Chromatin Supraorganization, Mitotic Abnormalities And Proliferation In Cells With Increased Or Down-regulated Lox Expression: Indirect Evidence Of A Lox-histone H1 Interaction In Vivo

FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOLysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX-H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA-H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX-H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and " cell death preceded by multinucleation" increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes. © 2010 Elsevier Ltd.Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX-H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA-H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX-H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and " cell death preceded by multinucleation" increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.421816FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2006/66-8306611/2006-6Archer, T.K., Cordingley, M.G., Wolford, R.G., Hager, G.L., Transcription factor access is mediated by accurately positioned nucleosomes on the mouse mammary tumor virus promoter (1991) Mol. Cell. Biol., 11, pp. 688-698Banks, G.C., Deterding, L.J., Tomer, K.B., Archer, T.K., Hormone-mediated dephosphorylation of specific histone H1 isoforms (2001) J. Biol. Chem., 276, pp. 36467-36473Belikov, S., Astrand, C., Wrange, Ö., Mechanism of histone H1-stimulated glucocorticoid receptor DNA binding in vivo (2007) Mol. Cell. Biol., 27, pp. 2398-2410Bhattacharjee, R.N., Banks, G.C., Trotter, K.W., Lee, H.-L., Archer, T.K., Histone H1 phosphorylation by Cdk2 selectively modulates Mouse Mammary Tumor Virus transcription through chromatin remodeling (2001) Mol. Cell. Biol., 21, pp. 5417-5425Calos, M.P., Lebkowski, J.S., Botchan, M.R., High mutation frequency in DNA transfected into mammalian cells (1983) Proc. Natl. Acad. Sci. U.S.A., 80, pp. 3015-3019Contente, S., Kenyon, K., Rimoldi, D., Friedman, R.M., Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras (1990) Science, 249, pp. 796-798Contente, S., Csiszar, K., Kenyon, K., Friedman, R.M., Structure of the mouse lysyl oxidase gene (1993) Genomics, 16, pp. 395-400Contente, S., Yeb, T.J.A., Friedman, R.M., Tumor suppressive effect of lysyl oxidase proenzyme (2009) Biochim. Biophys. Acta, 1793, pp. 1272-1278Di Donato, A., Lacal, J., Di Duca, M., Giampuzzi, M., Ghiggeri, G., Gusmano, R., Microinjection of recombinant lysyl oxidase blocks oncogenic p21-Ha-ras and progesterone effects on Xenopus laevis oocyte maturation (1997) FEBS Lett., 419, pp. 63-68Doudkine, A., MaCaulay, C., Poulin, N., Palcic, B., Nuclear texture measurements in image cytometry (1995) Pathologica, 87, pp. 286-299Fan, Y.H., Nikitina, T., Zhao, J., Fleury, T.J., Bhattacharyya, R., Bouhassira, E.E., Stein, A., Skoultchi, A.I., Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation (2005) Cell, 123, pp. 1199-1212Giampuzzi, M., Botti, G., Di Luca, M., Arata, L., Ghiggeri, G., Gusmano, R., Ravazzolo, R., Di Donato, A., Lysyl oxidase activates the transcription activity of human collagen III promoter. Possible involvement of Ku antigen (2000) J. Biol. Chem., 275, pp. 36341-36349Giampuzzi, M., Botti, G., Cilli, M., Gusmano, R., Borel, A., Sommer, P., Di Donato, A., Down-regulation of lysyl oxidase-induced tumorigenic transformation in NRK-49F cells characterized by constitutive activation of ras proto-oncogene (2001) J. Biol. Chem., 276, pp. 29226-29232Giampuzzi, M., Oleggini, R., Di Donato, A., Altered adhesion features and signal transduction in NRK-49F cells transformed by down-regulation of lysyl oxidase (2003) Biochim. Biophys. Acta: Proteins Proteomics, 1647, pp. 239-244Giampuzzi, M., Oleggini, R., Di Donato, A., Demonstration of in vitro interaction between tumor suppressor lysyl oxidase and