Calreticulin as a new histone binding protein in mitotic chromosomes

Calreticulin (CRT) is a multifunctional Ca(2+)-binding protein that mainly functions in the endoplasmic reticulum as a molecular chaperone for newly synthesized proteins. Recently we reported the protein composition of human metaphase chromosomes (Uchiyama et al., 2004), which included CRT. Here we describe new characteristics of CRT in vitro as well as its localization on the surface of metaphase chromosomes in vivo. CRT was detected in the chromosomal fraction by Western blotting and its binding partners were identified as core and linker histones by ligand overlay assay. Surface plasmon resonance sensor analyses revealed that CRT is bound to chromatin fibers. Moreover, we found that CRT has both supercoiling activity, which assists core histone assembly into chromatin fibers, and binding ability to histone H2A/H2B dimers and histone H3/H4 tetramers. Unlike the chromosome scaffold proteins, indirect immunofluorescent staining revealed that CRT is located on the surface of metaphase chromosomes. These results suggest that CRT plays a role which involves chromatin dynamics on the surface of mitotic chromosomes.     

Coiled Supercoiled DNA in Critical Point Dried and Thin Sectioned Human Chromosome Fibres

SUPERCOILING of the DNA double helix in the deoxynucleo-histone fibril (100 Â in diameter with a pitch of 120 Â) has been well documented by X-ray diffraction^1–3. The main structural element of eukaryotic metaphase chromosomes and inactive interphase chromatin, however, is a long, irregularly folded fibre, 200 to 300 Â in diameter, as electron microscopy has shown^4–10. The arrangement of DNA within this fibre has not yet been established clearly. I wish to present electron micrographs of critical point dried and thin sectioned fibres of human metaphase chromosomes, which demonstrate more or less regularly distributed electron densities which could be explained by supercoiling of the 100 Â deoxynucleohistone fibril.     

Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (Zea mays L. ssp. parviglumis and Zea nicaraguensis) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome.     

Isolation,fractionation and biochemical analysis of the metaphase chromosomes of Microtus agrestis

A method has been developed for isolating metaphase chromosomes from Microtus agrestis fibroblasts in relatively large quantities with recovery of about 50% of the chromosomes present in the metaphase cells. The method employs pressure homogenisation to release the chromosomes from the cells. The average chemical composition of the Microtus chromosome preparations is 24.6% DNA, 19.9% RNA and 55.5% protein. The isolated chromosomes were fractionated by sedimentation velocity in a density gradient into three size groups in one of which 75–80% of the chromosomes were the large sex-chromosomes. The relative composition of this fraction containing most of the heterochromatin of the cell was DNA: 100, RNA: 59, acid-soluble protein: 54, acid-insoluble protein: 178. — Disc electrophoresis studies revealed no significant difference in the histone patterns between the euchromatic and heterochromatic chromosomes of the three chromosome size-groups. Metaphase chromosomes appear to have a lower lysine-rich histone content than interphase nuclei.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

The structure of histone-depleted metaphase chromosomes

We have previously shown that histone-depleted metaphase chromosomes can be isolated by treating purified HeLa chromosomes with dextran sulfate and heparin (Adolph, Cheng and Laemmli, 1977a). The chromosomes form fast-sedimenting complexes which are held together by a few nonhistone proteins.In this paper, we have studied the histone-depleted chromosomes in the electron microscope. Our results show that: the histone-depleted chromosomes consist of a scaffold or core, which has the shape characteristic of a metaphase chromosome, surrounded by a halo of DNA; the halo consists of many loops of DNA, each anchored in the scaffold at its base; most of the DNA exists in loops at least 10–30 μm long (30–90 kilobases).We also show that the same results can be obtained when the histones are removed from the chromosomes with 2 M NaCl instead of dextran sulfate. Moreover, the histone-depleted chromosomes are extraordinarily stable in 2 M NaCI, providing further evidence that they are held together by nonhistone proteins.These results suggest a scaffolding model for metaphase chromosome structure in which a backbone of nonhistone proteins is responsible for the basic shape of metaphase chromosomes, and the scaffold organizes the DNA into loops along its length.     

