Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast

DNA double‐strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome‐wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.     

Spatial genome organization during T-cell differentiation

The spatial organization of genomes within the mammalian cell nucleus is non-random. The functional relevance of spatial genome organization might be in influencing gene expression programs as cells undergo changes during development and differentiation. To gain insight into the plasticity of genomes in space and time and to correlate the activity of specific genes with their nuclear position, we systematically analyzed the spatial genome organization in differentiating mouse T-cells. We find significant global reorganization of centromeres, chromosomes and gene loci during the differentiation process. Centromeres were repositioned from a preferentially internal distribution in undifferentiated cells to a preferentially peripheral position in differentiated CD4+ and CD8+ cells. Chromosome 6, containing the differentially expressed T-cell markers CD4 and CD8, underwent differential changes in position depending on whether cells differentiated into CD4+ or CD8+ thymocytes. Similarly, the two marker loci CD4 and CD8 showed distinct behavior in their position relative to the chromosome 6 centromere at various stages of differentiation. Our results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.     

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.     

Examination of interchromosomal interactions in vegetatively growing diploid Schizosaccharomyces pombe cells by Cre/loxP site-specific recombination

The probability with which different regions of a genome come in contact with one another is a question of general interest. The current study addresses this subject for vegetatively growing diploid cells of fission yeast Schizosaccharomyces pombe by application of the Cre/loxP site-specific recombination assay. High levels of allelic interactions imply a tendency for chromosomes to be colocalized along their lengths. Significant homology-dependent pairing at telomere proximal loci and robust nonspecific clustering of centromeres appear to be the primary determinants of this feature. Preference for direct homolog-directed interactions at interstitial chromosomal regions was ambiguous, perhaps as a consequence of chromosome flexibility and the constraints and dynamic nature of the nucleus. Additional features of the data provide evidence for chromosome territories and reveal an intriguing phenomenon in which interaction frequencies are favored for nonhomologous loci that are located at corresponding relative (rather than absolute) positions within their respective chromosome arms. The latter feature, and others, can be understood as manifestations of transient, variable, and/or occasional nonspecific telomeric associations. We discuss the factors whose interplay sets the probabilities of chromosomal interactions in this organism and implications of the inferred organization for ectopic recombination.     

Intrachromosomal rearrangements in avian genome evolution: evidence for regions prone to breakpoints

It is generally believed that the organization of avian genomes remains highly conserved in evolution as chromosome number is constant and comparative chromosome painting demonstrated there to be very few interchromosomal rearrangements. The recent sequencing of the zebra finch (Taeniopygia guttata) genome allowed an assessment of the number of intrachromosomal rearrangements between it and the chicken (Gallus gallus) genome, revealing a surprisingly high number of intrachromosomal rearrangements. With the publication of the turkey (Meleagris gallopavo) genome it has become possible to describe intrachromosomal rearrangements between these three important avian species, gain insight into the direction of evolutionary change and assess whether breakpoint regions are reused in birds. To this end, we aligned entire chromosomes between chicken, turkey and zebra finch, identifying syntenic blocks of at least 250 kb. Potential optimal pathways of rearrangements between each of the three genomes were determined, as was a potential Galliform ancestral organization. From this, our data suggest that around one-third of chromosomal breakpoint regions may recur during avian evolution, with 10% of breakpoints apparently recurring in different lineages. This agrees with our previous hypothesis that mechanisms of genome evolution are driven by hotspots of non-allelic homologous recombination.     

Centromere positioning and dynamics in living Arabidopsis plants

The organization and dynamics of the genome have been shown to influence gene expression in many organisms. Data from mammalian tissue culture cells have provided conflicting conclusions with regard to the extent to which chromatin organization is inherited from mother to daughter nuclei. To gain insight into chromatin organization and dynamics, we developed transgenic Arabidopsis lines in which centromeres were tagged with a green fluorescent protein fusion of the centromere-specific histone H3. Using four-dimensional (4-D) live cell imaging, we show that Arabidopsis centromeres are constrained at the nuclear periphery during interphase and that the organization of endoreduplicated sister centromeres is cell type dependent with predominant clustering in root epidermal cells and dispersion in leaf epidermal cells. 4-D tracking of the entire set of centromeres through mitosis, in growing root meristematic cells, demonstrated that global centromere position is not precisely transmitted from the mother cell to daughter cells. These results provide important insight into our understanding of chromatin organization among different cells of a living organism.     