histones H1 and H2: definition of the regions involved (2003) Biochim. Biophys. Acta: Proteins Proteomics, 1647, pp. 245-251Giampuzzi, M., Oleggini, R., Albanese, C., Pestell, R., Di Donato, A., Beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase (2005) Biochim. Biophys. Acta: Mol. Cell. Res., 1745, pp. 370-381Guo, Y., Pischon, N., Palamakumbura, A.H., Trackman, P.C., Intracellular distribution of the lysyl oxidase polypeptide in osteoblastic cells (2007) Am. J. Physiol.: Cell Physiol., 292, pp. C2095-C2102Happel, N., Doenecke, D., Histone H1 and its isoforms: contribution to chromatin structure and function (2009) Gene, 431, pp. 1-12He, S.H., Dunn, K.L., Espino, P.S., Drobic, B., Li, L., Yu, J., Sun, J.M., Davie, J.R., Chromatin organization and nuclear microenvironments in cancer cells (2008) J. Cell. Biochem., 104, pp. 2004-2015Hizume, K., Yoshimura, S.H., Takeyasu, K., Linker histone H1 per se can induce three-dimensional folding of chromatin fiber (2005) Biochemistry, 44, pp. 12978-12989Kagan, H.M., Li, W., Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell (2003) J. Cell. Biochem., 88, pp. 660-672Kagan, H.M., Trackman, P.C., Properties and function of lysyl oxidase (1991) Am. J. Resp. Cell Mol. Biol., 5, pp. 206-210Kagan, H.M., Williams, M.A., Calaman, S.D., Berkowitz, E.M., Histone H1 is a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible residues (1983) Biochem. Biophys. Res. Commun., 115, pp. 186-192Kaneda, A., Wakazono, K., Tsukamoto, T., Watanabe, N., Yagi, Y., Tatematsu, M., Kaminishi, M., Ushijima, T., Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers (2004) Cancer Res., 64, pp. 6410-6415Karymov, M.A., Tomschik, M., Leuba, S.H., Caiafa, P., Zlatanova, J., DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone (2001) FASEB J., 15, pp. 2631-2641Kirschmann, D.A., Seftor, E.A., Fong, S.F.T., Nieva, D.R.C., Sullivan, C.M., Edwards, E.M., Sommer, P., Hendrix, M.J.C., A molecular role for lysyl oxidase in breast cancer invasion (2002) Cancer Res., 62, pp. 4478-4483(1995) User's Guide, vol. 1, , Eching/Munich, Kontron Elektronic Imaging System KS400Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecice, E.H., Blagosklonny, M.V., Melino, G., Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009 (2009) Cell Death Differ., 16, pp. 3-11Li, W., Nellaiappan, K., Strassmaier, T., Graham, L., Thomas, K.M., Kagan, H.M., Localization and activity of lysyl oxidase within nuclei of fibrogenic cells (1997) Proc. Natl. Acad. Sci. U.S.A., 94, pp. 12817-12822Li, W., Nugent, M.A., Zhao, Y., Chau, A.N., Li, S.J., Chou, L.N., Liu, G., Kagan, H.M., Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential (2003) J. Cell. Biochem., 88, pp. 152-164Lison, L., (1960) Histochimie et Cytochimie Animales, , Gauthier-Villars, ParisLucero, H.A., Kagan, H.M., Lysyl oxidase: an oxidative enzyme and effector of cell function (2006) Cell. Mol. Life Sci., 63, pp. 2304-2316Maresca, T.J., Freedman, B.S., Heald, R., Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts (2005) J. Cell Biol., 169, pp. 859-869Mello, M.L.S., Cytochemistry of DNA, RNA, and nuclear proteins (1997) Braz. J. Genet., 20, pp. 257-264Mello, M.L.S., Barbisan, L.F., Lareef, M.H., Russo, J., Vidal, B.C., Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17 (2004) Mutat. Res., 546, pp. 39-43Mello, M.L.S., Contente, S., Vidal, B.C., Planding, W., Schenck, U., Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis (1995) Exp. Cell Res., 220, pp. 374-382Mello, M.L.S., Lin, T.Y., Russo, J., Scanning microphotometry image analysis of Ha-ras-transformed human breast epithelial cells (1994) Anal. Cell. Pathol., 7, pp. 301-309Mello, M.L.S., Russo, P., Russo, J., Vidal, B.C., Entropy of Feulgen-stained 17-beta-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis (2009) Oncol. Rep., 21, pp. 