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1.
Cell Rep ; 43(7): 114394, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-38923455

RESUMEN

The intricate interplay between resident cells and the extracellular matrix (ECM) profoundly influences cancer progression. In triple-negative breast cancer (TNBC), ECM architecture evolves due to the enrichment of lysyl oxidase, fibronectin, and collagen, promoting distant metastasis. Here we uncover a pivotal transcription regulatory mechanism involving the epigenetic regulator UBR7 and histone methyltransferase EZH2 in regulating transforming growth factor (TGF)-ß/Smad signaling, affecting the expression of ECM genes. UBR7 loss leads to a dramatic reduction in facultative heterochromatin mark H3K27me3, activating ECM genes. UBR7 plays a crucial role in matrix deposition in adherent cancer cells and spheroids, altering collagen content and lysyl oxidase activity, directly affecting matrix stiffness and invasiveness. These findings are further validated in vivo in mice models and TNBC patients, where reduced UBR7 levels are accompanied by increased ECM component expression and activity, leading to fibrosis-mediated matrix stiffness. Thus, UBR7 is a master regulator of matrix stiffening, influencing the metastatic potential of TNBC.


Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Matriz Extracelular , Transducción de Señal , Factor de Crecimiento Transformador beta , Neoplasias de la Mama Triple Negativas , Ubiquitina-Proteína Ligasas , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Ubiquitina-Proteína Ligasas/metabolismo
2.
Hum Mol Genet ; 32(5): 745-763, 2023 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-36124691

RESUMEN

Lamin B Receptor (LBR) is an inner nuclear membrane protein that assembles the nuclear envelope post mitosis. Here we show that LBR depletion induces mitotic defects accompanied by recurrent chromosomal losses. In addition, LBR knockdown results in nuclear aberrations such as nuclear blebs and micronuclei, with chromosomes showing higher frequency of losses, being enriched within the micronucleus. Furthermore, doxycycline-induced conditional depletion of LBR significantly increased tumor volumes that form within the subcutaneous xenografts of mice. Of note, the tumor-derived primary cells recapitulated chromosomal losses and gains, revealing a novel role for LBR as a tumor suppressor. Co-immunoprecipitation of LBR uncovered an association of LBR with telomere-associated factors. Interestingly, qPCR array-based gene expression profiling showed a significant upregulation of telomere repeat-binding factor 1 (TRF1) upon LBR depletion. Remarkably, TRF1 knockdown in the background of LBR depletion maintains chromosomal stability, unraveling a novel mechanism involving LBR and TRF in the maintenance of chromosomal stability in colorectal cancer cells.


Asunto(s)
Membrana Nuclear , Receptores Citoplasmáticos y Nucleares , Humanos , Animales , Ratones , Membrana Nuclear/metabolismo , Proteínas de la Membrana/metabolismo , Carcinogénesis , Inestabilidad Cromosómica , Lamina Tipo B/metabolismo , Receptor de Lamina B
3.
J Phys Chem Lett ; 13(28): 6427-6438, 2022 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-35816132

RESUMEN

The size of amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds dictates not only their cellular internalization and associated cell death but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small fibril seeds showed elongation possibly through monomer addition at the fibril termini, whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification and cellular uptake along with toxicity suggest that breakage of fibrils into seeds of different sizes determines the underlying pathological outcome of synucleinopathies.


Asunto(s)
Amiloide , alfa-Sinucleína , Amiloide/metabolismo , alfa-Sinucleína/metabolismo
4.
Front Cell Dev Biol ; 10: 1068347, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36589746

RESUMEN

In eukaryotic cells, the genome is organized in the form of chromatin composed of DNA and histones that organize and regulate gene expression. The dysregulation of chromatin remodeling, including the aberrant incorporation of histone variants and their consequent post-translational modifications, is prevalent across cancers. Additionally, nuclear envelope proteins are often deregulated in cancers, which impacts the 3D organization of the genome. Altered nuclear morphology, genome organization, and gene expression are defining features of cancers. With advances in single-cell sequencing, imaging technologies, and high-end data mining approaches, we are now at the forefront of designing appropriate small molecules to selectively inhibit the growth and proliferation of cancer cells in a genome- and epigenome-specific manner. Here, we review recent advances and the emerging significance of aberrations in nuclear envelope proteins, histone variants, and oncohistones in deregulating chromatin organization and gene expression in oncogenesis.

