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Find video protocols related to scientific articles indexed in Pubmed.
Specificity, propagation, and memory of pericentric heterochromatin.
Mol. Syst. Biol.
PUBLISHED: 08-18-2014
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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.
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Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells.
Nat Commun
PUBLISHED: 06-24-2014
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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.
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Nucleosome repositioning links DNA (de)methylation and differential CTCF binding during stem cell development.
Genome Res.
PUBLISHED: 05-08-2014
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During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type-specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning, and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we found that the interplay between these factors depends on their genomic context. The mostly unmethylated CpG islands have reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated, and the average methylation density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands, binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the TET1 and 5hmC levels go down. This process regulates a class of CTCF binding sites outside CpG islands that are occupied by CTCF in ESCs but lose the protein during differentiation. We rationalize this cell type-dependent targeting of CTCF with a quantitative biophysical model of competitive binding with the histone octamer, depending on the TET1, 5hmC, and 5mC state.
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Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6.
Int. J. Cancer
PUBLISHED: 04-17-2014
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Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.
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Establishing epigenetic domains via chromatin-bound histone modifiers.
Ann. N. Y. Acad. Sci.
PUBLISHED: 09-13-2013
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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 cells 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.
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Modeling nucleosome position distributions from experimental nucleosome positioning maps.
Bioinformatics
PUBLISHED: 07-11-2013
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Recent experimental advancements allow determining positions of nucleosomes for complete genomes. However, the resulting nucleosome occupancy maps are averages of heterogeneous cell populations. Accordingly, they represent a snapshot of a dynamic ensemble at a single time point with an overlay of many configurations from different cells. To study the organization of nucleosomes along the genome and to understand the mechanisms of nucleosome translocation, it is necessary to retrieve features of specific conformations from the population average.
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Evolutionary dynamics and information hierarchies in biological systems.
Ann. N. Y. Acad. Sci.
PUBLISHED: 05-20-2013
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The study of evolution has entered a revolutionary new era, where quantitative and predictive methods are transforming the traditionally qualitative and retrospective approaches of the past. Genomic sequencing and modern computational techniques are permitting quantitative comparisons between variation in the natural world and predictions rooted in neo-Darwinian theory, revealing the shortcomings of current evolutionary theory, particularly with regard to large-scale phenomena like macroevolution. Current research spanning and uniting diverse fields and exploring the physical and chemical nature of organisms across temporal, spatial, and organizational scales is replacing the model of evolution as a passive filter selecting for random changes at the nucleotide level with a paradigm in which evolution is a dynamic process both constrained and driven by the informational architecture of organisms across scales, from DNA and chromatin regulation to interactions within and between species and the environment.
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Hypermutation of the inactive X chromosome is a frequent event in cancer.
Cell
PUBLISHED: 04-27-2013
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Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.
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Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin.
Genes Dev.
PUBLISHED: 04-18-2013
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Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4-20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as "chromocenter scattering." We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4-20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation.
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Epigenetic upregulation of lncRNAs at 13q14.3 in leukemia is linked to the In Cis downregulation of a gene cluster that targets NF-kB.
PLoS Genet.
PUBLISHED: 04-01-2013
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Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
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Taking into account nucleosomes for predicting gene expression.
Methods
PUBLISHED: 03-10-2013
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The eukaryotic genome is organized in a chain of nucleosomes that consist of 145-147 bp of DNA wrapped around a histone octamer protein core. Binding of transcription factors (TF) to nucleosomal DNA is frequently impeded, which makes it a challenging task to calculate TF occupancy at a given regulatory genomic site for predicting gene expression. Here, we review methods to calculate TF binding to DNA in the presence of nucleosomes. The main theoretical problems are (i) the computation speed that is becoming a bottleneck when partial unwrapping of DNA from the nucleosome is considered, (ii) the perturbation of the binding equilibrium by the activity of ATP-dependent chromatin remodelers, which translocate nucleosomes along the DNA, and (iii) the model parameterization from high-throughput sequencing data and fluorescence microscopy experiments in living cells. We discuss strategies that address these issues to efficiently compute transcription factor binding in chromatin.
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SIRT6 recruits SNF2H to DNA break sites, preventing genomic instability through chromatin remodeling.
