Modeling properties of chromosome territories using polymer filaments in diverse confinement geometries.

Interphase chromosomes reside within distinct nuclear regions known as chromosome territories (CTs). Recent observations from Hi-C analyses, a method mapping chromosomal interactions, have revealed varied decay in contact probabilities among different chromosomes. Our study explores the relationship between this contact decay and the particular shapes of the chromosome territories they occupy. For this, we employed molecular dynamics (MD) simulations to examine how confined polymers, resembling chromosomes, behave within different confinement geometries similar to chromosome territory boundaries. Our simulations unveil so far unreported relationships between contact probabilities and end-to-end distances varying based on different confinement geometries. These findings highlight the crucial impact of chromosome territories on shaping the larger-scale properties of 3D genome organization. They emphasize the intrinsic connection between the shapes of these territories and the contact behaviors exhibited by chromosomes. Understanding these correlations is key to accurately interpret Hi-C and microscopy data, and offers vital insights into the foundational principles governing genomic organization.

Establishing of 3D-FISH on frozen section and its applying in chromosome territories analysis in Populus trichocarpa.

KEY MESSAGE: Fluorescence in situ hybridization with frozen sections of root tips showed difference of chromosome territories distribution between autosome and sex-chromosome homologous pairs in Populus trichocarpa. The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biologic processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. The distribution of chromosome pairs 17 and 19 in the 3D-preserved nuclei of P. trichocarpa root tip cells were analyzed and showed that the autosome pair 17 associated more often than sex chromosome 19. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar.

Time to match; when do homologous chromosomes become closer?

In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing process begins, it is still under investigation. Contrasting data exists in Mus musculus, since both leptotene DSB-dependent and preleptotene DSB-independent mechanisms have been described. To unravel this contention, we examined homologous pairing in pre-meiotic and meiotic Mus musculus cells using a three-dimensional fluorescence in situ hybridization-based protocol, which enables the analysis of the entire karyotype using DNA painting probes. Our data establishes in an unambiguously manner that 73.83% of homologous chromosomes are already paired at premeiotic stages (spermatogonia-early preleptotene spermatocytes). The percentage of paired homologous chromosomes increases to 84.60% at mid-preleptotene-zygotene stage, reaching 100% at pachytene stage. Importantly, our results demonstrate a high percentage of homologous pairing observed before the onset of meiosis; this pairing does not occur randomly, as the percentage was higher than that observed in somatic cells (19.47%) and between nonhomologous chromosomes (41.1%). Finally, we have also observed that premeiotic homologous pairing is asynchronous and independent of the chromosome size, GC content, or presence of NOR regions.

Large-scale topological disruption of chromosome territories 9 and 22 is associated with nonresponse to treatment in CML.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm defined by the presence of t(9;22) translocation whose origin has been associated with the tridimensional genome organization. This rearrangement leads to the fusion of BCR and ABL1 genes giving rise to a chimeric protein with constitutive kinase activity. Imatinib, a tyrosine kinase inhibitor (TKI), is used as a first-line treatment for CML, though ~40% of CML patients do not respond. Here, using structured illumination microscopy (SIM) and 3D reconstruction, we studied the 3D organization patterns of the ABL1 and BCR genes, and their chromosome territories (CTs) CT9 and CT22, in CD34+ cells from CML patients that responded or not to TKI. We found that TKI resistance in CML is associated with high levels of structural disruption of CT9 and CT22 in CD34+ cells, increased CT volumes (especially for CT22), intermingling between CT9 and CT22, and an open-chromatin epigenetic mark in CT22. Altogether our results suggest that large-scale disruption of CT9 and CT22 correlates with the clinical response of CML patients, which could be translated into a potential prognostic marker of response to treatment in this disease and provide novel insights into the mechanisms underlying resistance to TKI in CML.

Chromosomal positioning in spermatogenic cells is influenced by chromosomal factors associated with gene activity, bouquet formation and meiotic sex chromosome inactivation.

