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1.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
2.
Biophys J ; 120(7): 1288-1300, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33640380

RESUMO

Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage λ-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1-10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.


Assuntos
DNA , Proteínas , Cátions , DNA/genética , Humanos , Substâncias Macromoleculares
3.
Methods Mol Biol ; 2173: 171-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32651918

RESUMO

Blue light-induced chromatin recruitment (BLInCR) is a versatile optogenetic tool to enrich effector proteins at specific loci within the nucleus using illumination in the 400-500 nm range. The resulting chromatin binding reaction is reversible on the time scale of minutes. BLInCR is advantageous over ligand-binding induced methods since it does not require a change of growth medium for the relatively slow depletion of the inducer from the nucleus. However, applying BLInCR for reversibility experiments is challenging because of the need to spectrally separate light-induced activation from visualization of the chromatin locus and effector and/or reader proteins by light microscopy. Here, we describe an improved BLInCR protocol for light-dependent association and dissociation of effectors using the near-infrared fluorescent protein iRFP713. Due to its spectral properties, iRFP713 can be detected separately from the red fluorescent protein mCherry. Thus, it becomes possible to trace two proteins labeled with iRFP713 and mCherry independently of the light activation reaction. This approach largely facilitates applications of the BLInCR system for experiments that test the reversibility, persistence, and memory of chromatin states.


Assuntos
Cromatina/metabolismo , Luz , Optogenética/métodos , Humanos , Software , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
4.
Curr Opin Genet Dev ; 61: 62-68, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32408103

RESUMO

Cells compartmentalize their genome into active and inactive parts that contain distinct histone marks and are differently packaged. Recent work has shed new light on the mechanisms that drive this type of chromatin patterning and the properties of the resulting domains. Biophysical concepts such as liquid-liquid phase separation, polymer looping and collapse, molecular crowding and viscoelasticity are increasingly used to describe experimental observations. Accordingly, it is becoming clear that the physicochemical properties of the nuclear interior are relevant for understanding chromatin compartmentalization. Here, I discuss recent insights into the properties of chromatin subcompartments obtained with complementary techniques on different scales, and relate them to models for functional chromatin patterning.


Assuntos
Fenômenos Biofísicos , Compartimento Celular/genética , Núcleo Celular/genética , Cromatina/genética , Animais , Genoma/genética , Humanos
5.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
6.
Elife ; 92020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31995034

RESUMO

DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.


Assuntos
Quebras de DNA de Cadeia Dupla , Sirtuínas , Linhagem Celular , Reparo do DNA , Células HeLa , Humanos , Ligação Proteica , Transdução de Sinais/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Sirtuínas/fisiologia
7.
Methods Mol Biol ; 2038: 251-270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31407290

RESUMO

Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.


Assuntos
Luz , Microscopia Confocal , Microscopia de Fluorescência , Optogenética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Fatores de Tempo , Fatores de Transcrição/genética
8.
Mol Syst Biol ; 15(5): e8339, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118277

RESUMO

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B-cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease.


Assuntos
Cromatina/química , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Histonas/química , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Motivos de Aminoácidos , Sítios de Ligação , Fator de Ligação a CCCTC/genética , DNA/química , Metilação de DNA , Regulação para Baixo , Elementos Facilitadores Genéticos , Histona Desacetilases/genética , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genética
9.
Biophys J ; 114(10): 2262-2270, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29628210

RESUMO

Chromatin is partitioned on multiple length scales into subcompartments that differ from each other with respect to their molecular composition and biological function. It is a key question how these compartments can form even though diffusion constantly mixes the nuclear interior and rapidly balances concentration gradients of soluble nuclear components. Different biophysical concepts are currently used to explain the formation of "chromatin bodies" in a self-organizing manner and without consuming energy. They rationalize how soluble protein factors that are dissolved in the liquid nuclear phase, the nucleoplasm, bind and organize transcriptionally active or silenced chromatin domains. In addition to cooperative binding of proteins to a preformed chromatin structure, two different mechanisms for the formation of phase-separated chromatin subcompartments have been proposed. One is based on bridging proteins that cross-link polymer segments with particular properties. Bridging can induce a collapse of the nucleosome chain and associated factors into an ordered globular phase. The other mechanism is based on multivalent interactions among soluble molecules that bind to chromatin. These interactions can induce liquid-liquid phase separation, which drives the assembly of liquid-like nuclear bodies around the respective binding sites on chromatin. Both phase separation mechanisms can explain that chromatin bodies are dynamic spherical structures, which can coalesce and are in constant and rapid exchange with the surrounding nucleoplasm. However, they make distinct predictions about how the size, density, and stability of chromatin bodies depends on the concentration and interaction behavior of the molecules involved. Here, we compare the different biophysical mechanisms for the assembly of chromatin bodies and discuss experimental strategies to distinguish them from each other. Furthermore, we outline the implications for the establishment and memory of functional chromatin state patterns.


Assuntos
Cromatina/química , Cromatina/metabolismo , Modelos Biológicos
10.
J Cell Sci ; 130(24): 4213-4224, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29122982

RESUMO

Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.


