Organization Of Interphase Chromosomes 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.

Transcription-induced active forces suppress chromatin motion

The organization of interphase chromosomes in a number of species is starting to emerge thanks to advances in a variety of experimental techniques. However, much less is known about the dynamics, especially in the functional states of chromatin. Some experiments have shown that the motility of individual loci in human interphase chromosome decreases during transcription and increases upon inhibiting transcription. This is a counterintuitive finding because it is thought that the active mechanical force (F) on the order of ten piconewtons, generated by RNA polymerase II (RNAPII) that is presumably transmitted to the gene-rich region of the chromatin, would render it more open, thus enhancing the mobility. We developed a minimal active copolymer model for interphase chromosomes to investigate how F affects the dynamical properties of chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of F and are enhanced at small F values, which has also been observed in experiments. In the intermediate F, the bond length between consecutive loci increases, becoming commensurate with the distance at the minimum of the attractive interaction between nonbonded loci. This results in a transient disorder-to-order transition, leading to a decreased mobility during transcription. Strikingly, the F-dependent change in the locus dynamics preserves the organization of the chromosome at [Formula: see text]. Transient ordering of the loci, which is not found in the polymers with random epigenetic profiles, in the gene-rich region might be a plausible mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin.

Cyclic-polymer grafted colloids in spherical confinement: insights for interphase chromosome organization

Interphase chromosomes are known to organize non-randomly in the micron-sized eukaryotic cell nucleus and occupy certain fraction of nuclear volume, often without mixing. Using extensive coarse-grained simulations, we model such chromosome structures as colloidal particles whose surfaces are grafted by cyclic polymers. This model system is known as Rosetta. The cyclic polymers, with varying polymerization degrees, mimic chromatin loops present in interphase chromosomes, while the rigid core models the chromocenter section of the chromosome. Our simulations show that the colloidal chromosome model provides a well-separated particle distribution without specific attraction between the chain monomers. As the polymerization degree of the grafted cyclic chains decreases while maintaining the total chromosomal length (e.g. the more potent activity of condensin-family proteins), the average chromosomal volume becomes smaller, inter-chromosomal contacts decrease, and chromocenters organize in a quasi-crystalline order reminiscent of a glassy state. This order weakens for polymer chains with a characteristic size on the order of the confinement radius. Notably, linear-polymer grafted particles also provide the same chromocenter organization scheme. However, unlike linear chains, cyclic chains result in less contact between the polymer layers of neighboring chromosome particles, demonstrating the effect of DNA breaks in altering genome-wide contacts. Our simulations show that polymer-grafted colloidal systems could help decipher 3D genome architecture along with the fractal globular and loop-extrusion models.

Genomes, repeatomes and interphase chromosome organization in the meadowfoam family (Limnanthaceae, Brassicales)

SUMMARYThe meadowfoam family (Limnanthaceae) is one of the smallest and genomically underexplored families of the Brassicales. The Limnanthaceae harbor about seven species in the genus Limnanthes (meadowfoam) and Floerkea proserpinacoides (false mermaidweed), all native to North America. Because all Limnanthes and Floerkea species have only five chromosome pairs, i.e., a chromosome number rare in Brassicales and shared with Arabidopsis thaliana (Arabidopsis), we examined the Limnanthaceae genomes as a potential model system. Using low‐coverage whole‐genome sequencing data, we reexamined phylogenetic relationships and characterized the repeatomes of Limnanthaceae genomes. Phylogenies based on complete chloroplast and 35S rDNA sequences corroborated the sister relationship between Floerkea and Limnanthes and two major clades in the latter genus. The genome size of Limnanthaceae species ranges from 1.5 to 2.1 Gb, apparently due to the large increase in DNA repeats, which constitute 60–70% of their genomes. Repeatomes are dominated by long terminal repeat retrotransposons, while tandem repeats represent only less than 0.5% of the genomes. The average chromosome size in Limnanthaceae species (340–420 Mb) is more than 10 times larger than in Arabidopsis (32 Mb). A three‐dimensional fluorescence in situ hybridization analysis demonstrated that the five chromosome pairs in interphase nuclei of Limnanthes species adopt the Rabl‐like configuration.

