Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Cell ; 187(2): 331-344.e17, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38194964

RESUMO

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição SOXB1 , Super Intensificadores , Transcrição Gênica , DNA/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição SOXB1/genética , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Microscopia/métodos
3.
Mol Cell ; 79(6): 881-901, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32768408

RESUMO

Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of chromatin underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects, which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology and to test concepts such as phase separation of chromatin.


Assuntos
Cromatina/genética , Cromossomos/genética , DNA/genética , Nucleossomos/genética , Reparo do DNA/genética , Replicação do DNA/genética , Epigênese Genética/genética , Humanos
4.
Mol Cell ; 76(5): 753-766.e6, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31563432

RESUMO

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.


Assuntos
Elementos Facilitadores Genéticos/genética , Complexo Mediador/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo Mediador/fisiologia , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Proteínas da Superfamília de TGF-beta/metabolismo , Transcrição Gênica , Via de Sinalização Wnt , beta Catenina/metabolismo
5.
Nature ; 572(7770): 543-548, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391587

RESUMO

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex1-4. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7-12. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9-12. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.


Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Splicing de RNA , Transcrição Gênica , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Complexo Mediador/genética , Camundongos , Fosforilação , Domínios Proteicos , RNA Polimerase II/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
6.
Methods ; 153: 35-45, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30217531

RESUMO

The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Embrionárias Murinas/metabolismo , RNA Mensageiro/análise , Imagem Individual de Molécula/métodos , Animais , Células Cultivadas , Camundongos , RNA Mensageiro/química , RNA Mensageiro/metabolismo
7.
PLoS Genet ; 13(5): e1006783, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28489851

RESUMO

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.


Assuntos
Proteínas de Bactérias/genética , Quebras de DNA de Cadeia Dupla , Exodesoxirribonucleases/genética , Genoma Bacteriano , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo
8.
J Cell Sci ; 128(20): 3695-706, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26416818

RESUMO

RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.


Assuntos
Imagem Molecular/métodos , Poro Nuclear/metabolismo , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Humanos
9.
Development ; 141(3): 661-73, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24449842

RESUMO

The proper functioning of the dopaminergic system requires the coordinated formation of projections extending from dopaminergic neurons in the substantia nigra (SN), ventral tegmental area (VTA) and retrorubral field to a wide array of forebrain targets including the striatum, nucleus accumbens and prefrontal cortex. The mechanisms controlling the assembly of these distinct dopaminergic cell clusters are not well understood. Here, we have investigated in detail the migratory behavior of dopaminergic neurons giving rise to either the SN or the medial VTA using genetic inducible fate mapping, ultramicroscopy, time-lapse imaging, slice culture and analysis of mouse mutants. We demonstrate that neurons destined for the SN migrate first radially and then tangentially, whereas neurons destined for the medial VTA undergo primarily radial migration. We show that tangentially migrating dopaminergic neurons express the components of the reelin signaling pathway, whereas dopaminergic neurons in their initial, radial migration phase express CXC chemokine receptor 4 (CXCR4), the receptor for the chemokine CXC motif ligand 12 (CXCL12). Perturbation of reelin signaling interferes with the speed and orientation of tangentially, but not radially, migrating dopaminergic neurons and results in severe defects in the formation of the SN. By contrast, CXCR4/CXCL12 signaling modulates the initial migration of dopaminergic neurons. With this study, we provide the first molecular and functional characterization of the distinct migratory pathways taken by dopaminergic neurons destined for SN and VTA, and uncover mechanisms that regulate different migratory behaviors of dopaminergic neurons.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Quimiocina CXCL12/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem da Célula , Desenvolvimento Embrionário , Ligantes , Camundongos , Camundongos Knockout , Modelos Biológicos , Receptores CXCR4/metabolismo , Proteína Reelina , Transdução de Sinais , Substância Negra/citologia , Imagem com Lapso de Tempo , Área Tegmentar Ventral/citologia
10.
Nucleic Acids Res ; 43(2): e14, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25414330

RESUMO

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1-3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.


Assuntos
Microscopia de Fluorescência/métodos , RNA/análise , Algoritmos , Animais , Chironomidae , Puffs Cromossômicos , Corantes Fluorescentes , Membrana Nuclear/química , Fótons , RNA Mensageiro/análise , RNA Ribossômico 28S/análise , Lipossomas Unilamelares/química
11.
J Immunol ; 193(7): 3257-61, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25187660

RESUMO

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.


