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Masahiko Harata - One of the best experts on this subject based on the ideXlab platform.
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The linker histone in Saccharomyces cerevisiae interacts with Actin-Related Protein 4 and both regulate chromatin structure and cellular morphology.
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Milena Georgieva, Masahiko Harata, Dessislava Staneva, Katya Uzunova, Toni Efremov, Konstantin Balashev, George MiloshevAbstract:Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear Proteins that influence it. The intricate interaction among all these structural and functional nuclear Proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (Actin-Related Protein 4) which is a key subunit of three chromatin modifying complexes – INO80, SWR1 and NuA4. A single – point mutation in the Actin – fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization.
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dna binding properties of the Actin Related Protein arp8 and its role in dna repair
PLOS ONE, 2014Co-Authors: Akihisa Osakabe, Yuichiro Takahashi, Hirokazu Murakami, Kenji Otawa, Hiroaki Tachiwana, Hitoshi Nishijima, Kei Ich Shibahara, Hitoshi Kurumizaka, Masahiko HarataAbstract:Actin and Actin-Related Proteins (Arps), which are members of the Actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved Actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.
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Nuclear Actin filaments recruit cofilin and Actin-Related Protein 3, and their formation is connected with a mitotic block
Histochemistry and Cell Biology, 2014Co-Authors: Alžběta Kalendová, Masahiko Harata, Ilona Kalasová, Shota Yamazaki, Lívia Uličná, Pavel HozákAbstract:Although Actin monomers polymerize into filaments in the cytoplasm, the form of Actin in the nucleus remains elusive. We searched for the form and function of β-Actin fused to nuclear localization signal and to enhanced yellow fluorescent Protein (EN-Actin). Our results reveal that EN-Actin is either dispersed in the nucleoplasm (homogenous EN-Actin) or forms bundled filaments in the nucleus (EN-Actin filaments). Formation of such filaments was not connected with increased EN-Actin levels. Among numerous Actin-binding Proteins tested, only cofilin is recruited to the EN-Actin filaments. Overexpression of EN-Actin causes increase in the nuclear levels of Actin-Related Protein 3 (Arp3). Although Arp3, a member of Actin nucleation complex Arp2/3, is responsible for EN-Actin filament nucleation and bundling, the way cofilin affects nuclear EN-Actin filaments dynamics is not clear. While cells with homogenous EN-Actin maintained unaffected mitosis during which EN-Actin re-localizes to the plasma membrane, generation of nuclear EN-Actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-Actin manifests in two mitotic-inborn defects—formation of binucleic cells and generation of micronuclei—suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-Actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-Actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-Actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis.
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the human Actin Related Protein harp5 nucleo cytoplasmic shuttling and involvement in dna repair
Experimental Cell Research, 2009Co-Authors: Kumiko Kitayama, Barbara Winsor, Mariko Kamo, Ryo Matsuda, Takafumi Uchida, Tsuyoshi Ikura, Satoshi Tashiro, Takashi Ohyama, Masahiko HarataAbstract:Abstract Certain Actin-Related Proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) Proteins are localized in the nucleus, and that arp5Δ yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (γ-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced γ-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.
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the nuclear Actin Related Protein act3p arp4 influences yeast cell shape and bulk chromatin organization
Journal of Cellular Biochemistry, 2008Co-Authors: Milena Georgieva, Masahiko Harata, George MiloshevAbstract:: ACT3/ARP4 is an essential gene, coding for the Actin-Related Protein Act3p/Arp4 of Saccharomyces cerevisiae located within the nucleus. Act3p/Arp4 is a stoichiometric component of the NuA4, INO80, and SWR1 chromatin modulating complexes, and recruits these complexes onto chromatin for their proper chromatin functions. Mutated Act3p/Arp4 leads to impairment of the functions of these complexes and affects transcription of specific genes. Our results revealed significant disorder in the cell size and shape of act3/arp4 mutant cells, when grown at permissive temperature. act3/arp4 mutants have also demonstrated an increase in their nuclear diameters, thus suggesting that Act3p/Arp4 is a key regulator in the maintenance of cellular shape and nuclear organization. Furthermore, the use of Chromatin Yeast Comet Assay (ChYCA) for assessment of single-cell bulk chromatin organization in act3/arp4 mutant cells allowed us to detect an elevated sensitivity toward nuclease action, denoting differences in higher-order chromatin structure of the mutants.
