RESUMO
The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.
Assuntos
Cromatina/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Dano ao DNA , Eucromatina/metabolismo , Fluorescência , Heterocromatina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Lasers , Camundongos , Modelos Biológicos , Concentração Osmolar , FotodegradaçãoRESUMO
Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N3) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.
Assuntos
Corantes Fluorescentes , Imagem Individual de Molécula , Animais , Corantes Fluorescentes/química , Microscopia de Fluorescência , Imagem Individual de Molécula/métodos , Imagem Óptica , DNA , MamíferosRESUMO
Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.
Assuntos
Replicação do DNA , Animais , Camundongos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA/genética , Replicação do DNA/fisiologia , Epigênese Genética/fisiologia , Genoma/genética , Microscopia/métodosRESUMO
This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Heterocromatina/metabolismo , Humanos , Poro Nuclear/metabolismo , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
In recent years, our perception of chromatin structure and organization in the cell nucleus has changed in fundamental ways. The 30 nm chromatin fiber has lost its status as an essential in vivo structure. Hi-C and related biochemical methods, advanced electron and super-resolved fluorescence microscopy, together with concepts from soft matter physics, have revolutionized the field. A comprehensive understanding of the structural and functional interactions that regulate cell cycle and cell type specific nuclear functions appears within reach, but it requires the integration of top-down and bottom-up approachs. In this review, I present an update on nuclear architecture studies with an emphasis on organization and the controversy regarding the physical state of chromatin in cells.
Assuntos
Cromatina/química , Cromatina/metabolismo , Células Eucarióticas , Ciclo Celular , Núcleo Celular , Cromossomos , HumanosRESUMO
Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Histonas/metabolismo , Animais , Linhagem Celular , Dano ao DNA/efeitos da radiação , Reparo do DNA , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Lasers , Camundongos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.
Assuntos
Dano ao DNA/efeitos dos fármacos , Histonas/química , Indanos/química , Complexo Repressor Polycomb 1/antagonistas & inibidores , Piridinas/química , Ubiquitina/metabolismo , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Microscopia de Fluorescência , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.
Assuntos
Antígenos Nucleares/genética , Amplificação de Genes , Glioblastoma/genética , Proteínas Associadas à Matriz Nuclear/genética , Fuso Acromático , Fatores de Transcrição/genética , Apoptose , Northern Blotting , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Imunofluorescência , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Células Tumorais CultivadasRESUMO
Transmission electron microscopy (TEM) has been essential in defining the structural organization of the cell due to its ability to image cell structures at molecular resolution. However, the absence of colour has made it very difficult to compare the distributions and relationships of two or more types of biomolecules simultaneously if they lack clear morphological distinctions. Furthermore, single-channel information limits functional analysis, particularly in the nucleoplasm, where fibrillar material could be chromatin, ribonucleic acid or protein. Where specific stains exist to discriminate among these molecules, they cannot be combined because conventional TEM is a single-channel technology. A potential path around this barrier is through electron spectroscopic imaging (ESI). ESI can map the distributions of chemical elements within an ultrathin section. Here, we present methods to stain specific molecules with elements that ESI can visualize to enable multichannel electron microscopy.
Assuntos
Núcleo Celular , Cromatina , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Coloração e RotulagemRESUMO
Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (â¼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/µm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at â¼20 nm lateral and â¼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC.
Assuntos
Cromatina , DNA , Humanos , Animais , Camundongos , DNA/genética , Núcleo Celular/genética , Cromossomos , Microscopia de Fluorescência/métodosRESUMO
Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.