5.
J Mol Biol ; 434(3): 167256, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-34547328

RESUMEN

Chromosomal instability (CIN) is associated with the initiation and progression of gastrointestinal (GI) tract cancers. Cancers of the GI tract are typically characterized by altered chromosome numbers. While the dynamics of CIN have been extensively characterized in 2D monolayer cell cultures derived from GI tumors, the tumor microenvironment and 3D tumor architecture also contribute to the progression of CIN, which is not captured in 2D cell culture systems. To overcome these limitations, self-organizing cellular structures that retain organ-specific 3D architecture, namely organoids, have been derived from various tissues of the GI tract. Organoids derived from normal tissue and patient tumors serve as a useful paradigm to study the crosstalk between tumor cells in the context of a tissue microenvironment and its impact on chromosomal stability. Such a paradigm, therefore, has a considerable advantage over 2D cell culture systems in drug screening and personalized medicine. Here, we review the importance of patient-derived tumor organoids (PDTOs) as a model to study CIN in cancers of the GI tract.


Asunto(s)
Inestabilidad Cromosómica , Neoplasias Gastrointestinales , Organoides , Técnicas de Cultivo de Célula , Neoplasias Gastrointestinales/genética , Humanos , Microambiente Tumoral
6.
J Cell Sci ; 134(23)2021 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-34746948

RESUMEN

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating expression of the HOXA gene locus during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5' HOXA genes, respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3' and 5' HOXA genes in the undifferentiated human NT2/D1 cell line. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. Whereas Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation. This article has an associated First Person interview with the first authors of the paper.


Asunto(s)
Proteínas de Homeodominio , Proteínas de Complejo Poro Nuclear , Factor de Unión a CCCTC/genética , Diferenciación Celular/genética , Cromatina/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Complejo Poro Nuclear/genética
7.
Mol Cell Biol ; 41(8): e0038220, 2021 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-34060915

RESUMEN

The long noncoding RNA XIST is the master regulator for the process of X chromosome inactivation (XCI) in mammalian females. Here, we report the existence of a hitherto-uncharacterized cis regulatory element (cRE) within the first exon of human XIST, which determines the transcriptional status of XIST during the initiation and maintenance phases of XCI. In the initiation phase, pluripotency factors bind to this cRE and keep XIST repressed. In the maintenance phase of XCI, the cRE is enriched for CTCF, which activates XIST transcription. By employing a CRISPR-dCas9-KRAB-based interference strategy, we demonstrate that binding of CTCF to the newly identified cRE is critical for regulating XIST in a YY1-dependent manner. Collectively, our study uncovers the combinatorial effect of multiple transcriptional regulators influencing XIST expression during the initiation and maintenance phases of XCI.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Células Madre Embrionarias/metabolismo , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/fisiología , Factor de Unión a CCCTC/genética , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Inactivación del Cromosoma X/genética
8.
Biol Cell ; 113(7): 295-310, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-33638183

RESUMEN

Nuclear lamins are type V intermediate filament proteins that form a filamentous meshwork beneath the inner nuclear membrane. Additionally, a sub-population of A- and B-type lamins localizes in the nuclear interior. The nuclear lamina protects the nucleus from mechanical stress and mediates nucleo-cytoskeletal coupling. Lamins form a scaffold that partially tethers chromatin at the nuclear envelope. The nuclear lamina also stabilises protein-protein interactions involved in gene regulation and DNA repair. The lamin-based protein sub-complexes are implicated in both nuclear and cytoskeletal organisation, the mechanical stability of the nucleus, genome organisation, transcriptional regulation, genome stability and cellular differentiation. Here, we review recent research on nuclear lamins and unique roles of A- and B-type lamins in modulating various nuclear processes and their impact on cell function.