Mol. Cell
PUBLISHED: 01-29-2013
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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.
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De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation.
J. Cell. Sci.
PUBLISHED: 11-01-2011
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Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.
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Chromatin remodelling in mammalian cells by ISWI-type complexes--where, when and why?
FEBS J.
PUBLISHED: 09-02-2011
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The specific location of nucleosomes on DNA has important inhibitory or activating roles in the regulation of DNA-dependent processes as it affects the DNA accessibility. Nucleosome positions depend on the ATP-coupled activity of chromatin-remodelling complexes that translocate nucleosomes or evict them from the DNA. The mammalian cell harbors numerous different remodelling complexes that possess distinct activities. These can translate a variety of signals into certain patterns of nucleosome positions with specific functions. Although chromatin remodellers have been extensively studied in vitro, much less is known about how they operate in their cellular environment. Here, we review the cellular activities of the mammalian imitation switch proteins and discuss mechanisms by which they are targeted to sites where their activity is needed.
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Coding RNAs with a non-coding function: maintenance of open chromatin structure.
Nucleus
PUBLISHED: 09-01-2011
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The multi-layered organization of the genome in a large nucleoprotein complex termed chromatin regulates nuclear functions by establishing subcompartments with distinct DNA-associated activities. Here, we demonstrate that RNA plays an important role in maintaining a decondensed and biologically active interphase chromatin conformation in human and mouse cell lines. As shown by RNase A microinjection and fluorescence microscopy imaging, digestion of single-stranded RNAs induced a distinct micrometer scale chromatin aggregation of these decondensed regions. In contrast, pericentric heterochromatin was more resistant to RNase A treatment. We identified a class of coding RNA transcripts that are responsible for this activity, and thus termed these chromatin-interlinking RNAs or ciRNAs. The initial chromatin distribution could be restored after RNase A treatment with a purified nuclear RNA fraction that was analyzed by high-throughput sequencing. It comprised long > 500 nucleotides (nt) RNA polymerase II (RNAP II) transcripts that were spliced, depleted of polyadenylation and was enriched with long 3-untranslated regions (3-UTRs) above ~800 nt in length. Furthermore, similar reversible changes of the chromatin conformation and the RNAP II distribution were induced by either RNA depletion or RNAP II inhibition. Based on these results we propose that ciRNAs could act as genome organizing architectural factors of actively transcribed chromatin compartments.
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Calculating transcription factor binding maps for chromatin.
Brief. Bioinformatics
PUBLISHED: 07-06-2011
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Current high-throughput experiments already generate enough data for retrieving the DNA sequence-dependent binding affinities of transcription factors (TF) and other chromosomal proteins throughout the complete genome. However, the reverse task of calculating binding maps in a chromatin context for a given set of concentrations and TF affinities appears to be even more challenging and computationally demanding. The problem can be addressed by considering the DNA sequence as a one-dimensional lattice with units of one or more base pairs. To calculate protein occupancies in chromatin, one needs to consider the competition of TF and histone octamers for binding sites as well as the partial unwrapping of nucleosomal DNA. Here, we consider five different classes of algorithms to compute binding maps that include the binary variable, combinatorial, sequence generating function, transfer matrix and dynamic programming approaches. The calculation time of the binary variable algorithm scales exponentially with DNA length, which limits its use to the analysis of very small genomic regions. For regulatory regions with many overlapping binding sites, potentially applicable algorithms reduce either to the transfer matrix or dynamic programming approach. In addition to the recently proposed transfer matrix formalism for TF access to the nucleosomal organized DNA, we develop here a dynamic programming algorithm that accounts for this feature. In the absence of nucleosomes, dynamic programming outperforms the transfer matrix approach, but the latter is faster when nucleosome unwrapping has to be considered. Strategies are discussed that could further facilitate calculations to allow computing genome-wide TF binding maps.
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Nucleosome mediated crosstalk between transcription factors at eukaryotic enhancers.
