RESUMEN
The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells.
Asunto(s)
Imagen Individual de Molécula/métodos , Análisis de la Célula Individual/métodos , Expresión Génica/genética , Hibridación Fluorescente in Situ/métodos , Biosíntesis de Proteínas/genética , ARN/genética , ARN Mensajero/metabolismo , Transcriptoma/genéticaRESUMEN
In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.
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Núcleo Celular/virología , Genoma Viral/genética , VIH-1/genética , Macrófagos/virología , Transcripción Reversa/genética , Transporte Activo de Núcleo Celular/genética , Línea Celular , Análisis por Conglomerados , Citoplasma/virología , ADN Viral/genética , Células HEK293 , Infecciones por VIH/virología , Humanos , ARN Viral/genética , Células THP-1 , Replicación Viral/genéticaRESUMEN
BACKGROUND & AIMS: As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions, including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially resolved multimodal single-cell approach. METHODS: We applied single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and mass cytometry to profile the immune compartment in treatment-naïve PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma on single-cell level. RESULTS: We provided a spatially resolved fine map of the immune landscape in PDAC. We substantiated the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlighted immune subsets with potentially underappreciated roles in PDAC that diverged from immune populations within adjacent normal areas, particularly CD4 T cell subsets and natural killer T cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and lung adenocarcinoma revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. CONCLUSIONS: Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and lung adenocarcinoma, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.
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Adenocarcinoma del Pulmón , Carcinoma Ductal Pancreático , Enfermedades del Sistema Inmune , Neoplasias Pancreáticas , Humanos , Multiómica , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/tratamiento farmacológico , Análisis de la Célula Individual , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.
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ARN , Imagen Individual de Molécula , Hibridación Fluorescente in Situ/métodos , Nanotecnología , ARN/análisis , ARN/genética , ARN Mensajero/genética , Imagen Individual de Molécula/métodosRESUMEN
Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3'UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.
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Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.
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Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Extensiones de la Superficie Celular/metabolismo , Citoplasma/metabolismo , Cinesinas/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Células HeLa , Humanos , Cinesinas/genética , ARN Mensajero/genéticaRESUMEN
Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. In particular, for the HIV-1 genome to enter the nucleus, it must be led through the nuclear pore complex (NPC). During the HIV-1 cytoplasmic journey, the viral core acts as a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core is reshaped into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated with the preintegration complex (PIC) during and after nuclear translocation is still enigmatic. In this study, we analyzed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immunogold labeling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables the specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby offering the opportunity to uncover the architecture of the potential HIV-1 PIC. Thus, we combined the immunoelectron microscopy and ANCHOR technologies to reveal the presence of DNA- and CA-positive complexes by correlated light and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodeled in a pearl necklace-like shape. Thus, we could describe how CA proteins are reshaped around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed of the integrase and the retrotranscribed DNA leads the HIV-1 genome inside the host nucleus. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. Of note, we are now able to visualize the viral DNA in viral complexes, opening up new perspectives for future studies on virus's fate in the cell nucleus.IMPORTANCE How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of â¼100 nm, largely exceeding that of the NPC channel (â¼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.
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Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/metabolismo , Integrasa de VIH/metabolismo , VIH-1/metabolismo , Complejos Multiproteicos/metabolismo , Integración Viral , Transporte Activo de Núcleo Celular , Linfocitos T CD4-Positivos/virología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HEK293 , Infecciones por VIH/genética , Integrasa de VIH/genética , VIH-1/genética , Células HeLa , Humanos , Complejos Multiproteicos/genéticaRESUMEN
Literature on the air travel activities of patients supported by permanent mechanical assist devices is rare. To the best of our knowledge, no air travel guidelines or fitness prerequisites exist on whether and when ventricular assist device (VAD) patients are allowed to travel by plane after device implantation. In this study, we evaluated the topic of air travel after VAD implantation. This working group aimed to produce a report on air travel passengers supported by VADs, regarding their fitness to fly. Fifty left ventricular assist device (LVAD) patients were surveyed in a worldwide multicenter study. The single survey was performed with a multimethod design, including interviews conducted face-to-face, online, and on phone. Out of 50 patients, 97% described their traveling by aircraft as perfect and uneventful during the flight. Eighty-five percent of the study participants consulted their medical practitioner before the flight. No patient reported the occurrence of a severe condition associated with flying. LVAD alarms, especially low flow alarms, did not occur in any of the devices. Thirty-five percent of the surveyed patients, however, stated a major problem pertaining to the security check procedures at the airport. The results of this study suggest that commercial air travel is safe for stable patients on permanent VAD support and traveling can be resumed securely after VAD implantation. Conscientious preparation by packing necessary devices, fluids, medications, and careful preparation for the airport security check is recommended.