Asunto(s)
Laminas/fisiología , Lámina Nuclear/fisiología , Animales , Cromatina/metabolismo , Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Humanos , Relación Estructura-Actividad , Levaduras
9.
Hum Mol Genet ; 29(10): 1673-1688, 2020 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-32337580

RESUMEN

Twist1 is a basic helix-loop-helix transcription factor, essential during early development in mammals. While Twist1 induces epithelial-to-mesenchymal transition (EMT), here we show that Twist1 overexpression enhances nuclear and mitotic aberrations. This is accompanied by an increase in whole chromosomal copy number gains and losses, underscoring the role of Twist1 in inducing chromosomal instability (CIN) in colorectal cancer cells. Array comparative genomic hybridization (array CGH) analysis further shows sub-chromosomal deletions, consistent with an increased frequency of DNA double strand breaks (DSBs). Remarkably, Twist1 overexpression downmodulates key cell cycle checkpoint factors-Bub1, BubR1, Mad1 and Mad2-that regulate CIN. Mathematical simulations using the RACIPE tool show a negative correlation of Twist1 with E-cadherin and BubR1. Data analyses of gene expression profiles of patient samples from The Cancer Genome Atlas (TCGA) reveal a positive correlation between Twist1 and mesenchymal genes across cancers, whereas the correlation of TWIST1 with CIN and DSB genes is cancer subtype-specific. Taken together, these studies highlight the mechanistic involvement of Twist1 in the deregulation of factors that maintain genome stability during EMT in colorectal cancer cells. Twist1 overexpression enhances genome instability in the context of EMT that further contributes to cellular heterogeneity. In addition, these studies imply that Twist1 downmodulates nuclear lamins that further alter spatiotemporal organization of the cancer genome and epigenome. Notwithstanding their genetic background, colorectal cancer cells nevertheless maintain their overall ploidy, while the downstream effects of Twist1 enhance CIN and DNA damage enriching for sub-populations of aggressive cancer cells.


Asunto(s)
Cadherinas/genética , Inestabilidad Cromosómica/genética , Neoplasias Colorrectales/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 Relacionada con Twist/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Hibridación Genómica Comparativa , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas Mad2/genética
10.
J Cell Sci ; 133(4)2020 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-31988151

RESUMEN

The structure-function relationship of the nucleus is tightly regulated, especially during heat shock. Typically, heat shock activates molecular chaperones that prevent protein misfolding and preserve genome integrity. However, the molecular mechanisms that regulate nuclear structure-function relationships during heat shock remain unclear. Here, we show that lamin A and C (hereafter lamin A/C; both lamin A and C are encoded by LMNA) are required for heat-shock-mediated transcriptional induction of the Hsp70 gene locus (HSPA genes). Interestingly, lamin A/C regulates redistribution of nuclear myosin I (NM1) into the nucleus upon heat shock, and depletion of either lamin A/C or NM1 abrogates heat-shock-induced repositioning of Hsp70 gene locus away from the nuclear envelope. Lamins and NM1 also regulate spatial positioning of the SC35 (also known as SRSF2) speckles - important nuclear landmarks that modulates Hsp70 gene locus expression upon heat shock. This suggests an intricate crosstalk between nuclear lamins, NM1 and SC35 organization in modulating transcriptional responses of the Hsp70 gene locus during heat shock. Taken together, this study unravels a novel role for lamin A/C in the regulation of the spatial dynamics and function of the Hsp70 gene locus upon heat shock, via the nuclear motor protein NM1.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Lamina Tipo A , Miosina Tipo I , Núcleo Celular/genética , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Humanos , Lamina Tipo A/genética
11.
Methods Mol Biol ; 2038: 181-197, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31407285

RESUMEN

It is well established that the genome is non-randomly organized in the interphase nucleus with gene rich chromosome territories toward the nuclear interior, while gene poor chromosome territories are proximal to the nuclear periphery. In vivo tissue stiffness and architecture modulates cell type-specific genome organization and gene expression programs. However, the impact of external mechanical forces on the non-random organization of the genome is not completely understood. Here we describe a modified protocol for visualizing chromosome territories and gene loci positions in cells exposed to reduced matrix stiffness by employing soft polyacrylamide matrices. 3-Dimensional Fluorescence In Situ Hybridization (3D-FISH) protocol followed by image analyses performed on cells exposed to extracellular matrices of varying stiffness properties, enables the determination of the dynamics of chromosome territories as well as gene loci in the interphase nucleus. This will be useful in understanding how chromosome territories respond to changes in substrate stiffness and the potential correlation between the repositioning of chromosome territories and their respective transcriptional profiles.


Asunto(s)
Resinas Acrílicas/química , Cromosomas Humanos , Sitios Genéticos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Técnicas de Cultivo de Célula , Forma de la Célula , Células HCT116 , Dureza , Humanos
12.
BMC Mol Cell Biol ; 20(1): 11, 2019 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-31117946

RESUMEN

BACKGROUND: Nuclear lamins are type V intermediate filament proteins that maintain nuclear structure and function. Furthermore, Emerin - an interactor of Lamin A/C, facilitates crosstalk between the cytoskeleton and the nucleus as it also interacts with actin and Nuclear Myosin 1 (NM1). RESULTS: Here we show that the depletion of Lamin A/C or Emerin, alters the localization of the nuclear motor protein - Nuclear Myosin 1 (NM1) that manifests as an increase in NM1 foci in the nucleus and are rescued to basal levels upon the combined knockdown of Lamin A/C and Emerin. Furthermore, Lamin A/C-Emerin co-depletion destabilizes cytoskeletal organization as it increases actin stress fibers. This further impinges on nuclear organization, as it enhances chromatin mobility more toward the nuclear interior in Lamin A/C-Emerin co-depleted cells. This enhanced chromatin mobility was restored to basal levels either upon inhibition of Nuclear Myosin 1 (NM1) activity or actin depolymerization. In addition, the combined loss of Lamin A/C and Emerin alters the otherwise highly conserved spatial positions of chromosome territories. Furthermore, knockdown of Lamin A/C or Lamin A/C-Emerin combined, deregulates expression levels of a candidate subset of genes. Amongst these genes, both KLK10 (Chr.19, Lamina Associated Domain (LAD+)) and MADH2 (Chr.18, LAD-) were significantly repressed, while BCL2L12 (Chr.19, LAD-) is de-repressed. These genes differentially reposition with respect to the nuclear envelope. CONCLUSIONS: Taken together, these studies underscore a remarkable interplay between Lamin A/C and Emerin in modulating cytoskeletal organization of actin and NM1 that impinges on chromatin dynamics and function in the interphase nucleus.


Asunto(s)
Núcleo Celular/genética , Cromatina/metabolismo , Técnicas de Silenciamiento del Gen , Interfase/genética , Lamina Tipo A/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Actinas/metabolismo , Línea Celular Tumoral , Posicionamiento de Cromosoma/genética , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 19/genética , Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica , Sitios Genéticos , Humanos , Calicreínas/genética , Proteínas Musculares/genética , Miosina Tipo I/metabolismo , Membrana Nuclear/genética , Polimerizacion , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/genética , Proteína Smad2/genética , Transfección
13.
J Biosci ; 43(2): 417-419, 2018 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29872028

RESUMEN

The genome of higher eukaryotes is non-randomly organized in the interphase nucleus. However, notwithstanding the absence of membrane bound sub-compartments, the nucleus coordinates a number of functions largely by organizing chromatin in a non-random but dynamic manner. The plasticity of chromatin structure and function relies on epigenetic modifications as well as its association with nuclear landmarks such as the nuclear envelope, nuclear lamina, nuclear pore complex and nuclear bodies such as the nucleolus among others. In the absence of membrane-bound compartments, cells and the nucleus, in particular, employ phase-separation, which unmixes phases that constrain biochemical reactions in complex non-membranous sub-compartments such as the nucleolus or even the heterochromatin. This review attempts to provide a glimpse into the microcosm of phase-separated nuclear sub-compartments, that regulate nuclear structure- function relationships.


Asunto(s)
Epigénesis Genética , Genoma , Heterocromatina/genética , Compartimento Celular/genética , Núcleo Celular/genética , Cromatina/genética , Eucariontes/genética
14.
Nucleus ; 9(1): 350-367, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29943658

RESUMEN

Eukaryotic cells have 2 to ​3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus.