Assuntos
Complexo 1 de Proteínas Adaptadoras/imunologia , Complexo 2 de Proteínas Adaptadoras/imunologia , Linfócitos B/imunologia , Proteínas de Membrana Transportadoras/imunologia , Monócitos/imunologia , Receptores Toll-Like/imunologia , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Linfócitos B/citologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Monócitos/citologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Receptores Toll-Like/genética
12.
Biophys J ; 108(5): 1114-24, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25762323

RESUMO

The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Nisina/farmacologia , Lipossomas Unilamelares/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Lipossomas Unilamelares/química
13.
Methods Mol Biol ; 2563: 425-445, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227487

RESUMO

Fluorescence microscopy assays enable the investigation of endogenous biomolecular condensates directly in their cellular context. With appropriate experimental designs, these assays yield quantitative information on condensate material properties and inform on biophysical mechanisms of condensate formation. Single-molecule super-resolution and tracking experiments grant access to the smallest condensates and early condensation stages not resolved by conventional imaging approaches. Here, we discuss considerations for using single-molecule assays to extract quantitative information about biomolecular condensates directly in their cellular context.


Assuntos
Condensados Biomoleculares , Microscopia de Fluorescência
14.
Opt Express ; 20(18): 19697-707, 2012 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-23037022

RESUMO

Three-dimensional (3D) spatial information can be encoded in two-dimensional images of fluorescent nanoparticles by astigmatic imaging. We combined this method with light sheet microscopy for high contrast single particle imaging up to 200 µm deep within living tissue and real-time image analysis to determine 3D particle localizations with nanometer precision and millisecond temporal resolution. Axial information was instantly directed to the sample stage to keep a moving particle within the focal plane in an active feedback loop. We demonstrated 3D tracking of nanoparticles at an unprecedented depth throughout large cell nuclei over several thousand frames and a range of more than 10 µm in each spatial dimension, while simultaneously acquiring optically sectioned wide field images. We conclude that this 3D particle tracking technique employing light sheet microscopy presents a valuable extension to the nanoscopy toolbox.


Assuntos
Aumento da Imagem/instrumentação , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Nanopartículas/ultraestrutura , Desenho de Equipamento , Análise de Falha de Equipamento
15.
Biophys J ; 100(4): 1139-48, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21320460

RESUMO

The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α(2A) adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ∼50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557-573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113) and revealed a discrepancy of a factor between 10 and 100.


Assuntos
Arsenicais/metabolismo , Difusão , Fluoresceína/metabolismo , Fluoresceínas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Rotação , Coloração e Rotulagem , Animais , Anisotropia , Membrana Celular/metabolismo , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Camundongos , Modelos Moleculares , Fatores de Tempo
16.
Front Immunol ; 12: 682589, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084176

RESUMO

Vast repertoires of unique antigen receptors are created in developing B and T lymphocytes. The antigen receptor loci contain many variable (V), diversity (D) and joining (J) gene segments that are arrayed across very large genomic expanses and are joined to form variable-region exons of expressed immunoglobulins and T cell receptors. This process creates the potential for an organism to respond to large numbers of different pathogens. Here, we consider the possibility that genetic polymorphisms with alterations in a vast array of regulatory elements in the immunoglobulin heavy chain (IgH) locus lead to changes in locus topology and impact immune-repertoire formation.


Assuntos
Cromatina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Polimorfismo Genético , Hipermutação Somática de Imunoglobulina , Recombinação V(D)J , Animais , Elementos Facilitadores Genéticos , Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Loci Gênicos , Humanos , Regiões Promotoras Genéticas , Transcrição Gênica
17.
Elife ; 82019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30716021

RESUMO

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Assuntos
Agregados Proteicos/genética , Agregação Patológica de Proteínas/genética , Dobramento de Proteína , Linhagem Celular Tumoral , Humanos , Cinética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Transição de Fase , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas/patologia
18.
Science ; 361(6400): 412-415, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29930094

RESUMO

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.


Assuntos
Regulação da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Células Cultivadas , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Complexo Mediador/análise , Complexo Mediador/química , Camundongos , Imagem Molecular/métodos , RNA Polimerase II/análise , RNA Polimerase II/química
19.
Elife ; 52016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-27138339

RESUMO

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous ß-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.


Assuntos
Actinas/genética , Loci Gênicos , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Animais , Células Cultivadas , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Camundongos
20.
Methods Mol Biol ; 1042: 73-85, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23980001

RESUMO

Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the processing of RNA and the formation and fate of messenger ribonucleoprotein particles (mRNPs). For a long time, challenging imaging conditions limited the access to this system for in vivo fluorescence microscopy. Recent technical and methodical advantages now allow observing even single molecules in these cells. We describe here the experimental approach and the optical techniques required to analyze intranuclear trafficking and export of single native mRNPs across the nuclear envelope.


Assuntos
Chironomidae/metabolismo , Microscopia de Fluorescência/métodos , Transporte Proteico , Ribonucleoproteínas/metabolismo , Glândulas Salivares/metabolismo , Animais , Núcleo Celular/metabolismo , Chironomidae/citologia , Chironomidae/genética , Corantes Fluorescentes , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Microinjeções , Glândulas Salivares/citologia , Coloração e Rotulagem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA