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1.
Mol Cell ; 64(1): 176-188, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27716482

RESUMO

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.


Assuntos
Transformação Celular Neoplásica/genética , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/química , Cromatina/metabolismo , Ciclina D1/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Treonina/metabolismo , Ubiquitinação
2.
Cancer Sci ; 112(10): 4208-4219, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363714

RESUMO

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.


Assuntos
Neoplasias Ósseas/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Mitocôndrias/metabolismo , Osteossarcoma/patologia , Proteínas Repressoras/fisiologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Bases de Dados Factuais , Regulação para Baixo , Pontos de Checagem da Fase G2 do Ciclo Celular , Inativação Gênica , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Membranas Mitocondriais/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proibitinas , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo
3.
PLoS Biol ; 11(10): e1001697, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204212

RESUMO

The appearance of the notochord represented a milestone in Deuterostome evolution. The notochord is necessary for the development of the chordate body plan and for the formation of the vertebral column and numerous organs. It is known that the transcription factor Brachyury is required for notochord formation in all chordates, and that it controls transcription of a large number of target genes. However, studies of the structure of the cis-regulatory modules (CRMs) through which this control is exerted are complicated in vertebrates by the genomic complexity and the pan-mesodermal expression territory of Brachyury. We used the ascidian Ciona, in which the single-copy Brachyury is notochord-specific and CRMs are easily identifiable, to carry out a systematic characterization of Brachyury-downstream notochord CRMs. We found that Ciona Brachyury (Ci-Bra) controls most of its targets directly, through non-palindromic binding sites that function either synergistically or individually to activate early- and middle-onset genes, respectively, while late-onset target CRMs are controlled indirectly, via transcriptional intermediaries. These results illustrate how a transcriptional regulator can efficiently shape a shallow gene regulatory network into a multi-tiered transcriptional output, and provide insights into the mechanisms that establish temporal read-outs of gene expression in a fast-developing chordate embryo.


Assuntos
Ciona intestinalis/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Notocorda/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Sítios de Ligação , Ciona intestinalis/crescimento & desenvolvimento , Sequência Consenso/genética , Notocorda/crescimento & desenvolvimento , Ligação Proteica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes , Especificidade da Espécie , Fatores de Tempo
4.
PLoS Genet ; 8(3): e1002547, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22396663

RESUMO

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein-protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Desenvolvimento Embrionário , Músculos , Transativadores/genética , Proteína 1 Relacionada a Twist/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Músculos/anormalidades , Músculos/embriologia , Músculos/metabolismo , Mutação , Fatores de Regulação Miogênica/genética , Proteínas Nucleares , Fenótipo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo
5.
EMBO J ; 27(6): 898-909, 2008 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-18309295

RESUMO

The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. Here, we identify the Ebi protein as an essential Snail co-repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. We identify a Snail domain that is sufficient for Ebi binding, and which functions independently of another Snail co-repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail-related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT-HDAC3 (histone deacetylase 3) co-repressor complex. We found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail-mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail-dependent manner, which coincides with histone hypoacetylation. Our results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação para Baixo/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Histona Desacetilases/fisiologia , Histonas/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Drosophila/embriologia , Drosophila/enzimologia , Drosophila/genética , Feminino , Histona Desacetilases/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Cytokine ; 56(3): 564-72, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21890374

RESUMO

Interferon regulatory factor (IRF)-4 is a member of the IRF transcription factor family, whose expression is primarily restricted to lymphoid and myeloid cells. In T-cells, IRF-4 expression is induced by T-cell receptor (TCR) cross-linking or treatment with phorbol-12-myristate-13-acetate (PMA)/Ionomycin, and IRF-4 is thought to be a critical factor for various functions of T-cells. To elucidate the IRF-4 functions in human adult T-cell leukemia virus type 1 (HTLV-1)-infected T-cells, which constitutively express IRF-4, we isolated IRF-4-binding proteins from T-cells, using a tandem affinity purification (TAP)-mass spectrometry strategy. Fourteen proteins were identified in the IRF-4-binding complex, including endogenous IRF-4 and the nuclear factor-kappaB (NF-κB) family member, c-Rel. The specific association of IRF-4 with c-Rel was confirmed by immunoprecipitation experiments, and IRF-4 was shown to enhance the c-Rel-dependent binding and activation of the interleukin-4 (IL-4) promoter region. We also demonstrated that IL-2 production was also enhanced by exogenously-expressed IRF-4 and c-Rel in the presence of P/I, in T-cells, and that the optimal IL-2 and IL-4 productions in vivo was IRF-4-dependent using IRF-4-/- mice. These data provide molecular evidence to support the clinical observation that elevated expression of c-Rel and IRF-4 is associated with the prognosis in adult T-cell leukemia/lymphoma (ATLL) patients, and present possible targets for future gene therapy.