Asunto(s)
Nucléolo Celular/metabolismo , Histonas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ciclo Celular/efectos de los fármacos , Nucléolo Celular/efectos de los fármacos , Dactinomicina/farmacología , Células HCT116 , Histonas/efectos de los fármacos , Humanos , Células MCF-7 , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Nucleolina
15.
Nucleic Acids Res ; 46(11): 5561-5586, 2018 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-29684168

RESUMEN

Cells perceive and relay external mechanical forces into the nucleus through the nuclear envelope. Here we examined the effect of lowering substrate stiffness as a paradigm to address the impact of altered mechanical forces on nuclear structure-function relationships. RNA sequencing of cells on softer matrices revealed significant transcriptional imbalances, predominantly in chromatin associated processes and transcriptional deregulation of human Chromosome 1. Furthermore, 3-Dimensional fluorescence in situ hybridization (3D-FISH) analyses showed a significant mislocalization of Chromosome 1 and 19 Territories (CT) into the nuclear interior, consistent with their transcriptional deregulation. However, CT18 with relatively lower transcriptional dysregulation, also mislocalized into the nuclear interior. Furthermore, nuclear Lamins that regulate chromosome positioning, were mislocalized into the nuclear interior in response to lowered matrix stiffness. Notably, Lamin B2 overexpression retained CT18 near the nuclear periphery in cells on softer matrices. While, cells on softer matrices also activated emerin phosphorylation at a novel Tyr99 residue, the inhibition of which in a phospho-deficient mutant (emerinY99F), selectively retained chromosome 18 and 19 but not chromosome 1 territories at their conserved nuclear locations. Taken together, emerin functions as a key mechanosensor, that modulates the spatial organization of chromosome territories in the interphase nucleus.


Asunto(s)
Posicionamiento de Cromosoma , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fenómenos Biomecánicos , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas Humanos , Cromosomas Humanos Par 18 , Regulación de la Expresión Génica , Código de Histonas , Humanos , Lamina Tipo B/metabolismo , Laminas/análisis , Proteínas de la Membrana/química , Proteínas Nucleares/química , Fosforilación , Transcripción Genética , Tirosina/metabolismo
16.
Mol Cell Biol ; 37(24)2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-28993479

RESUMEN

The nucleolus is required for ribosome biogenesis. Human cells have 2 or 3 nucleoli associated with nucleolar organizer region (NOR)-bearing chromosomes. An increase in number and altered nucleolar morphology define cancer cells. However, the mechanisms that modulate nucleolar morphology and function are unclear. Here we show that in addition to localizing at the nuclear envelope, lamin B2 localizes proximal to nucleolin at the granular component (GC) of the nucleolus and associates with the nucleolar proteins nucleolin and nucleophosmin. Lamin B2 knockdown severely disrupted the nucleolar morphology, which was rescued to intact and discrete nucleoli upon lamin B2 overexpression. Furthermore, two mutually exclusive lamin B2 deletion mutants, ΔHead and ΔSLS, rescued nuclear and nucleolar morphology defects, respectively, induced upon lamin B2 depletion, suggesting independent roles for lamin B2 at the nucleolus and nuclear envelope. Lamin B2 depletion increased nucleolin aggregation in the nucleoplasm, implicating lamin B2 in stabilizing nucleolin within the nucleolus. Lamin B2 knockdown upregulated nucleolus-specific 45S rRNA and upstream intergenic sequence (IGS) transcripts. The IGS transcripts colocalized with aggregates of nucleolin speckles, which were sustained in the nucleoplasm upon lamin B2 depletion. Taken together, these studies uncover a novel role for lamin B2 in modulating the morphology, dynamics, and function of the nucleolus.


Asunto(s)
Lamina Tipo B/metabolismo , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Nucleolina
17.
Chromosoma ; 126(2): 223-244, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-26921073

RESUMEN

Chromosome territories assume non-random positions in the interphase nucleus with gene-rich chromosomes localized toward the nuclear interior and gene-poor chromosome territories toward the nuclear periphery. Lamins are intermediate filament proteins of the inner nuclear membrane required for the maintenance of nuclear structure and function. Here, we show using whole-genome expression profiling that Lamin A/C or Lamin B2 depletion in an otherwise diploid colorectal cancer cell line (DLD1) deregulates transcript levels from specific chromosomes. Further, three-dimensional fluorescence in situ hybridization (3D-FISH) analyses of a subset of these transcriptionally deregulated chromosome territories revealed that the diploid chromosome territories in Lamin-depleted cells largely maintain conserved positions in the interphase nucleus in a gene-density-dependent manner. In addition, chromosomal aneuploidies were induced in ~25 % of Lamin A/C or Lamin B2-depleted cells. Sub-populations of these aneuploid cells consistently showed a mislocalization of the gene-rich aneuploid chromosome 19 territory toward the nuclear periphery, while gene-poor aneuploid chromosome 18 territory was mislocalized toward the nuclear interior predominantly upon Lamin B2 than Lamin A/C depletion. In addition, a candidate gene locus ZNF570 (Chr.19q13.12) significantly overexpressed upon Lamin B2 depletion was remarkably repositioned away from the nuclear lamina. Taken together, our studies strongly implicate an overarching role for Lamin B2 in the maintenance of nuclear architecture since loss of Lamin B2 relieves the spatial positional constraints required for maintaining conserved localization of aneuploid chromosome territories in the interphase nucleus.


