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1.
Genes Dev ; 32(11-12): 806-821, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29899141

RESUMO

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.


Assuntos
Pareamento Incorreto de Bases/fisiologia , DNA Helicases/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Proteína 2 Homóloga a MutS/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Pareamento Incorreto de Bases/genética , Montagem e Desmontagem da Cromatina/genética , DNA/genética , DNA/metabolismo , DNA Helicases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xenopus laevis
2.
Development ; 149(15)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35831949

RESUMO

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.


Assuntos
Histonas , Inativação do Cromossomo X , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Fibroblastos/metabolismo , Camadas Germinativas/metabolismo , Histonas/metabolismo , Mamíferos/genética , Camundongos , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação do Cromossomo X/genética
3.
Genet Med ; 25(7): 100861, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37087635

RESUMO

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.


Assuntos
Homólogo 5 da Proteína Cromobox , Heterocromatina , Animais , Camundongos , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Histonas/metabolismo
4.
Nucleic Acids Res ; 49(21): 12152-12166, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34817054

RESUMO

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.


Assuntos
Ciclo Celular , DNA/metabolismo , Histonas/metabolismo , Replicação do DNA , Células HeLa , Humanos , Metilação
5.
PLoS Genet ; 15(6): e1008061, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170156

RESUMO

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Proteínas de Schizosaccharomyces pombe/genética , Transporte Ativo do Núcleo Celular/genética , Ciclo Celular/genética , Divisão Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Humanos , Meiose/genética , Microscopia de Fluorescência , Membrana Nuclear/genética , Poro Nuclear/ultraestrutura , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
6.
Development ; 145(18)2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30126901

RESUMO

X inactivation in mammals is regulated by epigenetic modifications. Functional deficiency of SmcHD1 has been shown to cause de-repression of X-inactivated genes in post-implantation female mouse embryos, suggesting a role of SmcHD1 in the maintenance of X inactivation. Here, we show that de-repression of X-inactivated genes accompanied a local reduction in the enrichment of H3K27me3 in mouse embryonic fibroblasts deficient for SmcHD1. Furthermore, many of these genes overlapped with those having a significantly lower enrichment of H3K27me3 at the blastocyst stage in wild type. Intriguingly, however, depletion of SmcHD1 did not compromise the X-inactivated state in immortalized female mouse embryonic fibroblasts, in which X inactivation had been established and maintained. Taking all these findings together, we suggest that SmcHD1 facilitates the incorporation of H3K27me3 and perhaps other epigenetic modifications at gene loci that are silenced even with the lower enrichment of H3K27me3 at the early stage of X inactivation. The epigenetic state at these loci would, however, remain as it is at the blastocyst stage in the absence of SmcHD1 after implantation, which would eventually compromise the maintenance of the X-inactivated state at later stages.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética/genética , Genes Ligados ao Cromossomo X/genética , Inativação do Cromossomo X/genética , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Embrião de Mamíferos/embriologia , Feminino , Fibroblastos/citologia , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
7.
Development ; 144(15): 2784-2797, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28684628

RESUMO

Xist RNA, which is responsible for X inactivation, is a key epigenetic player in the embryogenesis of female mammals. Of the several repeats conserved in Xist RNA, the A-repeat has been shown to be essential for its silencing function in differentiating embryonic stem cells. Here, we introduced a new Xist allele into mouse that produces mutated Xist RNA lacking the A-repeat (XistCAGΔ5' ). XistCAGΔ5' RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that XistCAGΔ5' RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on genome-wide gene expression. It is likely that dosage compensation is required not only for equalizing X-linked gene expression between the sexes but also for proper global gene regulation in differentiated female somatic cells.


Assuntos
Mecanismo Genético de Compensação de Dose/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Trofoblastos/metabolismo , Alelos , Animais , Células Cultivadas , Mecanismo Genético de Compensação de Dose/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Imunofluorescência , Camundongos , Cromossomo X/genética , Inativação do Cromossomo X/genética
8.
Cell Immunol ; 356: 104188, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32763501

RESUMO

Stimulator of interferon genes (STING) plays important roles in the DNA-mediated innate immune responses. However, the regulatory mechanism of STING in terms of stabilization is not fully understood. Here, we identified the chaperone protein Hsp90s as novel STING interacting proteins. Treatment with an Hsp90 inhibitor 17-AAG and knockdown of Hsp90ß but not Hsp90α reduced STING at protein level, resulted in the suppression of IFN induction in response to stimulation with cGAMP, and infections with HSV-1 and Listeria monocytogenes. Collectively, our results suggest that the control of STING protein by Hsp90ß is a critical biological process in the DNA sensing pathways.


