Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.     

Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies

The nuclear lamins are major components of a proteinaceous polymer that is located at the interface of the nuclear membrane and chromatin; these lamins are solubilized and dispersed throughout the cytoplasm during mitosis. It has been postulated that these proteins, assembled into the lamina, provide an architectural framework for the organization of the cell nucleus. To test this hypothesis we microinjected lamin antibodies into cultured PtK2 cells during mitosis, thereby decreasing the soluble pool of lamins. The antibody injected was identified, together with the lamins, in cytoplasmic aggregates by immunoelectron microscopy. We show that microinjected cells are not able to form normal daughter nuclei, in contrast to cells injected with other immunoglobulins. Although cells injected with lamin antibodies are able to complete cytokinesis, the chromatin of their daughter nuclei remains arrested in a telophase-like configuration, and the telophase-like chromatin remains inactive as judged from its condensed state and by the absence of nucleoli. These results indicate that lamins and the nuclear lamina structure are involved in the functional organization of the interphase chromatin.     

Organization of higher-level chromatin structures (chromomere,chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.     

Pathogenetic mechanisms of nuclear pleomorphism of tumour cells based on the mutator phenotype theory of carcinogenesis

The nuclei of the cells of most solid tumours in histopathologic preparations vary in size, shape and chromatin pattern, both from normal nuclei and from each other. These features have not been explained in terms of conventional concepts of nuclear structure and theories of carcinogenesis. In recent years, the unfolded chromosomes have been shown to occupy "domains" in the nucleus during interphase, providing a relatively uniform density of fine chromatin fibres throughout the nucleus in the living state. This is in contrast to the appearances of interphase chromatin existing as coarse clumps and fibres (heterochromatin and euchromatin respectively) as are seen in histologic preparations. Additionally, the binding of chromatin to nuclear membrane, the possible existence of a nuclear matrix, the functions of nuclear pores, and the attachments of cytoskeletal structures to the outer nuclear membrane are now recognised. Studies of genetic instability of cancer cells (many random mutations are present in the genome, which vary from nucleus-to-nucleus in individual tumours) have shown that this phenomenon occurs early in tumour formation, can be present in morphologically-normal cells adjacent to tumours, and can result in thousands of genomic events per tumour cell. These observations form the basis for the mutator phenotype/clonal selection theory of carcinogenesis, which proposes that genetic instability is an essential early part of carcinogenesis. Genetic instability has been used to explain significant cell-to-cell variability of behaviour (tumour cell heterogeneity) among cells of individual tumours. This paper proposes that a high incidence of nucleus-to-nucleus-variable mutation of the genes for factors controlling nuclear morphology in tumours can explain nucleus-to-nucleus variations of histopathologic appearance of these nuclei when some additional effects of histological processing are taken into account.     

爪蟾卵无细胞系统中核的自组装和组装核染色质纤维的研究

以人工促排的非洲爪蟾卵为材料制备体外无细胞系统,加入Lambda DNA或染色质,用荧光显微镜和电子显微镜观察核的自组装现象.在核的自组装过程中可以看出由丝状、不规则块状到小球状的形态变化,直至形成与真核细胞间期核的形态、结构相似的组装核.在加入染色质时,所观察到的现象和加入Lambda DNA时相似,但形成组装核的过程较快.组装核经过适当浓缩后,以低渗铺展法使其破裂,从核中释放的染色质纤维,电镜观察与细胞核内的染色质有相似之处也有其本身的特点.用蛋白酶处理,纤维变得光滑,其上的颗粒消失;DNase的作用则可使纤维消失,只剩下一些颗粒和片段;RNase处理时没见变化.     

Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.     

The pattern of chromosome folding in interphase is outlined by the linear gene density profile

Spatial organization of chromatin in the interphase nucleus plays a role in gene expression and inheritance. Although it appears not to be random, the principles of this organization are largely unknown. In this work, we show an explicit relationship between the intranuclear localization of various chromosome segments and the pattern of gene distribution along the genome sequence. Using a 7-megabase-long region of the Drosophila melanogaster chromosome 2 as a model, we observed that the six gene-poor chromosome segments identified in the region interact with components of the nuclear matrix to form a compact stable cluster. The six gene-rich segments form a spatially segregated unstable cluster dependent on nonmatrix nuclear proteins. The resulting composite structure formed by clusters of gene-rich and gene-poor regions is reproducible between the nuclei. We suggest that certain aspects of chromosome folding in interphase are predetermined and can be inferred through in silico analysis of chromosome sequence, using gene density profile as a manifestation of "folding code."     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several microm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of approximately 1 microm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.     

A Lamin-Associated Chromatin Model for Chromosome Organization

We propose a simple model for chromatin organization based on the interaction of the chromatin fibers with lamin proteins along the nuclear membrane. Lamin proteins are known to be a major factor that influences chromatin organization and hence gene expression in the cells. We provide a quantitative understanding of lamin-associated chromatin organization in a crowded macromolecular environment by systematically varying the heteropolymer segment distribution and the strength of the lamin-chromatin attractive interaction. Our minimal polymer model reproduces the formation of lamin-associated-domains and provides an in silico tool for quantifying domain length distributions for different distributions of heteropolymer segments. We show that a Gaussian distribution of heteropolymer segments, coupled with strong lamin-chromatin interactions, can qualitatively reproduce observed length distributions of lamin-associated-domains. Further, lamin-mediated interaction can enhance the formation of chromosome territories as well as the organization of chromatin into tightly packed heterochromatin and the loosely packed gene-rich euchromatin regions.     

The binding of ORC2 to chromatin from terminally differentiated cells

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.