Assuntos
Regulação da Expressão Gênica , Fatores Reguladores de Interferon/metabolismo , Interleucina-2/genética , Interleucina-4/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel/química
7.
Subcell Biochem ; 41: 319-36, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17484134

RESUMO

In eukaryotic cells, relaxed interphase chromatin undergoes pronounced changes resulting in formation of highly condensed mitotic chromosomes. Moreover, chromatin condensation is particularly evident during mitosis and apoptotic cell death, whereas chromatin relaxation is necessary for replication, repair, recombination and transcription. The post-translational modifications of histone tails such as reversible acetylation, phosphorylation and methylation play a critical role in dynamic condensation/relaxation that occurs during the cell cycle. Histone phosphorylation is believed to play a direct role in mitosis, cell death, repair, replication and recombination. However, definitive roles for this modification in these processes have not yet been elucidated. In this review, we discuss recent progress in studies of histone phosphorylation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Protamina Quinase/metabolismo , Processamento de Proteína Pós-Traducional , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação
8.
Sci Rep ; 5: 16567, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26568260

RESUMO

In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.


Assuntos
Epigênese Genética , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas de Ligação a RNA/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina , Expressão Gênica , Genes Controladores do Desenvolvimento , Histonas/metabolismo , Camundongos , Ligação Proteica , Ubiquitinação
9.
Mar Biotechnol (NY) ; 15(5): 520-5, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23592257

RESUMO

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.


Assuntos
Imunoprecipitação da Cromatina/métodos , Ciona intestinalis/química , Proteínas de Ligação a DNA/análise , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Anticorpos/genética , Primers do DNA/genética , DNA Complementar/genética , Imuno-Histoquímica , Imunoprecipitação , Biologia Marinha/métodos
10.
Mech Dev ; 126(1-2): 68-79, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18992810

RESUMO

C-terminal binding protein (CtBP) is an evolutionarily and functionally conserved transcriptional corepressor known to integrate diverse signals to regulate transcription. Drosophila CtBP (dCtBP) regulates tissue specification and segmentation during early embryogenesis. Here, we investigated the roles of dCtBP during development of the peripheral nervous system (PNS). Our study includes a detailed quantitative analysis of how altered dCtBP activity affects the formation of adult mechanosensory bristles. We found that dCtBP loss-of-function resulted in a series of phenotypes with the most prevalent being supernumerary bristles. These dCtBP phenotypes are more complex than those caused by Hairless, a known dCtBP-interacting factor that regulates bristle formation. The emergence of additional bristles correlated with the appearance of extra sensory organ precursor (SOP) cells in earlier stages, suggesting that dCtBP may directly or indirectly inhibit SOP cell fates. We also found that development of a subset of bristles was regulated by dCtBP associated with U-shaped through the PxDLS dCtBP-interacting motif. Furthermore, the double bristle with sockets phenotype induced by dCtBP mutations suggests the involvement of this corepressor in additional molecular pathways independent of both Hairless and U-shaped. We therefore propose that dCtBP is part of a gene circuitry that controls the patterning and differentiation of the fly PNS via multiple mechanisms.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Sistema Nervoso Periférico/crescimento & desenvolvimento , Sistema Nervoso Periférico/metabolismo , Oxirredutases do Álcool/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
11.
Dev Biol ; 305(2): 650-8, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17336283

RESUMO

The CBP protein is a transcriptional co-activator and histone acetyltransferase. Reduced expression of Drosophila CBP (dCBP) in the early embryo specifically impairs signaling by the TGF-beta molecules Dpp and Screw (Scw). This occurs by a failure to activate transcription of the tolloid (tld) gene, which codes for a protease that generates active Dpp and Scw ligands. We show that dCBP directly regulates this gene by binding to the tld enhancer, and that tld expression can be partially rescued with a dCBP transgene. At a slightly later stage of development, Dpp/Scw signaling recovers in mutant embryos, but is unable to turn on expression of the Dpp/Scw-target gene rhomboid (rho). Interestingly, an acetyltransferase (AT)-defective dCBP transgene rescued tld and rho gene expression to an extent comparable to the wild-type transgene, whereas a transgene containing a 130 amino acid deletion rescued tld but not late rho expression. A tracheal phenotype caused by the reduced dCBP levels was also rescued more efficiently with the wild-type dCBP transgene than with this mutant transgene. Our results indicate that separate parts of the dCBP protein are required on different promoters, and that the AT activity of dCBP is dispensable for certain aspects of Dpp signaling. We discuss the similarity of these results to the role of p300/CBP in TGF-beta signaling in the mouse.