Assuntos
Proteínas de Choque Térmico HSP90/imunologia , Proteínas de Membrana/imunologia , Animais , DNA Viral/imunologia , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Imunidade Inata , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Células RAW 264.7 , Transdução de Sinais , Proteínas Virais/metabolismo
9.
Proc Natl Acad Sci U S A ; 114(52): E11208-E11217, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29237752

RESUMO

Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Heterocromatina/genética , Domínios Proteicos , RNA Polimerase II/genética , RNA Fúngico/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Serina/genética , Serina/metabolismo
10.
J Cell Sci ; 130(14): 2416-2429, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28600324

RESUMO

The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental pericentromeric DNAs, which led to a significant mitotic delay and frequent chromosome mis-segregation upon the first mitotic division. A proteomic analysis identified nucleoplasmin 2 (NPM2) as a dominant component of the oocyte nucleolus. Consistently, Npm2-deficient oocytes, which lack a normal nucleolar structure, showed chromosome segregation defects similar to those in enucleolated oocytes, suggesting that nucleolar loss, rather than micromanipulation-related damage to the genome, leads to a disorganization of higher-order chromatin structure in pronuclei and frequent chromosome mis-segregation during the first mitosis. Strikingly, expression of NPM2 alone sufficed to reconstitute the nucleolar structure in enucleolated embryos, and rescued their first mitotic division and full-term development. The nucleolus rescue through NPM2 required the pentamer formation and both the N- and C-terminal domains. Our findings demonstrate that the NPM2-based oocyte nucleolus is an essential platform for parental chromatin organization in early embryonic development.


Assuntos
Nucleoplasminas/metabolismo , Oócitos/metabolismo , Sequência de Aminoácidos , Animais , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Feminino , Camundongos , Oócitos/citologia
11.
J Cell Sci ; 130(10): 1822-1834, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28386019

RESUMO

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins and subcellular localization analysis of GFP-fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-Nup160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either the MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on the compositional and structural specificity of NPCs.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Tetrahymena thermophila/metabolismo , Sequência Conservada , Macronúcleo/metabolismo , Micronúcleo Germinativo/metabolismo , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/química , Permeabilidade , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos
12.
Genes Cells ; 23(3): 122-135, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29292846

RESUMO

Inner nuclear membrane (INM) proteins are thought to play important roles in modulating nuclear organization and function through their interactions with chromatin. However, these INM proteins share redundant functions in metazoans that pose difficulties for functional studies. The fission yeast Schizosaccharomyces pombe exhibits a relatively small number of INM proteins, and molecular genetic tools are available to separate their redundant functions. In S. pombe, it has been reported that among potentially redundant INM proteins, Lem2 displays a unique genetic interaction with another INM protein, Bqt4, which is involved in anchoring telomeres to the nuclear envelope. Double mutations in the lem2 and bqt4 genes confer synthetic lethality during vegetative growth. Here, we show that Lem2 is retained at the nuclear envelope through its interaction with Bqt4, as the loss of Bqt4 results in the exclusive accumulation of Lem2 to the spindle pole body (SPB). An N-terminal nucleoplasmic region of Lem2 bears affinity to both Bqt4 and the SPB in a competitive manner. In contrast, the synthetic lethality of the lem2 bqt4 double mutant is suppressed by the C-terminal region of Lem2. These results indicate that the N-terminal and C-terminal domains of Lem2 show independent functions with respect to Bqt4.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Telômero/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Membrana Nuclear/genética , Domínios e Motivos de Interação entre Proteínas , Schizosaccharomyces/crescimento & desenvolvimento , Telômero/genética
13.
EMBO Rep ; 18(3): 403-419, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28077461

RESUMO

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N-terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1-PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1-PP1 protects the origin-binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1-depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1-targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.


