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.     

Bioflavonoids as poisons of human topoisomerase II alpha and II beta

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.     

Preparation of extended metaphase chromosomes from human cultured cells using a topoisomerase II inhibitor, ICRF-193.

Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.     

Proteomic analysis of complexes formed by human topoisomerase I

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.     

DNA ligation catalyzed by human topoisomerase II alpha

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.     

Isolation of specific human metaphase chromosomes

The human karyotype can be subdivided into seven fractions containing specific chromosomes to provide material for recombinant DNA research. The isolated metaphase chromosomes are sorted according to size by velocity zonal centrifugation, and specific chromosome groups are further purified by electrostatic deflection in a flow microfluorometer. Rapid improvements in technology should soon provide preparations of single chromosomes.     

Antibody labelling and flow cytometric analysis of metaphase chromosomes reveals two discrete structural forms

Metaphase chromosomes from cultured Chinese Hamster Ovary cells were labelled in suspension with a monoclonal antibody to histone 2B, counterstained with propidium iodide (PI) and analysed by flow cytometry. Contour plots of antibody binding (FITC fluorescence) against DNA content (PI fluorescence) revealed two discrete forms of each individual chromosome, showing high and low levels of antibody binding respectively. The two types of chromosome were easily distinguishable by immunofluorescence microscopy. The distribution of individual chromosomes between the two populations was related to chromosome size, with larger chromosomes predominating in the low-labelling population and vice versa. Variations in ionic strength, pH, divalent cation concentration or preparation procedure influenced the absolute level of antibody binding but did not eliminate the two populations. In contrast, preincubation with intercalating dyes, such as ethidium and propidium, inhibited antibody binding and generated a single, low-labelling population. Preliminary evidence suggests that in vivo changes in chromosome structure can affect the distribution of chromosomes between the two populations. Prolonged exposure of cells to Colcemid prior to chromosome isolation, a procedure known to increase chromosome condensation, resulted in a progressive shift into the low-labelling population. Our results suggest that chromosomes undergo a relatively rapid transition from the high-labelling to the low-labelling form during the prometaphase-metaphase stage of mitosis. The timing of this transition appears to be size dependent, with the larger chromosomes preceding the smaller. The transition may represent a change in chromosome condensation.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes.

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

Alteration of the metaphase checkpoint by B chromosomes

The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes.     

Banding and spiralization of human metaphase chromosomes

Summary A new technique is described which produces spiralization of human metaphase chromosomes. The important feature is heat followed by trypsin treatment. By varying conditions, it is possible to produce bands, spirals and intermediate stages. This provides a new approach to the understanding of banding and chromosome structure.     

Localization of topoisomerase II in mitotic chromosomes

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.     

Identification of isolated metaphase chromosomes. II. A comparison with the recognizability of chromosomes in metaphase plates

The metaphase chromosomes (MC) isolated from the Chinese hamster cells were identified with the aid of differential staining (G-bands). It was shown that differences in the relative recognizability of MC in metaphase plates and after their isolation are determined by changes in composition of isolated MC, rather than by those in staining capacity of MC after their isolation. The frequencies of identified MC are constant and independent upon the type of MC preparations and relation between identified and unidentified MC in certain preparations. At allows to apply the described method for the analysis of chromosome fractionation, using changes in frequencies of identified MC as a criterion of efficiency of the fractionation method. Possible ways of increasing the recognizability level of isolated MC are discussed.     

In situ nick translation of human metaphase chromosomes with the restriction enzymes MspI and HpaII reveals an R-band pattern.

Several restriction enzymes (HindIII, HaeIII, MspI, HpaII, EcoRI, KpnI, and NotI) were evaluated for their ability to induce bands in human metaphase chromosomes during in situ nick translation. MspI and HpaII were able to induce a completely developed R-band pattern. Preferential cleavage of R-band chromatin is due to the presence of unmethylated CpG-residues present in CpG-rich islands, which are apparently unevenly distributed and mainly concentrated in R-bands.     

Isolated metaphase chromosomes. II. Proteins of Chinese hamster chromosomes.

The proteins on metaphase chromosomes theoretically may be distributed ubiquitously throughout the karyotype, may be present uniquely on individual chromosomes or classes of chromosomes, or may exist in any combination of the above. Separation of chromosomes according to size using sucrose velocity gradients in high capacity zonal centrifuge rotors allows sufficient fractionation of the genome to indicate the distribution of proteins within the karyotype. Flow cytometric analysis and direct microscopic analysis were used to evaluate qualitatively the types of chromosomes present in the fractions obtained. This report is the first quantitative evidence that some of the chromosomal proteins are not distributed ubiquitously on all of the chromosomes of the karyotype.     

DNA catenation maintains structure of human metaphase chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.