effective and time-efficient protocol for isolation of circulating histones from serum of solid tumor, HCC, called Dual Acid Extraction (DAE) protocol and have confirmed by mass spectrometry. Also, we measured the activity of HDACs and HATs in serum samples. The serum purified histones were profiled for changes in histone PTMs and have shown a comparable pattern of modifications like acetylation (H4K16Ac), methylation (H4K20Me3, H3K27Me3, H3K9Me3) and phosphorylation (γ-H2AX and H3S10P) to paired cancer tissues. Profiling for the histone PTM changes in various other organs of normal and tumor bearing animal suggests that the changes in the histone PTMs observed in the tumor serum is indeed due to changes in the tumor tissue only. Further, we demonstrate that the observed hypo-acetylation of histone H4 in tissue and serum samples of tumor bearing animals corroborated with the elevated HDAC activity in both samples compared to normal. Interestingly, human normal and tumor serum samples also showed elevated HDAC activity with no significant changes in HAT activity. Our study provides the first evidence in the context of histone PTMs and modifiers that liquid biopsy is a valuable predictive tool for monitoring disease progression. Importantly, with the advent of drugs that target specific enzymes involved in the epigenetic regulation of gene expression, liquid biopsy-based 'real time' monitoring will be useful for

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

    Science.gov (United States)

    Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2015-01-01

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

  14. Coupling of glucose deprivation with impaired histone H2B monoubiquitination in tumors.

    Directory of Open Access Journals (Sweden)

    Yasuyo Urasaki

    Full Text Available Metabolic reprogramming is associated with tumorigenesis. However, glucose metabolism in tumors is poorly understood. Here, we report that glucose levels are significantly lower in bulk tumor specimens than those in normal tissues of the same tissue origins. We show that mono-ubiquitinated histone H2B (uH2B is a semi-quantitative histone marker for glucose. We further show that loss of uH2B occurs specifically in cancer cells from a wide array of tumor specimens of breast, colon, lung and additional 23 anatomic sites. In contrast, uH2B levels remain high in stromal tissues or non-cancerous cells in the tumor specimens. Taken together, our data suggest that glucose deficiency and loss of uH2B are novel properties of cancer cells in vivo, which may represent important regulatory mechanisms of tumorigenesis.

  15. Histone deacetylase 1, 2, 6 and acetylated histone H4 in B- and T-cell lymphomas

    DEFF Research Database (Denmark)

    Marquard, L.; Poulsen, C.B.; Gjerdrum, L.M.

    2009-01-01

    AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim was to det......AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim...... was to determine HDAC expression in DLBCL and PTCL which has not previously been investigated. METHODS AND RESULTS: The expression of HDAC1, HDAC2, HDAC6 and acetylated histone H4 was examined immunohistochemically in 31 DLBCL and 45 PTCL. All four markers showed high expression in both DLBCL and PTCL compared...

  16. Detection of histone acetylation levels in the dorsal hippocampus reveals early tagging on specific residues of H2B and H4 histones in response to learning.

    Directory of Open Access Journals (Sweden)

    Olivier Bousiges

    Full Text Available The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain--notably in the hippocampus--and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.

  17. DNA damage assessment by visualization and quantification of DNA damage response

    International Nuclear Information System (INIS)

    Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

    2017-01-01

    DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AXH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

  18. DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

    Energy Technology Data Exchange (ETDEWEB)

    Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J [MSKCC; (Rockefeller); (MRC)

    2013-01-24

    Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

  19. Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

    Directory of Open Access Journals (Sweden)

    Lam Hon-Ming

    2009-07-01

    Full Text Available Abstract Background Histone modifications and histone variants are of importance in many biological processes. To understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of Arabidopsis thaliana. Results In soybean leaves, mono-, di- and tri-methylation at Lysine 4, Lysine 27 and Lysine 36, and acetylation at Lysine 14, 18 and 23 were detected in HISTONE H3. Lysine 27 was prone to being mono-methylated, while tri-methylation was predominant at Lysine 36. We also observed that Lysine 27 methylation and Lysine 36 methylation usually excluded each other in HISTONE H3. Although methylation at HISTONE H3 Lysine 79 was not reported in A. thaliana, mono- and di-methylated HISTONE H3 Lysine 79 were detected in soybean. Besides, acetylation at Lysine 8 and 12 of HISTONE H4 in soybean were identified. Using a combination of mass spectrometry and nano-liquid chromatography, two variants of HISTONE H3 were detected and their modifications were determined. They were different at positions of A31F41S87S90 (HISTONE variant H3.1 and T31Y41H87L90 (HISTONE variant H3.2, respectively. The methylation patterns in these two HISTONE H3 variants also exhibited differences. Lysine 4 and Lysine 36 methylation were only detected in HISTONE H3.2, suggesting that HISTONE variant H3.2 might be associated with actively transcribing genes. In addition, two variants of histone H4 (H4.1 and H4.2 were also detected, which were missing in other organisms. In the histone variant H4.1 and H4.2, the amino acid 60 was isoleucine and valine, respectively. Conclusion This work revealed several distinct variants of soybean histone and their modifications that were different from A. thaliana, thus providing important biological information toward further understanding of the histone

  20. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    International Nuclear Information System (INIS)

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-01-01

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  1. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  2. Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns

    International Nuclear Information System (INIS)

    Gurley, L.R.; Prentice, D.A.; Valdez, J.G.; Spall, W.D.

    1983-01-01

    Previous work described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C 18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a μBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C 18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [ 3 H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species

  3. Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

    Science.gov (United States)

    Petter, Michaela; Lee, Chin Chin; Byrne, Timothy J.; Boysen, Katja E.; Volz, Jennifer; Ralph, Stuart A.; Cowman, Alan F.; Brown, Graham V.; Duffy, Michael F.

    2011-01-01

    Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are

  4. Global chromatin fibre compaction in response to DNA damage

    International Nuclear Information System (INIS)

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-01-01

    Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► 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 phosphorylationH2AX), 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 γH2AX 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 linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

  5. Control of radiation sensitivity of mammalian cells. Regulation of expression of DNA repair genes

    International Nuclear Information System (INIS)

    Yoshida, Kayo; Morita, Takashi

    2003-01-01

    This review describes authors' investigations concerning regulation of expression of DNA repair genes for the purpose of control of radiosensitivity of mammalian cells for cancer radiotherapy. One of their experiments concerns the enhancement of sensitivity to radiation and anti-tumor agents by suppressing the expression of mammalian Rad51 gene which playing a central role in recombination repair against DNA double-strand break, by RNA interference (RNAi). Described are the mode of action of RNAi, mechanism of suppression of Rad51 gene expression by it, enhancing effect in radiosensitivity, stable suppression and enhancement by hairpin RNA and its possible usefulness in cancer therapy. The other concerns the histone H2AX gene, which delivering the repair signal post phosphorylation in chromatin against the double-strand break. Experimental results of suppression of the histone H2AX gene by tet-off system, enhancement of radiosensitivity by the suppression and functional recovery by the gene transfer are described, and the radiosensitivity can be thus artificially controlled by tetracycline in authors' F9 2AX (tet/tet) cells. (N.I.)

  6. The histone variant macroH2A is an epigenetic regulator of key developmental genes

    DEFF Research Database (Denmark)

    Buschbeck, Marcus; Uribesalgo, Iris; Wibowo, Indra

    2009-01-01

    The histone variants macroH2A1 and macroH2A2 are associated with X chromosome inactivation in female mammals. However, the physiological function of macroH2A proteins on autosomes is poorly understood. Microarray-based analysis in human male pluripotent cells uncovered occupancy of both macroH2A ...

  7. Rtt109-dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana.

    Science.gov (United States)

    Cai, Qing; Wang, Juan-Juan; Shao, Wei; Ying, Sheng-Hua; Feng, Ming-Guang

    2018-04-27

    Rtt109 is a histone acetyltransferase that catalyzes histone H3K56 acetylation required for genomic stability, DNA damage repair and virulence-related gene activity in yeast-like human pathogens but remains functionally unknown in fungal insect pathogens. This study seeks to elucidate catalytic activity of Rtt109 orthologue and its possible role in sustaining biological control potential of Beauveria bassiana, a fungal entomopathogen. Deletion of rtt109 in B. bassiana abolished histone H3K56 acetylation and triggered histone H2A-S129 phosphorylation. Consequently, the deletion mutant showed increased sensitivities to the stresses of DNA damage, oxidation, cell wall perturbation, high osmolarity and heat shock during colony growth, severe conidiation defects under normal culture conditions, reduced conidial hydrophobicity, decreased conidial UV-B resistance, and attenuated virulence through normal cuticle infection. These phenotypic changes correlated well with reduced transcript levels of many genes, which encode the families of H2A-S129 dephosphorylation-related protein phosphotases, DNA damage-repairing factors, antioxidant enzymes, heat-shock proteins, key developmental activators, hydrophobins and cuticle-degrading Pr1 proteases respectively. Rtt109 can acetylate H3K56 and dephosphorylate H2A-S129 in direct and indirect manners respectively, and hence plays an essential role in sustaining genomic stability and global gene activity required for conidiation capacity, environmental fitness and pest-control potential in B. bassiana. This article is protected by copyright. All rights reserved.

  8. Mean frequency and relative fluorescence intensity measurement of γ-H2AX foci dose response in PBL exposed to γ-irradiation: An inter- and intra-laboratory comparison and its relevance for radiation triage.

    Science.gov (United States)

    Venkateswarlu, Raavi; Tamizh, Selvan G; Bhavani, Manivannan; Kumar, Arun; Alok, Amit; Karthik, Kanagaraj; Kalra, Namita; Vijayalakshmi, J; Paul, Solomon F D; Chaudhury, N K; Venkatachalam, Perumal

    2015-12-01

    Measurement of γ-H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of (60) Co γ-radiation at a dose rate of 1 Gy/min. Radiation induced γ-H2AX foci frequency (n = 3) and relative fluorescence intensity (n = 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for γ-H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with γ-H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r(2) = 0.918) than that obtained with automated (Metafer) scoring (r(2) = 0.690). It is noteworthy to mention that, the γ-H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the γ-H2AX foci frequency obtained by manual scoring and RFI (r(2) = 0.910). Kinetic studies showed that the γ-H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 37°C. Further, inter and intra-laboratory comparisons showed consistency in the scoring of γ-H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of γ-H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up

  9. Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

    Science.gov (United States)

    Earnshaw, W C; Allshire, R C; Black, B E; Bloom, K; Brinkley, B R; Brown, W; Cheeseman, I M; Choo, K H A; Copenhaver, G P; Deluca, J G; Desai, A; Diekmann, S; Erhardt, S; Fitzgerald-Hayes, M; Foltz, D; Fukagawa, T; Gassmann, R; Gerlich, D W; Glover, D M; Gorbsky, G J; Harrison, S C; Heun, P; Hirota, T; Jansen, L E T; Karpen, G; Kops, G J P L; Lampson, M A; Lens, S M; Losada, A; Luger, K; Maiato, H; Maddox, P S; Margolis, R L; Masumoto, H; McAinsh, A D; Mellone, B G; Meraldi, P; Musacchio, A; Oegema, K; O'Neill, R J; Salmon, E D; Scott, K C; Straight, A F; Stukenberg, P T; Sullivan, B A; Sullivan, K F; Sunkel, C E; Swedlow, J R; Walczak, C E; Warburton, P E; Westermann, S; Willard, H F; Wordeman, L; Yanagida, M; Yen, T J; Yoda, K; Cleveland, D W

    2013-04-01

    The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

  10. Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

    Science.gov (United States)

    Liu, X; Gorovsky, M A

    1996-01-01

    A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

  11. Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast

    International Nuclear Information System (INIS)

    Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung; Lee, Daeyoup

    2016-01-01

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5′ promoter and 3′ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.

  12. Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hanna [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kwon, Chang Seob [Department of Chemistry and Biology, Korea Science Academy of KAIST, Busan, 614-822 (Korea, Republic of); Choi, Yoonjung, E-mail: jjungii@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Daeyoup, E-mail: daeyoup@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2016-08-05

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5′ promoter and 3′ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.

  13. Biological significance of the focus on DNA damage checkpoint factors remained after irradiation of ionizing radiation

    International Nuclear Information System (INIS)

    Yamauchi, Motohiro; Suzuki, Keiji

    2005-01-01

    This paper reviews recent reports on the focus formation and participation to checkpoint of (such phosphorylated (P-d) as below) ATM and H2AX, MDC1, 53BP1 and NBS1, and discusses their role in DNA damage checkpoint induction mainly around authors' studies. When the cell is irradiated by ionizing radiation, the subtype histone like H2AX is P-d and the formed focus', seen in the nucleus on immuno-fluorographic observation, represents the P-d H2AX at the damaged site of DNA. The role of P-d ATM (the product of causative gene of ataxia-telangiectasia mutation, a protein kinase) has been first shown by laser beam irradiation. Described are discussions on the roles and functions after irradiation in focus formation and DNA damage checkpoint of P-d H2AX (a specific histone product by the radiation like γ-ray as above), P-d ATM, MDC1 (a mediator of DNA damage check point protein 1), 53BP1, (a p53 binding protein) and NBS1 (the product of the causative gene of Nijmegen Breakage Syndrome). Authors have come to point out the remained focal size increase as implications of the efficient repair of damaged DNA, and the second cycled p53 accumulation, of tumor suppression. Thus evaluation of biological significance of these aspects, scarcely noted hitherto, is concluded important. (S.I.)

  14. In vitro H2AX phosphorylation and micronuclei induction in human fibroblasts across the Bragg curve of a 577MeV/nucleon Fe incident beam

    Energy Technology Data Exchange (ETDEWEB)

    Desai, N. [NASA Johnson Space Center, 2101 NASA Parkway, Houston, TX (United States); Sodolak, J. [Prairie View A and M University, Prairie View, TX (United States); Gersey, B. [Prairie View A and M University, Prairie View, TX (United States); Durante, M. [University Federico II, Naples (Italy); Lin, Z.W. [University of Alabama in Huntsville, Huntsville, AL (United States); Rusek, A. [Brookhaven National Laboratory, Upton, NY (United States); Cucinotta, F.A. [NASA Johnson Space Center, 2101 NASA Parkway, Houston, TX (United States); Wu, H. [NASA Johnson Space Center, 2101 NASA Parkway, Houston, TX (United States)]. E-mail: honglu.wu-1@nasa.gov

    2006-10-15

    The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the only major protection to astronauts from harmful exposure. Unlike low-linear energy transfer (LET) {gamma} or X-rays, the presence of shielding does not always reduce the radiation risks for energetic charged particle exposure, since the dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. The Bragg curve does not necessarily represent the biological damage along the particle traversal, and the 'biological Bragg curve' is dependent on the energy and the type of the primary particle, and may vary for different biological endpoints. Here we used a unique irradiation geometry to measure the biological response across the Bragg curve in human fibroblasts exposed to 577MeV/nucleon incident Fe ions in vitro. Polyethylene shielding was used to achieve a Bragg curve distribution with the beam geometry parallel to a monolayer of fibroblast cells. Qualitative analyses of {gamma}-H2AX fluorescence, a known marker of DSBs, indicated increased clustering of DNA damage before the Bragg peak, enhanced homogenous distribution at the peak, and provided visual evidence of high-LET particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. A quantitative biological response curve generated for micronuclei induction across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the percentage of mononucleated cells, which indicates inhibition in cell progression, increased at the location of the peak. These results confirm the argument that severely damaged cells at the Bragg peak, as observed by increased {gamma}-H2AX formation, are likely to go through reproduction death. Depending on the LET value of the primary particles, the yield of

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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 μM) 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 phosphorylationH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 μM PM. Numbers of oocytes with ≥ 5 γH2AX foci were significantly increased both 1 and 8 days after exposure to ≥ 1 μM 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 μM) and increased γH2AX foci (≥ 3 μM) 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.

