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PubMed2002

Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation.

Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

Cell Cycle Proteins↗
PubMed2026

Mitotic chromosomes: from the chromosome scaffold model to condensins and physical forces.

Mitotic chromosome organization and assembly remain fundamental questions in genetics. Since the chromosome scaffold model proposed in 1977 highlighted the role of nonhistone proteins in determining chromosome shape and size, key nonhistone proteins, including condensins and topoisomerase IIα (topoIIα), have been shown to play critical roles in organizing chromosome axes and chromatin loops. Emerging evidence from biochemistry, imaging, and genomics suggests that mitotic chromosome assembly is a dynamic process driven by the interplay of condensin-mediated looping, topoIIα-dependent entanglement/disentanglement, and multiple physical forces, including electrostatic nucleosome interactions, linker histone H1, free Mg2+, and depletion attraction. In this review, we discuss how these mechanisms contribute to chromosome assembly and propose that interphase chromatin domains function as dynamic building blocks of mitotic chromosomes.

chromatin compaction↗
PubMed1998

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: factors affecting elution of DNA from nucleoids.

The nuclear matrix is increasingly identified with the processing of DNA damage. Previous work has suggested that association of DNA with the matrix can influence the repair of DNA double-strand breaks (DSBs) and the sensitivity of mammalian cells to ionizing radiation. By selectively examining DSBs that occur as multiples (multiple DSBs) within looped DNA structures, we have identified a subset of DSBs that repair with slow kinetics through the V(D)J recombination-associated DSB repair pathway. Enrichment of S-phase populations by centrifugal elutriation and selective examination of nascent DNA by pulse-labeling were used to demonstrate that elution of DNA from nucleoids is retarded by the presence of replicating DNA. Previously, application of a Poisson-based model of induction of multiple DSBs and DNA elution to a panel of mammalian cell lines indicated that the size of the looped chromatin domains varied between cell lines. The data presented here explain the range in domain sizes between cells as the result of differences in the percentage of cells actively replicating their DNA. Correction of the model to account for S-phase populations results in a looped domain size of 2.9 Mbp independent of cell type. Single-cell gel electrophoresis of nucleoids provides additional evidence for such sized structures. Stabilization of DNA to elution during S phase does not permit repair of DSBs in the DSB repair mutants xrs5 and St.SCID, both defective for the DSB repair pathway associated with V(D)J recombination.

Animals↗
PubMed1991

Chromosome structure and eukaryotic gene organization.

The DNA in the eukaryotic nucleus is highly compacted but well organized into distinct regional units. Chromosomal bands are characterized by their structure and distinctive replication time. They are subdivided into chromatin loops which serve as functional domains that have discrete boundary elements and can be regulated during development.

Animals↗
PubMed2004

Dynamics of DNA replication: an ultrastructural study.

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.

Binding Sites↗
PubMed2000

Androcam, a Drosophila calmodulin-related protein, is expressed specifically in the testis and decorates loop kl-3 of the Y chromosome.

The Drosophila genome encodes a protein that is 68% identical to Drosophila calmodulin (Cam). We show here that this Cam-related gene is specifically expressed in the germ-line of the testis, leading to the name Androcam (Acam). Early in spermatogenesis Acam accumulates on one of the chromatin loops of the Y chromosome, kl-3. This association with kl-3 may indicate an RNA processing-related role for Acam and/or could reflect an unusual storage/assembly function hypothesized for the Y loops. After meiosis Acam is detectable in developing sperm tail cytoplasm, where at least some of the protein is not tightly associated with tubulin. Late in spermiogenesis, some Acam staining overlaps the periphery of the investment cones, actin-containing structures hypothesized to support the motor function for cytoplasmic stripping of the tail. Acam cannot be detected in mature sperm by immunolocalization, but immunoblotting established that Acam is present in sperm stored in mated females, suggesting epitope masking during final maturation. Proteins more related to Acam than Cam are present in the testes of other Drosophila species and a mammalian species, the mouse.

Animals↗
PubMed2003

DNA supercoiling in apoptotic chromatin.

