Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids.

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin.

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/μm.

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis.

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery.

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus.

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis).

Nuclear compartmentalization, dynamics, and function of regulatory DNA sequences.

Transcription regulatory elements (TREs) have been extensively studied on the biochemical level with respect to their interactions with transcription factors (TFs), other DNA segments, and larger protein complexes. In this review, we describe concepts and preliminary experimental evidence for positional changes of TREs within a dynamic, functional nuclear architecture. We suggest a multilayered shell-like chromatin organization of chromatin domain clusters with increasing chromatin compaction levels from the periphery toward the interior with a decondensed transcriptionally active peripheral layer and compact repressed chromatin typically located in the interior.

Initial high-resolution microscopic mapping of active and inactive regulatory sequences proves non-random 3D arrangements in chromatin domain clusters.

Background: The association of active transcription regulatory elements (TREs) with DNAse I hypersensitivity (DHS[+]) and an 'open' local chromatin configuration has long been known. However, the 3D topography of TREs within the nuclear landscape of individual cells in relation to their active or inactive status has remained elusive. Here, we explored the 3D nuclear topography of active and inactive TREs in the context of a recently proposed model for a functionally defined nuclear architecture, where an active and an inactive nuclear compartment (ANC-INC) form two spatially co-aligned and functionally interacting networks.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction.

Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments.

Background: Previous studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization.

The 4D nucleome: Evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments.

Recent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC).

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs).

Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs).

Novel higher-order epigenetic regulation of the Bdnf gene upon seizures.

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have.

Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging.

The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.

Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution.

A top-down analysis of Xa- and Xi-territories reveals differences of higher order structure at ≥ 20 Mb genomic length scales.

The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb.

3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming.

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization.

3D-FISH on cultured cells combined with immunostaining.

Fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with immunocytochemistry and 3D fluorescence microscopy, is a key tool to analyze the functional organization of the interphase nucleus. In the last decade, 3D-FISH on cultured cells has become a routine technique and is now widely used in nuclear biology. This method allows visualization of chromosome territories, chromosome subregions, single genes, and RNA transcripts preserving their spatial positions in the cell nucleus.

Remodeling of nuclear architecture by the thiodioxoxpiperazine metabolite chaetocin.

Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite.

Chromosome territories.

Chromosome territories (CTs) constitute a major feature of nuclear architecture. In a brief statement, the possible contribution of nuclear architecture studies to the field of epigenomics is considered, followed by a historical account of the CT concept and the final compelling experimental evidence of a territorial organization of chromosomes in all eukaryotes studied to date. Present knowledge of nonrandom CT arrangements, of the internal CT architecture, and of structural interactions with other CTs is provided as well as the dynamics of CT arrangements during cell cycle and postmitotic terminal differentiation.

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development.

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative.

Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.

Fluorescence in situ hybridization (FISH) of specific DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specific DNA segments in metaphase preparations as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an efficient tool to analyze the spatial arrangement of targeted DNA sequences in the nucleus. Recent developments of a "new generation" of confocal microscopes that allow the distinct visualization of at least five different fluorochromes within one experiment opened the way for multicolor 3D-FISH experiments.

Replication-timing-correlated spatial chromatin arrangements in cancer and in primate interphase nuclei.

Using published high-resolution data on S-phase replication timing, we determined the three-dimensional (3D) nuclear arrangement of 33 very-early-replicating and 31 very-late-replicating loci. We analyzed diploid human, non-human primate and rearranged tumor cells by 3D fluorescence in situ hybridization with the aim of investigating the impact of chromosomal structural changes on the nuclear organization of these loci. Overall, their topology was found to be largely conserved between cell types, species and in tumor cells.

Radial chromatin positioning is shaped by local gene density, not by gene expression.

G- and R-bands of metaphase chromosomes are characterized by profound differences in gene density, CG content, replication timing, and chromatin compaction. The preferential localization of gene-dense, transcriptionally active, and early replicating chromatin in the nuclear interior and of gene-poor, later replicating chromatin at the nuclear envelope has been demonstrated to be evolutionary-conserved in various cell types. Yet, the impact of different local chromatin features on the radial nuclear arrangement of chromatin is still not well understood.

Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions.

The regulation of gene expression is mediated by interactions between chromatin and protein complexes. The importance of where and when these interactions take place in the nucleus is currently a subject of intense investigation. Increasing evidence indicates that gene activation or silencing is often associated with repositioning of the locus relative to nuclear compartments and other genomic loci.

Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes.

INTRODUCTIONDNA probes for fluorescence in situ hybridization (FISH) can be generated and labeled by various methods. This protocol describes the conjugation of dUTPs with haptens or fluorochromes, as well as the generation and labeling of DNA probes using those modified dUTPs. Sources of probe DNA include genomic DNA, DNA from flow-sorted chromosomes, bacterial artificial chromosomes (BACs), and cosmids.