Chromosome territoriality is not random along the cell cycle and it is mainly governed by intrinsic chromosome factors and gene expression patterns. Conversely, very few studies have explored the factors that determine chromosome territoriality and its influencing factors during meiosis. In this study, we analysed chromosome positioning in murine spermatogenic cells using three-dimensionally fluorescence in situ hybridization-based methodology, which allows the analysis of the entire karyotype. The main objective of the study was to decipher chromosome positioning in a radial axis (all analysed germ-cell nuclei) and longitudinal axis (only spermatozoa) and to identify the chromosomal factors that regulate such an arrangement. Results demonstrated that the radial positioning of chromosomes during spermatogenesis was cell-type specific and influenced by chromosomal factors associated to gene activity. Chromosomes with specific features that enhance transcription (high GC content, high gene density and high numbers of predicted expressed genes) were preferentially observed in the inner part of the nucleus in virtually all cell types. Moreover, the position of the sex chromosomes was influenced by their transcriptional status, from the periphery of the nucleus when its activity was repressed (pachytene) to a more internal position when it is partially activated (spermatid). At pachytene, chromosome positioning was also influenced by chromosome size due to the bouquet formation. Longitudinal chromosome positioning in the sperm nucleus was not random either, suggesting the importance of ordered longitudinal positioning for the release and activation of the paternal genome after fertilisation.

Microchromosomes Exhibit Distinct Features of Vertebrate Chromosome Structure and Function with Underappreciated Ramifications for Genome Evolution.

Microchromosomes are common yet poorly understood components of many vertebrate genomes. Recent studies have revealed that microchromosomes contain a high density of genes and possess other distinct characteristics compared with macrochromosomes. Whether distinctive characteristics of microchromosomes extend to features of genome structure and organization, however, remains an open question. Here, we analyze Hi-C sequencing data from multiple vertebrate lineages and show that microchromosomes exhibit consistently high degrees of interchromosomal interaction (particularly with other microchromosomes), appear to be colocalized to a common central nuclear territory, and are comprised of a higher proportion of open chromatin than macrochromosomes. These findings highlight an unappreciated level of diversity in vertebrate genome structure and function, and raise important questions regarding the evolutionary origins and ramifications of microchromosomes and the genes that they house.

Ribosomal DNA and the Nucleolus as Keystones of Nuclear Architecture, Organization, and Function.

The multicopy ribosomal DNA (rDNA) array gives origin to the nucleolus, a large nonmembrane-bound organelle that occupies a substantial volume within the cell nucleus. The rDNA/nucleolus has emerged as a coordinating hub in which seemingly disparate cellular functions converge, and from which a variety of cellular and organismal phenotypes emerge. However, the role of the nucleolus as a determinant and organizer of nuclear architecture and other epigenetic states of the genome is not well understood. We discuss the role of rDNA and the nucleolus in nuclear organization and function - from nucleolus-associated domains (NADs) to the regulation of imprinted loci and X chromosome inactivation, as well as rDNA contact maps that anchor and position the rDNA relative to the rest of the genome. The influence of the nucleolus on nuclear organization undoubtedly modulates diverse biological processes from metabolism to cell proliferation, genome-wide gene expression, maintenance of epigenetic states, and aging.

Functional interactions between scaffold proteins, noncoding RNAs, and genome loci induce liquid-liquid phase separation as organizing principle for 3-dimensional nuclear architecture: implications in cancer.

The eukaryotic cell nucleus consists of functionally specialized subcompartments. These nuclear subcompartments are biomolecular aggregates built of proteins, transcripts, and specific genome loci. The structure and function of each nuclear subcompartment are defined by the composition and dynamic interaction between these 3 components. The spatio-temporal localization of biochemical reactions into membraneless nuclear subcompartments can be achieved through liquid-liquid phase separation. Based on this organizing principle, nuclear subcompartments are droplet-like structures that adopt spherical shapes, flow, and fuse like liquids or gels. In the present review, we bring into the spotlight seminal works elucidating the functional interactions between scaffold proteins, noncoding RNAs, and genomic loci, thereby inducing liquid-liquid phase separation as an organizing principle for 3-dimensional nuclear architecture. We also discuss the implications in different cancer types as well as the potential use of this knowledge to develop novel therapeutic strategies against cancer.-Rubio, K., Dobersch, S., Barreto, G. Functional interactions between scaffold proteins, noncoding RNAs, and genome loci induce liquid-liquid phase separation as organizing principle for 3-dimensional nuclear architecture: implications in cancer.

Intermingling of chromosome territories.