  16. In vivo quantification of DNA double strand breaks

    International Nuclear Information System (INIS)

    Simonsson, M.; Qvarnstroem, F.; Turesson, I.; Johansson, K.-A.; Nyman, J.; Hermansson, I.; Oden, A.; Book, M.

    2003-01-01

    DNA double strand breaks (DSBs) can be introduced in the genome by exposure to exogenous agents such as ionising radiation and radio-mimetic chemicals. The biological importance of these breaks is significant even at low numbers. Inaccurate repair or lack of repair of a single DSB has the potential to kill a cell or lead to tumourigenesis. Thus the induction and repair of DSBs are crucial events in the onset of malignancies. Following the induction of DSBs, the core histone H2AX is rapidly phosphorylated at residue serine 139. This phosphorylated form of H2AX is referred to as gH2AX. Histones wrapped in megabase regions flanking these breaks are involved in this process, which results in the formation of discrete nuclear foci. It has previously been shown that a single DSB is sufficient to produce a detectable focus. So far there has been a lack of methods capable of measuring the amount of DSBs at clinically relevant quantities. Such a method would embrace a wide field of applications. It could be applied as a biological dosimeter when studying carcinogenic effects and provide the basis for an assay predicting individual radiosensitivity. We describe a measurement procedure that detects and quantifies small amounts of DSBs in vivo. This is accomplished using immunofluorescence detection of the molecular marker gH2AX. The gH2AX foci are quantified in histological sections using basic digital image analysis methods as the main component. In a primary assessment of the procedure we analysed the in vivo dose response of prostate cancer patients in clinical practice undergoing radiotherapy. Epidermal nucleated cells in skin biopsies taken 30 minutes following the first single dose delivered show linear dose response for low doses ranging from 0 - 1.2 Gy. The described procedure for double strand break quantification can detect dose changes as low as 0.18 Gy

  17. Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

    International Nuclear Information System (INIS)

    Gao, Zhen; Xu, Michael S.; Barnett, Tamara L.; Xu, C. Wilson

    2011-01-01

    Research highlights: → Resveratrol induces cellular senescence in glioma cell. → Resveratrol inhibits mono-ubiquitination of histone H2B at K120. → Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. → Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. → RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-β-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are

  18. Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhen; Xu, Michael S.; Barnett, Tamara L. [Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Xu, C. Wilson, E-mail: wxu@nvcancer.org [Nevada Cancer Institute, Las Vegas, NV 89135 (United States)

    2011-04-08

    Research highlights: {yields} Resveratrol induces cellular senescence in glioma cell. {yields} Resveratrol inhibits mono-ubiquitination of histone H2B at K120. {yields} Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. {yields} Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. {yields} RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-{beta}-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular

  19. Molecular recognition of H3/H4 histone tails by the tudor domains of JMJD2A: a comparative molecular dynamics simulations study.

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    Musa Ozboyaci

    Full Text Available BACKGROUND: Histone demethylase, JMJD2A, specifically recognizes and binds to methylated lysine residues at histone H3 and H4 tails (especially trimethylated H3K4 (H3K4me3, trimethylated H3K9 (H3K9me3 and di,trimethylated H4K20 (H4K20me2, H4K20me3 via its tandem tudor domains. Crystal structures of JMJD2A-tudor binding to H3K4me3 and H4K20me3 peptides are available whereas the others are not. Complete picture of the recognition of the four histone peptides by the tandem tudor domains yet remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We report a detailed molecular dynamics simulation and binding energy analysis of the recognition of JMJD2A-tudor with four different histone tails. 25 ns fully unrestrained molecular dynamics simulations are carried out for each of the bound and free structures. We investigate the important hydrogen bonds and electrostatic interactions between the tudor domains and the peptide molecules and identify the critical residues that stabilize the complexes. Our binding free energy calculations show that H4K20me2 and H3K9me3 peptides have the highest and lowest affinity to JMJD2A-tudor, respectively. We also show that H4K20me2 peptide adopts the same binding mode with H4K20me3 peptide, and H3K9me3 peptide adopts the same binding mode with H3K4me3 peptide. Decomposition of the enthalpic and the entropic contributions to the binding free energies indicate that the recognition of the histone peptides is mainly driven by favourable van der Waals interactions. Residue decomposition of the binding free energies with backbone and side chain contributions as well as their energetic constituents identify the hotspots in the binding interface of the structures. CONCLUSION: Energetic investigations of the four complexes suggest that many of the residues involved in the interactions are common. However, we found two receptor residues that were related to selective binding of the H3 and H4 ligands. Modifications or mutations

  20. Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Yong-Cheng [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Su, Nan [Department of Quality Detection and Management, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan (China); Shi, Xiao-Jing; Zhao, Wen; Ke, Yu [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China); Zi, Xiaolin [Department of Urology, University of California, Irvine, Orange, CA (United States); Department of Pharmacology, University of California, Irvine, Orange, CA (United States); Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA (United States); Zhao, Ning-Min; Qin, Yu-Hua; Zhao, Hong-Wei [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Liu, Hong-Min, E-mail: liuhm@zzu.edu.cn [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China)

    2015-01-15

    Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.

  1. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

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    Daisuke Yamamoto

    2010-09-01

    Full Text Available Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

  2. Purine receptor P2Y_6 mediates cellular response to γ-ray-induced DNA damage

    International Nuclear Information System (INIS)

    Ide, Shunta; Nishimaki, Naoko; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-01-01

    We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AXH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y_6 and P2Y_1_2 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y_6 receptor is highly expressed in A549 cells, but P2Y_1_2 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y_6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y_6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y_6 and P2Y_1_2 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y_6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage. (author)

  3. Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

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    Brigitte Müller

    2018-05-01

    Full Text Available Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30 in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL, suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.

  4. The histone H5 variant in Xenopus laevis

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Linders, M. T.; Charles, R.

    1984-01-01

    The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal

  5. ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

    International Nuclear Information System (INIS)

    Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

    2013-01-01

    Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

  6. Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

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    Hao Zhang

    Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

  7. Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

    Science.gov (United States)

    Zhang, Qian; Giebler, Holli A; Isaacson, Marisa K; Nyborg, Jennifer K

    2015-01-01

    In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo. These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M. [NIH; (UNC); (Columbia)

    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.

  9. DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.

    Science.gov (United States)

    Halicka, Dorota; Zhao, Hong; Li, Jiangwei; Garcia, Jorge; Podhorecka, Monika; Darzynkiewicz, Zbigniew

    2017-01-01

    Cell synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G 2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that synchronization of cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of cells synchronized this way since they do not represent unperturbed cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most cell synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for cell synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the cell cycle phase.

  10. Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

    Science.gov (United States)

    Ververis, Katherine; Rodd, Annabelle L; Tang, Michelle M; El-Osta, Assam; Karagiannis, Tom C

    2011-12-01

    Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

  11. Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

    Science.gov (United States)

    Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

    2017-10-16

    Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Effect of gamma irradiation on rat thymus arginine-rich H3 histone in vitro

    International Nuclear Information System (INIS)

    Patil, M.S.; Narasimhan, Saroja; Sreenivasan, A.

    1977-01-01

    Physicochemical properties of rat thymus H3 histone have been studied following gamma radiation (25-90 krad) in 0.2 N HCl. Polyacrylamide gel electrophoretic pattern (PGE) of H3 histone indicated that aggregates were formed in the histone fraction following gamma irradiation. The PGE pattern of the irradiated-histone fraction remained unaltered even after it was treated with 8.0 M urea to eliminate noncovalent bonding. On the other hand, the irradiated sample treated with β-mercaptoethanol exhibited the PGE pattern which was essentially similar to that of unirradiated sample. These results indicate that the aggregates seen in the PGE pattern of irradiated-H3 histone may be formed through interpolypeptide chain disulphide linkeges rather than by noncovalent bonding. This contention is also supported by the fact that irradiated-H3 histone exhibited hyperchromic shift at 240-250 nm region as well as increased disulphide content. Other results revealed that DNA-dependent RNA synthesis in vitro was inhibited to a greater extent by irradiated-H3 histone than by unirradiated-H3 histone. (author)

  13. In Vivo Acute on Chronic Ethanol Effects in Liver: A Mouse Model Exhibiting Exacerbated Injury, Altered Metabolic and Epigenetic Responses

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    Shivendra D. Shukla

    2015-11-01

    Full Text Available Chronic alcoholics who also binge drink (i.e., acute on chronic are prone to an exacerbated liver injury but its mechanism is not understood. We therefore investigated the in vivo effects of chronic and binge ethanol ingestion and compared to chronic ethanol followed by three repeat binge ethanol on the liver of male C57/BL6 mice fed ethanol in liquid diet (4% for four weeks followed by binge ethanol (intragastric administration, 3.5 g/kg body weight, three doses, 12h apart. Chronic followed by binge ethanol exacerbated fat accumulation, necrosis, decrease in hepatic SAM and SAM:SAH ratio, increase in adenosine levels, and elevated CYP2E1 levels. Histone H3 lysine acetylation (H3AcK9, dually modified phosphoacetylated histone H3 (H3AcK9/PS10, and phosphorylated H2AX increased after binge whereas phosphorylation of histone H3 ser 10 (H3S10 and H3 ser 28 (H3S28 increased after chronic ethanol-binge. Histone H3 lysine 4 and 9 dimethylation increased with a marked dimethylation in H3K9 in chronic ethanol binge group. Trimethylated histone H3 levels did not change. Nuclear levels of histone acetyl transferase GCN5 and histone deacetylase HDAC3 were elevated whereas phospho-CREB decreased in a distinctive manner. Taken together, acute on chronic ethanol ingestion caused amplification of liver injury and elicited characteristic profiles of histone modifications, metabolic alterations, and changes in nuclear protein levels. These findings demonstrate that chronic ethanol exposure renders liver more susceptible to repeat acute/binge ethanol induced acceleration of alcoholic liver disease.

  14. Radiation dose determines the method for quantification of DNA double strand breaks

    International Nuclear Information System (INIS)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-01-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AXH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  15. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AXH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  16. Nicotinamide N-Methyltransferase Suppression Participates in Nickel-Induced Histone H3 Lysine9 Dimethylation in BEAS-2B Cells

    Directory of Open Access Journals (Sweden)

    Qian Li

    2017-04-01

    Full Text Available Background: Nickel compounds are well-established human carcinogens with weak mutagenic activity. Histone methylation has been proposed to play an important role in nickel-induced carcinogenesis. Nicotinamide N-methyltransferase (NNMT decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine (SAM to S-adenosylhomocysteine (SAH. However, the role of NNMT in nickel-induced histone methylation remains unclear. Methods: BEAS-2B cells were exposed to different concentrations of nickel chloride (NiCl2 for 72 h or 200 μM NiCl2 for different time periods. Histone H3 on lysine 9 (H3K9 mono-, di-, and trimethylation and NNMT protein levels were measured by western blot analysis. Expressions of NNMT mRNA and the H3k9me2-associated genes, mitogen-activated protein kinase 3 (MAP2K3 and dickkopf1 (DKK1, were determined by qPCR analysis. The cellular ratio of nicotinamide adenine dinucleotide (NAD+ to reduced NAD (NADH and SAM/SAH ratio were determined. Results: Exposure of BEAS-2B cells to nickel increased H3K9 dimethylation (H3K9me2, suppressed the expressions of H3K9me2-associated genes (MAP2K3 and DKK1, and induced NNMT repression at both the protein and mRNA levels. Furthermore, over-expression of NNMT inhibited nickel-induced H3K9me2 and altered the cellular SAM/SAH ratio. Additionally, the NADH oxidant phenazine methosulfate (PMS not only reversed the nickel-induced reduction in NAD+/NADH but also inhibited the increase in H3K9me2. Conclusions: These findings indicate that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.

  17. Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.

    Science.gov (United States)

    Moraes, Izabel; Yuan, Zuo-Fei; Liu, Shichong; Souza, Glaucia Mendes; Garcia, Benjamin A; Casas-Mollano, J Armando

    2015-01-01

    Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in

  18. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...

  19. N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

    Science.gov (United States)

    Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas; Mizuguchi, Gaku; Wei, Debbie; Xiao, Hua; Luk, Ed; Ranjan, Anand; Wu, Carl

    2009-03-06

    Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

  20. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-α by decreasing levels of acetylated histone H3 and H4 at its promoter

    International Nuclear Information System (INIS)

    Engdahl, Ryan; Monroy, M. Alexandra; Daly, John M.

    2007-01-01

    Prostaglandin metabolite 15-Deoxy-Δ 12,14 -prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPARγ. We investigated the ability of 15d-PGJ2 to inhibit TNF-α gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 μM) inhibited LPS-stimulated TNF-α mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPARγ ligand, GW1929, failed to inhibit LPS-induced TNF-α mRNA expression nor did a PPARγ antagonist, GW9662, alter the repression of TNF-α mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPARγ-independent inhibition of TNF-α mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-α promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-α promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-α promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-α promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-α promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-α transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter

  1. Histone H1 heterogeneity in the midge, Chironomus thummi. Structural comparison of the H1 variants in an organism where their intrachromosomal localization is possible.

    Science.gov (United States)

    Hoyer-Fender, S; Grossbach, U

    1988-09-01

    1. Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I-1, II-1, II-2, II-3, III-1, III-2, and III-3, according to the order of migration in two steps of preparative electrophoresis. 2. The amino acid compositions are similar to those of other H1 histones. Subfractions I-1 and II-1 were found to contain one methionine and two tyrosine residues, II-2 contained two methionine and three tyrosine residues, and III-1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I-1 and II-1 a chain length of about 252 amino acids was estimated. 3. Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subfraction of histone H2A disappeared after this treatment. Most and very probably all subfractions are thus H1 sequence variants. 4. Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I-1 and at least some of the other variants. 5. H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison

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

    Science.gov (United States)

    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.

  3. Gamma-H2Ax quantification of low dose irradiation-induced DNA damage in patients receiving intensity modulated radiotherapy (IRMT)

    International Nuclear Information System (INIS)

    Sivabalasingham, S.; Short, S.; Worku, M.; Marks, G.; Guerrero-Urbano, T.

