Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells

Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.     

Reorganization of chromatin in Xenopus egg extracts: electron microscopic studies.

The structural basis of mitotic condensation of chromosomes is one of the problems of cell biology yet to be elucidated. A variety of approaches have been used to study this problem and a large number of hypotheses have been proposed to explain the different levels of compaction of chromatin. Xenopus egg extracts, now widely used to study various aspects of cell biology, provide a valuable tool to study mitotic condensation of chromosomes. No detailed study has however yet been reported on the submicroscopic organization of condensed chromosomes in vitro in egg extracts. We present here the results of our electron microscopic studies on the organization of condensed chromosomes in vitro, using demembranated sperm nuclei and mitotic (CSF-arrested) extracts of Xenopus laevis eggs, clarified by high speed centrifugation. Upon introduction of sperm nuclei in egg extracts, the nuclei swell and the chromatin undergoes a rapid decondensation; at this stage the chromatin is formed of 10 nm fibrils. After longer incubation, the chromatin condenses, and by 2 h chromosomal structures can be visualized by staining with DAPI or Hoechst 33258. Our results on the organization of chromosomes in different stages of condensation are discussed in relation to the different hypotheses proposed to explain the process of mitotic condensation of chromosomes. Finally, this study demonstrates the feasibility of high-resolution analysis of the process of chromosome condensation.     

Metaphase chromosome structure. Involvement of topoisomerase II

SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.     

Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.     

Chromosome Architecture and Genome Organization

How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase.     

The mitotic chromosome is an assembly of rigid elastic axes organized by structural maintenance of chromosomes (SMC) proteins and surrounded by a soft chromatin envelope

The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis.     

Condensin: crafting the chromosome landscape

The successful transmission of complete genomes from mother to daughter cells during cell divisions requires the structural re-organization of chromosomes into individualized and compact structures that can be segregated by mitotic spindle microtubules. Multi-subunit protein complexes named condensins play a central part in this chromosome condensation process, but the mechanisms behind their actions are still poorly understood. An increasing body of evidence suggests that, in addition to their role in shaping mitotic chromosomes, condensin complexes have also important functions in directing the three-dimensional arrangement of chromatin fibers within the interphase nucleus. To fulfill their different functions in genome organization, the activity of condensin complexes and their localization on chromosomes need to be strictly controlled. In this review article, we outline the regulation of condensin function by phosphorylation and other posttranslational modifications at different stages of the cell cycle. We furthermore discuss how these regulatory mechanisms are used to control condensin binding to specific chromosome domains and present a comprehensive overview of condensin’s interaction partners in these processes.     

The three-dimensional structure of in vitro reconstituted <Emphasis Type="Italic">Xenopus laevis</Emphasis> chromosomes by EM tomography

We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30–40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30–40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30–40-nm chromatin domains. These observations suggest that 30–40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation. Electronic supplementary material The online version of this article (doi ) contains supplementary material, which is available to authorized users.     

Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes

Chromatin organization spans a wide range of structural complexity. Substructures at the 10-200nm scale are poorly characterized, especially in living cells, due to the limitations of electron microscopy and standard optical microscopy. Recently developed super-resolution fluorescence microscopy methods represent an exciting opportunity to access those substructures, and recent progress with these techniques has yielded insights into chromatin organization at different condensation stages. Recent studies have focused on confronting the challenges that are specific to chromatin super-resolution imaging, such as the high packing density of mitotic chromosomes and difficulties in interpreting interphase chromatin images. Building on these first results and with ongoing rapid technical advances in super-resolution fluorescence imaging there is great potential to uncover new features with unprecedented detail.     

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.     

Mitotic chromosomes are constrained by topoisomerase II–sensitive DNA entanglements

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II–specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.     

Chromatin globules: a common motif of higher order chromosome structure?

Recent technological advances in the field of chromosome conformation capture are facilitating tremendous progress in the ability to map the three-dimensional (3D) organization of chromosomes at a resolution of several Kb and at the scale of complete genomes. Here we review progress in analyzing chromosome organization in human cells by building 3D models of chromatin based on comprehensive chromatin interaction datasets. We describe recent experiments that suggest that long-range interactions between active functional elements are sufficient to drive folding of local chromatin domains into compact globular states. We propose that chromatin globules are commonly formed along chromosomes, in a cell type specific pattern, as a result of frequent long-range interactions among active genes and nearby regulatory elements. Further, we speculate that increasingly longer range interactions can drive aggregation of groups of globular domains. This process would yield a compartmentalized chromosome conformation, consistent with recent observations obtained with genome-wide chromatin interaction mapping.     

Structural Organization of Multiple Alphoid Subsets Coexisting on Human Chromosomes 1, 4, 5, 7, 9, 15, 18, and 19

FISH experiments on metaphase chromosomes, interphase nuclei, and extended chromatin were performed to investigate the structural organization of alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Results indicate that multiple subsets present on chromosomes 5, 7, 15, 18, and 19 are organized in structurally distinct and contiguous domains, while those on chromosomes 4 and 9 give perfectly overlapping signals. Chromosome 1 shows a peculiar organization: probe pAL1, specific for this chromosome, detects two distinct domains separated by the subset identified by probe pZ5.1. The order along the chromosome of alphoid subsets lying on chromosomes 5, 7, 15, 18, and 19, organized in distinct blocks, has also been established. The relationship between the structural organization of these alphoid sequences and their evolutionary history in great apes is discussed.     

The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis

During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization, we depleted from S2 cells the Drosophila CAP-H homologue Barren, a subunit exclusively associated with condensin I. In the absence of Barren/CAP-H the condensin core subunits DmSMC4/2 still associate with chromatin, while the other condensin I non-structural maintenance of chromosomes family proteins do not. Immunofluorescence and in vivo analysis of Barren/CAP-H-depleted cells showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that do not appear to be due to abnormal kinetochore-microtubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows structural problems. After bipolar attachment, the centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. Taken together, our data suggest that the condensin I complex is required not only to promote sister chromatid resolution but also to maintain the structural integrity of centromeric heterochromatin during mitosis.     

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.     

Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase. Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (lambda, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (means, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.     

Ultrastructure of the mitotic chromosomes in pig embryonic kidney cells during their reversible artificial decondensation in vivo

Using methods of in vivo observation and ultrathin sectioning, it is shown that chromosomes of metaphase PE cells, previously treated with diluted Henk's solutions (70, 30 and 15%), undergo some structural transitions resulting in the formation of micronuclei. At the early stages of hypotonic treatment chromosomes are seen considerably swollen and losing the higher levels of organization, including the chromonema and chromomeres. The chromosomal bodies are formed by DNP fibers 10-25 nm in diameter making loops radiating from the central part of the chromatids. Chromosomes are capable of recondensing from this state by consecutive reconstitution of G-bands, chromomeres and the chromonema. The subsequent secondary decondensation of chromosomes is analogous to telophase decondensation at the normal mitosis, but it results in the formation of a great number of small nuclei (micronuclei). The chromatin structure in micronuclei as well as their ability to synthesize RNA and to replicate DNA show these effects to be reversible. It has been suggested that the loop organization of DNP may be essential for sustaining the structural integrity of the mitotic chromosome.