Ulrike Wintersberger - One of the best experts on this subject based on the ideXlab platform.
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the nuclear Actin Related Protein act3p arp4p is involved in the dynamics of chromatin modulating complexes
Yeast, 2005Co-Authors: Rie Sunada, Ulrike Wintersberger, Irene Gorzer, Takahito Yoshida, Noriyuki Suka, Masahiko HarataAbstract:Chromatin remodelling and histone-modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear Actin-Related Proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and Actin, sharing a common ancestor, are supposed to be highly similar in the three-dimensional structure of their core regions, including the ATP-binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP-binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPγS led to the release of wild-type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP-binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics. Copyright © 2005 John Wiley & Sons, Ltd.
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The nuclear Actin-Related Protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes†
Yeast, 2005Co-Authors: Rie Sunada, Ulrike Wintersberger, Irene Gorzer, Takahito Yoshida, Noriyuki Suka, Masahiko HarataAbstract:Chromatin remodelling and histone-modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear Actin-Related Proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and Actin, sharing a common ancestor, are supposed to be highly similar in the three-dimensional structure of their core regions, including the ATP-binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP-binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPγS led to the release of wild-type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP-binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics. Copyright © 2005 John Wiley & Sons, Ltd.
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the nuclear Actin Related Protein act3p arp4p of saccharomyces cerevisiae is involved in transcription regulation of stress genes
Molecular Microbiology, 2003Co-Authors: Irene Gorzer, Christoph Schuller, Erich Heidenreich, Ludmila Krupanska, Karl Kuchler, Ulrike WintersbergerAbstract:Summary A mutational analysis of the essential nuclear Actin-Related Protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between Actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of Actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide-sensitive which supports a structural Relatedness of Act3p/Arp4p to Actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob-served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome-wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress-responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress-induced genes under non-stress conditions.
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The nuclear Actin-Related Protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes.
Molecular Microbiology, 2003Co-Authors: Irene Gorzer, Christoph Schuller, Erich Heidenreich, Ludmila Krupanska, Karl Kuchler, Ulrike WintersbergerAbstract:Summary A mutational analysis of the essential nuclear Actin-Related Protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between Actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of Actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide-sensitive which supports a structural Relatedness of Act3p/Arp4p to Actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob-served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome-wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress-responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress-induced genes under non-stress conditions.
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the nuclear Actin Related Protein of saccharomyces cerevisiae act3p arp4 interacts with core histones
Molecular Biology of the Cell, 1999Co-Authors: Masahiko Harata, Shigeki Mizuno, Yi Wei Jiang, David J Stillman, Ulrike WintersbergerAbstract:Act3p/Arp4, an essential Actin-Related Protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the Actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin Protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a Protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.
Katsuhiko Nishimori - One of the best experts on this subject based on the ideXlab platform.
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the brain specific Actin Related Protein arpnα interacts with the transcriptional co repressor ctbp
Biochemical and Biophysical Research Communications, 2003Co-Authors: Katsuhiko Nishimori, Masahiko HarataAbstract:Abstract Actin-Related Protein (Arp) is found in many chromatin remodeling and histone acetyltransferase complexes. We previously identified ArpNα as an isoform of ArpNβ/BAF53, which is included in mammalian SWI/SNF chromatin remodeling complex, and showed that ArpNα is a potential component of the complex. Although it has a structure highly similar to ArpNβ/BAF53, ArpNα is expressed exclusively in brain and in neural differentiated embryonal carcinoma cells. Since ArpNα possesses a region that shows low similarity to ArpNβ/BAF53, we hypothesized that Proteins interActing with this region contribute to the ArpNα-specific function in brain. Here we showed that ArpNα, but not ArpNβ/BAF53, interacts with the transcriptional co-repressor CtBP (C-terminal binding Protein). Transactivation by the SWI/SNF complex and glucocorticoid receptor was repressed by the CtBP in the presence of ArpNα. These findings suggest that SWI/SNF complex containing ArpNα might regulate certain genes involved in brain development and/or its function differently from SWI/SNF complex containing ArpNβ/BAF53.