Phys Biol
PUBLISHED: 06-10-2011
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A recent study of transcription regulation in Drosophila embryonic development revealed a complex non-monotonic dependence of gene expression on the distance between binding sites of repressor and activator proteins at the corresponding enhancer cis-regulatory modules (Fakhouri et al 2010 Mol. Syst. Biol. 6 341). The repressor efficiency was high at small separations, low around 30 bp, reached a maximum at 50-60 bp, and decreased at larger distances to the activator binding sites. Here, we propose a straightforward explanation for the distance dependence of repressor activity by considering the effect of the presence of a nucleosome. Using a method that considers partial unwrapping of nucleosomal DNA from the histone octamer core, we calculated the dependence of activator binding on the repressor-activator distance and found a quantitative agreement with the distance dependence reported for the Drosophila enhancer element. In addition, the proposed model offers explanations for other distance-dependent effects at eukaryotic enhancers.
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Dissecting chromatin interactions in living cells from protein mobility maps.
Chromosome Res.
PUBLISHED: 05-27-2011
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The genome of eukaryotes is organized into a dynamic nucleoprotein complex referred to as chromatin, which can adopt different functional states. Both the DNA and the protein component of chromatin are subject to various post-translational modifications that define the cells gene expression program. Their readout and establishment occurs in a spatio-temporally coordinated manner that is controlled by numerous chromatin-interacting proteins. Binding to chromatin in living cells can be measured by a spatially resolved analysis of protein mobility using fluorescence microscopy based approaches. Recent advancements in the acquisition of protein mobility data using fluorescence bleaching and correlation methods provide data sets on diffusion coefficients, binding kinetics, and cellular concentrations on different time and length scales. The combination of different techniques is needed to dissect the complex interplay of diffusive translocations, binding events, and mobility constraints of the chromatin environment. While bleaching techniques have their strength in the characterization of particles that are immobile on the second/minute time scale, a correlation analysis is advantageous to characterize transient binding events with millisecond residence time. The application and synergy effects of the different approaches to obtain protein mobility and interaction maps in the nucleus are illustrated for the analysis of heterochromatin protein 1.
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Targeting chromatin remodelers: signals and search mechanisms.
Biochim. Biophys. Acta
PUBLISHED: 04-10-2011
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Chromatin remodeling complexes are ATP-driven molecular machines that change chromatin structure by translocating nucleosomes along the DNA, evicting nucleosomes, or changing the nucleosomal histone composition. They are highly abundant in the cell and numerous different complexes exist that display distinct activity patterns. Here we review chromatin-associated signals that are recognized by remodelers. It is discussed how these regulate the remodeling reaction via changing the nucleosome substrate/product binding affinity or the catalytic translocation rate. Finally, we address the question of how chromatin remodelers operate in the cell nucleus to find specifically marked nucleosome substrates via a diffusion driven target location mechanism, and estimate the search times of this process. This article is part of a Special Issue entitled:Snf2/Swi2 ATPase structure and function.
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A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway.
Biotechnol J
PUBLISHED: 03-03-2011
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A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.
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Dissecting DNA-histone interactions in the nucleosome by molecular dynamics simulations of DNA unwrapping.
Biophys. J.
PUBLISHED: 01-20-2011
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The nucleosome complex of DNA wrapped around a histone protein octamer organizes the genome of eukaryotes and regulates the access of protein factors to the DNA. We performed molecular dynamics simulations of the nucleosome in explicit water to study the dynamics of its histone-DNA interactions. A high-resolution histone-DNA interaction map was derived that revealed a five-nucleotide periodicity, in which the two DNA strands of the double helix made alternating contacts. On the 100-ns timescale, the histone tails mostly maintained their initial positions relative to the DNA, and the spontaneous unwrapping of DNA was limited to 1-2 basepairs. In steered molecular dynamics simulations, external forces were applied to the linker DNA to investigate the unwrapping pathway of the nucleosomal DNA. In comparison with a nucleosome without the unstructured N-terminal histone tails, the following findings were obtained: 1), Two main barriers during unwrapping were identified at DNA position ±70 and ±45 basepairs relative to the central DNA basepair at the dyad axis. 2), DNA interactions of the histone H3 N-terminus and the histone H2A C-terminus opposed the initiation of unwrapping. 3), The N-terminal tails of H2A, H2B, and H4 counteracted the unwrapping process at later stages and were essential determinants of nucleosome dynamics. Our detailed analysis of DNA-histone interactions revealed molecular mechanisms for modulating access to nucleosomal DNA via conformational rearrangements of its structure.
