Individual Chromosome Territories Research Articles

Analysis of long-range chromatin contacts, compartments and looping between mouse embryonic stem cells, lens epithelium and lens fibers

BackgroundNuclear organization of interphase chromosomes involves individual chromosome territories, “open” and “closed” chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei.ResultsChromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions.ConclusionsOur study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities.

Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains.

Continuing progress in super-resolution microscopy enables the study of sub-chromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell-impermeable fluorescent nucleotides for live-cell studies. Furthermore, we describe the application of (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) and 5-vinyl-2'-deoxyuridine (VdU) for the in situ detection of segregated chromosome territories and sister chromatids with minimized cytotoxic side effects.

Interphase Chromosomes in Replicative Senescence: Chromosome Positioning as a Senescence Biomarker and the Lack of Nuclear Motor-Driven Chromosome Repositioning in Senescent Cells.

This study demonstrates, and confirms, that chromosome territory positioning is altered in primary senescent human dermal fibroblasts (HDFs). The chromosome territory positioning pattern is very similar to that found in HDFs made quiescent either by serum starvation or confluence; but not completely. A few chromosomes are found in different locations. One chromosome in particular stands out, chromosome 10, which is located in an intermediate location in young proliferating HDFs, but is found at the nuclear periphery in quiescent cells and in an opposing location of the nuclear interior in senescent HDFs. We have previously demonstrated that individual chromosome territories can be actively and rapidly relocated, with 15 min, after removal of serum from the culture media. These chromosome relocations require nuclear motor activity through the presence of nuclear myosin 1β (NM1β). We now also demonstrate rapid chromosome movement in HDFs after heat-shock at 42°C. Others have shown that heat shock genes are actively relocated using nuclear motor protein activity via actin or NM1β (Khanna et al., 2014; Pradhan et al., 2020). However, this current study reveals, that in senescent HDFs, chromosomes can no longer be relocated to expected nuclear locations upon these two types of stimuli. This coincides with a entirely different organisation and distribution of NM1β within senescent HDFs.

In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains.

Recent advances in super-resolution microscopy enable the study of subchromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell impermeable fluorescent nucleotides for live cell studies. Furthermore we describe the application of (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) for the in situ detection of segregated chromosome territories with minimized cytotoxic side effects.

Large-scale probabilistic 3D organization of human chromosome territories.

There is growing evidence that chromosome territories (CT) have a probabilistic non-random arrangement within the cell nucleus of mammalian cells including radial positioning and preferred patterns of interchromosomal interactions that are cell-type specific. While it is generally assumed that the three-dimensional (3D) arrangement of genes within the CT is linked to genomic regulation, the degree of non-random organization of individual CT remains unclear. As a first step to elucidating the global 3D organization (topology) of individual CT, we performed multi-color fluorescence in situ hybridization using six probes extending across each chromosome in human WI38 lung fibroblasts. Six CT were selected ranging in size and gene density (1, 4, 12, 17, 18 and X). In-house computational geometric algorithms were applied to measure the 3D distances between every combination of probes and to elucidate data-mined structural patterns. Our findings demonstrate a high degree of non-random arrangement of individual CT that vary from chromosome to chromosome and display distinct changes during the cell cycle. Application of a classic, well-defined data mining and pattern recognition approach termed the 'k-means' generated 3D models for the best fit arrangement of each chromosome. These predicted models correlated well with the detailed distance measurements and analysis. We propose that the unique 3D topology of each CT and characteristic changes during the cell cycle provide the structural framework for the global gene expression programs of the individual chromosomes.

Gene density and chromosome territory shape.

Despite decades of study of chromosome territories (CT) in the interphase nucleus of mammalian cells, our understanding of the global shape and 3-D organization of the individual CT remains very limited. Past microscopic analysis of CT suggested that while many of the CT appear to be very regular ellipsoid-like shapes, there were also those with more irregular shapes. We have undertaken a comprehensive analysis to determine the degree of shape regularity of different CT. To be representative of the whole human genome, 12 different CT (~41% of the genome) were selected that ranged from the largest (CT 1) to the smallest (CT 21) in size and from the highest (CT 19) to lowest (CT Y) in gene density. Using both visual inspection and algorithms that measure the degree of shape ellipticity and regularity, we demonstrate a strong inverse correlation between the degree of regular CT shape and gene density for those CT that are most gene-rich (19, 17, 11) and gene-poor (18, 13, Y). CT more intermediate in gene density showed a strong negative correlation with shape regularity, but not with ellipticity. An even more striking correlation between gene density and CT shape was determined for the nucleolar-associated NOR-CT. Correspondingly, striking differences in shape between the X active and inactive CT implied that aside from gene density, the overall global level of gene transcription on individual CT is also an important determinant of chromosome territory shape.