Sperm Nuclear Architecture Is Locally Modified in Presence of a Robertsonian Translocation t(13;17)

In mammals, the non-random organization of the sperm nucleus supports an early function during embryonic development. Altering this organization may interfere with the zygote development and reduce fertility or prolificity. Thus, rare studies on sperm cells from infertile patients described an altered nuclear organization that may be a cause or a consequence of their respective pathologies. Thereby, chromosomal rearrangements and aneuploidy can be studied not only for their adverse effects on production of normal/balanced gametes at meiosis but also for their possible impact on sperm nuclear architecture and the epigenetic consequences of altered chromosome positioning. We decided to compare the global architecture of sperm nuclei from boars, either with a normal chromosome composition or with a Robertsonian translocation involving chromosomes 13 and 17. We hypothesized that the fusion between these chromosomes may change their spatial organization and we examined to what extend it could also modify the global sperm nuclear architecture. Analysis of telomeres, centromeres and gonosomes repartition does not support a global nuclear disorganization. But specific analysis of chromosomes 13 and 17 territories highlights an influence of chromosome 17 for the positioning of the fused chromosomes within the nucleus. We also observed a specific clustering of centromeres depending of the chromosome subtypes. Altogether our results showed that chromosome fusion does not significantly alter sperm nucleus architecture but suggest that centromere remodelling after chromosome fusion locally impacts chromosome positioning.     

Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast

Interactions between homologous chromosomes (pairing, recombination) are of central importance for meiosis. We studied entire chromosomes and defined chromosomal subregions in synchronous meiotic cultures of Schizosaccharomyces pombe by fluorescence in situ hybridization. Probes of different complexity were applied to spread nuclei, to delineate whole chromosomes, to visualize repeated sequences of centromeres, telomeres, and ribosomal DNA, and to study unique sequences of different chromosomal regions. In diploid nuclei, homologous chromosomes share a joint territory even before entry into meiosis. The centromeres of all chromosomes are clustered in vegetative and meiotic prophase cells, whereas the telomeres cluster near the nucleolus early in meiosis and maintain this configuration throughout meiotic prophase. Telomeres and centromeres appear to play crucial roles for chromosome organization and pairing, both in vegetative cells and during meiosis. Homologous pairing of unique sequences shows regional differences and is most frequent near centromeres and telomeres. Multiple homologous interactions are formed independently of each other. Pairing increases during meiosis, but not all chromosomal regions become closely paired in every meiosis. There is no detectable axial compaction of chromosomes in meiotic prophase. S. pombe does not form mature synaptonemal complexes, but axial element-like structures (linear elements), which were analyzed in parallel. Their appearance coincides with pairing of interstitial chromosomal regions. Axial elements may define minimal structures required for efficient pairing and recombination of meiotic chromosomes.     

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCF^Slimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.     

Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.     

Reactivation of an Inactive Centromere Reveals Epigenetic and Structural Components for Centromere Specification in Maize

Stable maize (Zea mays) chromosomes were recovered from an unstable dicentric containing large and small versions of the B chromosome centromere. In the stable chromosome, the smaller centromere had become inactivated. This inactive centromere can be inherited from one generation to the next attached to the active version and loses all known cytological and molecular properties of active centromeres. When separated from the active centromere by intrachromosomal recombination, the inactive centromere can be reactivated. The reactivated centromere regains the molecular attributes of activity in anaphase I of meiosis. When two copies of the dicentric chromosome with one active and one inactive centromere are present, homologous chromosome pairing reduces the frequency of intrachromosomal recombination and thus decreases, but does not eliminate, the reactivation of inactive centromeres. These findings indicate an epigenetic component to centromere specification in that centromere inactivation can be directed by joining two centromeres in opposition. These findings also indicate a structural aspect to centromere specification revealed by the gain of activity at the site of the previously inactive sequences.     

Chromosome arrangements in human fibroblasts at mitosis

Summary The positions of the centromeres of all 46 human chromosomes were analysed in three dimensional reconstructions of electron micrographs of 10 serially sectioned unpretreated human male fibroblast cells. The reconstructions show that the spatial positioning of the chromosomes during division is not random. The centromeres were arranged on a metaphase plate that was ellipsoidal and that tended to be flat. The distance of centromeres from the centre of the mitotic figure was correlated with chromosome size; small chromosomes tended to be central in all the metaphases. Large chromosomes were more peripheral, especially in cells that were more advanced in mitosis. Thus, there is a tendency for larger chromosomes to move outwards as metaphase advances. In many cells, the A group centromeres were overdispersed, whereas G group centromeres tended to be clustered. The acrocentric chromosomes (D and G groups) also tended to be clustered when analysed together, probably reflecting associations in nucleoli at the previous interphase. The results show that chromosome disposition is non-random and that it changes during division.     

Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.