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Force spectroscopy of chromatin fibers: extracting energetics and structural information from Monte Carlo simulations.
Biopolymers
PUBLISHED: 01-18-2011
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The folding of the nucleosome chain into a chromatin fiber is a central factor for controlling the DNA access of protein factors involved in transcription, DNA replication and repair. Force spectroscopy experiments with chromatin fibers are ideally suited to dissect the interactions that drive this process, and to probe the underlying fiber conformation. However, the interpretation of the experimental data is fraught with difficulties due to the complex interplay of the nucleosome geometry and the different energy terms involved. Here, we apply a Monte Carlo simulation approach to derive virtual chromatin fiber force spectroscopy curves. In the simulations, the effect of the nucleosome geometry, repeat length, nucleosome-nucleosome interaction potential, and the unwrapping of the DNA from the histone protein core on the shape of the force-extension curves was investigated. These simulations provide a framework for the evaluation of experimental data sets. We demonstrate how the relative contributions of DNA bending and twisting, nucleosome unstacking and unwrapping the nucleosomal DNA from the histone octamer can be dissected for a given fiber geometry.
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Binding kinetics of human ISWI chromatin-remodelers to DNA repair sites elucidate their target location mechanism.
Nucleus
PUBLISHED: 01-13-2011
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Chromatin remodelers translocate nucleosomes along the DNA chain in an ATP-dependent manner. This catalytic activity is particularly important for DNA replication and repair since both processes require a significant amount of nucleosome translocations and assembly during DNA synthesis. Recently, we have studied the mobility and interactions of the human ISWI family chromatin remodelers Snf2H and Snf2L as well as Acf1, one of the non-catalytic subunits present in the ACF and CHRAC complexes of Snf2H. We proposed that these protein complexes identify their nucleosomal substrates via a continuous sampling mechanism. It rationalizes the relatively high nuclear mobility and abundance observed for all ISWI proteins in terms of fast target location. According to our model a certain type of ISWI complex visits a given nucleosome in the human genome on the timescale of several seconds to a few minutes. Here, we show that the ISWI proteins Snf2H, Snf2L as well as Acf1 accumulate at UV-induced DNA damage sites within tens of seconds and reach a plateau after a few minutes. These findings corroborate the predictions of the continuous sampling mechanism as an efficient way for targeting chromatin remodelers to sites in the genome that require their activity. In comparison to the mobility of PCNA (proliferating cell nuclear antigen) that also accumulates at DNA repair sites the specifics of substrate location by chromatin remodelers are further characterized.
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Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-25-2010
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Chromatin remodeling complexes can translocate nucleosomes along the DNA in an ATP-dependent manner. Here, we studied autofluorescent protein constructs of the human ISWI family members Snf2H, Snf2L, the catalytically inactive Snf2L+13 splice variant, and the accessory Acf1 subunit in living human and mouse cells by fluorescence microscopy/spectroscopy. Except for Snf2L, which was not detected in the U2OS cell line, the endogenous ISWI proteins were abundant at nuclear concentrations between 0.14 and 0.83 ?M. A protein interaction analysis showed the association of multimeric Snf2H and Acf1 into a heterotetramer or higher-order ACF complex. During the G1/2 cell cycle phase, Snf2H and Snf2L displayed average residence times <150 ms in the chromatin-bound state. The comparison of active and inactive Snf2H/Snf2L indicated that an immobilized fraction potentially involved in active chromatin remodeling comprised only 1-3%. This fraction was largely increased at replication foci in S phase or at DNA repair sites. To rationalize these findings we propose that ISWI remodelers operate via a "continuous sampling" mechanism: The propensity of nucleosomes to be translocated is continuously tested in transient binding reactions. Most of these encounters are unproductive and efficient remodeling requires an increased binding affinity to chromatin. Due to the relatively high intranuclear remodeler concentrations cellular response times for repositioning a given nucleosome were calculated to be in the range of tens of seconds to minutes.
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Statistical-mechanical lattice models for protein-DNA binding in chromatin.
