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Distinct nuclear structures and bodies are involved in genome intranuclear positioning. Measuring proximity and relative distances of genomic loci to these nuclear compartments, and correlating this chromosome intranuclear positioning with epigenetic marks and functional readouts genome-wide, will be required to appreciate the true extent to which this nuclear compartmentalization contributes to regulation of genome functions. Here we present detailed protocols for TSA-seq, the first sequencing-based method for estimation of cytological proximity of chromosomal loci to spatially discrete nuclear structures, such as nuclear bodies or the nuclear lamina. TSA-seq uses Tyramide Signal Amplification (TSA) of immunostained cells to create a concentration gradient of tyramide-biotin free radicals which decays exponentially as a function of distance from a point-source target. Reaction of these free radicals with DNA deposits tyramide-biotin onto DNA as a function of distance from the point source. The relative enrichment of this tyramide-labeled DNA versus input DNA, revealed by DNA sequencing, can then be used as a "cytological ruler" to infer relative, or even absolute, mean chromosomal distances from immunostained nuclear compartments. TSA-seq mapping is highly reproducible and largely independent of the target protein or antibody choice for labeling a particular nuclear compartment. Our protocols include variations in TSA labeling conditions to provide varying spatial resolution as well as enhanced sensitivity. Our most streamlined protocol produces TSA-seq spatial mapping over a distance range of ~1 micron from major nuclear compartments using ~10-20 million cells.

TSA-seq mapping suggests that gene distance to nuclear speckles is more deterministic and predictive of gene expression levels than gene radial positioning. Gene expression correlates inversely with distance to nuclear speckles, with chromosome regions of unusually high expression located at the apex of chromosome loops protruding from the nuclear periphery into the interior. Genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping closest to nuclear speckles, suggesting the possibility of conservation of speckle-associated regions. To facilitate comparison of genome organization by TSA-seq, we reduced required cell numbers 10- to 20-fold for TSA-seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Only ∼10% of the genome shows statistically significant shifts in relative nuclear speckle distances in pair-wise comparisons between human cell lines (H1, HFF, HCT116, K562); however, these moderate shifts in nuclear speckle distances tightly correlate with changes in cell type–specific gene expression. Similarly, half of heat shock-induced gene loci already preposition very close to nuclear speckles, with the remaining positioned near or at intermediate distance (HSPH1) to nuclear speckles but shifting even closer with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely “hardwired” genome organization with specific genes moving small mean distances relative to speckles during cell differentiation or a physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.

We introduce Nucleome Browser (http://www.nucleome.org), an interactive, multimodal data visualization and exploration platform for 4D Nucleome research. Our tool effectively integrates heterogeneous datasets (e.g., genomics, imaging, 3D genome structure models, and single-cell data) and external data portals by a new adaptive communication mechanism. Nucleome Browser provides a scalable solution for integrating massive amounts of 4D Nucleome data to navigate multiscale nuclear structure and function in a wide range of biological contexts, enabling hypothesis generation and data sharing with the broad community.

Large-scale chromatin compaction is nonuniform across the human genome and correlates with gene expression and genome organization. Current methodologies for assessing large-scale chromatin compaction are indirect and largely based on assays that probe lower levels of chromatin organization, primarily at the level of the nucleosome and/or the local compaction of nearby nucleosomes. These assays assume a one-to-one correlation between local nucleosomal compaction and large-scale compaction of chromosomes that may not exist. Here we describe a method to identify interphase chromosome regions with relatively high levels of large-scale chromatin decondensation using TSA-seq, which produces a signal proportional to microscopic-scale distances relative to a defined nuclear compartment. TSA-seq scores that change rapidly as a function of genomic distance, detected by their higher slope values, identify decondensed large-scale chromatin domains (DLCDs), as then validated by 3D DNA-FISH. DLCDs map near a subset of chromatin domain boundaries, defined by Hi-C, which separate active and repressed chromatin domains and correspond to compartment, subcompartment, and some TAD boundaries. Most DLCDs can also be detected by high slopes of their Hi-C compartment score. In addition to local enrichment in cohesin (RAD21, SMC3) and CTCF, DLCDs show the highest local enrichment to super-enhancers, but are also locally enriched in transcription factors, histone-modifying complexes, chromatin mark readers, and chromatin remodeling complexes. The localization of these DLCDs to a subset of Hi-C chromatin domain boundaries that separate active versus inactive chromatin regions, as measured by two orthogonal genomic methods, suggests a distinct role for DLCDs in genome organization.

Chromosomes segregate differentially relative to distinct subnuclear structures, but this genome-wide compartmentalization, pivotal for modulating genome function, remains poorly understood. New genomic mapping methods can reveal chromosome positioning relative to specific nuclear structures. However, computational methods that integrate their results to identify overall intranuclear chromo-some positioning have not yet been developed. We report SPIN, a new method to identify genome-wide nuclear spatial localization patterns. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to genome function (transcription and replication timing). Comparisons of SPIN states with Hi-C sub-compartments and lamina-associated domains (LADs) from multiple cell types suggest constitutive compartmentalization patterns. By integrating different readouts of higher-order genome organization, SPIN provides critical insights into nuclear spatial and functional compartmentalization.

Achieving stable expression of a single transgene in mammalian cells remains challenging; even more challenging is obtaining simultaneous stable expression of multiple transgenes at reproducible, relative expression levels. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC "scaffold" containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this "BAC TG-EMBED" approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01- to 5-fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes. Second, we demonstrate little variation in expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making the choice of BAC scaffolds more flexible. Third, we present a novel BAC assembly scheme, "BAC-MAGIC", for inserting multiple transgenes into BAC scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved BAC TG-EMBED toolkit, we simultaneously fluorescently labeled three nuclear compartments at reproducible, relative intensity levels in 94% of stable clones after a single transfection using a DHFR BAC scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended BAC TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels.

Chen et al. present TSA-Seq, a new mapping method that measures cytological distances relative to spatially distinct nuclear subcompartments. From novel nuclear organization maps of human cells, they identify transcription hot zones of high gene density that are near nuclear speckles and enriched in highly expressed genes, housekeeping genes, and genes with low transcriptional pausing., While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a “cytological ruler” for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription “hot zones.” Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression., Graphical Abstract

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the “BAC TG-EMBED” method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.

This work reviews nuclear compartments, defined broadly to include distinct nuclear structures, bodies, and chromosome domains. It first summarizes original cytological observations before comparing concepts of nuclear compartments emerging from microscopy versus genomic approaches and then introducing new multiplexed imaging approaches that promise in the future to meld both approaches. I discuss how previous models of radial distribution of chromosomes or the binary division of the genome into A and B compartments are now being refined by the recognition of more complex nuclear compartmentalization. The poorly understood question of how these nuclear compartments are established and maintained is then discussed, including through the modern perspective of phase separation, before moving on to address possible functions of nuclear compartments, using the possible role of nuclear speckles in modulating gene expression as an example. Finally, the review concludes with a discussion of future questions for this field.

In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation.

Genome-wide mapping of chromosomal distances relative to nuclear compartments using TSA-Seq suggests a more deterministic relationship between intranuclear gene position and expression as a function of nuclear speckle distance than radial position. Gene activity increases overall with decreasing distance to nuclear speckles, with active chromosomal regions forming the apex of chromosome loops protruding from the nuclear periphery into the interior. Interestingly, genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping very close to nuclear speckles, suggesting the possibility of genomic “hardwiring” and conservation of speckle-associated regions. To facilitate comparison of genome organization relative to nuclear speckles in human K562, HCT116, HFFc6, and H1 cell lines, here we describe reducing the required cell number 10-20-fold for TSA-Seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Surprisingly, in pair-wise cell line comparisons, only ∼10% of the genome shows a statistically significant shift in relative nuclear speckle distances. These modest shifts in nuclear speckle distance, however, tightly correlate with changes in cell-type specific gene expression. Similarly, half of all loci that contain induced heat-shock protein genes appear pre-positioned close to nuclear speckles, with the remaining showing small shifts towards speckles with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely “hardwired” genome organization and specific genes moving small mean distances relative to speckles during cell differentiation or physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.