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Acetylation and deacetylation of histone H4 continue through metaphase with depletion of more-acetylated isoforms and altered site usage

Antibodies specific for acetylated isoforms of histone H4 have been used to compare acetylation of this histone in interphase and metaphase cells. Two rabbit antisera (R5 and R6) were used, each specific for H4 molecules acetylated at one of the four possible acetylation sites, namely Lys-5 (R6) and Lys-12 (R5). Both antisera bound preferentially to the more-acetylated H4 isoforms (H4Ac2-4). To test for continued H4 acetylation in metaphase chromosomes. Chinese hamster ovary cells were blocked in metaphase and treated for one hour with the deacetylase inhibitor sodium butyrate. Isolated chromosomes were assayed for H4 acetylation by antibody labeling and flow cytometry. H4 acetylation was increased several fold by this brief butyrate treatment. The increase was in direct proportion to DNA content, with no evidence for exceptionally high- or low-labeling chromosomes. The results demonstrate that a cycle of H4 acetylation and deacetylation continues within metaphase chromosomes. Immunofluorescence microscopy showed labeling to be distributed throughout the chromosome, but with variable intensity. Western blotting and immunostaining with R5 and R6 showed a net reduction in labeling of H4 from metaphase cells, with major reductions in the more-acetylated isoforms H4Ac3-4. In contrast, labeling of H4Ac1 was reduced to a lesser extent (R6) or increased (R5). This increase indicates more frequent use of the acetylation site at lysine 12 in H4Ac1 from metaphase cells.     

Delayed termination of nuclear histone doubling after premeiotic DNA synthesis inTriturus vulgaris male meiosis

It has been shown by means of double wavelength cytophotometry of DNA (Feulgen reaction) and histone (fast green, pH 8.2) inTriturus vulgaris spermatocytes that the doubling of DNA content in nuclei terminates at the end of preleptotene to beginning of leptotene whereas the doubling of histone content begun at premeiotic interphase is delayed and proceeds till the end of leptotene to beginning of zygotene. As a result preleptotene spermatocytes contain approximately 4C DNA and only 3C histone. Histone content in leptotene amounts to 93% of 4C, and in zygotene, pachytene and metaphase I both DNA and histone contents equal 4C. Thus, the temporal pattern of nucleo-histone doubling in meiotic chromosomes ofT. vulgaris differs from the synchronous DNA and histone doubling in mitotic chromosomes of all previously studied species. The delay of histone doubling inT. vulgaris meiocytes is less pronounced than in the previously studied insectsAcheta domestica andPyrrhocoris apterus where the histone content amounts to 3C in leptotene—zygotene and the equal histone/DNA ratio is restored only in pachytene.—Responsibilities for this phenomenon and its biolgoical sinnificance are discussed in connection with recent hypotheses concerning mechanisms of homologous chromosome pairing.     

Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes.

Histones have been extracted from isolated metaphase chromosomes prepared by the method of Wray and Sutbblefield [Exp. Cell Res 59, 469-478 (1970)] and by a Nonidet P-40 detergent procedure based on the method of Wigler and Axel [Nucleic Acids Res. 3, 1463-1471 (1976)]. Analysis of the densitometer profiles of long polyacrylamide gels shows that the mitotic phosphorylations of histone H1 (H1M) and histone H3 are extensively depleted during chromosome isolation. These data indicate that CHO metaphase chromosomes prepared by standard methodologies do not represent in vivo chromosomes with respect to their histone phosphorylations; therefore, current chemical and structural studies of isolated metaphase chromosomes may require further clarification.     

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.     

Phosphorylated histone H2AX foci persist on rejoined mitotic chromosomes in normal human diploid cells exposed to ionizing radiation

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.     

Presence of negative supercoiling in aggregates of histone H1-plasmidic polynucleosome complexes

It was shown in the past that in the presence of histone H1, plasmidic polynucleosomes formed densely packed aggregates. Our current studies demonstrate that these aggregates are susceptible to the actions of E. coli topoisomerase I, human topoisomerase I and DNA nicking enzyme, which is the indication that negative supercoiling is present in the condensed DNA-protein complexes. Since negative supercoiling leads to formation of highly curved and compact plectonemic and toroidal DNA structures, it would be reasonable to assume that DNA negative supercoils are responsible for aggregation of histone H1-plasmidic polynucleosome complexes.     

Effects of acetic acid-alcohol,trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes

Summary In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irrversibly changed the conformation of HI and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73–212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.     

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col E1 DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assembled in the presence of the H1 histone, polylysine (Mr 20 000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.