Innate Structure of DNA Foci Restricts the Mixing of DNA from Different Chromosome Territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.

Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cells

BackgroundHutchinson-Gilford progeria syndrome (HGPS) is a premature ageing syndrome that affects children leading to premature death, usually from heart infarction or strokes, making this syndrome similar to normative ageing. HGPS is commonly caused by a mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. Progerin cannot be processed as wild-type pre-lamin A and remains farnesylated, leading to its aberrant behavior during interphase and mitosis. Farnesyltransferase inhibitors prevent the accumulation of farnesylated progerin, producing a less toxic protein.ResultsWe have found that in proliferating fibroblasts derived from HGPS patients the nuclear location of interphase chromosomes differs from control proliferating cells and mimics that of control quiescent fibroblasts, with smaller chromosomes toward the nuclear interior and larger chromosomes toward the nuclear periphery. For this study we have treated HGPS fibroblasts with farnesyltransferase inhibitors and analyzed the nuclear location of individual chromosome territories. We have found that after exposure to farnesyltransferase inhibitors mis-localized chromosome territories were restored to a nuclear position akin to chromosomes in proliferating control cells. Furthermore, not only has this treatment afforded chromosomes to be repositioned but has also restored the machinery that controls their rapid movement upon serum removal. This machinery contains nuclear myosin 1β, whose distribution is also restored after farnesyltransferase inhibitor treatment of HGPS cells.ConclusionsThis study not only progresses the understanding of genome behavior in HGPS cells but demonstrates that interphase chromosome movement requires processed lamin A.

The cell nucleus taking centre stage. Workshop on the Functional Organization of the Cell Nucleus

The third European Molecular Biology Organization (EMBO) workshop on the Functional Organization of the Cell Nucleus was held in Prague between 5 and 8 May 2006, and was organized by U. Aebi, I. Raska and W. Earnshaw. ![][1] The cell nucleus is a highly complex organelle that hosts the chromosomes and many distinct macromolecular domains. Knowing the functional organization of the nucleus is essential for understanding many of the cell functions that are important for health and disease. This field has progressed considerably towards revealing the structure, dynamics and organization of the nucleus, as was evident from the excellent talks and discussions at the third European Molecular Biology Organization (EMBO) workshop on the Functional Organization of the Cell Nucleus held in the beautiful city of Prague. The workshop was attended by 115 participants, and included 31 speakers and 32 shorter talks chosen from the posters. The chromosomes form discrete domains within the nucleus. Outside these domains, a compartmentalization activity might dynamically organize nuclear bodies and speckles (Cremer & Cremer, 2001). T. Cremer (Munich, Germany) reported that increased osmolarity caused the condensation of interphase chromosomes and enlargement of the interchromatin compartment. A return to normal osmotic conditions caused chromosomes to occupy their previous positions, indicating a structural ‘memory’ of both chromosome positions and the interchromatin compartment containing the nuclear bodies. At present, it is not known which structural elements are engaged in, and necessary for, this remarkable preservation of the topography of the chromatin ‘network’. In particular, detailed structural information on the chromosome surface and the space between individual chromosome territories is missing. Therefore, new optical microscopy and cryo‐electron tomography methods are urgently needed to unravel the molecular organization of this part of the nuclear architecture. C. Cremer (Heidelberg, Germany) described an improvement in optical resolution by spatially modulated illumination microscopy, which … [1]: /embed/graphic-1.gif

Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells

Using sequential RNA–DNA fluorescence in situ hybridization, the nuclear arrangement of both the active and inactive c-myc gene as well as its transcription was investigated in colon cancer HT-29 cells induced to differentiate into enterocytes. Cytogenetic studies revealed the presence of two chromosomes 8 in HT-29 cells, of which the one containing c-myc gene amplicons was substantially larger and easily distinguished from the normal chromosome. This observation enabled detection of both activity and nuclear localization of c-myc genes in single cells and in individual chromosome territories. Similar transcriptional activity of the c-myc gene was observed in both the normal and derivative chromosome 8 territories showing no influence of the amplification on the c-myc gene expression. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography.