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brain specific expression of the nuclear Actin Related Protein arpnα and its involvement in mammalian swi snf chromatin remodeling complex
Biochemical and Biophysical Research Communications, 2002Co-Authors: Yukiko Kuroda, Katsuhiko Nishimori, Tsutomu Ohta, Masahiko HarataAbstract:Abstract Actin-Related Proteins share significant homology with conventional Actins and are classified into subfamilies based on the similarity of their sequences and functions. The Arp4 subfamily of Arps is localized in the nucleus, and a mammalian isoform, ArpNβ (also known as BAF53), is a component of the chromatin remodeling and histone acetyltransferase complexes. Another isoform identified in humans, ArpNα has scarcely been characterized yet. We identified mouse ArpNα, and showed that ArpNα is more similar between humans and mice than ArpNβ. No difference was observed between ArpNα and β in subcellular localization and interaction with BRM, which is an ATPase subunit of mammalian SWI/SNF chromatin remodeling complex. However, ArpNα was expressed exclusively in the brain and its expression was induced during neural differentiation of P19 mouse embryonic carcinoma cells. ArpNα is the first brain-specific component of a chromatin remodeling complex to be identified, suggesting that ArpNα has conserved and important roles in the differentiation of neural cells through regulation of chromatin structure.
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Brain-specific expression of the nuclear Actin-Related Protein ArpNα and its involvement in mammalian SWI/SNF chromatin remodeling complex
Biochemical and Biophysical Research Communications, 2002Co-Authors: Yukiko Kuroda, Katsuhiko Nishimori, Tsutomu Ohta, Masahiko HarataAbstract:Abstract Actin-Related Proteins share significant homology with conventional Actins and are classified into subfamilies based on the similarity of their sequences and functions. The Arp4 subfamily of Arps is localized in the nucleus, and a mammalian isoform, ArpNβ (also known as BAF53), is a component of the chromatin remodeling and histone acetyltransferase complexes. Another isoform identified in humans, ArpNα has scarcely been characterized yet. We identified mouse ArpNα, and showed that ArpNα is more similar between humans and mice than ArpNβ. No difference was observed between ArpNα and β in subcellular localization and interaction with BRM, which is an ATPase subunit of mammalian SWI/SNF chromatin remodeling complex. However, ArpNα was expressed exclusively in the brain and its expression was induced during neural differentiation of P19 mouse embryonic carcinoma cells. ArpNα is the first brain-specific component of a chromatin remodeling complex to be identified, suggesting that ArpNα has conserved and important roles in the differentiation of neural cells through regulation of chromatin structure.
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Alternative Splicing Products of the Gene for a Human Nuclear Actin-Related Protein, hArpNβ/Baf53, that Encode a Protein Isoform, hArpNβS, in the Cytoplasm
Bioscience Biotechnology and Biochemistry, 2002Co-Authors: Eri Ohfuchi, Katsuhiko Nishimori, Masahiko HarataAbstract:A human nuclear Actin-Related Protein, hArpNβ/Baf53, is a component of chromatin remodeling and histone acetyltransferase complexes. We identified two alternative splicing products of the gene for hArpNβ/Baf53. They encoded a Protein isoform, hArpNβS; and its fusion product with green fluorescent Protein was to be found in the cytoplasm, not the nucleus. The isoforms may contribute to functional regulation of these complexes.
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correlation between chromatin association and transcriptional regulation for the act3p arp4 nuclear Actin Related Protein of saccharomyces cerevisiae
Nucleic Acids Research, 2002Co-Authors: Masahiko Harata, Katsuhiko Nishimori, David J Stillman, Yan Zhang, Daisuke Matsui, Ryo MochizukiAbstract:Actin-Related Proteins (Arps), which share a basal structure with Actin but have distinct functions, have been found in a wide variety of organisms. While their functions are not yet clear, some Arps are localized in the nucleus and are suggested to contribute to the regulation of transcription. An essential gene of Saccharomyces cerevisiae, Act3p/Arp4, encodes the first identified nuclear Arp, which has been shown to bind to core histones in vitro. Here we have analyzed the in vivo function of Act3p/Arp4 on the his4-912δ promoter. Chromatin immunoprecipitation assays show that Act3p/Arp4 is bound to the entire his4-912δ promoter region. Conditional act3/arp4 mutations affect transcription from the his4-912δ promoter, where decreased Act3p/Arp4 binding and a change in nuclease sensitivity of chromatin were observed, showing the involvement of Act3p/Arp4 in the regulation of gene expression through the organization of chromatin structure. Taken together with the presence of Act3p/Arp4 in chromatin remodeling and histone acetyltransferase complexes, it is suggested that Act3p/Arp4 functions in transcriptional regulation to recruit chromatin remodeling and histone acetyltransferase complexes onto chromatin.