J Phys Condens Matter
PUBLISHED: 09-30-2010
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Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.
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Structure and function of the hetero-oligomeric cysteine synthase complex in plants.
J. Biol. Chem.
PUBLISHED: 08-18-2010
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Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (?G = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(D) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(D)) and the regulation of cysteine sensitivity of SAT within the CSC.
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Histone H2A C-terminus regulates chromatin dynamics, remodeling, and histone H1 binding.
PLoS Genet.
PUBLISHED: 06-21-2010
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The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.
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A lattice model for transcription factor access to nucleosomal DNA.
Biophys. J.
PUBLISHED: 06-04-2010
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Nucleosomes, the basic repeating unit of chromatin, consist of 147 basepairs of DNA that are wrapped in almost two turns around a histone protein octamer core. Because ?3/4 of the human genomic DNA is found within nucleosomes, their position and DNA interaction is an essential determinant for the DNA access of gene-specific transcription factors and other proteins. Here, a DNA lattice model was developed for describing ligand binding in the presence of a nucleosome. The model takes into account intermediate states, in which DNA is partially unwrapped from the histone octamer. This facilitates access of transcription factors to up to 60 DNA basepairs located in the outer turn of nucleosomal DNA, while the inner DNA turn was found to be more resistant to competitive ligand binding. As deduced from quantitative comparisons with recently published experimental data, our model provides a better description than the previously used all-or-none lattice-binding model. Importantly, nucleosome-occupancy maps predicted by the nucleosome-unwrapping model also differed significantly when partial unwrapping of nucleosomal DNA was considered. In addition, large effects on the cooperative binding of transcription factors to multiple binding sites occluded by the nucleosome were apparent. These findings indicate that partial unwrapping of DNA from the histone octamer needs to be taken into account in quantitative models of gene regulation in chromatin.
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Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase.
FEBS Lett.
PUBLISHED: 04-12-2010
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Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.
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Exploring the conformational space of chromatin fibers and their stability by numerical dynamic phase diagrams.
Biophys. J.
PUBLISHED: 03-23-2010
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The three-dimensional structure of chromatin affects DNA accessibility and is therefore a key regulator of gene expression. However, the path of the DNA between consecutive nucleosomes, and the resulting chromatin fiber organization remain controversial. The conformational space available for the folding of the nucleosome chain has been analytically described by phase diagrams with a two-angle model, which describes the chain trajectory by a DNA entry-exit angle at the nucleosome and a torsion angle between consecutive nucleosomes. Here, a novel type of numerical phase diagrams is introduced that relates the geometric phase space to the energy associated with a given chromatin conformation. The resulting phase diagrams revealed differences in the energy landscape that reflect the probability of a given conformation to form in thermal equilibrium. Furthermore, we investigated the effects of entropy and additional degrees of freedom in the dynamic phase diagrams by performing Monte Carlo simulations of the initial chain trajectories. Using our approach, we were able to demonstrate that conformations that initially were geometrically impossible could evolve into energetically favorable states in thermal equilibrium due to DNA bending and torsion. In addition, dynamic phase diagrams were applied to identify chromatin fibers that reflect certain experimentally determined features.
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Three-dimensional organization of promyelocytic leukemia nuclear bodies.
J. Cell. Sci.
PUBLISHED: 02-05-2010
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Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.
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Structural insights into tail-anchored protein binding and membrane insertion by Get3.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-30-2009
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Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and alpha-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the alpha-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the alpha-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg(2+)- and ADP-Mg(2+)-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP.AlF(4)(-)-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361-366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.
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Predicting nucleosome positions on the DNA: combining intrinsic sequence preferences and remodeler activities.
Nucleic Acids Res.