Nuclear organization in crucifer genomes: nucleolus-associated telomere clustering is not a universal interphase configuration in Brassicaceae.

Arabidopsis thaliana has become a major plant research model, where interphase nuclear organization exhibits unique features, including nucleolus-associated telomere clustering. The chromocenter (CC)-loop model, or rosette-like configuration, describes intranuclear chromatin organization in Arabidopsis as megabase-long loops anchored in, and emanating from, peripherally positioned CCs, with those containing telomeres associating with the nucleolus. To investigate whether the CC-loop organization is universal across the mustard family (crucifers), the nuclear distributions of centromeres, telomeres and nucleoli were analyzed by fluorescence in situ hybridization in seven diploid species (2n = 10-16) representing major crucifer clades with an up to 26-fold variation in genome size (160-4260 Mb). Nucleolus-associated telomere clustering was confirmed in Arabidopsis (157 Mb) and was newly identified as the major nuclear phenotype in other species with a small genome (215-381 Mb). In large-genome species (2611-4264 Mb), centromeres and telomeres adopted a Rabl-like configuration or dispersed distribution in the nuclear interior; telomeres only rarely associated with the nucleolus. In Arabis cypria (381 Mb) and Bunias orientalis (2611 Mb), tissue-specific patterns deviating from the major nuclear phenotypes were observed in anther and stem tissues, respectively. The rosette-like configuration, including nucleolus-associated telomere clustering in small-genome species from different infrafamiliar clades, suggests that genomic properties rather than phylogenetic position determine the interphase nuclear organization. Our data suggest that nuclear genome size, average chromosome size and degree of longitudinal chromosome compartmentalization affect interphase chromosome organization in crucifer genomes.

The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

Chromosome compaction and chromatin stiffness enhance diffusive loop extrusion by slip-link proteins.

We use Brownian dynamics simulations to study the formation of chromatin loops through diffusive sliding of slip-link-like proteins, mimicking the behaviour of cohesin molecules. We recently proposed that diffusive sliding is sufficient to explain the extrusion of chromatin loops of hundreds of kilo-base-pairs (kbp), which may then be stabilised by interactions between cohesin and CTCF proteins. Here we show that the flexibility of the chromatin fibre strongly affects this dynamical process, and find that diffusive loop extrusion is more efficient on stiffer chromatin regions. We also show that the dynamics of loop formation are faster in confined and collapsed chromatin conformations but that this enhancement is counteracted by the increased crowding. We provide a simple theoretical argument explaining why stiffness and collapsed conformations favour diffusive extrusion. In light of the heterogeneous physical and conformational properties of eukaryotic chromatin, we suggest that our results are relevant to understand the looping and organisation of interphase chromosomes in vivo.

Conformational heterogeneity in human interphase chromosome organization reconciles the FISH and Hi-C paradox

Hi-C experiments are used to infer the contact probabilities between loci separated by varying genome lengths. Contact probability should decrease as the spatial distance between two loci increases. However, studies comparing Hi-C and FISH data show that in some cases the distance between one pair of loci, with larger Hi-C readout, is paradoxically larger compared to another pair with a smaller value of the contact probability. Here, we show that the FISH-Hi-C paradox can be resolved using a theory based on a Generalized Rouse Model for Chromosomes (GRMC). The FISH-Hi-C paradox arises because the cell population is highly heterogeneous, which means that a given contact is present in only a fraction of cells. Insights from the GRMC is used to construct a theory, without any adjustable parameters, to extract the distribution of subpopulations from the FISH data, which quantitatively reproduces the Hi-C data. Our results show that heterogeneity is pervasive in genome organization at all length scales, reflecting large cell-to-cell variations.