    2013-01-01

    The full text of the publication follows. Purpose/Objective: IMRT (Intensity Modulated Radiotherapy) offers greater target dose compliance yet may produce a comparative higher whole body dose. The aim of this study is to quantify γH2Ax foci in lymphocytes (an established marker of DNA double strand breaks) in patients undergoing IMRT. Material/Methods: Radical inverse planned IMRT was delivered to patients with brain tumours. Peripheral blood samples were collected from each patient at the following time points: baseline; weekly- prior to and 30 minutes after one treatment fraction; 2 and 6 weeks following completion of treatment. Whole blood was centrifuged to separate lymphocytes, which were fixed and stained for fluorescent immunocytochemistry. 150 cells per sample were visualized. γH2Ax foci were identified and counted using confocal microscopy. Results A low basal level of foci was present in all samples prior to any radiation exposure (0.233, SD 0.028). There was a significant increase in mean foci per cell in post radiotherapy treatment samples(0.367 foci per cell pre-treatment and 0.612 foci per cell post treatment, p=0.000) and no significant difference between post-treatment foci numbers at different times during treatment(for example, 0.518 foci per cell at week 1 and 0.760 at week 6, p=0.279). Mean foci numbers returned to background levels at 6 weeks following completion of radiotherapy (0.239 foci per cell at baseline and 0.219 foci per cell at 6 weeks, p=0.529). Comparison between patients treated with different delivery methods is ongoing. Conclusion: γH2Ax is a feasible marker of DNA damage in lymphocytes during IMRT. These data demonstrate a reproducible level of foci induction in patients undergoing IMRT for tumour targets in brain. There is no significant accumulation of foci during treatment and foci numbers return to baseline post treatment. This assay may be useful to assess differences in whole body dose when different delivery methods

  4. Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C

    Directory of Open Access Journals (Sweden)

    Xi-yu Liu

    2017-01-01

    Full Text Available Aims. Latent autoimmune diabetes in adults (LADA is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods. Blood CD4+ T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results. Reduced global H3 lysine 9 methylation was observed in LADA patients’ CD4+ T lymphocytes, compared to healthy controls (P < 0.05. H3 lysine 4 methylation was not statistically different. The reduced H3 lysine 9 methylation was associated with GADA titer but not correlated with glycosylated hemoglobin (HbA1c. When the LADA patient group was divided into those with complication and those without, relatively reduced global H3 lysine 9 methylation was observed in LADA patients with complication (P < 0.05. The expression of histone methyltransferase SUV39H2 for H3 lysine 9 methylation was downregulated in LADA patients, and the expression of histone demethylase KDM4C which made H3 lysine 9 demethylation was upregulated. Conclusion. The reduction of histone H3 lysine 9 methylation which may due to the downregulation of methyltransferase SUV39H2 and the upregulation of demethylase KDM4C was found in CD4+ T lymphocytes of LADA patients.

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

    Science.gov (United States)

    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.

  6. Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

    Science.gov (United States)

    Kikuchi, Hidetomo; Yuan, Bo; Yuhara, Eisuke; Imai, Masahiko; Furutani, Ryota; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Takagi, Norio; Toyoda, Hiroo

    2014-08-01

    We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights

  7. Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress. PMID:26234813

  8. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  9. Impact of cigarette smoking on histone (H2B) to protamine ratio in human spermatozoa and its relation to sperm parameters.

    Science.gov (United States)

    Hamad, M F; Shelko, N; Kartarius, S; Montenarh, M; Hammadeh, M E

    2014-09-01

    Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 ± 58.24 ng/10(6)) were significantly higher (p = 0.001) than that of non-smokers (109.1 ± 43.70 ng/10(6)), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 ± 0.071) and non-smokers (1.05 ± 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 ± 0.71) of smokers were significantly higher (p = sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility. © 2014 American Society of Andrology and European Academy of Andrology.

  10. A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

    Energy Technology Data Exchange (ETDEWEB)

    Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

    2016-09-20

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

  11. Brain pattern of histone H3 phosphorylation after acute amphetamine administration: its relationship to brain c-fos induction is strongly dependent on the particular brain area.

    Science.gov (United States)

    Rotllant, David; Armario, Antonio

    2012-02-01

    Recent evidence strongly suggests a critical role of chromatin remodelling in the acute and chronic effects of addictive drugs. We reasoned that Immunohistochemical detection of certain histone modifications may be a more specific tool than induction of immediate early genes (i.e. c-fos) to detect brain areas and neurons that are critical for the action of addictive drugs. Thus, in the present work we studied in adult male rats the effects of a high dose of amphetamine on brain pattern of histone H3 phosphorylation in serine 10 (pH3S(10)) and c-fos expression. We firstly observed that amphetamine-induced an increase in the number of pH3S(10) positive neurons in a restricted number of brain areas, with maximum levels at 30 min after the drug administration that declined at 90 min in most areas. In a second experiment we studied colocalization of pH3S(10) immunoreactivity (pH3S(10)-IR) and c-fos expression. Amphetamine increased c-fos expression in medial prefrontal cortex (mPFC), dorsal striatum, nucleus accumbens (Acb), major Island of Calleja (ICjM), central amygdala (CeA), bed nucleus of stria terminalis lateral dorsal (BSTld) and paraventricular nucleus of the hypothalamus (PVN). Whereas no evidence for increase in pH3S(10) positive neurons was found in the mPFC and the PVN, in the striatum and the Acb basically all pH3S(10) positive neurons showed colocalization with c-fos. In ICjM, CeA and BSTld a notable degree of colocalization was found, but an important number of neurons expressing c-fos were negative for pH3S(10). The present results give support to the hypothesis that amphetamine-induced pH3S(10)-IR showed a more restricted pattern than brain c-fos induction, being this difference strongly dependent on the particular brain area studied. It is likely that those nuclei and neurons showing pH3S(10)-IR are more specifically associated to important effects of the drug, including neural plasticity. This article is part of a Special Issue entitled 'Post

  12. Absence of DNA double-strand breaks in human peripheral blood mononuclear cells after 3 Tesla magnetic resonance imaging assessed by γH2AX flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Fasshauer, Martin; Staab, Wieland; Sohns, Jan M.; Ritter, Christian; Lotz, Joachim [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Kruewel, Thomas; Stahnke, Vera C. [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); Zapf, Antonia [University Medical Center Goettingen, Department of Medical Statistics, Goettingen (Germany); Rave-Fraenk, Margret [University Medical Center Goettingen, Department of Radiotherapy and Radiooncology, Goettingen (Germany); Steinmetz, Michael [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Pediatric Cardiology and Intensive Care Medicine, University Medical Center Goettingen (Germany); Unterberg-Buchwald, Christina [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany); Schuster, Andreas [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany)

    2018-03-15

    Magnetic resonance imaging (MRI) is regarded as a non-harming and non-invasive imaging modality with high tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities, MRI does not use ionising radiation. Recently, however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double-strand breaks in human lymphocytes. In this study we investigated the impact of 3-T cardiac MRI examinations on the induction of DNA double-strand breaks in peripheral mononuclear cells by γH2AX staining and flow cytometry analysis. The study cohort consisted of 73 healthy non-smoking volunteers with 36 volunteers undergoing CMRI and 37 controls without intervention. Differences between the two cohorts were analysed by a mixed linear model with repeated measures. Both cohorts showed a significant increase in the γH2AX signal from baseline to post-procedure of 6.7 % (SD 7.18 %) and 7.8 % (SD 6.61 %), respectively. However, the difference between the two groups was not significant. Based on our study, γH2AX flow cytometry shows no evidence that 3-T MRI examinations as used in cardiac scans impair DNA integrity in peripheral mononuclear cells. (orig.)

  13. Histone deacetylase 1 phosphorylation at S421 and S423 is constitutive in vivo, but dispensable in vitro

    International Nuclear Information System (INIS)

    Karwowska-Desaulniers, Paulina; Ketko, Anastasia; Kamath, Nayana; Pflum, Mary Kay H.

    2007-01-01

    Histone Deacetylase 1 (HDAC1) is a transcriptional regulator associated with proliferation, apoptosis, and tumorigenesis, although its precise cellular role is unclear. HDAC1 was previously characterized as a phosphoprotein where mutation of phosphorylated S421 and S423 resulted in a loss of deacetylase activity and protein association. Here, the role of phosphorylation in regulating HDAC1 function was examined using phospho-specific antibodies. The antibody studies revealed that phosphorylation at S421 and S423 is constant during the cell cycle, under stress conditions, or in the presence of kinase or phosphatase inhibitors. Further, phosphorylation is dispensable for catalysis or protein association in vitro, as revealed by phosphatase studies. Truncation mutants of HDAC1 demonstrated that binding to Sin3A is promoted by S421 and S423 phosphorylation, while interaction with RbAp48 is not. Taken together, the data are consistent with constitutive phosphorylation of HDAC1 at S421 and S423 in vivo, which is dispensable for activity in vitro

  14. 3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation.

    Science.gov (United States)

    Hagiwara, Yoshihiko; Niimi, Atsuko; Isono, Mayu; Yamauchi, Motohiro; Yasuhara, Takaaki; Limsirichaikul, Siripan; Oike, Takahiro; Sato, Hiro; Held, Kathryn D; Nakano, Takashi; Shibata, Atsushi

    2017-12-12

    DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1-2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G 2 -phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm 3 . These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

  15. Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

    International Nuclear Information System (INIS)

    Jo, Hye-Ryeong; Kim, Yong-Seok; Son, Hyeon

    2016-01-01

    Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

  16. Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Hye-Ryeong [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Kim, Yong-Seok [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of); Son, Hyeon, E-mail: hyeonson@hanyang.ac.kr [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of)

    2016-01-29

    Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

  17. The influence of x-ray contrast agents in computed tomography on the induction of dicentrics and {gamma}-H2AX foci in lymphocytes of human blood samples

    Energy Technology Data Exchange (ETDEWEB)

    Jost, G; Golfier, S; Pietsch, H; Lengsfeld, P; Voth, M [Bayer Schering Pharma AG, 13353 Berlin (Germany); Schmid, T E [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universitaet Muenchen, 81675 Munich (Germany); Eckardt-Schupp, F [Institute of Radiation Biology, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, 85764 Neuherberg (Germany); Schmid, E [Institute for Cell Biology, Center for Integrated Protein Science, University of Munich, 80336 Muenchen (Germany)], E-mail: Ernst.Schmid@lrz.uni-muenchen.de

    2009-10-21

    The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and {gamma}-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{sup -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 {gamma}-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 {gamma}-H2AX foci were observed in the absence or presence of 5 mg iodine ml{sup -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{sup -1} serving as a positive control, a biological DEF of 9.5 {+-} 1.4 and 2.3 {+-} 0.5 was determined for dicentrics and {gamma}-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml{sup -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.

  18. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  19. 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

    Energy Technology Data Exchange (ETDEWEB)

    Goutham, Hassan Venkatesh; Mumbrekar, Kamalesh Dattaram [Division of Radiobiology and Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka (India); Vadhiraja, Bejadi Manjunath [Manipal Hospital, Bangalore, Karnataka (India); Fernandes, Donald Jerard; Sharan, Krishna [Department of Radiotherapy and Oncology, Shiridi Sai Baba Cancer Hospital and Research Centre, Kasturba Hospital, Manipal, Karnataka (India); Kanive Parashiva, Guruprasad; Kapaettu, Satyamoorthy [Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka (India); Bola Sadashiva, Satish Rao, E-mail: satishraomlsc@gmail.com [Division of Radiobiology and Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka (India)

    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.

  20. Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Hable, V. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)]. E-mail: volker.hable@unibw.de; Dollinger, G. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany); Greubel, C. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Hauptner, A. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Kruecken, R. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Dietzel, S. [Department Biologie II, LMU-Muenchen, 82152 Martinsried (Germany); Cremer, T. [Department Biologie II, LMU-Muenchen, 82152 Martinsried (Germany); Drexler, G.A. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Munich (Germany); Friedl, A.A. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Munich (Germany); Loewe, R. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Munich (Germany)

    2006-04-15

    Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone {gamma}-H2AX are studied. For this purpose cells are irradiated in line patterns. The {gamma}-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

  1. The COMPASS Family of Histone H3K4 Methylases: Mechanisms of Regulation in Development and Disease Pathogenesis

    Science.gov (United States)

    Shilatifard, Ali

    2014-01-01

    The Saccharomyces cerevisiae Set1/COMPASS was the first histone H3 lysine 4 (H3K4) methylase identified over ten years ago. Since then, it has been demonstrated that Set1/COMPASS and its enzymatic product, H3K4 methylation, is highly conserved across the evolutionary tree. Although there is only one COMPASS in yeast, human cells bear at least six COMPASS family members each capable of methylating H3K4 with non-redundant functions. In yeast, the monoubiquitination of histone H2B by Rad6/Bre1 is required for proper H3K4 and H3K79 trimethylations. This histone crosstalk and its machinery are also highly conserved from yeast to human. In this review, the process of histone H2B monoubiquitination-dependent and independent histone H3K4 methylation as a mark of active transcription, enhancer signatures, and developmentally poised genes will be discussed. The misregulation of histone H2B monoubiquitination and H3K4 methylation results in the pathogenesis of human diseases including cancer. Recent findings in this regard will also be examined. PMID:22663077

  2. About a significance of the avian linker histone (H1) polymorphic ...

    Indian Academy of Sciences (India)

    60

    structural disorder may specify histone H1 interaction with both DNA and partnering proteins through ... from the studies conducted on mammalian model, including the human H1 variants. However ..... Thus, the disparate layout of histone H1.

