Interphase Chromosomes Research Articles

CTCF and cohesin cooperate to organize the 3D structure of the mammalian genome

Genome function depends on its 3D organization. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct territories with respect to each other (1) (Fig. 1), and the spatial positioning of genomic segments affects ongoing DNA transactions. In recent years, the combination of advanced imaging techniques and new molecular approaches revealed the occurrence of previously underestimated long-range intrachromosomal and even interchromosomal interactions that have been linked to fundamental biological processes such as genomic imprinting (2), X-chromosome inactivation (3), and developmentally regulated programs (4). Therefore, considerable interest is growing around the study of 3D models of gene expression, focused on describing the fine-grain organization of chromatin architecture and the key players mediating and controlling long-range contacts. The development of Hi-C (high-throughput detection of chromosomal interactions) technology has markedly advanced the field and has revealed that chromosome territories are further arranged into large megabase-sized topological domains (5) that are highly conserved and stable across the cell type (6). In contrast, subregions within each domain are dynamic and may be responsible for cells' type-specific regulatory events (Fig. 1). As the boundaries between different domains dictate their spatial organization and function, understanding the contribution of factors that demarcate domain boundaries is of compelling importance. The work of Zuin et al. in PNAS explores the 3D architecture of the genome by studying the contributions of two leading players in loop organization: the CCCTC-binding factor (CTCF) and the cohesin complex (7). CTCF is a highly conserved protein with a unique structure that confers a versatile role in genome function (8). Through the 11 zinc fingers of its DNA-binding domain, …

Dissection of open chromatin domain formation by site specific recombination inDrosophila

Drosophila polytene interphase chromosomes provide an ideal test system to study the rules that define the structure of chromatin domains. We established a transgenic condensed chromatin domain cassette for the insertion of large pieces of DNA by site-specific recombination. Insertion of this cassette into open chromatin generated a condensed domain, visible as an extra band on polytene chromosomes. Site-specific recombination of DNA sequence variants into this ectopic band allowed us to compare their capacity for open chromatin formation by cytogenetic methods. We demonstrate that the 61C7-8 interband DNA maintains its open chromatin conformation and epigenetic state at an ectopic position. By deletion analysis, we mapped the sequences essential for open chromatin formation to a 490-bp fragment in the proximal part of the 17-kb interband sequence. This fragment overlaps binding sites for the chromatin protein Chriz (also known as Chro), the histone kinase Jil-1 and the boundary element protein CP190. It also overlaps a promoter region that locates between the Rev1 and Med30 transcription units.

Radiosensitivity of the moss Drepanocladus aduncus (Hedw.) Mnkm.

Radiosensitivity was determined in isolated fragments of <em>Drepanocladus aduncus</em> gametophytes cultured <em>in vitro</em>, on the basis of the growth reaction to acute gamma Co-60 radiation and postirradiation survival of the plants. A high resistivity of <em>D. aduncus</em> to this radiation was noted. At 12°C a 100 per cent LD was 120 kR and at 22°C it was 160 kR. The nuclear index of radiosensitivity (ICV - interphase chromosome volume) for various gametophyte cells has a value from 1.54 to 9.00 μm<sup>3</sup>. <em>Drepanocladus aduncus</em> plants exhibit postradiation developmental anomalies. In natural conditions they are characterised by an enhanced beta and gamma radiation activity. The plants contain Sr-90, Cs-137, much calcium, beryllium and lithium.

Polymer models of interphase chromosomes

Polymer models of interphase chromosomes

Chromosome conformation capture (3C) of tandem arrays in yeast.

Studying interphase chromosome arrangements at the molecular level can provide important details on the function and coordination of many metabolic processes that take place on DNA, such as transcription or DNA repair. The chromosome conformation capture (3C) methodology was originally developed in yeast to study the interphase organization of a single-yeast chromosome. 3C assays allow the identification of physical interactions between distant DNA segments and thus provide detailed information on the folding of chromatin in the native cellular state. Since its initial development, the technique has been used to advance our understanding of the folding of gene loci and has yielded important discoveries, like the demonstration that distant transcriptional regulatory elements interact with their target promoters through chromatin loops. The 3C method uses formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. After chromatin digestion and ligation of cross-linked fragments, the frequency of interaction between distant DNA elements can be quantified. However, the design, analysis, and controls used are critical when using 3C. In this chapter, we describe the general protocol for 3C analysis and demonstrate it through analysis of interactions in repetitive sequences of the ribosomal gene array of Saccharomyces cerevisiae.