Dopie et al. apply TSA-MS ratio, an approach that compares the abundance of proteins in nuclear speckles with centromeres to reduce nonspecific background, and show that MFAP1 levels modulate nuclear speckle size and MFAP1 is recruited early to reforming nuclear speckles after mitosis., We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

Achieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

A significant fraction of active chromosome regions and genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, we observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 °C. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through a “transcriptional amplification” mechanism distinct from transcriptional bursting.

Higher eukaryotic cell nuclei are highly compartmentalized into bodies and structural assemblies of specialized functions. Nuclear speckles/IGCs are one of the most prominent nuclear bodies, yet their functional significance remains largely unknown. Recent advances in sequence-based mapping of nuclear genome organization now provide genome-wide analysis of chromosome organization relative to nuclear speckles. Here we review older microscopy-based studies on a small number of genes with the new genomic mapping data suggesting a significant fraction of the genome is almost deterministically positioned near nuclear speckles. Both microscopy and genomic-based approaches support the concept of the nuclear speckle periphery as a major active chromosomal compartment which may play an important role in fine-tuning gene regulation.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. While links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions, but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared to non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism utilized by a transcription factor to boost gene expression.

Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green-fluorescent-protein (GFP) tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar long-range speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significant increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 um and with velocities between 0.2-1.5 μm/min. Frequently, 3 or even 4 speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer but larger speckles after transcriptional inhibition. These speckle movements are not actin-dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle-marker protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization.

SummaryWhile nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription “hot-zones”. Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

Due to a tight attachment of peripheral heterochromatin to the nuclear lamina its replication is connected with inevitable topological hindrances. Additional hindrances are caused by high stability of the lamina that complicates the access of replication factors to DNA duplication sites under conditions of highly condensed matrix with limited mobility. The work focuses on detailed study of structural organization and dynamics of the lamina in respect to replication of peripheral heterochromatin that is attached to it. The study of mobile properties of lamins at various stages of the S-phase using live cell imaging and super-resolution microscopy showed the absence of the dependence of lamins’ mobility on replicative status of attached heterochromatin. These data confirm the hypothesis on regulation of linkage between chromatin and lamina at the level of molecular intermediates. It has been shown at the ultrastructural level that possible temporary disruption in molecular bonds between the lamina and peripheral chromatin during replication does not cause movement of replicated domains from the nuclear periphery.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes. DOI: http://dx.doi.org/10.7554/eLife.20378.001

Traditionally large-scale chromatin structure has been studied by microscopic approaches, providing direct spatial information but limited sequence context. In contrast, newer 3C (chromosome capture conformation) methods provide rich sequence context but uncertain spatial context. Recent demonstration of large, topologically linked DNA domains, hundreds to thousands of kb in size, may now link 3C data to actual chromosome physical structures, as visualized directly by microscopic methods. Yet, new data suggesting that 3C may measure cytological rather than molecular proximity prompts a renewed focus on understanding the origin of 3C interactions and dissecting the biological significance of long-range genomic interactions.

Many active genes position near nuclear speckles. Kim et al. show that Hsp70 speckle association correlates with increased nascent transcripts. Live-cell imaging shows that increases in nascent Hsp70 transcripts strictly follow speckle association (∼0–2 min), suggesting that speckle association amplifies gene expression., Many active genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that HSPA1B BAC transgenes and endogenous Hsp70 genes turn on 2–4 min after heat shock (HS), irrespective of their distance to speckles. However, both total HSPA1B mRNA counts and nascent transcript levels measured adjacent to the transgene are approximately twofold higher for speckle-associated alleles 15 min after HS. Nascent transcript level fold-increases for speckle-associated alleles are 12–56-fold and 3–7-fold higher 1–2 h after HS for HSPA1B transgenes and endogenous genes, respectively. Severalfold higher nascent transcript levels for several Hsp70 flanking genes also correlate with speckle association at 37°C. Live-cell imaging reveals that HSPA1B nascent transcript levels increase/decrease with speckle association/disassociation. Initial investigation reveals that increased nascent transcript levels accompanying speckle association correlate with reduced exosome RNA degradation and larger Ser2p CTD-modified RNA polymerase II foci. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through “gene expression amplification.”