Assuntos
Replicação do DNA , Proteína Fosfatase 1/metabolismo , Origem de Replicação , Proteínas de Ligação a Telômeros/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas de Ligação a Telômeros/química
14.
Mol Cell ; 43(1): 45-56, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21726809

RESUMO

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding ß-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind ß-catenin. The parafibromin/ß-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/análise , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Tirosina/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
15.
Genes Dev ; 25(8): 863-74, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21498573

RESUMO

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. Here we report that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. We identified Thr 1415 of the CAP-D3 subunit as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and that the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/genética , Western Blotting , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Interferência de RNA
16.
Nucleic Acids Res ; 44(9): 4147-62, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-26792892

RESUMO

In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 and H3K36me3) via an interaction between its Set2-Rpb1 interaction (SRI) domain and C-terminal repeats of RNA polymerase II (Pol2) phosphorylated at Ser2 and Ser5 (CTD-S2,5-P). H3K36me2 is sufficient for recruitment of the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. In fission yeast, Set2 is also responsible for H3K36 methylation, which represses a subset of RNAs including heterochromatic and subtelomeric RNAs, at least in part via recruitment of Clr6 complex II, a homolog of Rpd3S. Here, we show that CTD-S2P-dependent interaction of fission yeast Set2 with Pol2 via the SRI domain is required for formation of H3K36me3, but not H3K36me2. H3K36me3 silenced heterochromatic and subtelomeric transcripts mainly through post-transcriptional and transcriptional mechanisms, respectively, whereas H3K36me2 was not enough for silencing. Clr6 complex II appeared not to be responsible for heterochromatic silencing by H3K36me3. Our results demonstrate that H3K36 methylation has multiple outputs in fission yeast; these findings provide insights into the distinct roles of H3K36 methylation in metazoans, which have different enzymes for synthesis of H3K36me1/2 and H3K36me3.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/ultraestrutura , Genes Fúngicos , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/fisiologia , Metilação , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Estabilidade de RNA , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/fisiologia , Telômero/genética , Transcrição Gênica
17.
J Immunol ; 195(10): 4933-42, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26466955

RESUMO

Compartmentalization of nucleic acid (NA)-sensing TLR3, 7, 8, and 9 is strictly regulated to direct optimal response against microbial infection and evade recognition of host-derived NAs. Uncoordinated 93 homolog B1 (UNC93B1) is indispensable for trafficking of NA-sensing TLRs from the endoplasmic reticulum (ER) to endosomes/lysosomes. UNC93B1 controls loading of the TLRs into COPII vesicles to exit from the ER and traffics with the TLRs in the steady state. Ligand-induced translocation also happens on NA-sensing TLRs. However, the molecular mechanism for ligand-dependent trafficking of TLRs from the ER to endosomes/lysosomes remains unclear. In this study, we demonstrated that leucine-rich repeat containing protein (LRRC) 59, an ER membrane protein, participated in trafficking of NA-sensing TLRs from the ER. Knockdown of LRRC59 reduced TLR3-, 8-, and 9-mediated, but not TLR4-mediated, signaling. Upon ligand stimulation, LRRC59 associated with UNC93B1 in a TLR-independent manner, which required signals induced by ligand internalization. Endosomal localization of endogenous TLR3 was decreased by silencing of LRRC59, suggesting that LRRC59 promotes UNC93B1-mediated translocation of NA-sensing TLRs from the ER upon infection. These findings help us understand how NA-sensing TLRs control their proper distribution in the infection/inflammatory state.


Assuntos
Retículo Endoplasmático/imunologia , Infecções/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Retículo Endoplasmático/genética , Endossomos/genética , Endossomos/imunologia , Células HEK293 , Humanos , Infecções/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Transdução de Sinais/genética , Receptores Toll-Like/genética
18.
J Biol Chem ; 290(49): 29375-88, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26491019

RESUMO

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/ß-mediated nuclear import. RBBP4 accelerates the release of importin ß1 from importin α via competitive binding to the importin ß-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/ß-mediated nuclear import. We showed that the importin α/ß pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin ß1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence.


Assuntos
Transporte Ativo do Núcleo Celular , Senescência Celular , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Fibroblastos/metabolismo , Glutationa Transferase/metabolismo , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
19.
Biochim Biophys Acta ; 1853(10 Pt A): 2676-83, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26220098

RESUMO

Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin ß1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin ß1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/ß1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin ß binding domain.


Assuntos
Núcleo Celular/metabolismo , Transdução de Sinais/fisiologia , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , beta Carioferinas/genética
20.
EMBO J ; 31(17): 3524-36, 2012 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-22828868

RESUMO

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.


Assuntos
Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Chaperonas de Histonas/metabolismo , Animais , Linhagem Celular , Galinhas , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo
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