1483-1487Nigro, S., Geido, E., Infusini, E., Orecchia, R., Giaretti, W., Transfection of human mutated K-ras in mouse NIH-3T3 cells is associated with increased cloning efficiency and DNA aneuploidization (1996) Int. J. Cancer, 67, pp. 871-875Oberholzer, M., Östreicher, M., Christen, H., Brühlmann, M., Methods in quantitative image analysis (1996) Histochem. Cell Biol., 105, pp. 333-355Pinnel, S.R., Martin, G.R., The cross-linking of collagen and elastin: enzymatic conversion of lysine in peptide linkage to alpha-aminoadipic-delta-semialdehyde (allysine) by an extract from bone (1968) Proc. Natl. Acad. Sci. USA, 61, pp. 708-716Rao, X.T., Zhang, Y.Y., Yi, Q.Y., Hou, H.L., Xu, B., Chu, L., Huang, Y., Shi, Q.H., Multiple origins of spontaneously arising micronuclei in HeLa cells: direct evidence from long-term live cell imaging (2008) Mut. Res.: Fund. Mol. Mech. Mutagen., 646, pp. 41-49Ren, C., Yang, G., Timme, T.L., Wheeler, T.M., Thompson, T.C., Reduced lysyl oxidase messenger RNA levels in experimental and human prostate cancer (1998) Cancer Res., 58, pp. 1285-1290Saad, F.A., Torres, M., Wang, H., Graham, L., Intracellular lysyl oxidase: effect of a specific inhibitor on nuclear mass in proliferating cells (2010) Biochem. Biophys. Res. Commun., 396, pp. 944-949Tang, S.S., Chichester, C.O., Kagan, H.M., Comparative sensitivities of purified preparations of lysyl oxidase and other amine oxidases to active site-directed enzyme inhibitors (1989) Connect. Tissue Res., 19, pp. 93-103Vidal, B.C., Birefringence measurements on toluidine blue-stained chromosomes during mitosis (1972) Ann. Histochim., 17, pp. 145-150Vidal, B.C., Doppelbrechungsdispersion und Lineardichroismus von Eu- und Heterochromatin nach Färbung mit Toluidinblau. Nachweis eines Cotton-Effektes (1972) Beitr. Pathol., 145, pp. 269-285Wu, G.J., Guo, Z.M., Chang, X.F., Kim, M.S., Nagpal, J.K., Liu, J.W., Maki, J.M., Sidransky, D., LOXL1 and LOXL4 are epigenetically silenced and can inhibit Ras/extracellular signal-regulated kinase signaling pathway in human bladder cancer (2007) Cancer Res., 67, pp. 4123-412

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (hemiptera, Reduviidae)

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A3/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4′,6-diamidino-2-phenylindole/actinomycin D and chromomycin A3/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.1147665672CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO47303/2009-7; 132341/2010-7; 301943/2009-52010/50015-

Histone epigenetic marks in heterochromatin and euchromatin of the Chagas' disease vector, Triatoma infestans

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOTriatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-alpha) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-alpha were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins. (C) 2016 Elsevier GmbH. All rights reserved.Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-alpha) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-alpha were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-alpha) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-alpha were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.1184401412FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2010/50015-6471303/2009-7; 304668/2014-1; 475261/2012-

