Preferential somatic pairing between homologous heterochromatic regions of human chromosomes.

The cytidine analog 5-azacytidine (5-azaC) induces an undercondensation of the heterochromatin in human chromosomes 1, 9, 15, 16, and Y when it is added in low concentrations to the late S-phase of growing lymphocyte cultures. In interphase nuclei, these heterochromatic regions are frequently somatically paired. The somatic pairing configurations are preserved up to metaphase stage in the 5-azaC-treated cultures and are thus susceptible to a direct microscopical examination. The statistical analysis of 1,000 somatic pairing configurations from 5-azaC-treated cells showed that the somatic pairing between the heterochromatic regions of homologous chromosomes is preferred over that between nonhomologous chromosomes.     

Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata

In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.     

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.     

Evidence for homologous pairing of chromosomes prior to meiotic prophase in maize

Relative positions of homologous heterochromatic regions of maize chromosomes were studied at premeiotic interphase, at tapetal mitotic interphase and at root tip mitotic interphase. In all three kinds of cell homologues were found to be situated significantly nearer to each other than to heterologues. It is concluded that some degree of homologous chromosome pairing may occur widely at anaphase or telophase (where it is easily overlooked) and that therefore, as has been previously suggested, homologues may be loosely aligned throughout premeiotic interphase in preparation for their subsequent synapsis.     

A direct demonstration of somatically paired heterochromatin of human chromosomes

Human lymphocyte cultures were treated with different concentrations of 5-azacytidine for various lengths of time. This cytosine analog induces very distinct undercondensation in the heterochromatin of chromosomes 1, 9, 15, 16, and Y if applied in low doses during the last hours of culture. These regions are further distinguished by their intense distamycin A/DAPI-staining and highly methylated DNA. In interphase nuclei, these heterochromatic regions are frequently somatically paired. These somatic pairings are preserved up to the metaphase stage in the 5-azacytidine-treated cultures and are thus susceptible to direct analysis. The specific effect of 5-azacytidine on the heterochromatin of these chromosomes, its conserving effect on somatic pairing, and some of the consequences of the somatic pairing on the development of human chromosome aberrations are discussed.     

Differing requirements for RAD51 and DMC1 in meiotic pairing of centromeres and chromosome arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.     

Centric-fusion translocation and whole-arm heterochromatin in the karyotype of the blue fox (Alopex lagopus L.): synaptonemal complex analysis.

Conventional observations of mitotic chromosomes from two male blue foxes, revealing a centric-fusion translocation and whole-arm heterochromatin, were verified by synaptonemal complex analysis. This analysis revealed that the centric fusion had been preceded by a conspicuous loss of chromosome material in the two one-armed chromosomes involved, but the chromosomal origin of the centric-fusion kinetochore could not be established. The nontranslocated chromosomes of the trivalent, which in all cells but one were in cis configuration, had reached by early pachytene a stage in which almost complete homologous pairing and nonhomologous association or pairing of the free ends of the chromosomes could be observed. In later stages, complete pairing of the nontranslocated chromosomes with the corresponding arms of the centric-fusion translocation was seen occasionally. One to six autosomal bivalents demonstrated unpaired heterochromatic arms in early pachytene, and the heterochromatic chromosome arms were sometimes unpaired even in late pachytene. Some of them showed a distinct size heteromorphism in late zygotene and early pachytene. In most late-pachytene cells, however, the heteromorphic chromosomes were completely length-adjusted. Only a small fraction of the cells showed pairing interference between nonhomologous chromosomes.     

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.     

Pairing of heterochromatin in response to cellular stress

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Discontinuities and unsynapsed regions in meiotic chromosomes have a trans effect on meiotic recombination of some chromosomes in human males

During meiosis, homologous chromosome pairing and synapsis are essential for subsequent meiotic recombination (crossing-over). Discontinuous regions (gaps) and unsynapsed regions (splits) were most frequently observed in the heterochromatic regions of bivalent synaptonemal complex (SC) 9, and we have previously demonstrated that gaps and splits significantly altered the distribution of MLH1 recombination foci on SC 9. Here, immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with a centromere-specific fluorescence in situ hybridization technique that allows identification of every individual chromosome. The effect of gaps/splits on meiotic recombination patterns in autosomes other than chromosome 9 during the pachytene stage of meiotic prophase was then examined in 6,026 bivalents from 262 pachytene cells from three human males. In 64 analyzed cells with a gapped SC 9, the frequency of MLH1 foci in SCs 5 and 10 and in SC arms 10q, 11p and 16q was decreased compared to 168 analyzed cells with a normally-synapsed SC 9 (controls). In 24 analyzed cells with splits in SC 9, there was a significant reduction in MLH1 focus frequency for SC 5q and the whole SC5 bivalent. The positioning of MLH1 foci on other SCs in cells with gapped/split SC 9 was not altered. These studies suggest that gaps and splits not only have a cis effect, but may also have a trans effect on meiotic recombination in humans.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