Assuntos
Cromatina/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Transcrição Genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Cromatina/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Genes Reporter/genética , Humanos , Cinética , Luz
11.
Bioessays ; 39(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29034500

RESUMO

Nucleosomes "talk" to each other about their modification state to form extended domains of modified histones independently of the underlying DNA sequence. At the same time, DNA elements promote modification of nucleosomes in their vicinity. How do these site-specific and histone-based activities act together to regulate spreading of histone modifications along the genome? How do they enable epigenetic memory to preserve cell identity? Many models for the dynamics of repressive histone modifications emphasize the role of strong positive feedback loops, which reinforce histone modifications by recruiting histone modifiers to preexisting modifications. Recent experiments question that repressive histone modifications are self-sustained independently of their genomic context, thereby indicating that histone-based feedback is relatively weak. In the present review, current models for the dynamics of histone modifications are compared and it is suggested that limitation of histone-based feedback is key to intrinsic confinement of spreading and coexistence of short- and long-term memory at different genomic loci. See also the video abstract here: https://youtu.be/3bxr_xDEZfQ.


Assuntos
Epigênese Genética , Genoma , Histonas/metabolismo , Modelos Genéticos , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Retroalimentação Fisiológica , Código das Histonas , Histonas/genética , Nucleossomos/química , Nucleossomos/ultraestrutura
12.
Nucleic Acids Res ; 45(18): 10534-10554, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977666

RESUMO

CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.


Assuntos
Autoantígenos/metabolismo , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Animais , Linhagem Celular Tumoral , Galinhas , Reparo do DNA , Humanos , Nucleossomos/metabolismo , Transcrição Genética
13.
Cell Rep ; 18(1): 41-53, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28052260

RESUMO

Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled shelterin complex are not well defined. Here, we characterize shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how shelterin recognizes and assembles onto telomeric repeats. We show that shelterin complexes can exist in solution and primarily locate telomeric DNA through a three-dimensional diffusive search. Shelterin can diffuse along non-telomeric DNA but is impeded by nucleosomes, arguing against extensive one-dimensional diffusion as a viable assembly mechanism. Our work supports a model in which individual shelterin complexes rapidly bind to telomeric repeats as independent functional units, which do not alter the DNA-binding mode of neighboring complexes but, rather, occupy telomeric DNA in a "beads on a string" configuration.


Assuntos
Mamíferos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , DNA/metabolismo , Difusão , Células HEK293 , Humanos , Cinética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo
14.
Biophys J ; 112(3): 473-490, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-28131315

RESUMO

Epigenetic modifications and other chromatin features partition the genome on multiple length scales. They define chromatin domains with distinct biological functions that come in sizes ranging from single modified DNA bases to several megabases in the case of heterochromatic histone modifications. Due to chromatin folding, domains that are well separated along the linear nucleosome chain can form long-range interactions in three-dimensional space. It has now become a routine task to map epigenetic marks and chromatin structure by deep sequencing methods. However, assessing and comparing the properties of chromatin domains and their positional relationships across data sets without a priori assumptions remains challenging. Here, we introduce multiscale correlation evaluation (MCORE), which uses the fluctuation spectrum of mapped sequencing reads to quantify and compare chromatin patterns over a broad range of length scales in a model-independent manner. We applied MCORE to map the chromatin landscape in mouse embryonic stem cells and differentiated neural cells. We integrated sequencing data from chromatin immunoprecipitation, RNA expression, DNA methylation, and chromosome conformation capture experiments into network models that reflect the positional relationships among these features on different genomic scales. Furthermore, we used MCORE to compare our experimental data to models for heterochromatin reorganization during differentiation. The application of correlation functions to deep sequencing data complements current evaluation schemes and will support the development of quantitative descriptions of chromatin networks.


Assuntos
Cromatina/genética , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Diferenciação Celular , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Heterocromatina/genética , Histonas/química , Histonas/metabolismo , Camundongos
15.
Proc Natl Acad Sci U S A ; 113(29): E4180-9, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27382173

RESUMO

Histone modifications can redistribute along the genome in a sequence-independent manner, giving rise to chromatin position effects and epigenetic memory. The underlying mechanisms shape the endogenous chromatin landscape and determine its response to ectopically targeted histone modifiers. Here, we simulate linear and looping-driven spreading of histone modifications and compare both models to recent experiments on histone methylation in fission yeast. We find that a generalized nucleation-and-looping mechanism describes key observations on engineered and endogenous methylation domains including intrinsic spatial confinement, independent regulation of domain size and memory, variegation in the absence of antagonists, and coexistence of short- and long-term memory at loci with weak and strong constitutive nucleation. These findings support a straightforward relationship between the biochemical properties of chromatin modifiers and the spatiotemporal modification pattern. The proposed mechanism gives rise to a phase diagram for cellular memory that may be generally applicable to explain epigenetic phenomena across different species.