  3. Application of machine learning methods to histone methylation ChIP-Seq data reveals H4R3me2 globally represses gene expression

    Science.gov (United States)

    2010-01-01

    Background In the last decade, biochemical studies have revealed that epigenetic modifications including histone modifications, histone variants and DNA methylation form a complex network that regulate the state of chromatin and processes that depend on it including transcription and DNA replication. Currently, a large number of these epigenetic modifications are being mapped in a variety of cell lines at different stages of development using high throughput sequencing by members of the ENCODE consortium, the NIH Roadmap Epigenomics Program and the Human Epigenome Project. An extremely promising and underexplored area of research is the application of machine learning methods, which are designed to construct predictive network models, to these large-scale epigenomic data sets. Results Using a ChIP-Seq data set of 20 histone lysine and arginine methylations and histone variant H2A.Z in human CD4+ T-cells, we built predictive models of gene expression as a function of histone modification/variant levels using Multilinear (ML) Regression and Multivariate Adaptive Regression Splines (MARS). Along with extensive crosstalk among the 20 histone methylations, we found H4R3me2 was the most and second most globally repressive histone methylation among the 20 studied in the ML and MARS models, respectively. In support of our finding, a number of experimental studies show that PRMT5-catalyzed symmetric dimethylation of H4R3 is associated with repression of gene expression. This includes a recent study, which demonstrated that H4R3me2 is required for DNMT3A-mediated DNA methylation--a known global repressor of gene expression. Conclusion In stark contrast to univariate analysis of the relationship between H4R3me2 and gene expression levels, our study showed that the regulatory role of some modifications like H4R3me2 is masked by confounding variables, but can be elucidated by multivariate/systems-level approaches. PMID:20653935

  4. Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells

    International Nuclear Information System (INIS)

    Collins, Hilary M; Kundu, Tapas K; Heery, David M; Abdelghany, Magdy K; Messmer, Marie; Yue, Baigong; Deeves, Sian E; Kindle, Karin B; Mantelingu, Kempegowda; Aslam, Akhmed; Winkler, G Sebastiaan

    2013-01-01

    Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. In

  5. Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells

    Directory of Open Access Journals (Sweden)

    Collins Hilary M

    2013-01-01

    Full Text Available Abstract Background Post-translational modifications (PTMs of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. Methods Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. Results Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2

  6. Study on characteristic differences of wheat 1Ax1 and 1Ax2* NILS obtained by transgenic HMW-GS 1Dx5 + 1Dy10 gene

    International Nuclear Information System (INIS)

    Sun Yan; Zhang Hongji; Liu Dongjun; Qi Qianqian; Yang Shuping; Guo Yipan; Ma Shumei; Liu Wenlin; Wang Guangjin; Zhao Wei

    2012-01-01

    Use wheat 1Ax1 and 1Ax2* NILS obtained by transgenic HMW-GS 1Dx5 + 1Dy10 gene, characteristics differences have been studied. The two-year results showed that the statistical differences in seed quality parameters, farinogram parameters, extensigram parameters, botany characters and main agricultural characters between 1Ax1 and 1Ax2* were not significant. We considered that 08K860 and 08K871 are similar in hereditary background, 1Ax1 and 1Ax2* have identical contribute on quality. Breeder should attach to 1Ax1 and 1Ax2* identically on wheat breeding. (authors)

  7. Study on characteristic differences of wheat 1Ax1 and 1Ax2* NILS obtained by transgenic HMW-GS 1Dx5+1Dy10 gene

    International Nuclear Information System (INIS)

    Sun Yan; Zhang Hongji; Liu Dongjun; Qi Qianqian; Yang Shuping; Guo Yifan; Ma Shumei; Liu Wenlin; Wang Guangjin; Zhao Wei

    2011-01-01

    Use wheat 1Ax1 and 1Ax2 * NILS obtained by transgenic HMW-GS 1Dx5+1Dy10 gene, characteristics differences have been studied. The two-year results showed that the statistical differences in seed quality parameters, farinogram parameters, extensigram parameters, botany characters and main agricultural characters between 1Ax1 and 1Ax2 * were not significant. We considered that 08K860 and 08K871 are similar in hereditary background, 1Ax1 and 1Ax2 * have identical contribute on quality. Breeder should atlach to 1Ax1 and 1Ax2 * identically on wheat breeding. (authors)

  8. Regulation of Cellular Dynamics and Chromosomal Binding Site Preference of Linker Histones H1.0 and H1.X.

    Science.gov (United States)

    Okuwaki, Mitsuru; Abe, Mayumi; Hisaoka, Miharu; Nagata, Kyosuke

    2016-11-01

    Linker histones play important roles in the genomic organization of mammalian cells. Of the linker histone variants, H1.X shows the most dynamic behavior in the nucleus. Recent research has suggested that the linker histone variants H1.X and H1.0 have different chromosomal binding site preferences. However, it remains unclear how the dynamics and binding site preferences of linker histones are determined. Here, we biochemically demonstrated that the DNA/nucleosome and histone chaperone binding activities of H1.X are significantly lower than those of other linker histones. This explains why H1.X moves more rapidly than other linker histones in vivo Domain swapping between H1.0 and H1.X suggests that the globular domain (GD) and C-terminal domain (CTD) of H1.X independently contribute to the dynamic behavior of H1.X. Our results also suggest that the N-terminal domain (NTD), GD, and CTD cooperatively determine the preferential binding sites, and the contribution of each domain for this determination is different depending on the target genes. We also found that linker histones accumulate in the nucleoli when the nucleosome binding activities of the GDs are weak. Our results contribute to understanding the molecular mechanisms of dynamic behaviors, binding site selection, and localization of linker histones. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Easy fix for clinical laboratories for the false-positive defect with the Abbott AxSym total beta-hCG test.

    Science.gov (United States)

    Cole, Laurence A; Khanlian, Sarah A

    2004-05-01

    False-positive hCG results can lead to erroneous diagnoses and needless chemotherapy and surgery. In the last 2 years, eight publications described cases involving false-positive hCG tests; all eight involved the AxSym test. We investigated the source of this abundance of cases and a simple fix that may be used by clinical laboratories. False-positive hCG was primarily identified by absence of hCG in urine and varying or negative hCG results in alternative tests. Seventeen false-positive serum samples in the AxSym test were evaluated undiluted and at twofold dilution with diluent containing excess goat serum or immunoglobulin. We identified 58 patients with false-positive hCG, 47 of 58 due to the Abbott AxSym total hCGbeta test (81%). Sixteen of 17 of these "false-positive" results (mean 100 mIU/ml) became undetectable when tested again after twofold dilution. A simple twofold dilution with this diluent containing excess goat serum or immunoglobulin completely protected 16 of 17 samples from patients having false-positive results. It is recommended that laboratories using this test use twofold dilution as a minimum to prevent false-positive results.

  10. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    Science.gov (United States)

    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

  11. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

  12. Abundance of intrinsic structural disorder in the histone H1 subtypes.

    Science.gov (United States)

    Kowalski, Andrzej

    2015-12-01

    The intrinsically disordered proteins consist of partially structured regions linked to the unstructured stretches, which consequently form the transient and dynamic conformational ensembles. They undergo disorder to order transition upon binding their partners. Intrinsic disorder is attributed to histones H1, perceived as assemblers of chromatin structure and the regulators of DNA and proteins activity. In this work, the comparison of intrinsic disorder abundance in the histone H1 subtypes was performed both by the analysis of their amino acid composition and by the prediction of disordered stretches, as well as by identifying molecular recognition features (MoRFs) and ANCHOR protein binding regions (APBR) that are responsible for recognition and binding. Both human and model organisms-animals, plants, fungi and protists-have H1 histone subtypes with the properties typical of disordered state. They possess a significantly higher content of hydrophilic and charged amino acid residues, arranged in the long regions, covering over half of the whole amino acid residues in chain. Almost complete disorder corresponds to histone H1 terminal domains, including MoRFs and ANCHOR. Those motifs were also identified in a more ordered histone H1 globular domain. Compared to the control (globular and fibrous) proteins, H1 histones demonstrate the increased folding rate and a higher proportion of low-complexity segments. The results of this work indicate that intrinsic disorder is an inherent structural property of histone H1 subtypes and it is essential for establishing a protein conformation which defines functional outcomes affecting on DNA- and/or partner protein-dependent cell processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123

    Energy Technology Data Exchange (ETDEWEB)

    An, Sojin [Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States; Yoon, Jungmin [Structural Biology Laboratory of Epigenetics, Department of Biological Sciences, Graduate school of Nanoscience and Technology (World Class University), KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, South Korea; Kim, Hanseong [Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States; Song, Ji-Joon [Structural Biology Laboratory of Epigenetics, Department of Biological Sciences, Graduate school of Nanoscience and Technology (World Class University), KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, South Korea; Cho, Uhn-soo [Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States

    2017-10-16

    Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

  14. Ethylene induces combinatorial effects of histone H3 acetylation in gene expression in Arabidopsis.

    Science.gov (United States)

    Wang, Likai; Zhang, Fan; Rode, Siddharth; Chin, Kevin K; Ko, Eun Esther; Kim, Jonghwan; Iyer, Vishwanath R; Qiao, Hong

    2017-07-17

    Histone acetylation and deacetylation are essential for gene regulation and have been implicated in the regulation of plant hormone responses. Many studies have indicated the role of histone acetylation in ethylene signaling; however, few studies have investigated how ethylene signaling regulates the genomic landscape of chromatin states. Recently, we found that ethylene can specifically elevate histone H3K14 acetylation and the non-canonical histone H3K23 acetylation in etiolated seedlings and the gene activation is positively associated with the elevation of H3K14Ac and H3K23Ac in response to ethylene. To assess the role of H3K9, H3K14, and H3K23 histone modifications in the ethylene response, we examined how ethylene regulates histone acetylation and the transcriptome at global level and in ethylene regulated genes both in wild type (Col-0) and ein2-5 seedlings. Our results revealed that H3K9Ac, H3K14Ac, and H3K23Ac are preferentially enriched around the transcription start sites and are positively correlated with gene expression levels in Col-0 and ein2-5 seedlings both with and without ethylene treatment. In the absence of ethylene, no combinatorial effect of H3K9Ac, H3K14Ac, and H3K23Ac on gene expression was detected. In the presence of ethylene, however, combined enrichment of the three histone acetylation marks was associated with high gene expression levels, and this ethylene-induced change was EIN2 dependent. In addition, we found that ethylene-regulated genes are expressed at medium or high levels, and a group of ethylene regulated genes are marked by either one of H3K9Ac, H3K14Ac or H3K23Ac. In this group of genes, the levels of H3K9Ac were altered by ethylene, but in the absence of ethylene the levels of H3K9Ac and peak breadths are distinguished in up- and down- regulated genes. In the presence of ethylene, the changes in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expressions. Our study reveals that

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

    Science.gov (United States)

    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

  16. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component.

    Science.gov (United States)

    Zheng, Lei; Roeder, Robert G; Luo, Yan

    2003-07-25

    We have isolated and functionally characterized a multicomponent Oct-1 coactivator, OCA-S which is essential for S phase-dependent histone H2B transcription. The p38 component of OCA-S binds directly to Oct-1, exhibits potent transactivation potential, is selectively recruited to the H2B promoter in S phase, and is essential for S phase-specific H2B transcription in vivo and in vitro. Surprisingly, p38 represents a nuclear form of glyceraldehyde-3-phosphate dehydrogenase, and binding to Oct-1, as well as OCA-S function, is stimulated by NAD(+) but inhibited by NADH. OCA-S also interacts with NPAT, a cyclin E/cdk2 substrate that is broadly involved in histone gene transcription. These studies thus link the H2B transcriptional machinery to cell cycle regulators, and possibly to cellular metabolic state (redox status), and set the stage for studies of the underlying mechanisms and the basis for coordinated histone gene expression and coupling to DNA replication.

  17. Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug

    International Nuclear Information System (INIS)

    Jafargholizadeh, Naser; Zargar, Seyed Jalal; Safarian, Shahrokh; Habibi-Rezaei, Mehran

    2012-01-01

    Highlights: ► For the first time, our results show mitoxantrone anticancer drug binds to histone H1, via hydrophobic, hydrogen, van der Waals and electrostatic interactions. ► Binding of mitoxantrone molecules to histone H1 is positive cooperative. ► Histone H1 may be considered as a new target for mitoxantrone at the chromatin level. - Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.

  18. Pretreatment of liver grafts in vivo by γ-aminobutyric acid receptor regulation reduces cold ischemia/warm reperfusion injury in rat

    Science.gov (United States)

    Hori, Tomohide; Gardner, Lindsay B.; Hata, Toshiyuki; Chen, Feng; Baine, Ann-Marie T.; Uemoto, Shinji; Nguyen, Justin H.

    2014-01-01

    Summary Background: Gamma-aminobutyric acid (GABA) is found throughout the body. The regulation of GABA receptor (GABAR) reduces oxidative stress (OS). Ischemia/reperfusion injury after orthotopic liver transplantation (OLT) causes OS-induced graft damage. The effects of GABAR regulation in donors in vivo were investigated. Material/Methods: Donor rats received saline, a GABAR agonist or GABAR antagonist 4 h before surgery. Recipient rats were divided into four groups according to the donor treatments: laparotomy, OLT with saline, OLT with GABAR agonist and OLT with GABAR antagonist. Histopathological, biochemical and immunohistological examinations were performed at 6, 12 and 24 h after OLT. Protein assays were performed at 6 h after OLT. The 4-hydroxynonenal (4-HNE), ataxia-telangiectasia mutated kinase (ATM), phosphorylated histone H2AXH2AX), phosphatidylinositol-3 kinase (PI3K), Akt and superoxide dismutase (SOD) were assessed by western blot analysis. Results: In the univariate analysis, histopathological and biochemical profiles verified that the GABAR agonist reduced graft damage. Immunohistology revealed that the GABAR agonist prevented the induction of apoptosis. Measurement of 4-4-HNE levels confirmed OS-induced damage after OLT, and the GABAR agonist improved this damage. In the γH2AX, PI3K, Akt and antioxidant enzymes (SODs), ATM and H2AX were greatly increased after OLT, and were reduced by the GABAR agonist. In the multivariate analyses between multiple groups, histopathological assessment, aspartate aminotransferase level, immunohistological examinations for apoptotic induction and γH2AX showed statistical differences. Conclusions: A specific agonist demonstrated regulation of GABAR in vivo in the liver. This activation in vivo reduced OS after OLT via the ATM/H2AX pathway. PMID:23792534

  19. Interactions of nickel(II) with histones. Stability and solution structure of complexes with CH3CO-Cys-Ala-Ile-His-NH2, a putative metal binding sequence of histone H3.

    Science.gov (United States)

    Bal, W; Lukszo, J; Jezowska-Bojczuk, M; Kasprzak, K S

    1995-01-01

    Nickel(II) compounds are established human carcinogens, but the molecular mechanisms underlying their activity are only partially known. One mechanism may include mediation by nickel of promutagenic oxidative DNA damage that depends on Ni(II) binding to chromatin. To characterize such binding at the histone moiety of chromatin, we synthesized the peptide CH3CO-Cys-Ala-Ile-His-NH2 (L), a model of the evolutionarily conserved motif in histone H3 with expected affinity for transition metals, and evaluated its reactivity toward Ni(II). Combined spectroscopic (UV/vis, CD, NMR) and potentiometric measurements showed that, at physiological pH, mixtures of Ni(II) and L yielded unusual macrochelate complexes, NiL and NiL2, in which the metal cation was bound through Cys and His side chains in a square-planar arrangement. Above pH 9, a NiH-3L complex was formed, structurally analogous to typical square-planar nickel complexes. These complexes are expected to catalyze oxidation reactions, and therefore, coordination of Ni(II) by the L motif in core histone H3 may be a key event in oxidative DNA base damage observed in the process of Ni(II)-induced carcinogenesis.

  20. Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms

    International Nuclear Information System (INIS)

    Johnson, E.M.; Sterner, R.; Allfrey, V.G.

    1987-01-01

    Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo[2-tritium]acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription

  1. Epigenetic-based combinatorial resveratrol and pterostilbene alters DNA damage response by affecting SIRT1 and DNMT enzyme expression, including SIRT1-dependent γ-H2AX and telomerase regulation in triple-negative breast cancer

    International Nuclear Information System (INIS)

    Kala, Rishabh; Shah, Harsh N.; Martin, Samantha L.; Tollefsbol, Trygve O.