The positioning of rye homologous chromosomes added to wheat through the cell cycle in somatic cells untreated and treated with colchicine

The arrangement of chromosome pairs 5RL and 7R added to the wild type and the ph1b mutant line of hexaploid wheat are analyzed in 2N somatic root tip cells during the cell cycle relative to the arrangement that chromosomes 5RL show in 4N tapetal cells produced after colchicine treatment. Both homologous chromosome pairs are identified at interphase and mitosis by fluorescence in situ hybridization. In nuclei at interphase, chromosomes appear as discrete domains that show the Rabl orientation. Homologous chromosomes are predominantly non-associated and their positioning seems not to be influenced by the Ph1 gene that suppresses homoeologous meiotic pairing. This pattern of arrangement contrasts with the high level of somatic pairing that sister chromosomes show in the interphase that follows chromosome duplication induced by colchicine. Disruption of pairing observed in some 4N nuclei is produced at c-anaphase which suggests no topological redistribution of homologues during conformation of the new nucleus. Homologous chromosomes show no predominant arrangement in ellipsoidal metaphase plates, which contrasts with the preferential opposite location of homologues in human prometaphase rosettes. Differences between chromosomes in the variation of the length through the cell cycle and in the chromatin morphology when the Ph1 is absent suggest different patterns of chromatin condensation in both chromosomes.     

Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

Chromosome pairing does not contribute to nuclear architecture in vegetative yeast cells

There are several reports of a closer-than-random colocalization of homologous chromosomes in the vegetative nuclei of diploid budding yeast. Here, we studied by fluorescence in situ hybridization (FISH) the nuclear distribution of chromosomes and found a slight tendency toward closer proximity between homologous (allelic) loci than between any nonhomologous chromosomal regions. We show that most of this preferential association is not due to vegetative (also known as somatic) pairing but is caused by the polar orientation of interphase chromosomes (Rabl orientation). We quantified the occasional loss of detectable fluorescence signals that is inherent to the FISH method. Signal loss leads to the occurrence of a single signal that may be misinterpreted as the close association of two homologous chromosomal sites. The nuclear distribution of homologous loci, when corrected for the influence of nuclear architecture and methodological faults, was not different or was only marginally different from a random relative positioning as predicted by computer simulation. We discuss here several possibilities for the residual homologous proximity that do not invoke homology-dependent vegetative pairing, and we conclude that, in diploid budding yeast, constitutive vegetative pairing is a negligible factor for the organization of the interphase nucleus.     

Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only ∼2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.     

Mitomycin C induced structural changes in heterochromatic regions of human chromosomes 1, 9, 16 and Y

Aberrations and variations in the heterochromatic blocks of chromosomes 1, 9, 16 and Y were found under the influence of mitomycin C in cultured lymphocytes of peripheral human blood. Lymphocytes were cultured during 96 hours, mitomycin C in final concentration of 0.3 mkg/ml was present in the culture during the latest 24 hours of culturing. Different changes in the heterochromatic regions of chromosomes were found in approximately 30% of cells: in 6.3% of cells mitotic chiasmata were indicated. In 9.5% of cells isolocus breaks were observed in heterochromatic region of chromosome 1 in segment 1q11. In the latter case this may be a fragile site detected under the influence of mitomycin C on the lymphocytes.     

Somatic synapsis of eu- and heterochromatic regions of Drosophila melanogaster chromosomes

Quantitative cytogenetical analysis has been used to study the synapsis of D. melanogaster neuroblast mitotic chromosomes from normal females, flies with heterozygous deletions, duplications or inversions in the heterochromatic regions of chromosome 2 and in triploid females. In all these genotypes chromocentric fusion of heterochromatic regions of heterologous chromosomes is observed. Eu- and heterochromatic regions of homologous chromosomes are intimately paired at the same time during the cell cycle. The structural rearrangements lead to reduced frequencies of chromocentric association as well as of homologous synapsis compared with the frequencies in the wild-type. The results obtained are discussed with respect to the general problem of the homologous interaction of chromosomes and the significance of heterochromatin for these processes.     

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.