Viscoelastic Behavior of Human Lamin A Proteins in the Context of Dilated Cardiomyopathy

Lamins are intermediate filament proteins of type V constituting a nuclear lamina or filamentous meshwork which lines the nucleoplasmic side of the inner nuclear membrane. This protein mesh provides a supporting scaffold for the nuclear envelope and tethers interphase chromosome to the nuclear periphery. Mutations of mainly A-type lamins are found to be causative for at least 11 human diseases collectively termed as laminopathies majority of which are characterised by aberrant nuclei with altered structural rigidity, deformability and poor mechanotransduction behaviour. But the investigation of viscoelastic behavior of lamin A continues to elude the field. In order to address this problem, we hereby present the very first report on viscoelastic properties of wild type human lamin A and some of its mutants linked with Dilated cardiomyopathy (DCM) using quantitative rheological measurements. We observed a dramatic strain-softening effect on lamin A network as an outcome of the strain amplitude sweep measurements which could arise from the large compliance of the quasi-cross-links in the network or that of the lamin A rods. In addition, the drastic stiffening of the differential elastic moduli on superposition of rotational and oscillatory shear stress reflect the increase in the stiffness of the laterally associated lamin A rods. These findings present a preliminary insight into distinct biomechanical properties of wild type lamin A protein and its mutants which in turn revealed interesting differences.

Models that include supercoiling of topological domains reproduce several known features of interphase chromosomes

Understanding the structure of interphase chromosomes is essential to elucidate regulatory mechanisms of gene expression. During recent years, high-throughput DNA sequencing expanded the power of chromosome conformation capture (3C) methods that provide information about reciprocal spatial proximity of chromosomal loci. Since 2012, it is known that entire chromatin in interphase chromosomes is organized into regions with strongly increased frequency of internal contacts. These regions, with the average size of ∼1 Mb, were named topological domains. More recent studies demonstrated presence of unconstrained supercoiling in interphase chromosomes. Using Brownian dynamics simulations, we show here that by including supercoiling into models of topological domains one can reproduce and thus provide possible explanations of several experimentally observed characteristics of interphase chromosomes, such as their complex contact maps.

Post-Irradiation Ageing Effect on Cytological Characters of Crotalaria Saltiana

Post-irradiation ageing effect on different cytological characters of Crotalaria saltiana were studied. Mitotic index followed no decreasing or increasing tendency based on the doses of gamma rays. Interphase chromosome volume was not found to increase with an increase of gamma rays. Percentage of pollen sterility was found to be lower in the lower doses and higher in the higher doses. Irregularities such as, formation of fragments, bridges, laggards, single and multiple chromatid bridges, unequal distribution and abnormal nuclear arrangement were recorded. This study reflects that irradiated seeds of Crotalaria saltiana showed no such dose wise effect and that might be due to ageing effect for long time. DOI: http://dx.doi.org/10.3329/rujs.v38i0.16551 Rajshahi University J. of Sci. 38, 87-95 (2010)

Architectural cohesin

Cohesin affects interphase chromosome architecture and transcription factor positioning.

Maintenance of Interphase Chromosome Compaction and Homolog Pairing in Drosophila Is Regulated by the Condensin Cap-H2 and Its Partner Mrg15

Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein–protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.