Assuntos
Epigenômica , Histonas/metabolismo , Modelos Biológicos , Metilação , Domínios Proteicos , Schizosaccharomyces/metabolismo
16.
J Phys Condens Matter ; 27(6): 064115, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25563347

RESUMO

The eukaryotic cell nucleus harbours the DNA genome that is organized in a dynamic chromatin network and embedded in a viscous crowded fluid. This environment directly affects enzymatic reactions and target search processes that access the DNA sequence information. However, its physical properties as a reaction medium are poorly understood. Here, we exploit mobility measurements of differently sized inert green fluorescent tracer proteins to characterize the viscoelastic properties of the nuclear interior of a living human cell. We find that it resembles a viscous fluid on small and large scales but appears viscoelastic on intermediate scales that change with protein size. Our results are consistent with simulations of diffusion through polymers and suggest that chromatin forms a random obstacle network rather than a self-similar structure with fixed fractal dimensions. By calculating how long molecules remember their previous position in dependence on their size, we evaluate how the nuclear environment affects search processes of chromatin targets.


Assuntos
Cromatina/metabolismo , Elasticidade , Movimento , Linhagem Celular , Sobrevivência Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Citoplasma/metabolismo , Difusão , Proteínas de Fluorescência Verde/metabolismo , Humanos , Porosidade , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Viscosidade
17.
Mol Syst Biol ; 10: 746, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25134515

RESUMO

The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.


Assuntos
Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Inativação Gênica , Marcadores Genéticos , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Mitose , Células NIH 3T3 , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade
18.
Nat Commun ; 5: 4494, 2014 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-25058002

RESUMO

In living cells, most proteins diffuse over distances of micrometres within seconds. Protein translocation is constrained due to the cellular organization into subcompartments that impose diffusion barriers and guide enzymatic activities to their targets. Here, we introduce an approach to retrieve structural features from the scale-dependent mobility of green fluorescent protein monomer and multimers in human cells. We measure protein transport simultaneously between hundreds of positions by multi-scale fluorescence cross-correlation spectroscopy using a line-illuminating confocal microscope. From these data we derive a quantitative model of the intracellular architecture that resembles a random obstacle network for diffusing proteins. This topology partitions the cellular content and increases the dwell time of proteins in their local environment. The accessibility of obstacle surfaces depends on protein size. Our method links multi-scale mobility measurements with a quantitative description of intracellular structure that can be applied to evaluate how drug-induced perturbations affect protein transport and interactions.


Assuntos
Proteínas/análise , Proteínas/metabolismo , Espectrometria de Fluorescência/métodos , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Difusão , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Transporte Proteico , Pontos Quânticos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Espectrometria de Fluorescência/instrumentação
19.
Ann N Y Acad Sci ; 1305: 29-43, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24033539

RESUMO

The eukaryotic nucleus harbors the DNA genome, which associates with histones and other chromosomal proteins into a complex referred to as chromatin. It provides an additional layer of so-called epigenetic information via histone modifications and DNA methylation on top of the DNA sequence that determines the cell's active gene expression program. The nucleus is devoid of internal organelles separated by membranes. Thus, free diffusive transport of proteins and RNA can occur throughout the space accessible for a given macromolecule. At the same time, chromatin is partitioned into different specialized structures such as nucleoli, chromosome territories, and heterochromatin domains that serve distinct functions. Here, we address the question of how the activity of chromatin-modifying enzymes is confined to chromatin subcompartments. We discuss mechanisms for establishing activity gradients of diffusive chromatin-modifying enzymes that could give rise to distinct chromatin domains within the cell nucleus. Interestingly, such gradients might directly result from immobilization of the enzymes on the flexible chromatin chain. Thus, locus-specific tethering of these enzymes to chromatin could have the potential to establish, maintain, or modulate epigenetic patterns of characteristic domain size.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Histonas/metabolismo , Animais , DNA/genética , DNA/metabolismo , Epigenômica , Loci Gênicos , Humanos , Nucleossomos/metabolismo
20.
Mol Cell ; 51(4): 454-68, 2013 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-23911928

RESUMO

DNA damage is linked to multiple human diseases, such as cancer, neurodegeneration, and aging. Little is known about the role of chromatin accessibility in DNA repair. Here, we find that the deacetylase sirtuin 6 (SIRT6) is one of the earliest factors recruited to double-strand breaks (DSBs). SIRT6 recruits the chromatin remodeler SNF2H to DSBs and focally deacetylates histone H3K56. Lack of SIRT6 and SNF2H impairs chromatin remodeling, increasing sensitivity to genotoxic damage and recruitment of downstream factors such as 53BP1 and breast cancer 1 (BRCA1). Remarkably, SIRT6-deficient mice exhibit lower levels of chromatin-associated SNF2H in specific tissues, a phenotype accompanied by DNA damage. We demonstrate that SIRT6 is critical for recruitment of a chromatin remodeler as an early step in the DNA damage response, indicating that proper unfolding of chromatin plays a rate-limiting role. We present a unique crosstalk between a histone modifier and a chromatin remodeler, regulating a coordinated response to prevent DNA damage.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Instabilidade Genômica , Sirtuínas/metabolismo , Sirtuínas/fisiologia , Adenosina Trifosfatases/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Hipocampo/citologia , Hipocampo/metabolismo , Histonas/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Sirtuínas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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