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Viaje en Avión/estadística & datos numéricos , Insuficiencia Cardíaca/cirugía , Corazón Auxiliar/normas , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Guías como Asunto , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Encuestas y Cuestionarios/estadística & datos numéricos , Resultado del Tratamiento , Adulto JovenRESUMEN
Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required â¼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.
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Hibridación Fluorescente in Situ/métodos , Virus de la Coriomeningitis Linfocítica/genética , ARN Viral/genética , Células A549 , Línea Celular , Genoma Viral/genética , Humanos , Sondas ARN/genética , Coloración y Etiquetado/métodos , Replicación Viral/genéticaRESUMEN
Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor occupancy, but give no indication of kinetics and are poor predictors of transcription factor function at a given locus. To measure transcription-factor-binding dynamics across the genome, we performed competition ChIP (refs 6, 7) with a sequence-specific Saccharomyces cerevisiae transcription factor, Rap1 (ref. 8). Rap1-binding dynamics and Rap1 occupancy were only weakly correlated (R(2) = 0.14), but binding dynamics were more strongly linked to function than occupancy. Long Rap1 residence was coupled to transcriptional activation, whereas fast binding turnover, which we refer to as 'treadmilling', was linked to low transcriptional output. Thus, DNA-binding events that seem identical by conventional ChIP may have different underlying modes of interaction that lead to opposing functional outcomes. We propose that transcription factor binding turnover is a major point of regulation in determining the functional consequences of transcription factor binding, and is mediated mainly by control of competition between transcription factors and nucleosomes. Our model predicts a clutch-like mechanism that rapidly engages a treadmilling transcription factor into a stable binding state, or vice versa, to modulate transcription factor function.
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ADN de Hongos/metabolismo , Genoma Fúngico , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Inmunoprecipitación de Cromatina , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Unión Proteica , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/clasificación , Complejo Shelterina , Factores de TiempoRESUMEN
Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.
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ARN Mensajero/biosíntesis , Animales , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Límite de Detección , Ratones , Microscopía Fluorescente , Células Madre Embrionarias de Ratones , ARN Mensajero/genéticaRESUMEN
In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.
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Cromatina/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Imagen Molecular/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/genética , Histonas/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Transcription factor (TF) recruitment to chromatin is central to activation of transcription. TF-chromatin interactions are highly dynamic, which are evaluated by recovery half time (t1/2) in seconds, determined by fluorescence recovery experiments in living cells, and chromatin immunoprecipitation (ChIP) analysis, measured in minutes. These two states are related: the larger the t1/2, the longer the ChIP occupancy resulting in increased transcription. Here we present data showing that this relationship does not always hold. We found that histone deacetylase inhibitors (HDACis) significantly increased t1/2 of green fluorescent protein (GFP) fused androgen receptor (AR) on a tandem array of positive hormone response elements (HREs) in chromatin. This resulted in increased ChIP signal of GFP-AR. Unexpectedly, however, transcription was inhibited. In contrast, the GFP-fused glucocorticoid receptor (GR), acting through the same HREs, displayed a profile consistent with current models. We provide evidence that these differences are mediated, at least in part, by HDACs. Our results provide insight into TF action in living cells and show that very closely related TFs may trigger significantly divergent outcomes at the same REs.
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Adenocarcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta/genética , Activación Transcripcional/genética , Adenocarcinoma/genética , Anciano , Animales , Inmunoprecipitación de Cromatina , Femenino , Técnica del Anticuerpo Fluorescente , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/química , Humanos , Neoplasias Mamarias Animales/genética , Ratones , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Androgénicos/genética , Receptores de Glucocorticoides/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.
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Caenorhabditis elegans/citología , Rastreo Celular , Imagenología Tridimensional/métodos , Microscopía Fluorescente , Neuronas/citología , Saccharomyces cerevisiae/citología , Animales , Neoplasias Óseas/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Osteosarcoma/enzimología , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.
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VIH-1/metabolismo , Carioferinas/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Empalme Alternativo/fisiología , Sitios de Unión , Células Cultivadas , VIH-1/genética , Humanos , Complejos Multiproteicos/genética , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , ARN Viral/genética , ARN Viral/metabolismo , Proteína Exportina 1RESUMEN
Transcription factors (TFs) interact dynamically in vivo with chromatin binding sites. Here we summarize and compare the four different techniques that are currently used to measure these kinetics in live cells, namely fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single molecule tracking (SMT) and competition ChIP (CC). We highlight the principles underlying each of these approaches as well as their advantages and disadvantages. A comparison of data from each of these techniques raises an important question: do measured transcription kinetics reflect biologically functional interactions at specific sites (i.e. working TFs) or do they reflect non-specific interactions (i.e. playing TFs)? To help resolve this dilemma we discuss five key unresolved biological questions related to the functionality of transient and prolonged binding events at both specific promoter response elements as well as non-specific sites. In support of functionality, we review data suggesting that TF residence times are tightly regulated, and that this regulation modulates transcriptional output at single genes. We argue that in addition to this site-specific regulatory role, TF residence times also determine the fraction of promoter targets occupied within a cell thereby impacting the functional status of cellular gene networks. Thus, TF residence times are key parameters that could influence transcription in multiple ways.
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Factores de Transcripción/metabolismo , Animales , Humanos , Cinética , Regiones Promotoras Genéticas/genética , Unión ProteicaRESUMEN
BACKGROUND INFORMATION: Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined. RESULTS: Fluorescently tagged PcG proteins CBXs and their partners BMI1 and RING1 form foci of different sizes and intensities in human U2OS cells. Fluorescence recovery after photobleaching (FRAP) analysis reveals that PcG dynamics outside foci is governed by diffusion as complexes and transient binding. In sharp contrast, recovery curves inside foci are substantially slower and exhibit large variability. The slow binding component amplitudes correlate with the intensities and sizes of these foci, suggesting that foci contained varying numbers of binding sites. CBX4-green fluorescent protein (GFP) foci were more stable than CBX8-GFP foci; yet the presence of CBX4 or CBX8-GFP in the same focus had a minor impact on BMI1 and RING1 recovery kinetics. CONCLUSION: We propose that FRAP recovery curves provide information about PcG binding to their target genes outside foci and about PcG spreading onto chromatin inside foci.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Grupo Polycomb/química , Proteínas del Grupo Polycomb/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Humanos , CinéticaRESUMEN
The linker histone H1 has a fundamental role in DNA compaction. Although models for H1 binding generally involve the H1 C-terminal tail and sites S1 and S2 within the H1 globular domain, there is debate about the importance of these binding regions and almost nothing is known about how they work together. Using a novel fluorescence recovery after photobleaching (FRAP) procedure, we have measured the affinities of these regions individually, in pairs, and in the full molecule to demonstrate for the first time that binding among several combinations is cooperative in live cells. Our analysis reveals two preferred H1 binding pathways and we find evidence for a novel conformational change required by both. These results paint a complex, highly dynamic picture of H1-chromatin binding, with a significant fraction of H1 molecules only partially bound in metastable states that can be readily competed against. We anticipate the methods we have developed here will be broadly applicable, particularly for deciphering the binding kinetics of other nuclear proteins that, similar to H1, interact with and modify chromatin.