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOHeterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA3/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation. © 2011 Elsevier Ltd.Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA3/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.426568578CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO47303/2009-72010-50015-6132341/2010-7503702/2007-2301943/2009-5Adams, M.D., The genome sequence of Drosophila melanogaster (2000) Science, 287, pp. 2185-2195Allis, C.D., Jenuwein, T., Reinberg, D., Caparros, M.L., (2007) Epigenetics, , Cold Spring Harbor Lab. Press, Cold Spring HarborAndrade, L.M., Fernandes, R., Mondin, M., Immunodetection of methylcytosine in maize chromatin by a denaturating protocol (2009) Maize Genet. Coop. Newslett., 83, pp. 39-40Bardella, V.B., Gaeta, M.L., Vanzela, A.L.L., Azeredo-Oliveira, M.T.V., Chromosomal location of heterochromatin and 45S rDNA sites in four South American triatomines (Heteroptera: Reduviidae) (2010) Comp. Cytogenet., 4, pp. 141-149Bartova, E., Krejci, J., Harnicarova, A., Galiova, G., Kozubek, S., Histone modifications and nuclear architecture: a review (2008) J. Histochem. Cytochem., 56, pp. 711-721Bianchi, N.O., Vidal-Rioja, L., Cleaver, J.E., Direct visualization of the sites of DNA methylation in human and mosquito chromosomes (1986) Chromosoma, 94, pp. 362-366Cabral-de-Mello, D.C., Moura, R.C., Carvalho, R., Souza, M.J., Cytogenetic analysis of two related Deltochilum (Coleoptera, Scarabaeidae) species: diploid number reduction, extensive heterochromatin addition and differentiation (2010) Micron, 41, pp. 112-117Campos, S.G.P., Rodrigues, V.L.C.C., Mello, M.L.S., Changes in nuclear phenotype frequencies following sequential cold shocks in Triatoma infestans (Hemiptera, Reduviidae) (2002) Mem. Inst. Oswaldo Cruz, 97, pp. 857-864Chaves, R., Heslop-Harrison, J.S., Guedes-Pinto, H., Centromeric heterochromatin in the cattle rob (129) translocation: α-satellite I sequences, in situ Msp I digestion patterns, chromomycin staining and C-bands (2000) Chromosome Res., 8, pp. 621-626Clouaire, T., Stancheva, I., Methyl-CpG binding proteins: specialized transcriptional repressors or structural components of chromatin? (2008) Cell Mol. Life Sci., 65, pp. 1509-1522Cremer, T., Cremer, C., Chromosome territories, nuclear architecture and gene regulation in mammalian cells (2001) Nat. Rev. Genet., 2, pp. 292-301Cremer, T., Kupper, K., Dietzel, S., Kakan, S., Higher order chromatin architecture in the cell nucleus: on the way from structure to function (2004) Biol. Cell, 96, pp. 555-567Das, J.K., Khuda-Bukhsh, A.R., GC-rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A(3) (CMA(3)) staining in three species of teleostean fishes (Pisces) (2007) Ind. J. Exp. Biol., 45, pp. 413-418Derenzini, M., Ploton, D., Interphase nuclear regions in cancer cells (1991) Int. Rev. Exp. Path., 32, pp. 149-192Friebe, B., Endo, T.R., Gill, B.S., Chromosome-banding methods (1996) Plant Chromosomes: Laboratory Methods, pp. 123-154. , CRC Press, Boca Raton, K. Fukui, N. Shigeki (Eds.)Gilbert, N., Gilchrist, S., Bickmore, W.A., Chromatin organization in the mammalian nucleus (2005) Int. Rev. Cytol., 242, pp. 283-336Gilbert, N., Thomson, I., Boyle, S., Allan, J., Ramsahoye, B., Bickmore, W.A., DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction (2007) J. Cell Biol., 177, pp. 401-411Golub, N.V., Nokkala, S., Kuznetsova, V.G., Holocentric chromosomes of psocids (Insecta, Psocoptera) analysed by C-banding, silver impregnation and sequence specific fluorochromes CMA(3) and DAPI (2004) Folia Biol. (Krakow), 52, pp. 143-149Gonsálvez, J., López-Fernández, C., Fernández, J.L., Goyanes, V.L., Buño, I., Digital image analysis of chromatin fibre phenotype after " in situ" digestion with restriction endonucleases (1995) Cell Biol. Int., 19, pp. 827-832Grewal, S.I., Moazed, D., Heterochromatin and epigenetic control of gene expression (2003) Science, 301, pp. 798-802Hirai, H., Shono, Y., Arias, A.R., Tada, I., Constitutive heterochromatin polymorphism of a Triatoma infestans strain, a main vector insect of Chagas' disease (1991) Jpn. J. Sanit. Zool., 42, pp. 301-303Jiang, Z., Li, X., Hu, J., Zhou, W., Jiang, Y., Li, G., Lu, D., Promoter hypermethylation-mediated down-regulation of LATS1 and LATS2 in human astrocytoma (2006) Neurosci. Res., 