In a previous paper, we have reported that in rat thymocyte apoptosis chromatin undergoes a specific structural change as well as an appreciable increase in the unacetylated forms of histones H3 and H4. Here, we show that H3 and H4 deacetylation bears no relation to chromatin condensation, and present new ultrastructural and topological observations that largely clarify the organization of the condensed state. The texture of the latter corresponds to a closely woven network of negatively supercoiled 11 nm fibers, as shown by both ultrastructural observations and relaxation experiments using ethidium bromide. Circularly closed chromatin loops undergoing apoptotic condensation, clearly showing nucleosome compact dimers or higher oligomers, as well as long stretches of supercoiled DNA, have also been detected. All of these modifications are strongly reminiscent of the alterations induced in nucleosome bearing plasmids by the chromatin remodeling factors SWI/SNF and RSC.

Animals↗
PubMed2006

Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Animals↗
PubMed2003

Micromechanical studies of mitotic chromosomes.

We review micromechanical experiments studying mechanoelastic properties of mitotic chromosomes. We discuss the history of this field, starting from the classic in vivo experiments of Nicklas (1983). We then focus on experiments where chromosomes were extracted from prometaphase cells and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have a well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic "spraying" of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e., that the native state is not maximally compacted. We compare our results for chromosomes from cells to results of experiments by Houchmandzadeh and Dimitrov (1999) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of reconstituted chromosomes is similar to that observed for chromosomes from cells, reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure that is quite different from chromosomes in somatic cells. Finally, we discuss microspraying experiments of DNA-cutting enzymes, which reveal that the element that gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous "scaffold," but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction enzyme digestion experiments indicate a spacing between chromatin "cross-links" of roughly 15 kb, a size similar to that inferred from classical chromatin loop isolation studies. These results suggest a general strategy for the use of micromanipulation methods for the study of chromosome structure.

Animals↗
PubMed2025

SHICEDO: single-cell Hi-C data enhancement with reduced over-smoothing.

MOTIVATION: Single-cell Hi-C (scHi-C) technologies have significantly advanced our understanding of the 3D genome organization. However, scHi-C data are often sparse and noisy, leading to substantial computational challenges in downstream analyses. RESULTS: In this study, we introduce SHICEDO, a novel deep-learning model specifically designed to enhance scHi-C contact matrices by imputing missing or sparsely captured chromatin contacts through a generative adversarial framework. SHICEDO leverages the unique structural characteristics of scHi-C matrices to derive customized features that enable effective data enhancement. Additionally, the model incorporates a channel-wise attention mechanism to mitigate the over-smoothing issue commonly associated with scHi-C enhancement methods. Through simulations and real-data applications, we demonstrate that SHICEDO outperforms the state-of-the-art methods, achieving superior quantitative and qualitative results. Moreover, SHICEDO enhances key structural features in scHi-C data, thus enabling more precise delineation of chromatin structures such as A/B compartments, TAD-like domains, and chromatin loops. AVAILABILITY AND IMPLEMENTATION: SHICEDO is publicly available at https://github.com/wmalab/SHICEDO.

Single-Cell Analysis↗
PubMed1996

DNA supercoiling changes and nucleoid protein composition in a group of L5178Y cells of varying radiosensitivity.

Cell of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-1 and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage.

Animals↗
PubMedDate not supplied

[Acquired radioresistance of progeny of irradiated cells is accompanied by rearrangements in chromatin organization].

gamma-Irradiation action within a dose range of 0-20 Gy on parental djungarian hamster fiborblasts, DH-TK- cell line, and the progenies of these irradiated cells, surviving acute exposure to 20 Gy irradiation, PIC-20 cell line, was examined. The PICs were 3 times more radioresistant than the parental cells as calculated from D0. Using a method of anomalous viscosity time dependence (AVTD) it was revealed that starting (initial) level (in untreated cells) of chromatin compactness in radioresistant progenies was more than 1.4 times as high as for parental cells. The analysis of dose dependence has shown that irradiation with a dose of 5 Gy resulted in complete chromatin loop relaxation in radiosensitive DH-TK- cells and partial one in radioresistant PIC-20 cells. Besides, the beginning of DNA-membrane complexes degradation following the irradiation with doses over 15 Gy in DH-TK- cells was observed. It was shown that the increased state of relative chromatin relaxation in PIC-20 cells determines an increasing in reparation effectiveness that resulted in lower percent of residual damages in these cells. Using the Nosern hybridization method the expression level of mts 1, tag 7 and vseap 1 genes was studied. It is revealed that tag 7 and vseap 1 gene expression in radioresistant cells were correspondingly 6 and 10 times higher than in radiosensitive parental cells and the level of mts 1 gene expression was not changed. So, based on the results obtained we suggest that acquired radioresistance in progenies of irradiated cells is determined by rearrangements in chromatin structure and accompanied constitutive changes of gene expression.