    2015-01-01

    Nutrition is believed to be a primary contributor in regulating gene expression by affecting epigenetic pathways such as DNA methylation and histone modification. Resveratrol and pterostilbene are phytoalexins produced by plants as part of their defense system. These two bioactive compounds when used alone have been shown to alter genetic and epigenetic profiles of tumor cells, but the concentrations employed in various studies often far exceed physiologically achievable doses. Triple-negative breast cancer (TNBC) is an often fatal condition that may be prevented or treated through novel dietary-based approaches. HCC1806 and MDA-MB-157 breast cancer cells were used as TNBC cell lines in this study. MCF10A cells were used as control breast epithelial cells to determine the safety of this dietary regimen. CompuSyn software was used to determine the combination index (CI) for drug combinations. Combinatorial resveratrol and pterostilbene administered at close to physiologically relevant doses resulted in synergistic (CI <1) growth inhibition of TNBCs. SIRT1, a type III histone deacetylase (HDAC), was down-regulated in response to this combinatorial treatment. We further explored the effects of this novel combinatorial approach on DNA damage response by monitoring γ-H2AX and telomerase expression. With combination of these two compounds there was a significant decrease in these two proteins which might further resulted in significant growth inhibition, apoptosis and cell cycle arrest in HCC1806 and MDA-MB-157 breast cancer cells, while there was no significant effect on cellular viability, colony forming potential, morphology or apoptosis in control MCF10A breast epithelial cells. SIRT1 knockdown reproduced the effects of combinatorial resveratrol and pterostilbene-induced SIRT1 down-regulation through inhibition of both telomerase activity and γ-H2AX expression in HCC1806 breast cancer cells. As a part of the repair mechanisms and role of SIRT1 in recruiting DNMTs

  2. Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation.

    Science.gov (United States)

    Asghar, Adeel; Lajeunesse, Audrey; Dulla, Kalyan; Combes, Guillaume; Thebault, Philippe; Nigg, Erich A; Elowe, Sabine

    2015-09-24

    During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.

  3. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    Energy Technology Data Exchange (ETDEWEB)

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges [Mayo

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

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

    Science.gov (United States)

    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

  5. Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3

    OpenAIRE

    Keith, Kevin C.; Baker, Richard E.; Chen, Yinhuai; Harris, Kendra; Stoler, Sam; Fitzgerald-Hayes, Molly

    1999-01-01

    Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions o...

  6. Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

    Science.gov (United States)

    Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

    2009-05-01

    Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

  7. Radiation response and chromatin dynamics

    International Nuclear Information System (INIS)

    Ikura, Tsuyoshi

    2009-01-01

    Described is a recent progress in studies of chromatin structural alterations induced by DNA damage by radiation. DNA in eukaryotes exists in the chromatin structure and different mechanisms of response to damage and repair of DNA from those in prokaryotes have been recognized. Chromatin is composed from its unit structure of mono-nucleosome, which is formed from DNA and an octamer of core histones of H2A, H2B, H3 and H4. When DNA is damaged, histone structural alterations are required for repair factors and checkpoint proteins to access the damaged site. At the actual genome damage, chemical modification of histone to work as a code occurs dependently on the damage where chromatin remodeling factors and histone chaperone participate for structural alteration and remodeling. As well, the exchange of histone variants and fluidization of histones are recently reported. Known chemical modification involves phosphorylation, acetylation and ubiquitination of H2AX (a variant of H2A), and acetylation and methylation of H3. Each complex of TIP60, NuA4 and INO80 is known to be included in the regulation of chromatin with damaged/repaired DNA for remodeling, but little is known about recruitment of the factors concerned at the damage site. Regulatory mechanisms in above chromatin dynamics with consideration of quality and timing of radiation should be further elucidated for understanding the precise response to DNA damage. (K.T.)

  8. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    Science.gov (United States)

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Genetic engineering of cotton with a novel cry2AX1 gene to impart insect resistance against Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Karunamurthy Dhivya

    2016-09-01

    Full Text Available Embryogenic calli of cotton (Coker310 were cocultivated with the Agrobacterium tumefaciens strain LBA4404 harbouring the codon-optimised, chimeric cry2AX1 gene consisting of sequences from cry2Aa and cry2Ac genes isolated from Indian strains of Bacillus thuringiensis. Forty-eight putative transgenic plants were regenerated, and PCR analysis of these plants revealed the presence of the cry2AX1 gene in 40 plants. Southern blot hybridisation analysis of selected transgenic plants confirmed stable T-DNA integration in the genome of transformed plants. The level of Cry2AX1 protein expression in PCR positive plants ranged from 4.9 to 187.5 ng g-1 of fresh tissue. A transgenic cotton event, TP31, expressing the cry2AX1 gene showed insecticidal activity of 56.66 per cent mortality against Helicoverpa armigera in detached leaf disc bioassay. These results indicate that the chimeric cry2AX1 gene expressed in transgenic cotton has insecticidal activity against H. armigera.

  10. Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 phosphorylation and Ca2+ mobilisation

    DEFF Research Database (Denmark)

    Varela, Diego; Simon, Felipe; Olivero, Pablo

    2007-01-01

    )R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCgamma1 activation and/or [Ca(2+)](i) rise, abolished H(2)O(2)-induced VSOR Cl(-) currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCgamma1, H(2)O(2) did not induce activation......Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H(2)O(2)) in VSOR Cl(-) channel...... activation. The aim of this study was to determine the signalling pathways responsible for H(2)O(2)-induced VSOR Cl(-) channel activation. In rat hepatoma (HTC) cells, H(2)O(2) elicited a transient increase in tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) that was blocked by PP2, a Src...

  11. Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

    Science.gov (United States)

    Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel

    2013-01-01

    Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…

  12. Repair response for DNA double-strand damage through ubiquitylation of chromatin

    International Nuclear Information System (INIS)

    Nakada, Shinichiro

    2011-01-01

    The chromatin modulation (remodeling) via lysine63 (K63)-linked ubiquitin (U) has been found important in the repair response for DNA double-strand damage, and the sequential signaling events at the damage site are explained. As the first step of the repair, MRN (MRE11, RAD50 and nibrin) complex recognizes the damage site and binds to it followed by many linked reactions by recruited and activated enzymes of various protein kinases and phosphatases, which resulting in the enhanced early signaling. As well, gamma-H2AX (phosphorylated histone H2AX) is yielded by the process, to which phosphorylated MDC1 (mediator of DNA-damage checkpoint 1) binds to produce their complex. Then further binding of RNF8-HERC2-UBC13 (ring finger protein 8, hect domain and RCC1 (CHC1)-like domain, and U conjugating enzyme E2N, respectively) occurs for starting the cumulative ubiquitylation of H2AX via K63 as the middle phase response. Signaling in the late phase occurs on the U chain formed at the damage site by binding of RAP (receptor-associated protein) 80 and other recruited 5 proteins like BRCA1 (breast cancer 1, early onset) to repair DNA by the homologous recombination after 53BP1 (tumor protein p53 binding protein) binding followed by methylation of histone H4. In a case of human compound heterozygous RNF168 defect, RIDDLE syndrome (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties), cells have no and slight abnormality of G2/M and intra-S checkpoint, respectively. Another defecting case with homozygous nonsense mutation has high radiosensitivity, intra-S checkpoint abnormality and others. Abnormality of immuno-globulins observed in both cases is similar to that in the RNF8-knockout mouse. Many tasks in chromatin ubiquitylation in the repair are still remained to be solved for protection and treatment of related diseases. (T.T.)

  13. Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

    International Nuclear Information System (INIS)

    Ke Qingdong; Ellen, Thomas P.; Costa, Max

    2008-01-01

    Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

  14. Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

    KAUST Repository

    Chauhan, Sakshi; Mandal, Papita; Tomar, Raghuvir S.

    2016-01-01

    Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have

  15. Gadolinium-enhanced cardiac MR exams of human subjects are associated with significant increases in the DNA repair marker 53BP1, but not the damage marker γH2AX.

    Directory of Open Access Journals (Sweden)

    Jennifer S McDonald

    Full Text Available Magnetic resonance imaging is considered low risk, yet recent studies have raised a concern of potential damage to DNA in peripheral blood leukocytes. This prospective Institutional Review Board-approved study examined potential double-strand DNA damage by analyzing changes in the DNA damage and repair markers γH2AX and 53BP1 in patients who underwent a 1.5 T gadolinium-enhanced cardiac magnetic resonance (MR exam. Sixty patients were enrolled (median age 55 years, 39 males. Patients with history of malignancy or who were receiving chemotherapy, radiation therapy, or steroids were excluded. MR sequence data were recorded and blood samples obtained immediately before and after MR exposure. An automated immunofluorescence assay quantified γH2AX or 53BP1 foci number in isolated peripheral blood mononuclear cells. Changes in foci number were analyzed using the Wilcoxon signed-rank test. Clinical and MR procedural characteristics were compared between patients who had a >10% increase in γH2AX or 53BP1 foci numbers and patients who did not. The number of γH2AX foci did not significantly change following cardiac MR (median foci per cell pre-MR = 0.11, post-MR = 0.11, p = .90, but the number of 53BP1 foci significantly increased following MR (median foci per cell pre-MR = 0.46, post-MR = 0.54, p = .0140. Clinical and MR characteristics did not differ significantly between patients who had at least a 10% increase in foci per cell and those who did not. We conclude that MR exposure leads to a small (median 25% increase in 53BP1 foci, however the clinical relevance of this increase is unknown and may be attributable to normal variation instead of MR exposure.

  16. Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

    International Nuclear Information System (INIS)

    Callaway, J.E.; Ho, Y.S.; DeLange, R.J.

    1985-01-01

    The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. The authors also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly

  17. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

    DEFF Research Database (Denmark)

    Gotfryd, Kamil; Skladchikova, Galina; Lepekhin, Eugene E

    2010-01-01

    lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3). Results: VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1......ABSTRACT: BACKGROUND: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods: The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell....../2 phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK...

  18. X-ray-induced DNA double-strand breaks after angiographic examinations of different anatomic regions; Strahleninduzierte DNA-Doppelstrangbrueche nach Angiografien verschiedener Koerperregionen

    Energy Technology Data Exchange (ETDEWEB)

    Kuefner, M.A.; Schwab, S.A.; Azoulay, S.; Heckmann, M.; Heinrich, M.C.; Uder, M. [Universitaetsklinikum Erlangen (Germany). Radiologisches Inst.; Grudzenski, S.; Lobrich, M. [Technische Univ. Darmstadt (Germany). Strahlenbiologie und DNA-Reparatur

    2009-04-15

    Purpose: The aim of this study was to investigate DNA double-strand breaks (DSBs) in blood lymphocytes as markers of the biological radiation effects in angiography patients. Materials and Methods: The method is based on the phosphorylation of the histone variant H 2AX ({gamma}-H2AX) after formation of DSBs. Blood samples were collected before and up to 24 hours after exposure of 31 patients undergoing angiographies of different body regions. Blood lymphocytes were isolated, fixed, and stained with a specific {gamma}-H2AX antibody. Distinct foci representing DSBs were enumerated using fluorescence microscopy. Additional in-vitro experiments (10 - 100 mGy) were performed for evaluation of DBS repair. Results: 15 minutes after the end of fluoroscopy values between 0.01 and 1.50 DSBs per cell were obtained. The DNA damage level normalized to the dose area product was 0.099 (cardiac angiographies), 0.053 (abdominal angiographies), 0.023 (pelvic/leg angiographies) and 0.004 excess foci/cell/mGym{sup 2} (cerebrovascular angiographies). A linear correlation was found between {gamma}-H2AX foci levels and the dose area product (abdomen: R2 = 0.96; pelvis/legs: R2 = 0.71). In-vivo on average 46 % of DSBs disappeared within 1 hour and 70 % within 2.5 hours. Conclusion: {gamma}-H2AX immunofluorescence microscopy is a sensitive and reliable method for the determination of X-ray-induced DSBs during angiography. The DNA damage level depends on the dose, the exposed anatomic region, and the duration/fractionation of the X-ray exposure. (orig.)

  19. Covalent binding of benzo(a)pyrene-diol-epoxide to histone H2A in rat liver nuclei: target site specificity

    International Nuclear Information System (INIS)

    Kurokawa, M.; MacLeod, M.C.

    1986-01-01

    The authors have recently found that 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I), a strong carcinogen, binds selectively to histone H2A-2 variant in rat liver nuclei, using a high performance liquid chromatography (HPLC) system which can separate H4, H2B, 3 different fractions of H2A variants and 3 different H3 variants in an hour. Here the authors examined the binding site of BPDE-I to the H2A-2 variant. The H2A-2 variants were purified from the acid extracted core histones of rat liver nuclei treated with ( 3 H)-BPDE-I by the HPLC system with a semi-preparative Aquapore RP-300 column. HPLC analysis of cyanogen bromide treated-H2A-2, which has one methionine residue, showed that the binding site is located in C-terminal half of H2A-2. In addition, digestions with V8-protease, trypsin and different types of carboxypeptides suggested that there are some target amino acid residues for BPDE-I in the V8-proteolytic C-terminal octapeptide which contains 2 histadine and 3 lysine residues. Currently identification of the target amino acid is proceeding, using amino acid-BPDE adducts prepared in vitro

  20. Modification of the histone tetramer at the H3-H3 interface impacts tetrasome conformations and dynamics

    Science.gov (United States)

    Ordu, Orkide; Kremser, Leopold; Lusser, Alexandra; Dekker, Nynke H.

    2018-03-01

    Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.

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

    Science.gov (United States)

    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.

  2. Opposite replication polarities of transcribed and nontranscribed histone H5 genes

    International Nuclear Information System (INIS)

    Trempe, J.P.; Lindstrom, Y.I.; Leffak, M.

    1988-01-01

    The authors used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocyctes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, the authors conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction

  3. Intermediate frequency magnetic field generated by a wireless power transmission device does not cause genotoxicity in vitro.

    Science.gov (United States)

    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. © 2014 Wiley Periodicals, Inc.

  4. Simplified Method for Rapid Purification of Soluble Histones

    Directory of Open Access Journals (Sweden)

    Nives Ivić

    2016-06-01

    Full Text Available Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H42 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research. This work is licensed under a Creative Commons Attribution 4.0 International License.

  5. Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.

    OpenAIRE

    Hays, Shan M; Swanson, Johanna; Selker, Eric U

    2002-01-01

    We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged ...

  6. Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability.

    Science.gov (United States)

    Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Jesús Cañal, Maria; Rodríguez, Roberto

    2010-03-15

    Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs. Copyright 2009 Elsevier GmbH. All rights reserved.

  7. Utility of γH2AX as a molecular marker of DNA double-strand breaks in nuclear medicine: applications to radionuclide therapy employing auger electron-emitting isotopes.

    Science.gov (United States)

    Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-01-01

    There is an intense interest in the development of radiopharmaceuticals for cancer therapy. In particular, radiopharmaceuticals which involve targeting radionuclides specifically to cancer cells with the use of monoclonal antibodies (radioimmunotherapy) or peptides (targeted radiotherapy) are being widely investigated. For example, the ultra-short range Auger electron-emitting isotopes, which are discussed in this review, are being considered in the context of DNAtargeted radiotherapy. The efficient quantitative evaluation of the levels of damage caused by such potential radiopharmaceuticals is required for assessment of therapeutic efficacy and determination of relevant doses for successful treatment. The DNA double-strand break surrogate marker, γH2AX, has emerged as a useful biomonitor of damage and thus effectiveness of treatment, offering a highly specific and sensitive means of assessment. This review will cover the potential applications of γH2AX in nuclear medicine, in particular radionuclide therapy.

  8. Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research.

    Science.gov (United States)

    Howe, Elizabeth S; Clemente, Thomas E; Bass, Hank W

    2012-06-01

    Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

  9. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  10. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    International Nuclear Information System (INIS)

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  11. High mobility group protein number17 cross-links primarily to histone H2A in the reconstituted HMG 17 - nucleosome core particle complex

    International Nuclear Information System (INIS)

    Cook, G.R.; Yau, P.; Yasuda, H.; Traut, R.R.; Bradbury, E.M.

    1986-01-01

    The neighbor relationship of lamb thymus High Mobility Group (HMG) protein 17 to native HeLa nucleosome core particle histones in the reconstituted complex has been studied. 125 I-labeled HMG 17 was cross-linking to core histones using the protein-protein cross-linking reagent 2-iminothiolane. Specific cross-linked products were separated on a two-dimensional Triton-acid-urea/SDS gel system, located by autoradiography, excised and quantified. Disulfide bonds in the cross links were then cleaved and the protein constituents were identified by SDS gel electrophoresis. HMG 17 cross-linked primarily to histone H2A while lower levels of cross-linking occurred between HMG 17 and the other histones. In contrast, cross-linking between two HMG 17 molecules bound on the same nucleosome was relatively rare. It is concluded that the same nucleosome was relatively rare. It is concluded that H2A comprises part of the HMG 17 binding site but that HMG 17 is sufficiently elongated and mobile to permit cross-linking to the other histones and to a second HMG 17 molecule. These results are in agreement with the current model for the structure of the nucleosome and the proposed binding sites for HMG 17

  12. Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

    Institute of Scientific and Technical Information of China (English)

    Chengkun Wang; Jie Shen; Zhongzheng Yang; Ping Chen; Bin Zhao; Wei Hu; Wenxian Lan

    2011-01-01

    Dear Editor,We report two NMR complex structures of PHDUHRF1 binding to unmodified or K9 trimethylated histone tails,which clarify a controversy regarding how the binding of UHRF1 to H3 tails is mediated.Based on our structures,H3R2,not H3K9,mediates PHD binding.

  13. Syntheses and modulations in the chromatin contents of histones H1/sup o/ and H1 during G1 and S phases in Chinese hamsters cells

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Tobey, R.A.

    1982-01-01

    Flow cytometry, conventional autoradiography, and autoradiography employing high concentrations of high specific activity [ 3 H]thymidine indicate that (1) treatment of Chinese hamster ovary (line CHO) cells with butyrate truly blocks cells in G 1 and (2) cells blocked in G 1 by isoleucine deprivation remain blocked in G 1 when they are released into complete medium containing butyrate. Measurements of H1/sup o/ content relative to core histones and H1/sup o/:H1 ratios indicate that H1/sup o/ is enhanced somewhat in G 1 cells arrested by isoleucine deprivation; however, (1) treatment with butyrate greatly increases the H1/sup o/ content in G 1 -blocked cells, and (2) the enhancement is very sensitive to butyrate concentration. Measurements of relative histone contents in the isolated chromatin of synchronized cultures also suggest that the acid-soluble content of histone H1 (relative to core histones) becomes greatly depleted in the isolated chromatin when synchronized cells are blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade. We also have measured [ 3 H]lysine incorporation, various protein ratios, and relative rates of deposition of newly synthesized H1/sup o/, H1, and H4 onto chromatin during G 1 and S in the absence of butyrate. The results suggest a dynamic picture of chromatin organization in which (1) newly synthesized histone H1/sup o/ binds to chromatin during traverse of G 1 and S phases and (2) histone H1 dissociates from (or becomes loosely bound to) chromatin during prolonged early S-phase block with hydroxyurea

  14. Dynamic behavior of histone H1 microinjected into HeLa cells

    International Nuclear Information System (INIS)

    Wu, L.H.; Kuehl, L.; Rechsteiner, M.

    1986-01-01

    Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125 I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that of endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of ∼100h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts < 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis

  15. Histone H1x is highly expressed in human neuroendocrine cells and tumours

    International Nuclear Information System (INIS)

    Warneboldt, Julia; Haller, Florian; Horstmann, Olaf; Danner, Bernhard C; Füzesi, László; Doenecke, Detlef; Happel, Nicole

    2008-01-01

    Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation. Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study. We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues. We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate

  16. Visualization of DNA clustered damage induced by heavy ion exposure

    International Nuclear Information System (INIS)

    Tomita, M.; Yatagai, F.

    2003-01-01

    Full text: DNA double-strand breaks (DSBs) are the most lethal damage induced by ionizing radiations. Accelerated heavy-ions have been shown to induce DNA clustered damage, which is two or more DNA lesions induced within a few helical turns. Higher biological effectiveness of heavy-ions could be provided predominantly by induction of complex DNA clustered damage, which leads to non-repairable DSBs. DNA-dependent protein kinase (DNA-PK) is composed of catalytic subunit (DNA-PKcs) and DNA-binding heterodimer (Ku70 and Ku86). DNA-PK acts as a sensor of DSB during non-homologous end-joining (NHEJ), since DNA-PK is activated to bind to the ends of double-stranded DNA. On the other hand, NBS1 and histone H2AX are essential for DSB repair by homologous recombination (HR) in higher vertebrate cells. Here we report that phosphorylated H2AX at Ser139 (named γ-H2AX) and NBS1 form large undissolvable foci after exposure to accelerated Fe ions, while DNA-PKcs does not recognize DNA clustered damage. NBS1 and γ-H2AX colocalized with forming discrete foci after exposure to X-rays. At 0.5 h after Fe ion irradiation, NBS1 and γ-H2AX also formed discrete foci. However, at 3-8 h after Fe ion irradiation, highly localized large foci turned up, while small discrete foci disappeared. Large NBS1 and γ-H2AX foci were remained even 16 h after irradiation. DNA-PKcs recognized Ku-binding DSB and formed foci shortly after exposure to X-rays. DNA-PKcs foci were observed 0.5 h after 5 Gy of Fe ion irradiation and were almost completely disappeared up to 8 h. These results suggest that NBS1 and γ-H2AX can be utilized as molecular marker of DNA clustered damage, while DNA-PK selectively recognizes repairable DSBs by NHEJ

  17. Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

    Science.gov (United States)

    Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

    2011-01-01

    To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories. PMID:21508930

  18. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

  19. Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis.

    Directory of Open Access Journals (Sweden)

    Jiang-Li Wu

    Full Text Available During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae. The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.

  20. Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis.

    Science.gov (United States)

    Wu, Jiang-Li; Kang, Xian-Jiang; Guo, Ming-Shen; Mu, Shu-Mei; Zhang, Zhao-Hui

    2015-01-01

    During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.

  1. Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

    Czech Academy of Sciences Publication Activity Database

    Vilasová, Z.; Řezáčová, M.; Vávrová, J.; Tichý, Adam; Vokurková, D.; Zoelzer, F.; Řeháková, Z.; Osterreicher, J.; Lukášová, Emilie

    2008-01-01

    Roč. 55, č. 2 (2008), s. 381-390 ISSN 0001-527X Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : apoptosis * DNA damage * phytohemagglutinin Subject RIV: BO - Biophysics Impact factor: 1.448, year: 2008

  2. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

    International Nuclear Information System (INIS)

    Waterborg, J.H.; Robertson, A.J.; Tatar, D.L.; Borza, C.M.; Davie, J.R.

    1995-01-01

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

  3. Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

    Science.gov (United States)

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

    2015-01-15

    Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to

  4. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang, E-mail: songyangwenrong@hotmail.com

    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.

  5. Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes

    OpenAIRE

    Jufvas, ?sa; Sj?din, Simon; Lundqvist, Kim; Amin, Risul; Vener, Alexander V; Str?lfors, Peter

    2013-01-01

    BACKGROUND: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies. RESULTS: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation com...

  6. Tank 241-AX-101, grab samples, 1AX-97-1 through 1AX-97-3 analytical results for the final report

    International Nuclear Information System (INIS)

    Esch, R.A.

    1997-01-01

    This document is the final report for tank 241-AX-101 grab samples. Four grab samples were collected from riser 5B on July 29, 1997. Analyses were performed on samples 1AX-97-1, 1AX-97-2 and 1AX-97-3 in accordance with the Compatibility Grab Sampling and Analysis Plan (TSAP) and the Data Quality Objectives for Tank Farms Waste Compatibility Program (DQO) (Rev. 1: Fowler, 1995; Rev. 2: Mulkey and Miller, 1997). The analytical results are presented in Table 1. No notification limits were exceeded. All four samples contained settled solids that appeared to be large salt crystals that precipitated upon cooling to ambient temperature. Less than 25 % settled solids were present in the first three samples, therefore only the supernate was sampled and analyzed. Sample 1AX-97-4 contained approximately 25.3 % settled solids. Compatibility analyses were not performed on this sample. Attachment 1 is provided as a cross-reference for relating the tank farm customer identification numbers with the 222-S Laboratory sample numbers and the portion of sample analyzed. Table 2 provides the appearance information. All four samples contained settled solids that appeared to be large salt crystal that precipitated upon cooling to ambient temperature. The settled solids in samples 1AX-97-1, 1AX-97-2 and 1AX-97-3 were less than 25% by volume. Therefore, for these three samples, two 15-mL subsamples were pipetted to the surface of the liquid and submitted to the laboratory for analysis. In addition, a portion of the liquid was taken from each of the these three samples to perform an acidified ammonia analysis. No analysis was performed on the settled solid portion of the samples. Sample 1AX-97-4 was reserved for the Process Chemistry group to perform boil down and dissolution testing in accordance with Letter of Instruction for Non-Routine Analysis of Single-Shell Tank 241-AX-101 Grab Samples (Field, 1997) (Correspondence 1). However, prior to the analysis, the sample was inadvertently

  7. Tank 241-AX-101 grab samples 1AX-97-1 through 1AX-97-3 analytical results for the final report

    Energy Technology Data Exchange (ETDEWEB)

    Esch, R.A.

    1997-11-13

    This document is the final report for tank 241-AX-101 grab samples. Four grab samples were collected from riser 5B on July 29, 1997. Analyses were performed on samples 1AX-97-1, 1AX-97-2 and 1AX-97-3 in accordance with the Compatibility Grab Sampling and Analysis Plan (TSAP) and the Data Quality Objectives for Tank Farms Waste Compatibility Program (DQO) (Rev. 1: Fowler, 1995; Rev. 2: Mulkey and Miller, 1997). The analytical results are presented in Table 1. No notification limits were exceeded. All four samples contained settled solids that appeared to be large salt crystals that precipitated upon cooling to ambient temperature. Less than 25 % settled solids were present in the first three samples, therefore only the supernate was sampled and analyzed. Sample 1AX-97-4 contained approximately 25.3 % settled solids. Compatibility analyses were not performed on this sample. Attachment 1 is provided as a cross-reference for relating the tank farm customer identification numbers with the 222-S Laboratory sample numbers and the portion of sample analyzed. Table 2 provides the appearance information. All four samples contained settled solids that appeared to be large salt crystal that precipitated upon cooling to ambient temperature. The settled solids in samples 1AX-97-1, 1AX-97-2 and 1AX-97-3 were less than 25% by volume. Therefore, for these three samples, two 15-mL subsamples were pipetted to the surface of the liquid and submitted to the laboratory for analysis. In addition, a portion of the liquid was taken from each of the these three samples to perform an acidified ammonia analysis. No analysis was performed on the settled solid portion of the samples. Sample 1AX-97-4 was reserved for the Process Chemistry group to perform boil down and dissolution testing in accordance with Letter of Instruction for Non-Routine Analysis of Single-Shell Tank 241-AX-101 Grab Samples (Field, 1997) (Correspondence 1). However, prior to the analysis, the sample was inadvertently

  8. Mitotic accumulation of dimethylated lysine 79 of histone H3 is important for maintaining genome integrity during mitosis in human cells.

    Science.gov (United States)

    Guppy, Brent J; McManus, Kirk J

    2015-02-01

    The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is altered in many tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential prerequisite for subsequent downstream dimethylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2, and H3K79me2 throughout the cell cycle, with a particular focus on mitosis. We show that H2Bub1, H3K4me2, and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone post-translational modification that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as a histone methyltransferase required for the mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors

  9. Imaging Features that Discriminate between Foci Induced by High-and Low-LET Radiation in Human Fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano,Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-10-08

    In this study, we investigated the formation ofradiation-induced foci in normal human fibroblasts exposed to X rays or130 keV/mum nitrogen ions using antibodies to phosphorylated proteinkinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantifythe immunofluorescence of radiation-induced foci. The size ofradiation-induced foci increased for both proteins over a 2-h periodafter nitrogen-ion irradiation, while the size of radiation-induced focidid not change after exposure to low-LET radiation. The number ofradiation-induced ATMp foci showed a more rapid rise and greaterfrequency after X-ray exposure and was resolved more rapidly such thatthe frequency of radiation-induced foci decreased by 90 percent comparedto 60 percent after exposure to high-LET radiation 2 h after 30 cGy. Incontrast, the kinetics of radiation-induced gamma-H2AX focus formationwas similar for high- and low-LET radiation in that it reached a plateauearly and remained constant for up to 2 h. High-resolution 3D images ofradiation-induced gamma-H2AX foci and dosimetry computation suggest thatmultiple double-strand breaks from nitrogen ions are encompassed withinlarge nuclear domains of 4.4 Mbp. Our work shows that the size andfrequency of radiation-induced foci vary as a function of radiationquality, dose, time and protein target. Thus, even though double-strandbreaks and radiation-induced foci are correlated, the dynamic nature ofboth contradicts their accepted equivalence for low doses of differentradiation qualities.

  10. A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation

    Directory of Open Access Journals (Sweden)

    Kumar Sonia

    2011-02-01

    Full Text Available Abstract Background The mammalian DNA-damage response (DDR has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs, mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+ with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37. Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

  11. 3-phosphorylated and -thiophosphorylated 2-thiazolidine- and 2-oxazolidine-thiones

    International Nuclear Information System (INIS)

    Vorob'eva, N.N.; Razvodovskaya, L.V.; Negrebetskii, V.V.; Grapov, A.F.; Mel'nikov, N.N.