Multiple cis-elements surrounding the β-globin gene locus combine to target this locus to the nuclear periphery through at least two different epigenetic marks., Increasing evidence points to nuclear compartmentalization as a contributing mechanism for gene regulation, yet mechanisms for compartmentalization remain unclear. In this paper, we use autonomous targeting of bacterial artificial chromosome (BAC) transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs) within an HBB BAC bias a competition between pericentric versus peripheral heterochromatin targeting toward the nuclear periphery, which correlates with increased H3K9me3 across the β-globin gene cluster and locus control region. Targeting to both heterochromatin compartments is dependent on Suv39H-mediated H3K9me3 methylation. In different chromosomal contexts, PTRs confer no targeting, targeting to pericentric heterochromatin, or targeting to the periphery. A combination of fluorescent in situ hybridization, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over hundreds of kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent lamin-associated domain. Our results demonstrate that multiple cis-elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Summary In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication.

International audience; Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression , but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pair-ing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation.

An Hsp70 transgene system is used to identify cis-elements required for gene-specific association with nuclear speckles., Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid–processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association.

A paper appearing in late 2008 attracted considerable attention with its description of a dramatic juxtaposition of two estrogen responsive genes on different chromosomes within 15-60 minutes of adding estradiol. These results challenged a growing consensus of limited chromosome mobility within interphase nuclei, while raising questions of whether a hitherto unknown molecular mechanism might exist to move chromosomes long distances within the nucleus. These results also raised the fascinating question of how two genes on widely separated chromosomes might find each other over such a short time span. Now, a more recent paper reports no such long-range interaction or chromosome movements in the same cell types under what appear to be well replicated conditions, forcing a reexamination of the prior results.

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an approximately 1 microm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to approximately 300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.

Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophilaphosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a `kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the `kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophilain vivo.

Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization.

Summary Increasing evidence suggests functional compartmentalization of interphase nuclei [1]. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions [2, 3], association of some active genes with nuclear speckles [4, 5] or transcription "factories" [6], and association of transcriptionally repressed genes with heterochromatic regions [7]. Dynamic changes in chromosome compartmentalization [7–9] imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility [10], described as "constrained diffusion" [11]. None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1–2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1–0.9 μm/min over distances of 1–5 μm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1alpha or HP1beta we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1alpha and HP1beta to the chromosome region. We show that CD-less HP1alpha can induce chromatin condensation, whereas the effect of truncated HP1beta is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization.

Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle-tracking method in a two-photon microscope that provides ∼10-fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac-operator repeat in cells stably expressing lac repressor fused with enhanced green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of ∼150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy-dependent mechanism driving fast motion of chromatin in interphase cells. © 2005 by the Biophysical Society.

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”

Previous work has demonstrated a more decondensed large-scale chromatin structure and a more internal nuclear position for gene-rich versus gene-poor chromosome regions. Here, we show that large-scale chromatin opening and changes in intranuclear positioning of chromosome regions can be induced by normal levels of endogenous transcription factors acting on mammalian regulatory sequences. We transfected mouse erythroleukemia cells with a 15 kbp plasmid containing a lac operator repeat plus β-globin regulatory sequences driving a β-galactosidase reporter gene. After green-fluorescent-protein/lac-repressor fusion-protein binding or after fluorescence in situ hybridization, the volume and location of the transgene array signal were measured. With both detection methods, we found that the volume was severalfold larger when transcription was on. While silent transgene arrays were located close to the nuclear membrane, we observed a significantly more internal position for the transcriptionally active state. Our results indicate that both large-scale chromatin decondensation and changes in nuclear positioning as observed for large, complex gene-rich chromosome regions can be reproduced by endogenous regulatory sequences acting within simple repetitive transgene arrays.