The importance of higher order nuclear structure and compartmentalization for the control of the cell life is now indisputable. The genome of higher eukaryotes is organized into definite chromosome territories, and the three-dimensional organization of these territories may be intently related to genomic function, global regulation of gene expression, and even formation of exchange aberrations. In this review, we discuss our current understanding of the chromosome territories phenomenon and briefly describe how genes relocation in three-dimensional arrangement of the genome may influence their functioning. We explain how the intermingling of the edges of chromosome territories allows the formation of rare long-range interchromosomal interactions. Moreover, we illustrate recent discoveries describing the mechanisms of physical proximity-based chromosome translocations and its clinical consequence for fusion genes formation and tumor development. Finally, we characterize the inner structure of the intermingled chromosomes briefly, and explain how chromosome intermingling affects gene expression regulation.

Chromatin dynamics governed by a set of nuclear structural proteins.

During the past three decades, the study of nuclear and chromatin organization has become of great interest. The organization and dynamics of chromatin are directly responsible for many functions including gene regulation, genome replication, and maintenance. In order to better understand the details of these mechanisms, we need to understand the role of specific proteins that take part in these processes. The genome in the nucleus is organized in different length scales, ranging from the bead-like nucleosomes through topological associated domains up to chromosome territories. The mechanisms that maintain these structures, however, remain to be fully elucidated. Previous works highlighted the significance of lamin A, an important nucleoplasmic protein; however, there are other nuclear structural proteins that are also important for chromatin organization. Studying the organizational aspects of the nucleus is a complex task, and different methods have been developed and adopted for this purpose, including molecular and imaging methods. Here we describe the use of the live-cell imaging method and demonstrate that the dynamics of the nucleus is strongly related to its organizational mechanisms. We labeled different genomic sites in the nucleus and measured the effect of nuclear structural proteins on their dynamics. We studied lamin A, BAF, Emerin, lamin B, CTCF, and Cohesin and discuss how each of them affect chromatin dynamics. Our findings indicate that lamin A and BAF have a significant effect on chromosomes dynamics, while other proteins mildly affect the type of the diffusion while the volume of motion is not affected.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells.

We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is three-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis, and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

Chromosomal aneuploidies induced upon Lamin B2 depletion are mislocalized in the interphase nucleus.

Chromosome territories assume non-random positions in the interphase nucleus with gene-rich chromosomes localized toward the nuclear interior and gene-poor chromosome territories toward the nuclear periphery. Lamins are intermediate filament proteins of the inner nuclear membrane required for the maintenance of nuclear structure and function. Here, we show using whole-genome expression profiling that Lamin A/C or Lamin B2 depletion in an otherwise diploid colorectal cancer cell line (DLD1) deregulates transcript levels from specific chromosomes. Further, three-dimensional fluorescence in situ hybridization (3D-FISH) analyses of a subset of these transcriptionally deregulated chromosome territories revealed that the diploid chromosome territories in Lamin-depleted cells largely maintain conserved positions in the interphase nucleus in a gene-density-dependent manner. In addition, chromosomal aneuploidies were induced in ~25 % of Lamin A/C or Lamin B2-depleted cells. Sub-populations of these aneuploid cells consistently showed a mislocalization of the gene-rich aneuploid chromosome 19 territory toward the nuclear periphery, while gene-poor aneuploid chromosome 18 territory was mislocalized toward the nuclear interior predominantly upon Lamin B2 than Lamin A/C depletion. In addition, a candidate gene locus ZNF570 (Chr.19q13.12) significantly overexpressed upon Lamin B2 depletion was remarkably repositioned away from the nuclear lamina. Taken together, our studies strongly implicate an overarching role for Lamin B2 in the maintenance of nuclear architecture since loss of Lamin B2 relieves the spatial positional constraints required for maintaining conserved localization of aneuploid chromosome territories in the interphase nucleus.

Three-dimensional organization and dynamics of the genome.

Genome is a complex hierarchical structure, and its spatial organization plays an important role in its function. Chromatin loops and topological domains form the basic structural units of this multiscale organization and are essential to orchestrate complex regulatory networks and transcription mechanisms. They also form higher-order structures such as chromosomal compartments and chromosome territories. Each level of this intrinsic architecture is governed by principles and mechanisms that we only start to understand. In this review, we summarize the current view of the genome architecture on the scales ranging from chromatin loops to the whole genome. We describe cell-to-cell variability, links between genome reorganization and various genomic processes, such as chromosome X inactivation and cell differentiation, and the interplay between different experimental techniques.

Chromosome positioning and male infertility: it comes with the territory.

The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility.

Reorganization of the interchromosomal network during keratinocyte differentiation.

The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression.

Altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers.