    1987-01-01

    We investigated the phosphorylation and thiophosphorylation of 2-thiazolidine- and 2-oxazolidine-thiones. The presence in the heterocycle of the ambident triad HN-C=S can also lead to two series of phosphorylation products formed at the nitrogen and at the sulfur atom. It was therefore of interest to determine the dependence of the site of the phosphorylation on the structures of the heterocycle and off the phosphorylating agent. The formation of the N-phosphorylation products is confirmed by the 1 H NMR spectra, in which the signals of protons of the methylene group of the heteroring (C 4 H 2 ) are split on account of interaction with the phosphorus atom ( 3 JPH 0.5-2.3 Hz). We observed analogous values of 3 JPH constants for 2-aminothiazolines phosphorylated on the endocyclic nitrogen atom. In the 13 C NMR spectra of these compounds there are also coupling constants for the interaction of the carbon atoms C 4 and C 5 of the heterocycle with the phosphorus atom. The existence of the compounds as N-phosphorylated heterocycles is evidenced also by the 31 P chemical shifts

  12. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  13. Synergistic Effect of Combination Topotecan and Chronomodulated Radiation Therapy on Xenografted Human Nasopharyngeal Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, YanLing; Chen, Xin; Ren, PeiRong; Su, Zhou; Cao, HongYing; Zhou, Jie; Zou, XiaoYan; Fu, ShaoZhi; Lin, Sheng; Fan, Juan; Yang, Bo; Sun, XiaoYang [Department of Oncology, Affiliated Hospital of Luzhou Medical College, Luzhou (China); Zhou, Yan; Chen, Yue [Department of Medical Imaging, Luzhou Medical College, Luzhou (China); Yang, LingLin, E-mail: yanglinglin2003@tom.com [Department of Oncology, Affiliated Hospital of Luzhou Medical College, Luzhou (China); Wu, JingBo, E-mail: wjb6147@163.com [Department of Oncology, Affiliated Hospital of Luzhou Medical College, Luzhou (China)

    2013-10-01

    Purpose: To investigate the in vivo chronomodulated radiosensitizing effect of topotecan (TPT) on human nasopharyngeal carcinoma (NPC) and its possible mechanisms. Methods and Materials: Xenografted BALB/c (nu/nu) NPC mice were synchronized with an alternation of 12 hours of light from 0 to 12 hours after light onset (HALO) and 12 hours of darkness to establish a unified biological rhythm. Chronomodulated radiosensitization of TPT was investigated by analysis of tumor regrowth delay (TGD), pimonidazole hydrochloride, histone H2AX phosphorylation, (γ-H2AX) topoisomerase I (Top I), cell cycle, and apoptosis after treatment with (1) TPT (10 mg/kg) alone; (2) radiation therapy alone (RT); and (3) TPT+RT at 3, 9, 15, and 21 HALO. The tumor-loaded mice without any treatment were used as controls. Results: The TPT+RT combination was more effective than TPT or RT as single agents. The TPT+RT combination at 15 HALO was best (TGD = 58.0 ± 3.6 days), and TPT+RT at 3 HALO was worst (TGD = 35.0 ± 1.5 days) among the 4 TPT+RT groups (P<.05). Immunohistochemistry analysis revealed a significantly increased histone H2AX phosphorylation expression and decreased pimonidazole hydrochloride expression in the TPT+RT group at the same time point. The results suggested that the level of tumor hypoxia and DNA damage varied in a time-dependent manner. The expression of Top I in the TPT+RT group was also significantly different from the control tumors at 15 HALO (P<.05). Cell apoptosis index was increased and the proportion of cells in S phase was decreased (P<.05) with the highest value in 15 HALO and the lowest in 3 HALO. Conclusions: This study suggested that TPT combined with chronoradiotherapy could enhance the radiosensitivity of xenografted NPC. The TPT+RT group at 15 HALO had the best therapeutic effect. The chronomodulated radiosensitization mechanisms of TPT might be related to circadian rhythm of tumor hypoxia, cell cycle redistribution, DNA damage, and expression of Top I.

  14. Synergistic Effect of Combination Topotecan and Chronomodulated Radiation Therapy on Xenografted Human Nasopharyngeal Carcinoma

    International Nuclear Information System (INIS)

    Zhang, YanLing; Chen, Xin; Ren, PeiRong; Su, Zhou; Cao, HongYing; Zhou, Jie; Zou, XiaoYan; Fu, ShaoZhi; Lin, Sheng; Fan, Juan; Yang, Bo; Sun, XiaoYang; Zhou, Yan; Chen, Yue; Yang, LingLin; Wu, JingBo

    2013-01-01

    Purpose: To investigate the in vivo chronomodulated radiosensitizing effect of topotecan (TPT) on human nasopharyngeal carcinoma (NPC) and its possible mechanisms. Methods and Materials: Xenografted BALB/c (nu/nu) NPC mice were synchronized with an alternation of 12 hours of light from 0 to 12 hours after light onset (HALO) and 12 hours of darkness to establish a unified biological rhythm. Chronomodulated radiosensitization of TPT was investigated by analysis of tumor regrowth delay (TGD), pimonidazole hydrochloride, histone H2AX phosphorylation, (γ-H2AX) topoisomerase I (Top I), cell cycle, and apoptosis after treatment with (1) TPT (10 mg/kg) alone; (2) radiation therapy alone (RT); and (3) TPT+RT at 3, 9, 15, and 21 HALO. The tumor-loaded mice without any treatment were used as controls. Results: The TPT+RT combination was more effective than TPT or RT as single agents. The TPT+RT combination at 15 HALO was best (TGD = 58.0 ± 3.6 days), and TPT+RT at 3 HALO was worst (TGD = 35.0 ± 1.5 days) among the 4 TPT+RT groups (P<.05). Immunohistochemistry analysis revealed a significantly increased histone H2AX phosphorylation expression and decreased pimonidazole hydrochloride expression in the TPT+RT group at the same time point. The results suggested that the level of tumor hypoxia and DNA damage varied in a time-dependent manner. The expression of Top I in the TPT+RT group was also significantly different from the control tumors at 15 HALO (P<.05). Cell apoptosis index was increased and the proportion of cells in S phase was decreased (P<.05) with the highest value in 15 HALO and the lowest in 3 HALO. Conclusions: This study suggested that TPT combined with chronoradiotherapy could enhance the radiosensitivity of xenografted NPC. The TPT+RT group at 15 HALO had the best therapeutic effect. The chronomodulated radiosensitization mechanisms of TPT might be related to circadian rhythm of tumor hypoxia, cell cycle redistribution, DNA damage, and expression of Top I

  15. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...

  16. Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

    Science.gov (United States)

    Bheda, Poonam; Swatkoski, Stephen; Fiedler, Katherine L; Boeke, Jef D; Cotter, Robert J; Wolberger, Cynthia

    2012-04-17

    Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

  17. Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid ‘96 well lyse/fix’ protocol with a routine method

    Directory of Open Access Journals (Sweden)

    Jayne Moquet

    2014-03-01

    Full Text Available Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes in larger blood volumes are typically separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid ‘96 well lyse/fix’ method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples. Similar foci yields were scored for both protocols and consistent dose estimates were obtained for samples exposed to 0, 0.2, 0.6, 1.1, 1.2, 2.1 and 4.3 Gy of 250 kVp X-rays at 0.5 Gy/min and incubated for 2 h. Linear regression coefficients were 0.87 ± 0.06 (R2 = 97.6% and 0.85 ± 0.05 (R2 = 98.3% for estimated versus actual doses for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

  18. Characterization of histone H3K27 modifications in the β-globin locus

    International Nuclear Information System (INIS)

    Kim, Yea Woon; Kim, AeRi

    2011-01-01

    Research highlights: → The β-globin locus control region is hyperacetylated and monomethylated at histone H3K27. → Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. → Association of PRC2 subunits is comparable with H3K27me3 pattern. → Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human β-globin locus using the ChIP assay. The LCR of the human β-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the β-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  19. Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells.

    Directory of Open Access Journals (Sweden)

    Yukina Kawada

    Full Text Available Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic β cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic β cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2 expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic β cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

  20. Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

    Science.gov (United States)

    Ma, Zhaowu; Yu, Guanghui

    2010-02-15

    The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

  1. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    Science.gov (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  2. Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Maroschik, Belinda; Gürtler, Anne; Krämer, Anne; Rößler, Ute; Gomolka, Maria; Hornhardt, Sabine; Mörtl, Simone; Friedl, Anna A

    2014-01-01

    Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. In vitro studies for the introduction of γ-H2AX foci as an indicator of radiation damage in peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Mandina, Tania; Garcia, Omar; Roch-Lefevre, Sandrine; Voisin, Pascale; Voisin, Philippe; Roy, Laurence

    2008-01-01

    Biological indicators are used for assessing DNA damage and repair in cells exposed to ionising radiation. DNA Double-strand breaks (DSBs) have been known as one of the most significant lesion producing lethal and mutagenic effects in irradiated cells. A new biological marker for DSBs is the presence of γ-H2AX foci in cells nucleus after exposure to ionising radiation. γ-H2AX formation was analysed in human lymphocytes. The blood was obtained from a same donor in three different occasions and exposed to doses of 0, 0.2 and 0.5 Gy of gamma rays with a dose-rate of 1.2 Gy/min. After blood irradiation the lymphocytes were incubated 30 minutes at 37 C degrees, isolated, fixed with paraformaldehyde, and spread on a microscope slide using a Cytospin. The slides were stored at -20 C degrees and immuno-stained the next day and 14, 16, 27 and 37 days after irradiation to test the influence of the storage time on results. The number of foci per cell was scored automatically, in about 200 cells per dose using HISTOLAB and CARTOGRAPH software. The mean of number of foci/cell for samples was as follow 0 Gy= 0.15 ± 0.04, 0.2 Gy =2.06 ± 0.25 and 0.5 Gy=5.21 ± 0.36 without appreciable effect of the storage time on the final results. Nevertheless some aspects require additional research, particularly the background values of the assay. A higher variability of DNA damage was observed for control samples, than for exposed ones. The influence of the number of cells scored on this variability should be tested. (author)

  5. Hyperacetylation and differential deacetylation of histones H4 and H3 define two distinct classes of acetylated SV40 chromosomes early in infection

    International Nuclear Information System (INIS)

    Milavetz, Barry

    2004-01-01

    SV40 chromosomes undergoing encapsidation late in infection and SV40 chromatin in virions are hyperacetylated on histones H4 and H3. However, the fate of the SV40 chromosomes containing hyperacetylated histones in a subsequent round of infection has not been determined. In order to determine if SV40 chromosomes undergo changes in the extent of histone acetylation during early infection, we have analyzed SV40 chromosomes isolated 30 min and 3 h postinfection by quantitative ChIP assays, depletion ChIP assays, competitive ChIP assays, and ChIP assays combined with restriction endonuclease sensitivity using antibodies to hyperacetylated histones H4 and H3. We have shown that at 30 min postinfection, the hyperacetylated histones are associated with two distinct classes of SV40 chromosomes. One form is hyperacetylated specifically on histone H4 while a second form is hyperacetylated on both H4 and H3. Both forms of chromosomes appear to contain a nucleosome-free promoter region. Over the course of the next few hours of infection, the class of SV40 chromosomes hyperacetylated on only H4 is reduced or completely eliminated through deacetylation

  6. Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Yang, Zhihong [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516 (United States); Huang, Yi [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035 (China); Bennett, Jason [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Wang, Li, E-mail: li.wang@uconn.edu [Department of Physiology and Neurobiology and The Institute for Systems Genomics, University of Connecticut, Storrs, CT 062696 (United States); Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516 (United States); School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035 (China); Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT 06520 (United States)

    2016-08-01

    It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposure decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. - Graphical abstract: Schematic showing arsenic-mediated epigenetic pathway that inhibits PDK4 expression. (A) BRD induces PDK4 expression by decreasing G9a protein and histone H3K9me2 and H3K9me3 levels as well as diminishing their recruitment to the PDK4 promoter. (B) Arsenic counteracts the effect of BRD by increasing histone H3K9me2 and H3K9me3 levels as well as enhancing their enrichment to the PDK4 promoter. Display Omitted - Highlights: • Histone methyltrasferase G9a inhibitor BRD induces PDK4 expression. • Arsenic decreases PDK4 expression and increases H3K9me2 and me3 levels. • Arsenic enhances H3K9me2/me3 enrichment in the PDK4 promoter. • Arsenic antagonizes the activation of

  7. Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a

    International Nuclear Information System (INIS)

    Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan; Yang, Zhihong; Huang, Yi; Bennett, Jason; Wang, Li

    2016-01-01

    It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposure decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. - Graphical abstract: Schematic showing arsenic-mediated epigenetic pathway that inhibits PDK4 expression. (A) BRD induces PDK4 expression by decreasing G9a protein and histone H3K9me2 and H3K9me3 levels as well as diminishing their recruitment to the PDK4 promoter. (B) Arsenic counteracts the effect of BRD by increasing histone H3K9me2 and H3K9me3 levels as well as enhancing their enrichment to the PDK4 promoter. Display Omitted - Highlights: • Histone methyltrasferase G9a inhibitor BRD induces PDK4 expression. • Arsenic decreases PDK4 expression and increases H3K9me2 and me3 levels. • Arsenic enhances H3K9me2/me3 enrichment in the PDK4 promoter. • Arsenic antagonizes the activation of

  8. Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

    Science.gov (United States)

    Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

    2014-01-01

    Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR

  9. In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

    Science.gov (United States)

    Bruhat, A; Jost, J P

    1995-01-01

    We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7731964

  10. Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes

    Directory of Open Access Journals (Sweden)

    Yuki Hatanaka

    2017-09-01

    Full Text Available At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT, which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a, as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression. Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.

  11. Alterations of global histone H4K20 methylation during prostate carcinogenesis

    Directory of Open Access Journals (Sweden)

    Behbahani Turang E

    2012-03-01

    Full Text Available Abstract Background Global histone modifications have been implicated in the progression of various tumour entities. Our study was designed to assess global methylation levels of histone 4 lysine 20 (H4K20me1-3 at different stages of prostate cancer (PCA carcinogenesis. Methods Global H4K20 methylation levels were evaluated using a tissue microarray in patients with clinically localized PCA (n = 113, non-malignant prostate disease (n = 27, metastatic hormone-naive PCA (mPCA, n = 30 and castration-resistant PCA (CRPC, n = 34. Immunohistochemistry was performed to assess global levels of H4K20 methylation levels. Results Similar proportions of the normal, PCA, and mPCA prostate tissues showed strong H4K20me3 staining. CRPC tissue analysis showed the weakest immunostaining levels of H4K20me1 and H4K20me2, compared to other prostate tissues. H4K20me2 methylation levels indicated significant differences in examined tissues except for normal prostate versus PCA tissue. H4K20me1 differentiates CRPC from other prostate tissues. H4K20me1 was significantly correlated with lymph node metastases, and H4K20me2 showed a significant correlation with the Gleason score. However, H4K20 methylation levels failed to predict PSA recurrence after radical prostatectomy. Conclusions H4K20 methylation levels constitute valuable markers for the dynamic process of prostate cancer carcinogenesis.

  12. Evidence for gene-specific rather than transcription rate-dependent histone H3 exchange in yeast coding regions.

    Science.gov (United States)

    Gat-Viks, Irit; Vingron, Martin

    2009-02-01

    In eukaryotic organisms, histones are dynamically exchanged independently of DNA replication. Recent reports show that different coding regions differ in their amount of replication-independent histone H3 exchange. The current paradigm is that this histone exchange variability among coding regions is a consequence of transcription rate. Here we put forward the idea that this variability might be also modulated in a gene-specific manner independently of transcription rate. To that end, we study transcription rate-independent replication-independent coding region histone H3 exchange. We term such events relative exchange. Our genome-wide analysis shows conclusively that in yeast, relative exchange is a novel consistent feature of coding regions. Outside of replication, each coding region has a characteristic pattern of histone H3 exchange that is either higher or lower than what was expected by its RNAPII transcription rate alone. Histone H3 exchange in coding regions might be a way to add or remove certain histone modifications that are important for transcription elongation. Therefore, our results that gene-specific coding region histone H3 exchange is decoupled from transcription rate might hint at a new epigenetic mechanism of transcription regulation.

