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1.
Nucleus ; 10(1): 7-20, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30663495

RESUMO

Immunosuppressive drugs such as cyclosporin A (CsA) can elicit hepatotoxicity by affecting gene expression. Here, we address the link between CsA and large-scale chromatin organization in HepG2 hepatocarcinoma cells. We show the existence of lamina-associated domains (LADs) interacting with lamin A, lamin B, or both. These 'A-B', 'A-only' and 'B-only' LADs display distinct fates after CsA treatment: A-B LADs remain constitutive or lose A, A-only LADs mainly lose A or switch to B, and B-only LADs remain B-only or acquire A. LAD rearrangement is overall uncoupled from changes in gene expression. Three-dimensional (3D) genome modeling predicts changes in radial positioning of LADs as LADs switch identities, which are corroborated by fluorescence in situ hybridization. Our results reveal interplay between A- and B-type lamins on radial locus positioning, suggesting complementary contributions to large-scale genome architecture. The data also unveil a hitherto unsuspected impact of cytotoxic drugs on genome conformation.Abbreviations: ChIP-seq: chromatin immunoprecipitation sequencing; CsA: cyclosporin A; FISH; fluorescence in situ hybridization; ICMT: isoprenylcysteine methyltransferase; LAD: lamina-associated domain; TAD: topologically-associated domain.


Assuntos
Cromatina/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Lâmina Nuclear/metabolismo , Cromatina/efeitos dos fármacos , Ciclosporina/farmacologia , Células Hep G2 , Humanos , Hibridização in Situ Fluorescente , Lamina Tipo A/antagonistas & inibidores , Lamina Tipo B/antagonistas & inibidores , Modelos Genéticos , Lâmina Nuclear/efeitos dos fármacos , Células Tumorais Cultivadas
2.
Sci Rep ; 7(1): 4897, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28687747

RESUMO

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development. Ablation of endogenous Lamin B1 (Lmnb1) in the mouse strongly impairs embryonic brain development and corticogenesis. However, the mechanisms underlying these neurodevelopmental effects are unknown. Here, we report that Lamin B1 levels modulate the differentiation of murine neural stem cells (NSCs) into neurons and astroglial-like cells. In vitro, endogenous Lmnb1 depletion favors NSC differentiation into glial fibrillar acidic protein (GFAP)-immunoreactive cells over neurons, while overexpression of human Lamin B1 (LMNB1) increases the proportion of neurons. In Lmnb1-null embryos, neurogenesis is reduced, while in vivo Lmnb1 silencing in mouse embryonic brain by in utero electroporation of a specific Lmnb1 sh-RNA results in aberrant cortical positioning of neurons and increased expression of the astrocytic marker GFAP in the cortex of 7-day old pups. Together, these results indicate that finely tuned levels of Lamin B1 are required for NSC differentiation into neurons, proper expression of the astrocytic marker GFAP and corticogenesis.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Proteína Glial Fibrilar Ácida/genética , Lamina Tipo B/genética , Neurogênese/genética , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Diferenciação Celular , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida/metabolismo , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Gravidez , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
3.
Sci Rep ; 5: 17186, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26603343

RESUMO

In the interphase nucleus, chromatin is organized into three-dimensional conformation to coordinate genome functions. The lamina-chromatin association is important to facilitate higher-order chromatin in mammalian cells, but its biological significances and molecular mechanisms remain poorly understood. One obstacle is that the list of lamina-associated proteins remains limited, presumably due to the inherent insolubility of lamina proteins. In this report, we identified 182 proteins associated with lamin B1 (a constitutive component of lamina) in mouse hepatocytes, by adopting virus-based proximity-dependent biotin identification. These proteins are functionally related to biological processes such as chromatin organization. As an example, we validated the association between lamin B1 and core histone macroH2A1, a histone associated with repressive chromatin. Furthermore, we mapped Lamina-associated domains (LADs) in mouse liver cells and found that boundaries of LADs are enriched for macroH2A. More interestingly, knocking-down of macroH2A1 resulted in the release of heterochromatin foci marked by histone lysine 9 trimethylation (H3K9me3) and the decondensation of global chromatin structure. However, down-regulation of lamin B1 led to redistribution of macroH2A1. Taken together, our data indicated that macroH2A1 is associated with lamina and is required to maintain chromatin architecture in mouse liver cells.


Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Lâmina Nuclear/metabolismo , Animais , Cromatina/química , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Hepatócitos/citologia , Hepatócitos/metabolismo , Heterocromatina/metabolismo , Histonas/antagonistas & inibidores , Histonas/genética , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Metilação , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Lâmina Nuclear/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espectrometria de Massas em Tandem
4.
FASEB J ; 28(9): 3906-18, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24858279

RESUMO

The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage-step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.