Wapl is an essential regulator of chromatin structure and chromosome segregation

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

Structural features of 3C6/C7 interband chromatin organization in Drosophila melanogaster polytene chromosomes

Mechanisms of chromatin decompaction in interbands of Drosophila polytene chromosomes have been studied. Using the example of interband 3C6/C7 of the X chromosome, we investigated the ability of different DNA segments to form an interband in a new genetic environment. We applied site-specific FLP recombination between two transposons with FRT-sites that allows introducing the DNA fragments from the interband 3C6/C7 into pICon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region caused by insertion of the DNA fragments into the transposon. It was shown that the insertion of a 276-bp DNA fragment from the 3C6/C7 region into the pICon(dv) transposon leads to the formation of a new interband between two thin bands represented by the transposon material. This DNA fragment is the known minimal sequence that is necessary and sufficient for interband generation. In addition, the sequence containing three copies repeated in tandem of 0.9 kb DNA from the interband 3C6/C7, including the 276-bp fragment, were integrated in the transposon. The presence of introduced DNA fragments did not change the morphology of the resulting interband. It was shown that the sites of DNase I hypersensitivity were saved in transposon sequences introduced into a new genetic environment. The data obtained allow analysis to be started of specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin in a certain interband region and chromomeric organization of interphase chromosomes in Drosophila as a whole.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells

The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500μm3), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000μm3) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255μm3 were detected on day 7. The data suggest a radiation-induced generation of large intra-nuclear chromosome territories and their repositioning prior to genome reduction.

The genome folding mechanism in yeast

The structure of the nucleosome has been solved at atomic resolution, and the genome-wide nucleosome positions have been clarified for the budding yeast Saccharomyces cerevisiae. However, the genome-wide three-dimensional arrangement of nucleosomal arrays in the nucleus remains unclear. Several studies simulated overall interphase chromosome architectures by introducing the putative persistence length of the controversial 30-nm chromatin fibres into the modelling and using data-fitting approaches. However, the genome-folding mechanism still could not be linked with the chromosome shapes, to identify which structures or properties of chromatin fibres or DNA sequences determine the overall interphase chromosome architectures. Here we demonstrate that the paths of nucleosomal arrays and the chromatin architectures themselves are determined principally by the physical properties of genomic DNA and the nucleus size in yeast. We clarified the flexibilities and persistence lengths of all linker DNAs of the organism, deduced their spatial expanses and simulated the architectures of all 16 interphase chromosomes in the nucleus, at a resolution of beads-on-a-string chromatin fibre. For the average spatial distance between two given loci in a chromosome, the model predictions agreed well with all experimental data reported to date. These findings suggest a general mechanism underlying the folding of eukaryotic genomes into interphase chromosomes.

SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2.

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF(Slimb) ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF(Slimb) function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF(Slimb)-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.

Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation—preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse β-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.

Effects of Fluctuation of Chromosome Conformation and Spatial Arrangement of Genes on the Pattern of Gene Expression

Recently, conformation of all chromosomes in the interphase nucleus of budding yeast has been inferred from the Chromosome Conformation Capture-on-Chip (4C) data (Duan et al., Nature (2010)). However, it has not yet been ascertained how the conformational fluctuation around their mean structures affects the regulation of gene expression. To clarify this issue, we developed a dynamical structural model of interphase chromosomes in budding yeast (Tokuda et al., Biophys. J. (2012)). In the present paper, the effects of the conformational fluctuation and the arrangement of genes on the pattern of gene expression are discussed by using this coarse-grained chromosome model. In particular, the observed difference in the pattern of gene expression between the yku70 esc1 mutant which abrogates telomere anchoring and the wild-type strain (Taddei et al., Genome Res. (2009)) is studied. In the data of Taddei et al., 32 genes are expressed at higher levels and 28 genes are expressed at lower levels in the yku70 esc1 mutant than in the wild-type strain. We examine the reason of this misregulation by comparing the fluctuation of the chromosome conformation and spatial arrangement of genes in the case that telomeres are not anchored to the nuclear periphery with that in the anchored case.

Role of polyteny and chromosome-membrane interactions in plant genetic processes

Existing data in the literature on polyteny in plants and in eukaryotes in general, data on the structure of interphase chromosomes and their association with the nuclear membrane are analyzed in the paper. In light of these data, the results of our studies on the ratios of phenotypic classes of marker enzymes in agamospermy (apomictic) progeny of sugar beet are considered. The given data allow to state that chromosome polyteny and their association with nuclear membrane provide not only hereditary information, but also determine the ratio of genotypes and phenotypes in progeny.