  13. The histones of the endosymbiont alga of Peridinium balticum (Dinophyceae).

    Science.gov (United States)

    Rizzo, P J; Morris, R L; Zweidler, A

    1988-01-01

    The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively.

  14. Biological significance of lysine mono-, di- and trimethylation on histone and non-histone proteins

    International Nuclear Information System (INIS)

    Perez-Burgos, L.

    2006-01-01

    Histones are the proteins that compact DNA into the repeating unit of chromatin known as the nucleosome. The N-termini of histones are subject to a series of post-translational modifications, one of which is methylation. This modification is termed 'epigenetic' because it extends the information encoded in the genome. Lysines can be mono-, di- or tri-methylated at different positions on histones H1, H3 and H4. In order to study the biological role of histone lysine methylation, antibodies were generated against mono-, di- and trimethylated H3-K9 and H3-27. Indeed, different chromatin domains in the mouse nucleus are enriched in distinct forms of histone lysine methylation, such as pericentric heterochromatin and the inactive X chromosome. Interestingly, heterochromatin in Arabidopsis thaliana is enriched in the mono- and di-, but not the trimethylated form of H3-K9. Furthermore, there exists a hierarchy of epigenetic modifications in which H3-K9 trimethylation is found to be upstream of DNA methylation on mouse major satellites. Histone lysine methylation is also involved in gene regulation upon development. One example is the chicken 61538;-globin locus, a region of facultative chromatin that undergoes a loss of di- and trimethylated H3-K27 in mature red blood cells, concomitant with expression of the 61538;-globin genes. SET-domain proteins are enzymes that methylate histones, but some of them are also able to methylate non-histone substrates. In particular, p53 is methylated by Set9 on lysine 372, G9a and Glp-1 on lysine 373 and by Smyd2 on lysine 370. Smyd2 transcript levels are greatly increased upon irradiation and dimethylated p53-370 specifically binds to 53BP1, a protein involved in recognizing DNA double-stranded breaks upon ionizing radiation. These results argue for a novel role of p53-K370 methylation in the biology of DNA damage. In summary, lysine methylation is a post-translational modification that can occur both on histone and non-histone proteins

  15. The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

    Energy Technology Data Exchange (ETDEWEB)

    Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.; Ahn, Natalie G.; Luger, Karolin

    2017-04-18

    Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

  16. Establishment of a semi-biological phantom model for the study of the effect of dose reducing measures on radiation-induced DNA double strand breaks in CT using the example of risk organ based tube current modulation; Etablierung eines semibiologischen Phantommodells zur Untersuchung des Effekts dosisreduzierender Massnahmen auf strahleninduzierte DNA-Doppelstrangbrueche in der CT am Beispiel der risikoorganbasierten Roehrenstrommodulation

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, Matthias

    2013-12-12

    The number of computed tomography (CT) examinations has been rising during the last decades. Therefore techniques for dose reduction receive increasing attention. Risk organ-based tube current modulation (RCM) in CT is a new approach and works by lowering the tube current, while the tube is in front of the patient's body. Therefore it should lead to a dose reduction for radiosensitive organs like the female breast, the eye lenses and the thyroid gland. Biological radiation effects cannot be estimated by physical-based dose measurements. γ-H2AX is a sensitive marker for the determination of x-ray induced DNA double-strand breaks (DSB). Hence the aim of this study was to establish a biological phantom model based on the γ-H2AX immunofluorescence microscopy method and to investigate the effect of RCM on radiation induced DNA damages. The γ-H2AX method is based on the phosphorylation of the histone variant H2AX. The phosphorylated histone γ-H2AX can be visualised using antibodies and is specific for radiation induced DSB. Blood lymphocytes from healthy volunteers, skin fibroblasts (LN) and mammary epithelial cells (HMEpC-p) were placed in different positions of an Alderson-phantom and exposed to x-rays using a 128-slice dual-source CT scanner. Standard head, neck and chest-CT scan protocols either with or without risk-organ based tube current modulation were used. RCM reduces the tube current to 20 percent at an angle of 130 degree anterior to the body, whereas tube current is increased at an angle of 230 degree posterior to the body. Afterwards cells were isolated, fixed on slides und stained with specific primary γ-H2AX antibodies and fluorescent secondary antibodies. Tiny green dots (named foci) can be detected and quantified with a fluorescence microscope and represent distinct DSB. Non-irradiated samples served as controls and CT-induced DSB were calculated by subtraction of pre- from post-exposure values. In this study a semibiological phantom model

  17. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    Science.gov (United States)

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  18. Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation

    International Nuclear Information System (INIS)

    Alamdar, Ambreen; Xi, Guochen; Huang, Qingyu; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

    2017-01-01

    Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. - Highlights: • Epigenetic mechanisms of arsenic-induced male reproductive toxicity remain unclear. • Arsenic disturbs the expression of key steroidogenic genes in MLTC-1 cells. • Histone H3K9 di- and tri-methylation was suppressed in arsenic-exposed cells. • Arsenic activates 3β-HSD expression through repression of histone H3K9 methylation.

  19. Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation

    Energy Technology Data Exchange (ETDEWEB)

    Alamdar, Ambreen; Xi, Guochen [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Huang, Qingyu, E-mail: qyhuang@iue.ac.cn [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Centre for Epigenetics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M (Denmark); Tian, Meiping [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Eqani, Syed Ali Musstjab Akber Shah [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Public Health and Environment Division, Department of Biosciences, COMSAT Institute of Information & Technology, Islamabad (Pakistan); Shen, Heqing, E-mail: hqshen@iue.ac.cn [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China)

    2017-07-01

    Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. - Highlights: • Epigenetic mechanisms of arsenic-induced male reproductive toxicity remain unclear. • Arsenic disturbs the expression of key steroidogenic genes in MLTC-1 cells. • Histone H3K9 di- and tri-methylation was suppressed in arsenic-exposed cells. • Arsenic activates 3β-HSD expression through repression of histone H3K9 methylation.

  20. The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.

    Science.gov (United States)

    Nakata, Shiori; Watanabe, Tadashi; Nakagawa, Koji; Takeda, Hiroshi; Ito, Akihiro; Fujimuro, Masahiro

    2016-03-25

    Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter

  1. Sequence of cDNAs for mammalian H2A. Z, an evolutionarily diverged but highly conserved basal histone H2A isoprotein species

    Energy Technology Data Exchange (ETDEWEB)

    Hatch, C L; Bonner, W M

    1988-02-11

    The nucleotide sequences of cDNAs for the evolutionarily diverged but highly conserved basal H2A isoprotein, H2A.Z, have been determined for the rat, cow, and human. As a basal histone, H2A.Z is synthesized throughout the cell cycle at a constant rate, unlinked to DNA replication, and at a much lower rate in quiescent cells. Each of the cDNA isolates encodes the entire H2A.Z polypeptide. The human isolate is about 1.0 kilobases long. It contains a coding region of 387 nucleotides flanked by 106 nucleotides of 5'UTR and 376 nucleotides of 3'UTR, which contains a polyadenylation signal followed by a poly A tail. The bovine and rat cDNAs have 97 and 94% nucleotide positional identity to the human cDNA in the coding region and 98% in the proximal 376 nucleotides of the 3'UTR which includes the polyadenylation signal. A potential stem-forming sequence imbedded in a direct repeat is found centered at 261 nucleotides into the 3'UTR. Each of the cDNA clones could be transcribed and translated in vitro to yield H2A.Z protein. The mammalian H2A.Z cDNA coding sequences are approximately 80% similar to those in chicken and 75% to those in sea urchin.

  2. Imaging Features that Discriminate between Foci Induced by High- and Low-LET Radiation in Human Fibroblasts

    International Nuclear Information System (INIS)

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano, Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-01-01

    In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90 percent compared to 60 percent after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities

  3. Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

    Directory of Open Access Journals (Sweden)

    Buro Lauren J

    2010-09-01

    Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

  4. Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

    Directory of Open Access Journals (Sweden)

    Ysabel Montoya

    2000-08-01

    Full Text Available Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V. peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V. braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

  5. Study of hTERT and Histone 3 Mutations in Medulloblastoma.

    Science.gov (United States)

    Viana-Pereira, Marta; Almeida, Gisele Caravina; Stavale, João Norberto; Malheiro, Susana; Clara, Carlos; Lobo, Patrícia; Pimentel, José; Reis, Rui Manuel

    2017-01-01

    Hotspot activating mutations of the telomerase reverse transcriptase (hTERT) promoter region were recently described in several tumor types. These mutations lead to enhanced expression of telomerase, being responsible for telomere maintenance and allowing continuous cell division. Additionally, there are alternative telomere maintenance mechanisms, associated with histone H3 mutations, responsible for disrupting the histone code and affecting the regulation of transcription. Here, we investigated the clinical relevance of these mechanistically related molecules in medulloblastoma. Sixty-nine medulloblastomas, formalin fixed and paraffin embedded, from a cohort of patients aged 1.5-70 years, were used to investigate the hotspot mutations of the hTERT promoter region, i.e. H3F3A and HIST1H3B, using Sanger sequencing. We successfully sequenced hTERT in all 69 medulloblastoma samples and identified a total of 19 mutated cases (27.5%). c.-124:G>A and c.-146:G>A mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p < 0.0001), being found only in 5 patients <20 years of age. In addition, hTERT-mutated tumors were more frequently recurrent (p = 0.026) and hTERT mutations were significantly enriched in tumors located in the right cerebellar hemisphere (p = 0.039). No mutations were found on the H3F3A or HIST1H3B genes. hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in medulloblastoma. © 2016 S. Karger AG, Basel.

  6. Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C

    OpenAIRE

    Liu, Xi-yu; Li, Hong

    2017-01-01

    Aims. Latent autoimmune diabetes in adults (LADA) is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods. Blood CD4+ T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results. Reduced global H3 lysine 9 methylation was observed in LADA patients’ CD4+ T lymphocytes, compared to healthy controls (P < 0.05). H3 l...

  7. Role of H1 linker histones in mammalian development and stem cell differentiation.

    Science.gov (United States)

    Pan, Chenyi; Fan, Yuhong

    2016-03-01

    H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Arginine-rich histones have strong antiviral activity for influenza A viruses.

    Science.gov (United States)

    Hoeksema, Marloes; Tripathi, Shweta; White, Mitchell; Qi, Li; Taubenberger, Jeffery; van Eijk, Martin; Haagsman, Henk; Hartshorn, Kevan L

    2015-10-01

    While histones are best known for DNA binding and transcription-regulating properties, they also have antimicrobial activity against a broad range of potentially pathogenic organisms. Histones are abundant in neutrophil extracellular traps, where they play an important role in NET-mediated antimicrobial killing. Here, we show anti-influenza activity of histones against both seasonal H3N2 and H1N1, but not pandemic H1N1. The arginine rich histones, H3 and H4, had greater neutralizing and viral aggregating activity than the lysine rich histones, H2A and H2B. Of all core histones, histone H4 is most potent in neutralizing IAV, and incubation with IAV with histone H4 results in a decrease in uptake and viral replication by epithelial cells when measured by qRT-PCR. The antiviral activity of histone H4 is mediated principally by direct effects on viral particles. Histone H4 binds to IAV as assessed by ELISA and co-sedimentation of H4 with IAV. H4 also induces aggregation, as assessed by confocal microscopy and light transmission assays. Despite strong antiviral activity against the seasonal IAV strains, H4 was inactive against pandemic H1N1. These findings indicate a possible role for histones in the innate immune response against IAV. © The Author(s) 2015.

  9. The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes.

    Science.gov (United States)

    Ji, Guang; Zhang, Weiwei; Quan, Moyuan; Chen, Yang; Qu, Hui; Hu, Zhiping

    2016-12-01

    This study aimed to investigate the effects of H 2 O 2 -induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H 2 O 2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H 2 O 2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6 h after H 2 O 2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H 2 O 2 treatment, peaked at 3 h, and finally dropped at 6 h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H 2 O 2 treatment, while its role in H 2 O 2 -induced Golgi morphological changes may be complex.

  10. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Francesca Rappa

    Full Text Available Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously.We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry.Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2.These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

  11. Radiation damage to histones

    International Nuclear Information System (INIS)

    Mee, L.K.; Adelstein, S.J.

    1985-01-01

    The damage to histones irradiated in isolation is being elaborated to aid the identification of the crosslinking sites in radiation-induced DNA-histone adducts. Histones are being examined by amino acid analysis to determine the destruction of residues and by polyacrylamide gel electrophoresis to delineate changes in conformation. For the slightly lysine-rich histone, H2A, a specific attack on selective residues has been established, the aromatic residues, tyrosine and phenylalanine, and the heterocyclic residue, histidine, being significantly destroyed. In addition, a significant increase in aspartic acid was found; this may represent a radiation product from scission of the ring in the histidine residues. The similarity of the effects on residues in nitrous oxide-saturated and nitrogen-saturated solutions suggests that OH . and e/sub aq//sup -/ are equally efficient and selective in their attack. On gel electrophoresis degradation of the histone H2A was found to be greatest for irradiations in nitrous oxide-saturated solutions, suggesting CH . is the most effective radical for producing changes in conformation; O/sub 2//sup -/ was essentially ineffective. Other histones are being examined for changes in amino acid composition, conformation, and for the formation of radiation products

  12. High-resolution structure of the native histone octamer

    International Nuclear Information System (INIS)

    Wood, Christopher M.; Nicholson, James M.; Lambert, Stanley J.; Chantalat, Laurent; Reynolds, Colin D.; Baldwin, John P.

    2005-01-01

    The high-resolution (1.90 Å) model of the native histone octamer allows structural comparisons to be made with the nucleosome-core particle, along with an identification of a likely core-histone binding site. Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an R work value of 18.7% and an R free of 22.2%. The crystal space group is P6 5 , the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle

  13. CFP1 Regulates Histone H3K4 Trimethylation and Developmental Potential in Mouse Oocytes

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    Chao Yu

    2017-08-01

    Full Text Available Trimethylation of histone H3 at lysine-4 (H3K4me3 is associated with eukaryotic gene promoters and poises their transcriptional activation during development. To examine the in vivo function of H3K4me3 in the absence of DNA replication, we deleted CXXC finger protein 1 (CFP1, the DNA-binding subunit of the SETD1 histone H3K4 methyltransferase, in developing oocytes. We find that CFP1 is required for H3K4me3 accumulation and the deposition of histone variants onto chromatin during oocyte maturation. Decreased H3K4me3 in oocytes caused global downregulation of transcription activity. Oocytes lacking CFP1 failed to complete maturation and were unable to gain developmental competence after fertilization, due to defects in cytoplasmic lattice formation, meiotic division, and maternal-zygotic transition. Our study highlights the importance of H3K4me3 in continuous histone replacement for transcriptional regulation, chromatin remodeling, and normal developmenta