David J Stillman - One of the best experts on this subject based on the ideXlab platform.
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correlation between chromatin association and transcriptional regulation for the act3p arp4 nuclear Actin Related Protein of saccharomyces cerevisiae
Nucleic Acids Research, 2002Co-Authors: Masahiko Harata, Katsuhiko Nishimori, David J Stillman, Yan Zhang, Daisuke Matsui, Ryo MochizukiAbstract:Actin-Related Proteins (Arps), which share a basal structure with Actin but have distinct functions, have been found in a wide variety of organisms. While their functions are not yet clear, some Arps are localized in the nucleus and are suggested to contribute to the regulation of transcription. An essential gene of Saccharomyces cerevisiae, Act3p/Arp4, encodes the first identified nuclear Arp, which has been shown to bind to core histones in vitro. Here we have analyzed the in vivo function of Act3p/Arp4 on the his4-912δ promoter. Chromatin immunoprecipitation assays show that Act3p/Arp4 is bound to the entire his4-912δ promoter region. Conditional act3/arp4 mutations affect transcription from the his4-912δ promoter, where decreased Act3p/Arp4 binding and a change in nuclease sensitivity of chromatin were observed, showing the involvement of Act3p/Arp4 in the regulation of gene expression through the organization of chromatin structure. Taken together with the presence of Act3p/Arp4 in chromatin remodeling and histone acetyltransferase complexes, it is suggested that Act3p/Arp4 functions in transcriptional regulation to recruit chromatin remodeling and histone acetyltransferase complexes onto chromatin.
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Correlation between chromatin association and transcriptional regulation for the Act3p/Arp4 nuclear Actin-Related Protein of Saccharomyces cerevisiae
Nucleic Acids Research, 2002Co-Authors: Masahiko Harata, Katsuhiko Nishimori, David J Stillman, Yan Zhang, Daisuke Matsui, Ryo MochizukiAbstract:Actin-Related Proteins (Arps), which share a basal structure with Actin but have distinct functions, have been found in a wide variety of organisms. While their functions are not yet clear, some Arps are localized in the nucleus and are suggested to contribute to the regulation of transcription. An essential gene of Saccharomyces cerevisiae, Act3p/Arp4, encodes the first identified nuclear Arp, which has been shown to bind to core histones in vitro. Here we have analyzed the in vivo function of Act3p/Arp4 on the his4-912δ promoter. Chromatin immunoprecipitation assays show that Act3p/Arp4 is bound to the entire his4-912δ promoter region. Conditional act3/arp4 mutations affect transcription from the his4-912δ promoter, where decreased Act3p/Arp4 binding and a change in nuclease sensitivity of chromatin were observed, showing the involvement of Act3p/Arp4 in the regulation of gene expression through the organization of chromatin structure. Taken together with the presence of Act3p/Arp4 in chromatin remodeling and histone acetyltransferase complexes, it is suggested that Act3p/Arp4 functions in transcriptional regulation to recruit chromatin remodeling and histone acetyltransferase complexes onto chromatin.
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the nuclear Actin Related Protein of saccharomyces cerevisiae act3p arp4 interacts with core histones
Molecular Biology of the Cell, 1999Co-Authors: Masahiko Harata, Shigeki Mizuno, Yi Wei Jiang, David J Stillman, Ulrike WintersbergerAbstract:Act3p/Arp4, an essential Actin-Related Protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the Actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin Protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a Protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.
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The nuclear Actin-Related Protein of Saccharomyces cerevisiae, Act3p/Arp4, interacts with core histones.
Molecular Biology of the Cell, 1999Co-Authors: Masahiko Harata, Shigeki Mizuno, Yi Wei Jiang, David J Stillman, Ulrike WintersbergerAbstract:Act3p/Arp4, an essential Actin-Related Protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the Actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin Protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a Protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.