Assuntos
Proteína de Ligação a CREB/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Histona Acetiltransferases/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Proteína de Ligação a CREB/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Mutação Puntual , Regiões Promotoras Genéticas , Transdução de Sinais/genética
12.
Dev Genes Evol ; 217(11-12): 759-69, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17972097

RESUMO

ANGUSTIFOLIA (AN) controls leaf morphology in the plant Arabidopsis thaliana. Previous studies on sequence similarity demonstrated that the closest proteins to AN are members of animal C-terminal-binding proteins (CtBPs) found in nematodes, arthropods, and vertebrates. Drosophila CtBP (dCtBP) functions as a transcriptional corepressor for deoxyribonucleic acid (DNA)-binding repressors containing the short amino acid motif, PXDLS, to regulate tissue specification and segmentation during early embryogenesis. It has previously been shown that AN was thought to repress transcription similar to the function of CtBPs; however, AN lacks some of the structural features that are conserved in animal CtBPs. In this paper, we examined whether AN is functionally related to dCtBP. Firstly, we re-examined sequence similarity among AN and various CtBPs from several representative species in the plant and animal kingdoms. Secondly, yeast two-hybrid assays demonstrated that AN failed to interact with an authentic CtBP-interacting factor, adenovirus E1A oncoprotein bearing the PXDLS motif. Thirdly, AN tethered to DNA was unable to repress the expression of reporter genes in transgenic Drosophila embryos. Fourthly, overexpression assays suggested that dCtBP and AN function differently in Drosophila tissues. Finally, AN failed to rescue the zygotic lethality caused by dCtBP loss-of-function. These data, taken together, suggest that AN is functionally distinct from dCtBP. Likely, ancestral CtBPs acquired corepressor function (capability of both repression and binding to repressors containing the PXDLS motif) after the animal-plant divergence but before the protostome-deuterostome split. We therefore propose to categorize AN as a subfamily member within the CtBP/BARS/RIBEYE/AN superfamily.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Homologia Estrutural de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , DNA/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Teste de Complementação Genética , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transgenes , Zigoto/metabolismo
13.
Proc Natl Acad Sci U S A ; 102(16): 5697-702, 2005 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-15821029

RESUMO

Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency.


Assuntos
Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Histonas/metabolismo , Conformação de Ácido Nucleico , Animais , Proteínas de Ciclo Celular , Células HeLa , Histonas/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares , Proteína 1 de Modelagem do Nucleossomo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo
14.
Genes Dev ; 18(8): 877-88, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15078818

RESUMO

Posttranslational histone modifications are important for the regulation of many biological phenomena. Here, we show the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression.


Assuntos
Drosophila/metabolismo , Histonas/metabolismo , Mitose/fisiologia , Protamina Quinase/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Animais , Drosophila/embriologia , Imunofluorescência , Humanos , Dados de Sequência Molecular , Fosforilação , Protamina Quinase/isolamento & purificação
15.
Biochem Biophys Res Commun ; 317(1): 259-64, 2004 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-15047177

RESUMO

The isoflavones genistein and daidzein and the daidzein metabolite equol have been reported to interact with estrogen receptors (ERs). Some studies indicate that they behave clinically like estrogen in some estrogen-deficiency diseases. However, the detailed molecular mechanism used by these compounds to create beneficial effects in patients with estrogen-related diseases has not been clarified. Using histone acetyltransferase (HAT) assay, we found that equol, genistein, and AglyMax had significant effects on ERalpha-mediated histone acetylation. Although 17beta-estradiol (E2)-dependent HAT activity of steroid receptor coactivators 2 (SRC2) and p300 mediated by ERbeta could be detected, it was weaker than that mediated by ERalpha. Equol, genistein, AglyMax, and daidzein all markedly stimulated ERbeta-mediated histone acetylation. On the other hand, anti-estrogenic compounds ICI 182,780 (ICI) and tamoxifen (TA) did not have an effect on HAT activity mediated by either ERalpha or ERbeta. Our data indicate that estrogenic ligands exert their effects by elevating histone acetylation and coactivator activity of ER, and suggest that the risk of estrogen-related diseases might be reduced by a sufficient amount of genistein or AglyMax supplements.


Assuntos
Estradiol/análogos & derivados , Histonas/metabolismo , Isoflavonas/farmacologia , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Transativadores/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Drosophila/química , Equol , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Fulvestranto , Genisteína/farmacologia , Histona Acetiltransferases , Histonas/química , Isoflavonas/química , Coativador 2 de Receptor Nuclear , Receptores de Estrogênio/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citologia , Tamoxifeno/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica
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