Gene dunce localization in the polytene chromosome of Drosophila melanogaster long span batch of adjacent chromosomal structures

The molecular and chromosomal localization of the dunce gene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is located. On the basis of the results of FISH analysis of the gene fragments, we established that the dunce gene is located within nine chromosomal structures (four bands and five interbands), which contradicts the common idea that genes are located in only one structure (band or interband) or at the boundary of these structures. Our results are quite unexpected and original and greatly expand the current understanding of the genetic organization of interphase chromosomes.

Gene dunce Localization in the Polytene Chromosome of Drosophila melanogaster Long Span Batch of Adjacent Chromosomal Structures.

The molecular and chromosomal localization of the dunce gene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is located. On the basis of the results of FISH analysis of the gene fragments, we established that the dunce gene is located within nine chromosomal structures (four bands and five interbands), which contradicts the common idea that genes are located in only one structure (band or interband) or at the boundary of these structures. Our results are quite unexpected and original and greatly expand the current understanding of the genetic organization of interphase chromosomes.

Banding Pattern of Polytene Chromosomes as a Representation of Universal Principles of Chromatin Organization into Topological Domains.

Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.

Structural-Functional Domains of the Eukaryotic Genome.

It is well known that DNA folding in the eukaryotic cell nucleus is tightly coupled with the operation of epigenetic mechanisms defining the repertoires of the genes expressed in different types of cells. To understand these mechanisms, it is important to know how DNA is packaged in chromatin. About 30 years ago a hypothesis was formulated, according to which epigenetic mechanisms operate not at the level of individual genes, but rather groups of genes localized in structurally and functionally isolated genomic segments that were called structural and functional domains. The question of what exactly these domains constitute has been re-examined multiple times as our knowledge of principles of chromatin folding has changed. In this review, we discuss structural and functional genomic domains in light of the current model of interphase chromosome organization based on the results of analysis of spatial proximity between remote genomic elements.

BIANCA, a biophysical model of cell survival and chromosome damage by protons, C-ions and He-ions at energies and doses used in hadrontherapy

An upgraded version of the BIANCA II biophysical model, which describes more realistically interphase chromosome organization and the link between chromosome aberrations and cell death, was applied to V79 and AG01522 cells exposed to protons, C-ions and He-ions over a wide LET interval (0.6–502 keV µm−1), as well as proton-irradiated U87 cells. The model assumes that (i) ionizing radiation induces DNA ‘cluster lesions’ (CLs), where by definition each CL produces two independent chromosome fragments; (ii) fragment (distance-dependent) mis-rejoining, or un-rejoining, produces chromosome aberrations; (iii) some aberrations lead to cell death. The CL yield, which mainly depends on radiation quality but is also modulated by the target cell, is an adjustable parameter. The fragment un-rejoining probability, f, is the second, and last, parameter. The value of f, which is assumed to depend on the cell type but not on radiation quality, was taken from previous studies, and only the CL yield was adjusted in the present work.Good agreement between simulations and experimental data was obtained, suggesting that BIANCA II is suitable for calculating the biological effectiveness of hadrontherapy beams. For both V79 and AG01522 cells, the mean number of CLs per micrometer was found to increase with LET in a linear-quadratic fashion before the over-killing region, where a less rapid increase, with a tendency to saturation, was observed. Although the over-killing region deserves further investigation, the possibility of fitting the CL yields is an important feature for hadrontherapy, because it allows performing predictions also at LET values where experimental data are not available.Finally, an approach was proposed to predict the ion-response of the cell line(s) of interest from the ion-response of a reference cell line and the photon response of both. A pilot study on proton-irradiated AG01522 and U87 cells, taking V79 cells as a reference, showed encouraging results.

The 10-nm chromatin fiber and its relationship to interphase chromosome organization.