The genome and the nucleus: a marriage made by evolution

Genomes are housed within cell nuclei as individual chromosome territories. Nuclei contain several architectural structures that interact and influence the genome. In this review, we discuss how the genome may be organised within its nuclear environment with the position of chromosomes inside nuclei being either influenced by gene density or by chromosomes size. We compare interphase genome organisation in diverse species and reveal similarities and differences between evolutionary divergent organisms. Genome organisation is also discussed with relevance to regulation of gene expression, development and differentiation and asks whether large movements of whole chromosomes are really observed during differentiation. Literature and data describing alterations to genome organisation in disease are also discussed. Further, the nuclear structures that are involved in genome function are described, with reference to what happens to the genome when these structures contain protein from mutant genes as in the laminopathies.

Non-random positioning of chromosomes in human sperm nuclei.

In human spermatozoa, the arrangement of chromosomes is non-random. Characteristic features are association of centromeres in the interior chromocenter and peripheral location of telomeres. In this paper, we have investigated the highest level of order in DNA packing in sperm--absolute and relative intranuclear chromosome positioning. Asymmetrical nuclear shape, existence of a defined spatial marker, and the haploid complement of chromosomes facilitated an experimental approach using in situ hybridization. Our results showed the tendency for non-random intranuclear location of individual chromosome territories. Moreover, centromeres demonstrated specific intranuclear position, and were located within a limited area of nuclear volume. Additionally, the relative positions of centromeres were non-random; some were found in close proximity, while other pairs showed significantly greater intercentromere distances. Therefore, a unique and specific adherence may exist between chromosomes in sperm. The observed chromosome order is discussed in relation to sperm nuclei decondensation, and reactivation during fertilization.

Visualizing chromatin and chromosomes in living cells

The dynamic organization of eukaryotic genomes in cell nuclei recently came into the focus of research interest. The kinetics of genome dynamics can be addressed only by approaches involving live cell microscopy. Different methods are available to visualize chromatin, specific chromatin fractions, or individual chromosome territories within nuclei of living mammalian cells. Appropriate labeling procedures as well as cell chamber systems and important controls for live cell microscopy are described.

Conservation of Relative Chromosome Positioning in Normal and Cancer Cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM−/− mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.

Spatial Distribution of GC- and AT-Rich DNA Sequences within Human Chromosome Territories

Previous topological analyses of DNA sequence organization in the interphase chromosome mainly focused on the spatial distribution of individual gene copies within chromosome territories. In order to achieve a more comprehensive view into the subchromosomal arrangement of DNA, we isolated the GC-richest/gene-richest fraction (H3 isochores) as well as AT-richest/gene-poorest fraction of human genomic DNA (L1+L2 isochores) and visualized the respective DNA within individual chromosome territories by means of dual-color FISH. Application of confocal laser scanning microscopy and dedicated 3D image analysis software, which differentiated territory subvolumes by peeling shells one voxel in width, revealed a significant difference in the intraterritorial distribution of these two DNA sequence classes. While the H3 isochores were found localized in all subvolumes of the territories at similar frequency, simultaneously detected L1+L2 isochores were observed more to the interior of the same chromosome territories. Thus the GC-rich sequences display a much higher variability in their intraterritorial localization than AT-rich DNA fragments.

Structure and dynamics of human interphase chromosome territories in vivo.

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5-10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.

Spatial organization of large-scale chromatin domains in the nucleus: a magnified view of single chromosome territories.

We have analyzed the spatial organization of large scale chromatin domains in chinese hamster fibroblast, human lymphoid (IM-9), and marsupial kidney epithelial (PtK) cells by labeling DNA at defined stages of S phase via pulsed incorporation of halogenated deoxynucleosides. Most, if not all, chromosomes contribute multiple chromatin domains to both peripheral and internal nucleoplasmic compartments. The peripheral compartment contains predominantly late replicating G/Q bands, whereas early replicating R bands preferentially localize to the internal nucleoplasmic compartment. During mitosis, the labeled chromatin domains that were separated in interphase form a pattern of intercalated bands along the length of each metaphase chromosome. The transition from a banded (mitotic) to a compartmentalized (interphasic) organization of chromatin domains occurs during the late telophase/early G1 stage and is independent of transcriptional activation of the genome. Interestingly, generation of micronuclei with a few chromosomes showed that the spatial separation of early and late replicating chromatin compartments is recapitulated independently of chromosome number, even in micronuclei containing only a single chromosome. Our data strongly support the notion that the compartmentalization of large-scale (band size) chromatin domains seen in the intact nucleus is a magnified image of a similar compartmentalization occurring in individual chromosome territories.