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.     

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.     

A search for protein cores in chromosomes: Is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.     

A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.     

Higher order metaphase chromosome structure: Evidence for metalloprotein interactions

One level of DNA organization in metaphase chromosomes is brought about by a scaffolding structure that is stabilized by metalloprotein interactions. Fast-sedimenting, histone-depleted structures (4000–7000 S), derived from metaphase chromosomes by extraction of the histones, are dissociated by metal chelators or by thiol reagents. The chromosomal (scaffolding) proteins responsible for constraining the DNA in this fast-sedimenting form are solubilized under the same conditions. Chromosomes isolated in a metal-depleted form, which generate slow-sedimenting, histone-depleted structures, can be specifically and reversibly stabilized by Cu²⁺, but not by Mn²⁺, Co²⁺, Zn²⁺ or Hg²⁺. Metal-depleted chromosomes can also be stabilized by Ca²⁺ (at 37°C), but this effect is less specific than that of Cu²⁺. The scaffolding protein pattern that is reproducibly generated following treatment with Cu²⁺ is composed primarily of two high molecular weight proteins—Sc1 and Sc2 (170,000 and 135,000 daltons). The identification of this simple protein pattern has depended upon the development of new chromosome isolation methods that are highly effective in eliminating cytoskeletal contamination.     

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.     

Metaphase chromosome structure: evidence for a radial loop model.

Electron micrographs of thin sections of metaphase chromosomes isolated from HeLa cells provide new insight into the higher-order arrangement of the nucleoprotein fiber. Micrographs obtained from chromosomes swollen by chelation of the divalent cation are particularly revealing. Under these conditions, chromosomes swell in width by a factor of about 4 and the basic, thick nucleoprotein fiber (200–300 Å) relaxes to the thin fiber (100 Å), which is probably a linear array of nucleosomes. Cross sections show a central area from which the fibers emerge in a radial fashion, often forming loops which are 3–4 μm long. Chromosomes fixed in the presence of 1 mM MgCl₂ are more compact, having an average chromatid diameter of about 1 μm, and consist of the thick (200–300 Å) fiber. Radial loops of about 0.6 μm can be observed frequently in these chromosomes, although the loops are more difficult to visualize due to the compactness of the structure and the material contaminating the periphery. Chromosomes isolated with the help of hexylene glycol are extremely compact (diameter about 0.6 μm) but quite free of cytoplasmic material. They consist of a 500 Å fiber that forms rather regular projections at the periphery. These projections appear to be loops of the thick fiber (200–300 Å), possibly shortened by twisting into a short supercoil. The chromatin loops observed in the intact chromosomes are thought to be structurally related to the DNA loops observed previously in the histone-depleted chromosomes (Paulson and Laemmli, 1977). In this paper, we discuss a model in which the nucleoprotein fiber is folded into loops which are arranged in the chromatid in radial fashion, in such a way that their bases become the central axis of the chromatid.     

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.     

Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.     

ADP-ribosylation of metaphase and interphase nonhistones using [3H]adenosine as a radioactive label

ADP-ribosylation of HeLa nonhistone proteins was investigated by using [3H]adenosine as an in vivo radioactive label. The aim was to determine basic differences in the patterns of modification of interphase and metaphase nonhistones. Fluorography revealed a relatively small number of modified proteins for isolated metaphase chromosomes. In addition to the core histones, a protein of 116 kDa, which is identified as poly-(ADP-ribose) polymerase, was a primary acceptor of [3H]adenosine. Two-dimensional gels revealed a profound difference in the modification of metaphase and interphase nonhistones. For interphase nuclei, 3H label was distributed among a large number of nonhistone acceptors.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.     

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.     

The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.     

Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000-52 000 and 65 000-72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Electron microscopic study of changes in chromosomal structural organization under the effect of formamide]

Isolated human metaphase chromosomes were treated with formamide at different (0-70%) concentrations and examined electronmicroscopically by protein monolayer technique. At increasing formamide concentration chromosomes gradually decondense, the scaffold becomes more clearly visible, the loops of chromatin fibres coming off the central part of chromosomes lose their nucleosomal appearance. Electrophoretic analysis of chromosomal proteins data show that formamide-treated chromosomes have approximately the same histone content as those before treatment, although chromosomes treated with 70% formamide look very similar to histone-depleted ones described elsewhere.     

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge <Emphasis Type="Italic">Chironomus plumosus</Emphasis> determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl₂, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.