Assuntos
Cromossomos/efeitos da radiação , Dano ao DNA/efeitos da radiação , Inativação Gênica/efeitos da radiação , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Feminino , Imunofluorescência , Raios gama/efeitos adversos , Histonas/genética , Humanos , Metilação/efeitos da radiação , Microscopia Confocal , Osteossarcoma/genética , Osteossarcoma/patologia , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Chromatin is thought to regulate the accessibility of the underlying DNA sequence to machinery that transcribes and repairs the DNA. Heterochromatin is chromatin that maintains a sufficiently high density of DNA packing to be visible by light microscopy throughout the cell cycle and is thought to be most restrictive to transcription. Several studies have suggested that larger proteins and protein complexes are attenuated in their access to heterochromatin. In addition, heterochromatin domains may be associated with phase separated liquid condensates adding further complexity to the regulation of protein concentration within chromocenters. This provides a solvent environment distinct from the nucleoplasm, and proteins that are not size restricted in accessing this liquid environment may partition between the nucleoplasm and heterochromatin based on relative solubility. In this study, we assessed the accessibility of constitutive heterochromatin in mouse cells, which is organized into large and easily identifiable chromocenters, to fluorescently tagged DNA damage response proteins. We find that proteins larger than the expected 10 nm size limit can access the interior of heterochromatin. We find that the sensor proteins Ku70 and PARP1 enrich in mouse chromocenters. At the same time, MRE11 shows variability within an asynchronous population that ranges from depleted to enriched but is primarily homogeneously distribution between chromocenters and the nucleoplasm. While larger downstream proteins such as ATM, BRCA1, and 53BP1 are commonly depleted in chromocenters, they show a wide range of concentrations, with none being depleted beyond approximately 75%. Contradicting exclusively size-dependent accessibility, many smaller proteins, including EGFP, are also depleted in chromocenters. Our results are consistent with minimal size-dependent selectivity but a distinct solvent environment explaining reduced concentrations of diffusing nucleoplasmic proteins within the volume of the chromocenter.
RESUMO
Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored.
Assuntos
Núcleo Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/fisiologia , Antioxidantes/farmacologia , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Histonas/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Piperazinas/metabolismo , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Transcrição Gênica , Inativação do Cromossomo XRESUMO
Ring1-YY1-binding protein (RYBP) is a member of the non-canonical polycomb repressive complex 1 (PRC1), and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF) domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs), we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR) repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP) inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding.
Assuntos
Reparo do DNA/genética , Recombinação Homóloga/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas RepressorasRESUMO
The identification of a vast array of posttranslational modifications of histone proteins during cell cycle, repair, replication, and transcription has created the challenge of determining structure-function relationships for individual modifications and combinations of modifications. Some of this information can be gathered from indirect immunofluorescence, where the location and cell cycle relationships can be readily identified. Here we present an immunofluorescence protocol that is adapted for the use in histone modifications.
Assuntos
Imunofluorescência/métodos , Histonas/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Código das Histonas , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Ideally, disease modeling using patient-derived induced pluripotent stem cells (iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome-wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient-derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages.
Assuntos
Reprogramação Celular , Epigênese Genética , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lamina Tipo A/genética , Progéria/genética , Sequência de Bases , Estudos de Casos e Controles , Diferenciação Celular , Senescência Celular , Fibroblastos/patologia , Perfilação da Expressão Gênica , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Cariótipo , Lamina Tipo A/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Cultura Primária de Células , Progéria/metabolismo , Progéria/patologiaRESUMO
The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 µm, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.
Assuntos
Reparo do DNA , Microscopia Eletrônica de Transmissão por Filtração de Energia/métodos , Proteínas/análise , Oxigênio Singlete/química , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Humanos , Microscopia Eletrônica de TransmissãoRESUMO
Recent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC). The INC comprises the compact, transcriptionally inactive core of chromatin domain clusters (CDCs). The ANC is formed by the transcriptionally active periphery of CDCs, called the perichromatin region (PR), and the interchromatin compartment (IC). The IC is connected to nuclear pores and serves nuclear import and export functions. The ANC is the major site of RNA synthesis. It is highly enriched in epigenetic marks for transcriptionally competent chromatin and RNA Polymerase II. Marks for silent chromatin are enriched in the INC. Multi-scale cross-correlation spectroscopy suggests that nuclear architecture resembles a random obstacle network for diffusing proteins. An increased dwell time of proteins and protein complexes within the ANC may help to limit genome scanning by factors or factor complexes to DNA exposed within the ANC.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/fisiologia , Animais , Núcleo Celular/fisiologia , Cromatina/ultraestrutura , Reparo do DNA , Regulação da Expressão Gênica , Humanos , Transcrição GênicaRESUMO
DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , DNA de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA de Neoplasias/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Autoantígeno Ku , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Microscopia Confocal , Mutação , Ligação Proteica , Interferência de RNA , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.