Asunto(s)
Aneuploidia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Interfase/genética , Lamina Tipo B/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Diploidia , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transcripción Genética
18.
Artículo en Inglés | MEDLINE | ID: mdl-27980680

RESUMEN

BACKGROUND: The nuclear pore complex (NPC) mediates nuclear transport of RNA and proteins into and out of the nucleus. Certain nucleoporins have additional functions in chromatin organization and transcription regulation. Nup93 is a scaffold nucleoporin at the nuclear pore complex which is associated with human chromosomes 5, 7 and 16 and with the promoters of the HOXA gene as revealed by ChIP-on-chip studies using tiling microarrays for these chromosomes. However, the functional consequences of the association of Nup93 with HOXA is unknown. RESULTS: Here, we examined the association of Nup93 with the HOXA gene cluster and its consequences on HOXA gene expression in diploid colorectal cancer cells (DLD1). Nup93 showed a specific enrichment ~1 Kb upstream of the transcription start site of each of the HOXA1, HOXA3 and HOXA5 promoters, respectively. Furthermore, the association of Nup93 with HOXA was assisted by its interacting partners Nup188 and Nup205. The depletion of the Nup93 sub-complex significantly upregulated HOXA gene expression levels. However, expression levels of a control gene locus (GLCCI1) on human chromosome 7 were unaffected. Three-dimensional fluorescence in situ hybridization (3D-FISH) analyses revealed that the depletion of the Nup93 sub-complex (but not Nup98) disengages the HOXA gene locus from the nuclear periphery, suggesting a potential role for Nup93 in tethering and repressing the HOXA gene cluster. Consistently, Nup93 knockdown increased active histone marks (H3K9ac), decreased repressive histone marks (H3K27me3) on the HOXA1 promoter and increased transcription elongation marks (H3K36me3) within the HOXA1 gene. Moreover, the combined depletion of Nup93 and CTCF (a known organizer of HOXA gene cluster) but not Nup93 alone, significantly increased GLCCI1 gene expression levels. Taken together, this suggests a novel role for Nup93 and its interactors in repressing the HOXA gene cluster. CONCLUSIONS: This study reveals that the nucleoporin Nup93 assisted by its interactors Nup188 and Nup205 mediates the repression of HOXA gene expression.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Células HeLa , Histonas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Hibridación Fluorescente in Situ , Familia de Multigenes , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Elongación de la Transcripción Genética , Sitio de Iniciación de la Transcripción
19.
Org Lett ; 18(6): 1274-7, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26933774

RESUMEN

2-Methyl-3-[1-(N,N-dimethylamino)diazen-1-ium-1,2-diol-2-ato-methyl]-naphthalene-1,4-dione 1 (HyPR-1), a small molecule containing a superoxide generator strategically linked to a diazeniumdiolate-based nitric oxide donor, is reported. Evidence for HyPR-1's ability to generate peroxynitrite in the presence of an enzyme as well as enhance peroxynitrite within cells is provided. The utility of this tool in generating peroxynitrite for cellular studies is demonstrated.


Asunto(s)
Naftoquinonas/síntesis química , Ácido Peroxinitroso/química , Compuestos Azo/química , Estructura Molecular , Naftoquinonas/química , Donantes de Óxido Nítrico/química
20.
Chem Commun (Camb) ; 50(78): 11533-5, 2014 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-25133745

RESUMEN

We describe here hitherto unexplored chemistry of the sulfinate ester functional group as being highly selective towards nucleophilic substitution by thiols at physiological pH. Using this cleavable trigger, an optical thiol probe that is suitable for thiol bioimaging has been developed.


Asunto(s)
Compuestos de Sulfhidrilo/química , Ácidos Sulfínicos/química , Línea Celular Tumoral , Ésteres , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Microscopía Confocal , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/metabolismo
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