Assuntos
Núcleo Celular/patologia , Embrião de Mamíferos/citologia , Fibroblastos/patologia , Lamina Tipo B/metabolismo , Doença de Pelizaeus-Merzbacher/patologia , Pele/citologia , Adulto , Animais , Western Blotting , Estudos de Casos e Controles , Permeabilidade da Membrana Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/metabolismo , Fenótipo , RNA Interferente Pequeno/genética , Pele/metabolismo
5.
OMICS ; 18(1): 34-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24199607

RESUMO

Integrative genomics has the potential to uncover relevant loci, as clinical outcome and response to chemotherapies are most likely not due to a single gene (or data type) but rather a complex relationship involving genetic variation, mRNA, DNA methylation, and copy number variation. In addition to this complexity, many complex phenotypes are thought to be controlled by the interplay of multiple genes within the same molecular pathway or gene set (GS). To address these two challenges, we propose an integrative gene set analysis approach and apply this strategy to a cisplatin (CDDP) pharmacogenomics study involving lymphoblastoid cell lines for which genome-wide SNP and mRNA expression data was collected. Application of the integrative GS analysis implicated the role of the RNA binding and cytoskeletal part GSs. The genes LMNB1 and CENPF, within the cytoskeletal part GS, were functionally validated with siRNA knockdown experiments, where the knockdown of LMNB1 and CENPF resulted in CDDP resistance in multiple cancer cell lines. This study demonstrates the utility of an integrative GS analysis strategy for detecting novel genes associated with response to cancer therapies, moving closer to tailored therapy decisions for cancer patients.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Farmacogenética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Família Multigênica , Polimorfismo de Nucleotídeo Único , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcriptoma/efeitos dos fármacos
6.
PLoS One ; 7(11): e49692, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226217

RESUMO

Theoretical models suggest that gene silencing at the nuclear periphery may involve "closing" of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.


Assuntos
Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilases/genética , Histonas/genética , Lâmina Nuclear/genética , Acetilação , Animais , Western Blotting , Linhagem Celular , Cromatina/enzimologia , Imunoprecipitação da Cromatina , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Inativação Gênica , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Família Multigênica , Lâmina Nuclear/enzimologia , RNA de Cadeia Dupla/genética , Transcrição Gênica
7.
Nucleic Acids Symp Ser (Oxf) ; (53): 293-4, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19749376

RESUMO

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analyses. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Our present finding provides an interesting possibility that lamin-B1 may have an important role in regulating cell-death morphology.


Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Apoptose , Floxuridina/toxicidade , Lamina Tipo B/fisiologia , Animais , Linhagem Celular Tumoral , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Camundongos , Necrose , Interferência de RNA
8.
Nucleic Acids Symp Ser (Oxf) ; (52): 627-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776536

RESUMO

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.


Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Apoptose/fisiologia , Floxuridina/toxicidade , Necrose/metabolismo , Animais , Linhagem Celular Tumoral , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Camundongos , Necrose/genética , Interferência de RNA
9.
Nucleosides Nucleotides Nucleic Acids ; 27(5): 433-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18569782

RESUMO

We report that anticancer 5-fluoro-2 '-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Lamina Tipo B/metabolismo , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Floxuridina/farmacologia , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Camundongos , Necrose , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
10.
Leuk Res ; 29(12): 1443-53, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16242777

RESUMO

Viscum album agglutinin-I (VAA-I) is a potent inducer of cell apoptosis and possesses anti-tumoral activity. Using PLB-985 and chronic granulomatous disease (X-CGD) cells, which lack expression of gp91(phox), VAA-I was found to induce apoptosis in both cell lines as assessed by cytology, DNA laddering and degradation of the cytoskeletal protein gelsolin. Both cell lines expressed caspase-3 and -8 and VAA-I activated these caspases. We demonstrated that lamin B(1) is a novel target to VAA-I and its degradation was reversed by a pan-caspase inhibitor and by a caspase-6, but not a caspase-8, inhibitor.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas do Citoesqueleto/efeitos dos fármacos , Leucemia/patologia , Lectinas de Plantas/farmacologia , Preparações de Plantas/farmacologia , Proteínas de Plantas/farmacologia , Toxinas Biológicas/farmacologia , Caspase 3 , Caspase 8 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Gelsolina/antagonistas & inibidores , Humanos , Lamina Tipo B/antagonistas & inibidores , Leucemia/tratamento farmacológico , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos Tipo 2 , Viscum album/química
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