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epigenetic effects on yeast transcription caused by mutations in an Actin Related Protein present in the nucleus
Genes & Development, 1996Co-Authors: Yi Wei Jiang, David J StillmanAbstract:: Gene inactivation can result when a delta element of the Ty1 transposon inserted into the yeast HIS4 promoter (his4-912delta) alters the transcription initiation site. Previous work has identified mutations that suppress this transcriptional defect by restoring the transcription start site to the native position, and these mutations have been implicated in transcriptional regulation and chromatin structure. We show that in a sin4 mutant such suppression is incompletely penetrant, such that genetically identical yeast cells (sin4 his4-912delta) show either of two distinct phenotypic states, His+ or His-. To study this type of potential epigenetic control of gene expression, we constructed a strain with ADE2 expression under the control of the his4-912delta promoter, as colony color provides a convenient assay for ADE2 expression. We isolated mutations in the ACT3 gene that show variegated expression of this ADE2 reporter. The act3 his4delta-ADE2 colonies display both white and red sectors, showing that the two different phenotypes are possible in a single colony. The two phenotypic states can be inherited during clonal growth, yet are reversible. Analysis of RNA isolated from individual colonies of either red or white color demonstrates that it is the state of the promoter, as either On or Off, that is inherited and is responsible for the colony color. An act3 mutation also affects expression of the HIS4 and LYS2 genes; thus, Act3p is not a delta element-specific transcriptional regulator. Immunofluorescence microscopy experiments demonstrate that the Act3p Protein is present in the nucleus. Act3p shows clear homology to Actin, and possible roles for an Actin-Related Protein in transcription are discussed.
Brian T Chait - One of the best experts on this subject based on the ideXlab platform.
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subunits of the yeast swi snf complex are members of the Actin Related Protein arp family
Journal of Biological Chemistry, 1998Co-Authors: Craig L Peterson, Yingming Zhao, Brian T ChaitAbstract:Abstract The yeast SWI/SNF chromatin remodeling complex is comprised of 11 tightly associated polypeptides (SWI1, SWI2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73, SWP59, SWP61, and SWP29). We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the genes that encode the SWP59 and SWP61 subunits. Surprisingly, we find that SWP59 and SWP61 are encoded by theARP9 and ARP7 genes, respectively, which encode members of the Actin-Related Protein (ARP) family. Sequence analyses have shown that ARP9 and ARP7 are 24–26% identical (48–51% similar) to yeast Actin and that they are likely to maintain the overall Actin fold. Deletion of either the ARP9 or ARP7 gene causes typical swi/snf phenotypes, including growth defects on media containing galactose, glycerol, or sucrose as sole carbon sources. ARP9 and ARP7 are also required for expression of an HO-lacZ fusion gene and for full transcriptional enhancement by the GAL4 activator. The identification of two ARP family members as crucial subunits of the SWI/SNF complex suggests that the complex may contain a total of three different ATPase subunits; furthermore, the similarity of ARP7 and ARP9 to the HSP and HSC family of ATPases suggests the possibility that chromatin remodeling by SWI/SNF may involve chaperone-like activities.
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Subunits of the Yeast SWI/SNF Complex Are Members of the Actin-Related Protein (ARP) Family
Journal of Biological Chemistry, 1998Co-Authors: Craig L Peterson, Yingming Zhao, Brian T ChaitAbstract:Abstract The yeast SWI/SNF chromatin remodeling complex is comprised of 11 tightly associated polypeptides (SWI1, SWI2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73, SWP59, SWP61, and SWP29). We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the genes that encode the SWP59 and SWP61 subunits. Surprisingly, we find that SWP59 and SWP61 are encoded by theARP9 and ARP7 genes, respectively, which encode members of the Actin-Related Protein (ARP) family. Sequence analyses have shown that ARP9 and ARP7 are 24–26% identical (48–51% similar) to yeast Actin and that they are likely to maintain the overall Actin fold. Deletion of either the ARP9 or ARP7 gene causes typical swi/snf phenotypes, including growth defects on media containing galactose, glycerol, or sucrose as sole carbon sources. ARP9 and ARP7 are also required for expression of an HO-lacZ fusion gene and for full transcriptional enhancement by the GAL4 activator. The identification of two ARP family members as crucial subunits of the SWI/SNF complex suggests that the complex may contain a total of three different ATPase subunits; furthermore, the similarity of ARP7 and ARP9 to the HSP and HSC family of ATPases suggests the possibility that chromatin remodeling by SWI/SNF may involve chaperone-like activities.