PURPOSE: The study aims to determine whether there is an altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers. METHODS: Metaphase I human spermatocytes from three 45,XY,der(13;14)(q10;q10) individuals and a 45,XY,der(14;15)(q10;q10) individual were analyzed. Proximity relationships of bivalents were established by analyzing meiotic preparations combining Leishman staining and multiplex-FISH procedures. Poisson regression model was used to determine proximity frequencies between bivalents and to assess associations with chromosome size, gene density, acrocentric morphology, and chromosomes with heterochromatic blocks. The hierarchical cluster Ward method was used to characterize the groups of bivalents with preferred proximities in a cluster analysis. Bivalent groups obtained were individually compared with those obtained in normal karyotype individuals evaluated in a previous study. RESULTS: A total of 1288 bivalents were examined, giving a total of 2289 proximity data. Only four positive significant proximities were detected for each type of Robertsonian translocation. Significant bivalent associations were only observed by small-size chromosomes for MI,22,XY,III(13q14q). These results were clearly divergent from 46,XY individuals. Moreover, cluster analysis revealed that about 30 % of the bivalents showed changes in their proximity relationships in metaphase I. CONCLUSIONS: The territorial organization of bivalents in metaphase I human spermatocytes changes in the presence of a Robertsonian translocation.

Aneuploidy: the impact of chromosome imbalance on nuclear organization and overall genome expression.

The organization and dynamics of chromatin within the interphase nucleus as chromosome territories (CTs) and the relationship with transcriptional regulation are not fully understood. We studied a natural example of chromosomal disorganization: aneuploidy due to trisomies 13, 18 and 21. We hypothesized that the presence of an extra copy of one chromosome alters the CT distribution, which perturbs transcriptional activity. We used 3D-FISH to study the position of the chromosomes of interest (18 and 21) in cultured amniocytes and chorionic villus cells from pregnancies with a normal or aneuploid karyotype. We studied the volumes of nuclei and CTs in both conditions and performed a compared transcriptome analysis. We did not observe any differences between euploid and aneuploid cells in terms of the radial and relative CT positions, suggesting that the same rules govern nuclear organization in cases of trisomy. We observed lower volumes for CTs 18 and 21. Overall genome expression profiles highlighted changes in the expression of a subset of genes in trisomic chromosomes, while the majority of transcriptional changes concerned genes located on euploid chromosomes. Our results suggest that a dosage imbalance of the genes on trisomic chromosomes is associated with a disturbance of overall genomic expression.

The arrangement of Brachypodium distachyon chromosomes in interphase nuclei.

The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biological processes, such as DNA replication, transcription, and repair. However, detailed data about the CT arrangement in monocotyledonous plants are scarce. In this study, chromosome painting was used to analyse the distribution and associations of individual chromosomes in the 3-D preserved nuclei of Brachypodium distachyon root cells in order to determine the factors that may have an impact on the homologous CT arrangement. It was shown that the frequency of CT association is linked to the steric constraints imposed by the limited space within the nucleus and may depend on chromosome size and morphology as well as on the nuclear shape. Furthermore, in order to assess whether the distribution of interphase chromosomes is random or is subject to certain patterns, a comparison between the experimental data and the results of a computer simulation (ChroTeMo), which was based on a fully probabilistic distribution of the CTs, was performed. This comparison revealed that homologous chromosome arm CTs associate more often than if they were randomly arranged inside the interphase nucleus.

Profiling Chromosome Topological Features by Super-Resolution 3D Structured Illumination Microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) and fluorescence in situ hybridization on three-dimensional preserved cells (3D-FISH) have proven to be robust and efficient methodologies for analyzing nuclear architecture and profiling the genome's topological features. These methods have allowed the simultaneous visualization and evaluation of several target structures at super-resolution. In this chapter, we focus on the application of 3D-SIM for the visualization of 3D-FISH preparations of chromosomes in interphase, known as Chromosome Territories (CTs). We provide a workflow and detailed guidelines for sample preparation, image acquisition, and image analysis to obtain quantitative measurements for profiling chromosome topological features. In parallel, we address a practical example of these protocols in the profiling of CTs 9 and 22 involved in the translocation t(9;22) in Chronic Myeloid Leukemia (CML). The profiling of chromosome topological features described in this chapter allowed us to characterize a large-scale topological disruption of CTs 9 and 22 that correlates directly with patients' response to treatment and as a possible potential change in the inheritance systems. These findings open new insights into how the genome structure is associated with the response to cancer treatments, highlighting the importance of microscopy in analyzing the topological features of the genome.