A chromosome is a single long DNA molecule assembled along its length with nucleosomes and proteins. During interphase, a mammalian chromosome exists as a highly organized supramolecular globule in the nucleus. Here, we discuss new insights into how genomic DNA is packaged and organized within interphase chromosomes. Our emphasis is on the structural principles that underlie chromosome organization, with a particular focus on the intrinsic contributions of the 10-nm chromatin fiber, but not the regular 30-nm fiber. We hypothesize that the hierarchical globular organization of an interphase chromosome is fundamentally established by the self-interacting properties of a 10-nm zig-zag array of nucleosomes, while histone post-translational modifications, histone variants, and chromatin-associated proteins serve to mold generic chromatin domains into specific structural and functional entities.

Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo.

High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.

Chromosome biology: How to build a cohesive genome in 3D.

Removing the protein complex cohesin from chromosomes destroys one layer of the genome's 3D structure but leaves another intact. Genome structure is therefore built by independent processes that work together. See Letter p.51 The nuclear organization of interphase chromosomes is thought to be mediated by architectural protein complexes such as CTCF and cohesin, which are found at loops and at the boundaries of topological domains (TADs). However, experimental depletion of these proteins has shown limited impact on chromosome organization. Here, Francois Spitz and colleagues perform an inducible deletion of the cohesin-loading factor Nipbl in liver cells in mice. They find that depletion of chromosome-associated cohesin leads to the loss of TADs and TAD-associated loops, but segregation of the genome into compartments is preserved and transcription is affected only at a subset of genes. The disappearance of TADs unmasks a finer compartment structure that reflects local transcriptional activity. Genome organization therefore seems to result from two distinct mechanisms with different requirements for cohesin.

Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation.

Mammalian chromosomes are organized in structural and functional domains of 0.1–10 Mb, which are characterized by high self-association frequencies in the nuclear space and different contact probabilities with nuclear sub-compartments. They exhibit distinct chromatin modification patterns, gene expression levels and replication timing. Recently, nucleolus-associated chromosomal domains (NADs) have been discovered, yet their precise genomic organization and dynamics are still largely unknown. Here, we use nucleolus genomics and single-cell experiments to address these questions in human embryonic fibroblasts during replicative senescence. Genome-wide mapping reveals 1,646 NADs in proliferating cells, which cover about 38% of the annotated human genome. They are mainly heterochromatic and correlate with late replicating loci. Using Hi-C data analysis, we show that interactions of NADs dominate interphase chromosome contacts in the 10–50 Mb distance range. Interestingly, only minute changes in nucleolar association are observed upon senescence. These spatial rearrangements in subdomains smaller than 100 kb are accompanied with local transcriptional changes. In contrast, large centromeric and pericentromeric satellite repeat clusters extensively dissociate from nucleoli in senescent cells. Accordingly, H3K9me3-marked heterochromatin gets remodelled at the perinucleolar space as revealed by immunofluorescence analyses. Collectively, this study identifies connections between the nucleolus, 3D genome structure, and cellular aging at the level of interphase chromosome organization.

Nucleosomal arrays self-assemble into supramolecular globular structures lacking 30-nm fibers.

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg2+‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in the absence of MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.

Chromosomes at Work: Organization of Chromosome Territories in the Interphase Nucleus.

The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear-structure building blocks in directly supporting nuclear function in a physiologically responsive manner.

Chromosome Conformation of Human Fibroblasts Grown in 3-Dimensional Spheroids

In the study of interphase chromosome organization, genome-wide chromosome conformation capture (Hi-C) maps are often generated using 2-dimensional (2D) monolayer cultures. These 2D cells have morphological deviations from cells that exist in 3-dimensional (3D) tissues in vivo, and may not maintain the same chromosome conformation. We used Hi-C maps to test the extent of differences in chromosome conformation between human fibroblasts grown in 2D cultures and those grown in 3D spheroids. Significant differences in chromosome conformation were found between 2D cells and those grown in spheroids. Intra-chromosomal interactions were generally increased in spheroid cells, with a few exceptions, while inter-chromosomal interactions were generally decreased. Overall, chromosomes located closer to the nuclear periphery had increased intra-chromosomal contacts in spheroid cells, while those located more centrally had decreased interactions. This study highlights the necessity to conduct studies on the topography of the interphase nucleus under conditions that mimic an in vivo environment.