PUBLISHED: 07-22-2009
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Nucleosome positions on the DNA are determined by the intrinsic affinities of histone proteins to a given DNA sequence and by the ATP-dependent activities of chromatin remodeling complexes that can translocate nucleosomes with respect to the DNA. Here, we report a theoretical approach that takes into account both contributions. In the theoretical analysis two types of experiments have been considered: in vitro experiments with a single reconstituted nucleosome and in vivo genome-scale mapping of nucleosome positions. The effect of chromatin remodelers was described by iteratively redistributing the nucleosomes according to certain rules until a new steady state was reached. Three major classes of remodeler activities were identified: (i) the establishment of a regular nucleosome spacing in the vicinity of a strong positioning signal acting as a boundary, (ii) the enrichment/depletion of nucleosomes through amplification of intrinsic DNA-sequence-encoded signals and (iii) the removal of nucleosomes from high-affinity binding sites. From an analysis of data for nucleosome positions in resting and activated human CD4(+) T cells [Schones et al., Cell 132, p. 887] it was concluded that the redistribution of a nucleosome map to a new state is greatly facilitated if the remodeler complex translocates the nucleosome with a preferred directionality.
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X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases.
FEBS Lett.
PUBLISHED: 06-01-2009
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The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.
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Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy.
Biophys. J.
PUBLISHED: 04-08-2009
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Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1alpha and HP1beta was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1alpha/beta in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times t(res)
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Assembly and mobility of exon-exon junction complexes in living cells.
RNA
PUBLISHED: 03-26-2009
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The exon-exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.
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Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy.
Eur. Biophys. J.
PUBLISHED: 03-03-2009
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The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 mus time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.
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Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line.
Mol. Biol. Cell
PUBLISHED: 02-11-2009
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Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs.
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Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis.
Proc. Natl. Acad. Sci. U.S.A.
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Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ?1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.
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Genome-wide nucleosome positioning during embryonic stem cell development.
Nat. Struct. Mol. Biol.
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We determined genome-wide nucleosome occupancies in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell-type- and protein-specific binding preferences of transcription factors to sites with either low (Myc, Klf4 and Zfx) or high (Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome-depleted regions around transcription start and transcription termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to CpG content or histone methylation marks. Throughout the genome, nucleosome occupancy was correlated with certain histone methylation or acetylation modifications. In addition, the average nucleosome repeat length increased during differentiation by 5-7 base pairs, with local variations for specific regions. Our results reveal regulatory mechanisms of cell differentiation that involve nucleosome repositioning.
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Loss of the abundant nuclear non-coding RNA MALAT1 is compatible with life and development.
RNA Biol
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The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer metastasis. It is highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. The nuclear transcript MALAT-1 has been functionally associated with gene regulation and alternative splicing and its regulation has been shown to impact proliferation, apoptosis, migration and invasion.   Here, we have developed a human and a mouse knockout system to study the loss-of-function phenotypes of this important ncRNA. In human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases. Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation nor cell cycle progression nor nuclear architecture in human lung or liver cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not give rise to any obvious phenotype or histological abnormalities in Malat1-null compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA MALAT1 is compatible with cell viability and normal development.
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PML body meets telomere: the beginning of an ALTernate ending?
Nucleus
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The unlimited proliferation potential of cancer cells requires the maintenance of their telomeres. This is frequently accomplished by reactivation of telomerase. However, in a significant fraction of tumors an alternative lengthening of telomeres (ALT) mechanism is active. The molecular mechanism of the ALT pathway remains elusive. In particular, the role of characteristic complexes of promyelocytic leukemia nuclear bodies (PML-NBs) with telomeres, the ALT-associated PML-NBs (APBs), is currently under investigation. Here, we review recent findings on the assembly, structure and functions of APBs. It is discussed how genomic aberrations in ALT-positive cancer cells could result in the formation of APBs and in ALT activity. We conclude that they are important functional intermediates in what is considered the canonical ALT pathway and discuss deregulations of cellular pathways that contribute to the emergence of the ALT phenotype.
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Nuclear architecture by RNA.
Curr. Opin. Genet. Dev.
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The dynamic organization of the cell nucleus into subcompartments with distinct biological activities represents an important determinant of cell function. Recent studies point to a crucial role of RNA as an architectural factor for shaping the genome and its nuclear environment. Here, we outline general principles by which RNA organizes functionally different nuclear subcompartments in mammalian cells. RNA is a structural component of mobile DNA-free nuclear bodies like paraspeckles or Cajal bodies, and is involved in establishing specific chromatin domains. The latter group comprises largely different structures that require RNA for the formation of active or repressive chromatin compartments with respect to gene expression as well as separating boundaries between these.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.