56, pp. 450-458Joffe, B., Leonhardt, H., Solovei, I., Differential and large scale spatial organization of the genome (2010) Curr. Opin. Genet. Dev., 20, pp. 562-569Kuznetsova, V.G., Maryanska-Nadachowska, A., Nokkala, S., A new approach to the Auchenorrhyncha (Hemiptera, Insecta) cytogenetics: chromosomes of the meadow spittle bug Philaenus spumarius (L.) examined using various chromosome banding techniques (2003) Folia Biol. (Praha), 51, pp. 33-40Lima-de-Faria, A., Isaksson, M., Olsson, E., Action of restriction endonucleases on the DNA and chromosomes of Muntiacus muntjak (1980) Hereditas, 92, pp. 267-273Liu, J., Benbrahim-Tallaa, L., Qian, X., Yu, L., Xie, Y., Boos, J., Qu, W., Waalkes, M.P., Further studies on aberrant gene expression associated with arsenic-induced malignant transformation in rat liver TRL1215 cells (2006) Toxicol. Appl. Pharmacol., 216, pp. 407-415Malatesta, M., Bertoni-Freddari, C., Fattoretti, P., Caporaloni, C., Fakan, S., Gazzanelli, G., Altered RNA structural constituents in aging and vitamin E deficiency (2003) Mech. Ageing Dev., 124, pp. 175-181Mampumbu, A.R., Mello, M.L.S., DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis (2006) Cytometry Part A, 69, pp. 986-991Mandrioli, M., Bizzaro, D., Manicardi, G.C., Gionghi, D., Bassoli, L., Bianchi, U., Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae (1999) Chromosoma, 108, pp. 436-442Marcilla, A., Bargues, M.D., Ramsey, J.M., Magallon-Gastelum, E., Salazar-Schettino, P.M., Abad-Franch, F., Dujardin, J.P., Mas-Coma, S., The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease (2001) Mol. Phylogen. Evol., 18, pp. 136-142Mello, M.L.S., Nuclear behavior in the Malpighian tubes of Triatoma infestans (1971) Cytologia (Tokyo), 36, pp. 42-49Mello, M.L.S., Feulgen-DNA values and ploidy degrees in the Malpighian tubes of some triatomids (1975) Rev. Brasil. Pesq. Méd. Biol., 8, pp. 101-107Mello, M.L.S., Computer analysis of stained chromatin on Malpighian tubes of Triatoma infestans Klug (Hemiptera, Reduviidae) (1978) Mikroskopie, 34, pp. 285-299Mello, M.L.S., Patterns of lability towards acid hydrolysis in heterochromatins and euchromatins of Triatoma infestans Klug (1979) Cell Mol. Biol., 24, pp. 1-16Mello, M.L.S., Nuclear fusion and change in chromatin packing state in response to starvation in Triatoma infestans (1989) Rev. Brasil. Genét., 12, pp. 485-498Mello, M.L.S., Recco-Pimentel, S.M., Response to banding and Hoechst 33258 treatment in chromocenters of the Malpighian tubule cells of Triatoma infestans (1987) Cytobios, 52, pp. 175-184Mello, M.L.S., Vidal, B.C., A reação de Feulgen (1978) Ciênc. Cult., 30, pp. 665-676Mello, M.L.S., Aldrovani, M., Moraes, A.S., Guaraldo, A.M.A., Vidal, B.C., DNA content, chromatin supraorganization, nuclear glycoproteins and RNA amounts in hepatocytes of mice expressing insulin-dependent diabetes (2009) Micron, 40, pp. 577-585Mello, M.L.S., Chambers, A.F., Vidal, B.C., Planding, W., Schenck, U., Restriction enzyme analysis of DNA methylation in " condensed" chromatin of Ha-ras-transformed NIH 3T3 cells (2000) Anal. Cell. Pathol., 20, pp. 163-171Mello, M.L.S., Dolder, H., Dias, C.A., Nuclear ultrastructure of Malpighian tubule cells in Triatoma infestans (Hemiptera, Reduviidae) under conditions of full nourishment and starvation (1990) Rev. Brasil. Genét., 13, pp. 5-17Mello, M.L.S., Kubrusly, F.S., Randi, M.A.F., Rodrigues, V.L.C.C., Ferraz-Filho, A.N., Effect of heavy metals on chromatin supraorganization, nuclear phenotypes, and survival of Triatoma infestans (1995) Entom. Exp. Appl., 74, pp. 209-218Mello, M.L.S., Russo, P., Russo, J., Vidal, B.C., Entropy of Feulgen-stained 17-β-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis (2009) Oncol. Rep., 21, pp. 1483-1487Mello, M.L.S., Tavares, M.C.H., Dantas, M.M., Rodrigues, V.L.C.C., Maria-Engler, S.S., Campos, S.P., Garcia, N.L., Cell