Adaptation, Physiological↗
PubMed1995

SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein.

SARs are candidate DNA elements for defining the bases of chromatin loops and possibly for serving as cis elements of chromosome dynamics. SARs contain numerous A tracts, whose altered DNA structure is recognized by cooperatively interacting proteins such as topoisomerase II. We constructed multi-AT hook (MATH) proteins and demonstrate that they specifically bind the clustered A tracts of SARs in chromatin and chromosomes. They are also potent inhibitors of chromosome assembly in mitotic Xenopus extracts, demonstrating the importance of SARs in this process. Titration of SARs with MATH20 (20 hooks) blocks shape determination of chromatids but not chromatin condensation per se. SARs are also required for shape maintenance of chromosomes. If MATH20 is added after formation of chromatids, they collapse and are reshaped by an active, mitotic process into spherical chromatid balls.

Adenine↗
PubMed1986

Organization within the mammalian kinetochore.

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25-30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.

Animals↗
PubMed2005

In silico and wet-bench identification of nuclear matrix attachment regions.

Chromatin loops are tethered at discrete regions that are approx 100-1000 bp in length. These regions of attachment serve as specific sequence landmarks, anchoring the DNA to the fibers of the chromosomal scaffold. It has been estimated that our genome contains 70,000 nuclear matrix attachment sites that serve as a dynamic nuclear organizer in both the interphase and metaphase cell. Approximately 30,000-40,000 matrix attachment regions (MARs) serve as origins of replication. MARs can also be associated with chromosomal segments densely populated with transcription factor-binding sites. This may facilitate transcription that is initiated within the region of the chromosome coincident with the surface of the nuclear matrix. Assuming an average somatic loop size of 100 kb, it is reasonable to propose that each cell utilizes 30,000 MARs to anchor each of the approx 20,000 active genic domains. This is sufficient to encompass the 30,000 functional genes in our genome that exist as members of single or multigenic families, each constituting a single chromatin domain. With the sequencing phase of various genome projects complete, in silico tools are being developed to identify the long-range control elements that modulate gene expression. This information is necessary to specifically target the time-intensive wet-bench verification and expression experiments that will provide a unified understanding of gene regulation. In this chapter we review some of the in silico strategies that are currently available and a new in vivo method based on the real-time polymerase chain reaction, to assess regions of matrix association.

Actins↗
PubMed1994

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.

Animals↗
PubMed2004

Progress towards understanding the nature of chromatid breakage.

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.

Animals↗
PubMed2000

XMR is associated with the asynapsed segments of sex chromosomes in the XY body of mouse primary spermatocytes.

The XMR (Xlr-related, meiosis-regulated) protein is an M(r) 30,000 nuclear protein closely associated with the XY body in mouse primary spermatocytes. It shows sequence similarity with several other meiosis-specific proteins. In the present study, we investigated the fine immunolocalization of XMR in the XY body by laser confocal and electron microscopy. It was found that XMR was associated with the asynapsed segments of sex chromosomes, including their axes and the surrounding chromatin loops. In contrast, the pseudoautosomal region and the opposite free end of the X were unlabeled for XMR. In mice with the reciprocal T(X;16)16H translocation, XMR was also associated with the heterochromatic translocation product that emerges from the XY body. These findings at the subchromosomal level point to a role for XMR in chromatin condensation and transcriptional inactivation. XMR is unique among proteins in being capable of association with the XY body. It could play a specific role in a mechanism of male X-chromosome inactivation in mammals.

Animals↗