RESUMO
Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.
Assuntos
Proteínas de Ciclo Celular , Cromátides , Coesinas , Troca de Cromátide Irmã , Humanos , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina/metabolismo , Coesinas/metabolismo , DNA/genética , DNA/metabolismo , Linhagem Celular TumoralRESUMO
Transcription must be tightly controlled to regulate gene expression and development. However, our understanding of the molecular mechanisms that influence transcription and how these are coordinated in cells to ensure normal gene expression remains rudimentary. Here, by dissecting the function of the SET1 chromatin-modifying complexes that bind to CpG island-associated gene promoters, we discover that they play a specific and essential role in enabling the expression of low to moderately transcribed genes. Counterintuitively, this effect can occur independently of SET1 complex histone-modifying activity and instead relies on an interaction with the RNA Polymerase II-binding protein WDR82. Unexpectedly, we discover that SET1 complexes enable gene expression by antagonising premature transcription termination by the ZC3H4/WDR82 complex at CpG island-associated genes. In contrast, at extragenic sites of transcription, which typically lack CpG islands and SET1 complex occupancy, we show that the activity of ZC3H4/WDR82 is unopposed. Therefore, we reveal a gene regulatory mechanism whereby CpG islands are bound by a protein complex that specifically protects genic transcripts from premature termination, effectively distinguishing genic from extragenic transcription and enabling normal gene expression.
Assuntos
Histonas , Transcrição Gênica , Ilhas de CpG/genética , Histonas/metabolismo , Cromatina/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Metilação de DNA/genéticaRESUMO
The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The inter-relatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics, we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.
Assuntos
Histonas/genética , Complexo Repressor Polycomb 1/genética , Iniciação da Transcrição Genética , Animais , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Histonas/metabolismo , Lisina/genética , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Polimerase II/metabolismo , Análise de Célula ÚnicaRESUMO
How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Cromatina/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , CoesinasRESUMO
DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.
Assuntos
Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Simples , Replicação do DNA , Proteína da Leucemia Promielocítica/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Células Cultivadas , Reparo do DNA , Células HeLa , Humanos , Complexo de Reconhecimento de Origem/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Domínios ProteicosRESUMO
Structured Illumination Microscopy (SIM) is a super-resolution microscopy method that has significantly advanced studies of cellular structures. It relies on projection of illumination patterns onto a fluorescently labelled biological sample. The information derived from the sample is then shifted to a detectable band, and in the process of image calculation in Fourier space the resolution is doubled. Refractive index homogeneity along the optical path is crucial to maintain a highly modulated illumination pattern necessary for high-quality SIM. This applies in particular to thick samples consisting of large cells and tissues. Surprisingly, sample mounting media for SIM have not undergone a significant evolution for almost a decade. Through identification and systematic evaluation of a number of non-hazardous, water-soluble chemical components of mounting media, we demonstrate an unprecedented improvement in SIM-image quality. Mounting solutions presented in this research are capable of reducing abundant light scattering which constitutes the limiting factor in 3D-SIM imaging of large Hodgkin's lymphoma and embryonic stem cells as well as 10 µm tissue sections. Moreover, we demonstrate usefulness of some of the media in single molecule localisation microscopy. The results presented here are of importance for standardisation of 3D-SIM data acquisition pipelines for an expanding community of users.
Assuntos
Doença de Hodgkin/patologia , Processamento de Imagem Assistida por Computador/métodos , Iluminação/instrumentação , Linfócitos/ultraestrutura , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Baço/ultraestrutura , Animais , Humanos , Imageamento Tridimensional/métodos , Camundongos , Melhoria de Qualidade , Células Tumorais CultivadasRESUMO
Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.
Assuntos
Núcleo Celular/química , DNA/química , Conformação de Ácido Nucleico , Imagem Individual de Molécula , Sítios de Ligação , Núcleo Celular/metabolismo , DNA/metabolismoRESUMO
The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.
Assuntos
Carbocianinas/química , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência/instrumentação , Mioblastos/metabolismo , Mioblastos/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions.
Assuntos
Cromatina/ultraestrutura , DNA/ultraestrutura , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Ácido Ascórbico/farmacologia , Benzoxazóis/química , Sítios de Ligação , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/metabolismo , Glucose/deficiência , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Concentração de Íons de Hidrogênio , Substâncias Intercalantes/química , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Compostos de Quinolínio/químicaRESUMO
Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50-100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field.
RESUMO
Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Zurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.
RESUMO
Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.
Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Microscopia Confocal/métodos , Raios Ultravioleta , Bromodesoxiuridina/metabolismo , Núcleo Celular/efeitos da radiação , Células Cultivadas , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Fluorescência , Humanos , Desnaturação de Ácido Nucleico/efeitos da radiação , Frações Subcelulares/metabolismo , Frações Subcelulares/efeitos da radiação , Fatores de TempoRESUMO
Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy.
Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência/métodos , Nanoestruturas/química , Imagem Individual de Molécula/métodos , Animais , Chlorocebus aethiops , Cromossomos/metabolismo , Drosophila melanogaster , Células VeroRESUMO
BACKGROUND: Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones. RESULTS: Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. CONCLUSIONS: These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Isquemia/genética , Hipóxia Celular/genética , Linhagem Celular , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/genética , Recuperação de Fluorescência Após Fotodegradação , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histonas/genética , Histonas/metabolismo , Humanos , Isquemia/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ligação Proteica , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until "bleached". This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.
Assuntos
Cromatina/metabolismo , Corantes Fluorescentes , Microscopia/métodos , Animais , Cromatina/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Humanos , Microscopia de FluorescênciaRESUMO
Heterochromatin protein 1 (HP1), a small non-histone chromosomal protein, was recently shown to form a complex in vivo with Proliferating Cell Nuclear Antigen (PCNA), a key factor in DNA replication. The complex, which requires HP1ß in a form of a dimer, is engaged in DNA repair and replication. We now provide further evidence based on FRET-FLIM live cell studies confirming the association and close proximity between HP1ß and PCNA in the complex. We also demonstrate using FRAP, that although HP1ß-PCNA complexes are highly mobile in nonreplicating nuclei, when engaged in DNA replication, they become bound and do not exchange with the mobile pool. These observations are in agreement with a notion that a subpopulation of HP1 molecules interact with PCNA in vivo during DNA replication. Similarly, HP1ß which is associated with PCNA in regions of DNA repair, is bound and does not exchange with the mobile pool, suggesting that HP1ß in association with PCNA may be a component of a DNA repair complex.