death and survival alterations in Malpighian tubules of Triatoma infestans following heat shock (2001) Biochem. Cell Biol., 79, pp. 709-717Mello, M.L.S., Vidal, B.C., Planding, W., Schenck, U., Image analysis: video system adequacy for the assortment of nuclear phenotypes based on chromatin texture evaluation (1994) Acta Histochem. Cytochem., 27, pp. 23-31Mezzanotte, R., Bianchi, U., Vanni, R., Ferrucci, L., Chromatin organization and restriction endonuclease activity on human metaphase chromosomes (1983) Cytogenet. Cell Genet., 36, pp. 562-566Morielle-Souza, A., Azeredo-Oliveira, M.T.V., Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization (2007) Genet. Mol. Res., 6, pp. 713-720Nelson, M., McCleland, M., Site-specific methylation: Effect on DNA modification methyltransferases and restriction endonucleases (1991) Nucleic Acids Res., 19, pp. 2045-2071Panzera, F., Dujardin, J.P., Nicolini, P., Caraccio, M.N., Rose, V., Tellez, T., Bermudez, H., Perez, R., Genomic changes of Chagas' disease vector, South America (2004) Emerg. Infect. Dis., 10, pp. 438-446Panzera, F., Ferrandis, I., Ramsey, J., Salazar-Schettino, P.M., Cabrera, M., Monroy, C., Bargues, M.D., Perez, R., Genome size determination in Chagas disease transmitting bugs (Hemiptera - Triatominae) by flow cytometry (2007) Am. J. Trop. Med. Hyg., 76, pp. 516-521Panzera, F., Perez, R., Panzera, Y., Ferrandis, I., Ferreiro, M.J., Calleros, L., Cytogenetics and genome evolution in the subfamily Triatominae (Hemiptera, Reduviidae) (2010) Cytogen. Genome Res., 128, pp. 77-87Pardo, M.C., Viseras, E., Cabrero, J., Camacho, J.P.M., A supernumerary chromosome segment in Locusta migratoria (1993) Genome, 36, pp. 919-923Ploton, D., Menager, M., Jeannesson, P., Himber, G., Pigeon, F., Adnet, J.J., Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level (1986) Histochem. J., 18, pp. 5-14Rippe, K., Dynamic organization of the cell nucleus (2007) Curr. Opin. Genet. Dev., 17, pp. 373-380Sabater-Muñoz, B., Legeai, F., Rispe, C., Bonhomme, J., Dearden, P., Dossat, C., Duclert, A., Tagu, D., Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera) (2006) Genome Biol., 7, pp. R21Schreiber, G., Bogliolo, A.R., Pinto, A.C., Cytogenetics of Triatominae: caryotype, DNA content, nuclear size and heteropyknosis of autosomes (1972) Braz. J. Biol., 32, pp. 255-263Schweizer, D., Reverse fluorescent chromosome banding with chromomycin and DAPI (1976) Chromosoma, 58, pp. 307-324Segura, E.L., Torres, A.G., Fusco, O., Garcia, B.A., Mitochondrial 16S DNA variation in populations of Triatoma infestans from Argentina (2009) Med. Vet. Entomol., 23, pp. 34-40Sentis, C., Santos, J., Fernández-Piqueras, J., Breaking up the chromosomes of Baetica ustulata by in situ treatments with restriction endonucleases (1989) Genome, 32, pp. 208-215Skalnikova, M., Bartova, E., Ulman, V., Matula, P., Svoboda, D., Hranicarova, A., Kozubek, M., Kozubek, S., Distinct patterns of histone methylations and acetylation in human interphase nuclei (2007) Physiol. Res., 56, pp. 797-806Solari, J., Autosomal synaptonemal complexes and sex chromosomes without axes in Triatoma infestans (Reduvidae: Hemiptera) (1979) Chromosoma, 72, pp. 225-240Vidal, B.C., Polyploidy and nuclear phenotypes in salivary glands of the rat (1984) Biol. Cell, 50, pp. 137-146Vidal, B.C., Planding, W., Mello, M.L.S., Schenck, U., Quantitative evaluation of AgNOR in liver cells by high-resolution image cytometry (1994) Anal. Cell. Pathol., 7, pp. 27-41Vidal, B.C., Schlüter, G., Moore, G.W., Cell nucleus pattern recognition: influence of staining (1973) Acta Cytol., 17, pp. 510-521Zhang, W.L., Wang, X., Yu, Q., Ming, R., Jiang, J., DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya (2008) Genome Res., 18, pp. 1938-194

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved