Nuclear Matrix Structure Research Articles

Localization of Arg-X proteolysis in the supramolecular structures of cell nuclei during the induction of growth in mature wheat germs

Proteolysis is a form of the biological control providing fast physiological response to changing conditions of an environment. The goal of the present work was to analyze the localization of the Arg-X proteolysis in complexes of histones and non-histone proteins isolated from supra-molecular structures (nucleoplasm, chromatin, the nuclear matrix) of cell nuclei of mature wheat germs during induction of growth. Cell nuclei were isolated from germs, cleared, and then nucleoplasm, chromatin, nuclear matrix were extracted by increasing ionic strength of solution. From isolated supra-molecular structures, non-histone proteins were separated from histones by using ion exchange chromatography. The Arg-X proteolytic activity was assessed by cleavage of Arg-X bonds in the arginine-enriched protein protamine in all nuclear fractions. It was shown that in the coleoptile, which grows by cell elongation, Arg-X proteolysis activity was at the level of nuclear matrix structures, namely the linker histone HI and core histones, and in mesocotyls, Arg-X proteolysis activity was found in the core histones of the fraction of chromatin tightly bound to the nuclear matrix. A possible role of Arg-X proteolysis in the fine regulation of morphogenetic mechanisms occurring during seed germination is suggested.

TRIM19/PML Restricts HIV Infection in a Cell Type-Dependent Manner.

The promyelocytic leukemia protein (PML) is the main structural component of the nuclear matrix structures termed nuclear domain 10 (ND10) or PML nuclear bodies (PML-NBs). PML and ND10 structures have been shown to mediate an intrinsic immune response against a variety of different viruses. Their role during retroviral replication, however, is still controversially discussed. In this study, we analyzed the role of PML and the ND10 components Daxx and Sp100 during retroviral replication in different cell types. Using cell lines exhibiting a shRNA-mediated knockdown, we found that PML, but not Daxx or Sp100, inhibits HIV and other retroviruses in a cell type-dependent manner. The PML-mediated block to retroviral infection was active in primary human fibroblasts and murine embryonic fibroblasts but absent from T cells and myeloid cell lines. Quantitative PCR analysis of HIV cDNA in infected cells revealed that PML restricts infection at the level of reverse transcription. Our findings shed light on the controversial role of PML during retroviral infection and show that PML contributes to the intrinsic restriction of retroviral infections in a cell type-dependent manner.

P108 PMLIV inhibits VSV replication through the specific induction of IFN-beta and IFN-lambda

Promyelocytic Leukemia (PML) protein (also named TRIM19 for TRIpartite Motif protein 19), the organizer of small nuclear-matrix structures named nuclear bodies, has been implicated in the antiviral response toward diverse cytoplasmic replicating RNA viruses through different mechanisms. PML is covalently conjugated to the small ubiquitin-like modifier (SUMO). Splice variants transcribed from the PML gene yield several PML isoforms (PMLI to PMLVII) that share the same N-terminal region but differ in their C-termini. In consequence, each PML isoform is able to interact with specific partners and to display different functions. We have previously shown that PMLIII confers resistance to vesicular stomatitis virus (VSV) [1] . The antiviral activity of PML has been validated in vivo since knock out PML mice are more sensitive to VSV than wild-type mice [2] . The role of the other PML isoforms in VSV-infected cells is unknown. Therefore, we studied the implication of all PML isoforms during VSV infection. Viral proteins synthesis was determined in MEFs derived from knock out PML mice (MEFs PML-/-) and in human cells down-regulated for PML. U373MG cells stably expressing each PML isoform (PMLI to PMLVII) were infected with VSV at different MOIs, viral protein synthesis and VSV growth were determined. To investigate which viral step is targeted by PMLIV, we studied VSV entry, primary and secondary transcription. To determine whether PMLIV affects the expression of IFNs and/or pro-inflammatory cytokines, we quantified IFN-a, IFN-b, IFN-l, TNF-a and IL8 mRNAs in extracts from U373MG-EV and U373MG-PMLIV cells infected with VSV. We have shown that VSV protein synthesis were boosted in MEFs PML-/- or in human cells down-regulated for PML expression. Our comparative study performed with cells stably expressing each of PML isoform revealed that both PMLIII and PMLIV conferred resistance to VSV, whereas other nuclear isoforms (PMLI, II, V and VI) and cytoplasmic PMLVII failed to do so. The antiviral activity of PMLIV was higher than that of PMLIII. PMLIV did not alter VSV entry but inhibited viral mRNA and protein synthesis. PMLIV SUMOylation was required as the antiviral effect was not observed with PMLIV-3KR, in which lysines involved in conjugation to SUMO were mutated. The protective effect of PMLIII was independent of IFN. In contrast, PMLIV expression led to the induction of type I IFNs, particularly IFN-band IFN-lduring VSV infection. Here, we demonstrate for the first time that one particular isoform of PML (PMLIV) was implicated in the induction of type I IFNs, particularly IFN-band IFN-l, during VSV infection, suggesting a new role for PMLIV in the antiviral innate immunity.

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

Evidence for the structural stability of ribonucleoprotein LMG160 under ribonuclease-A treatment

Low mobility group nonhistone protein, LMG(160), is a ribonucleoprotein particle of the nuclear matrix with an inhibitory effect on transcription. Through the current study, we have investigated comparatively the effect and behavior of the protein in the absence and presence of its RNA moiety. Analysis was performed with the intact LMG(160) and its RNase-treated form using native and denatured gel electrophoresis as well as fluorescence spectroscopy and trypsin digestion techniques. The results show that the RNA moiety of LMG(160) plays a key role in maintaining the overall structure and conformation of this RNP particle, in the way that RNA removal causes some alterations in the structural stability of the protein, leading it to become self-associated. This finding can easily explain the loss of function of LMG(160) after RNase-treatment, the effect that may influence the biological activity of the molecule in the nuclear matrix structure.

Chromatin movement visualized with photoactivable GFP-labeled histone H4

The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

Structure and function of nuclear matrix associated regions (S/MARs)

Modern concepts on the chromatin loop-domain organization and the role of the DNA regions specifically binding the nuclear matrix (nuclear scaffold, or S/MARs) in its formation, maintenance, and regulation are discussed. Some S/MAR structural features, properties of binding the nuclear matrix, and probable mechanisms of their involvement in regulation of gene activity are considered. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

A new look at the cellular scaffold by embedment‐free electron microscopy method

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.

Characterization of a nuclear matrix-associated endonuclease

The nuclear matrix is a proteinaceous network that serves as a structural framework of the eukaryotic nucleus. It was isolated by treating purified nuclei with nucleases, followed by extractions with high-salt and non ionic detergent (B ere z n e y and C 0 f fey 1974). Aside from its welldefined structural role, the nuclear matrix is involved in many nuclear functions. It participates in chromatin organization and every aspect of DNA metabolism: from DNA synthesis, gene transcription to DNA degradation (B ere z n e y et at. 1995; Get zen b erg 1994;G rom 0 v a et at. 1995). DNA degradation during apoptosis starts with the release of high-molecular weight DNA fragments (50-300 kb) from their points of attachment on the nuclear matrix structure. Although the nuclease that catalyses this process is still unidentified, the biochemical requirements of initial apoptotic DNA degradation are well established (S u nand C 0 hen 1994; K 0 k i I e v a 1995). Recently, it was shown that nonapoptotic cells contain a constitutive high-molecular weight fragmentation activity that is localized on the isolated nuclear matrix (S 0 I 0 v y a n et at. 2002). In this paper we describe for the first time an endonuclease that is associated with the rat hepatocyte nuclear matrix. At present, a role for such a nuclear matrix-associated enzymatic activity is unclear. One possibility is that the nuclear matrix-associated endonuclease is involved in apoptotic DNA degradation. In order to verify this assumption, the biochemical requirements of the nuclear matrixassociated nuclease were examined. Nuclear matrices were isolated from purified hepatocyte nuclei essentially as described previously (P 0 z nan 0 vic et at. 1996) except that the nuclei were subjected to endogenous nuclease digestion. Endonuclease activities in the protein fractions were established by activity-gel analysis. The method is based on the electrophoretic separation of proteins in 10% SDS-polyacrylamide gels containing 100 ~/mL salmon sperm DNA (R a u c h et at. 1997). After electrophoresis the gels were washed in renaturation buffer (50 mM Tris pH 7.0, 1 mM DTT) overnight at 4°C, and incubated for 24 h in the same buffer with the addition of different cations in order to activate nuclease activity/-ies. The gels were then stained with ethidium bromide and nuclease activities were detected as dark areas on a fluorescent background after transillumination of the gels with UV light. Using this method we examined the presence of endonucleases in rat hepatocyte nuclei and their redistribution in protein fractions released during the isolation of the nuclear matrix (Fig. lB). Initially, activity-gel analysis was performed at

Changes in nuclear matrix proteins during the senescence-like phenomenon induced by 5-chlorodeoxyuridine in HeLa cells

5-Chlorodeoxyuridine (CldU) immediately induces a senescence-like phenomenon in any type of mammalian cells probably due to a change in nuclear matrix structure. We thus examined nuclear matrix proteins in HeLa cells cultured with CldU by high-resolution two-dimensional gel electrophoresis and peptide mass spectrometry. Three proteins were found to be down-regulated and one protein up-regulated by addition of CldU. In addition, one acidic protein accumulated in the nuclear matrix, although not quantitatively changed in the nuclei. Since these alterations were observed within 24 h after addition of CldU, these proteins may be involved in an early step of the senescence-like phenomenon.

Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides.

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.

Changes in the ultrastructure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation.

The cellular scaffold that comprises nuclear matrix and cytoskeleton provides mechanical support for the cell and plays a crucial role in motility, cellular signaling, regulation of gene transcription and DNA replication. In this study we examined the structure of cytoskeleton and nuclear matrix in the keratinocyte cell line HaCaT using a recently developed technique, embedment-free electron microscopy. With this method the three-dimensional structure of cellular scaffold is visualized in the cells extracted from soluble proteins and the chromatin. In actively proliferating cells the cytoskeleton appeared to consist of a continuous meshwork of 10--15 nm filaments with a smaller amount of thin (5 nm) and ultrathin (1--2 nm) filaments. In contrast to what could be expected from earlier immunofluorescence and electron microscopy studies, the cytoskeleton in HaCaT keratinocytes did not consist of superposed autonomous networks of different filaments but was a highly integrated, continuous structure filling whole cytoplasmic territory. Moreover, cytoskeletons of adjacent cells were in a direct physical contact. Nuclear matrix consisted of globular ribonucleoprotein aggregates attached to the meshwork of 20--40 nm filaments. Nuclear envelope was firmly fastened to the cytoskeleton. In keratinocytes induced to differentiation by calcium switch both the cytoskeleton and nuclear matrix were drastically rearranged and comprised a monomorphic, dense and regular meshwork of 10--15 nm filaments. Our data underscore the fact that in HaCaT keratinocyte monolayer in vitro, and probably also in the epidermis in vivo, the nuclear matrices and the cytoskeletons of adjacent cells seemed to form a continuous, highly ordered structure which is rapidly rearranged during cell differentiation. This feature may be crucial for the understanding of how the signal initiated by, e.g. mechanical forces generated through the cell--cell and cell--matrix interaction is transmitted to the nucleus producing ultimately changes in the pattern of gene expression.

Experimental observations of a nuclear matrix.

Nuclei are intricately structured, and nuclear metabolism has an elaborate spatial organization. The architecture of the nucleus includes two overlapping and nucleic-acid-containing structures - chromatin and a nuclear matrix. The nuclear matrix is observed by microscopy in live, fixed and extracted cells. Its ultrastructure and composition show it to be, in large part, the ribonucleoprotein (RNP) network first seen in unfractionated cells more than 30 years ago. At that time, the discovery of this RNP structure explained surprising observations that RNA, packaged in proteins, is attached to an intranuclear, non-chromatin structure. Periodic and specific attachments of chromatin fibers to the nuclear matrix create the chromatin loop domains that can be directly observed by microscopy or inferred from biochemical experiments. The ultrastructure of the nuclear matrix is well characterized and consists of a nuclear lamina and an internal nuclear network of subassemblies linked together by highly structured fibers. These complex fibers are built on an underlying scaffolding of branched 10-nm filaments that connect to the nuclear lamina. The structural proteins of the nuclear lamina have been well characterized, but the structural biochemistry of the internal nuclear matrix has received less attention. Many internal matrix proteins have been identified, but far less is known about how these proteins assemble to make the fibers, filaments and other assemblies of the internal nuclear matrix. Correcting this imbalance will require the combined application of biochemistry and electron microscopy. The central problem in trying to define nuclear matrix structure is to identify the proteins that assemble into the 10-nm filaments upon which the interior architecture of the nucleus is constructed. Only by achieving a biochemical characterization of the nuclear matrix will we advance beyond simple microscopic observations of structure to a better understanding of nuclear matrix function, regulation and post-mitotic assembly.

ChemInform Abstract: Functional Arrangement of Genomic DNA and Structure of Nuclear Matrix

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

C/EBPα AND C/EBPβ ARE PERSISTENTLY ASSOCIATED WITH THE RAT LIVER NUCLEAR MATRIX THROUGHOUT DEVELOPMENT AND THE ACUTE PHASE RESPONSE

The partitioning of C/EBPα and C/EBPβ on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPα and C/EBPβ was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage- and AP-related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPα decreased and C/EBPβ increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.

Structure of cytomatrix and nuclear matrix revealed by embedment-free electron microscopy

Embedment-free electron microscopy (EFEM) is a new method which allows the visualisation of cytoskeleton in whole-mounted cells. In this study we employed EFEM to investigate the structure of cellular scaffolds in glioma C6 cell line. The cells were extracted with Triton X-100 that dissolves phospholipids in the membranes and removes most of cytoplasmic soluble proteins. The DNA and nuclear histones were removed with DNase I and high-salt buffer, respectively. The remaining cellular frameworks were temporary embedded in diethylene glycol distearate (DGD), sectioned and observed in transmission and scanning electron microscope after the removal of DGD. The predominant structure was the extensive meshwork of 10-20 nm filaments in the cytoplasm (cytomatrix) and 15-30 nm filaments in the nucleus (nuclear matrix). The 5 nm filaments, presumably corresponding to the actin filaments, were present in the cytomatrix, but not in the nuclear matrix. Moreover, the ultrathin (3 nm) filaments, connecting other cytoskeletal components were detected. Those are possibly identical with the previously described plectin filaments. For the first time we report the occurrence of ultrathin filaments in the nuclear matrix. Thus, in a addition to the well known cytoskeletal components (microtubules, intermediate filaments, actin microfilaments) EFEM showed a new type of filaments (the ultrathin filaments) in the cytomatrix and nuclear matrix. Further immunocytochemical studies are needed to determine the biochemical identity of the filaments observed in EFEM.

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracilis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix.

Significance of protein kinase CK2 nuclear signaling in neoplasia.

Many stimuli play a role in influencing the structure and function of chromatin and nuclear matrix through post-translational modifications of the component proteins in these dynamic structures. We propose that the protein serine/threonine kinase CK2 (formerly casein kinase II) is one such agent that is involved in signal transduction in the nuclear matrix and chromatin in response to a variety of stimuli. Protein kinase CK2 appears to undergo rapid modulations in its association with nuclear matrix and nucleosomes in response to mitogenic signals and is involved in the phosphorylation of a variety of intrinsic proteins in these structures depending on the state of genomic activity. In addition, its association or loss from the nuclear matrix may also influence the apoptotic activity in the cell. CK2 has been found to be dysregulated in virtually all the neoplasias examined and nuclear association appears to be an important facet of its expression in tumor cells. We hypothesize that CK2 provides a functional paradigm linking the nuclear matrix and chromatin structures. Identification of precise loci of action of CK2 in these structures and how they influence the morphological appearance of the nucleus under normal and abnormal growth conditions would be an important future direction of investigation. J. Cell. Biochem. Suppl. 35:130-135, 2000. Published 2001 Wiley-Liss, Inc.

DNA loop domain organization: the three-dimensional genomic code.

It is well known that aberrations in the nuclear matrix contribute to the development of cancer, but many aspects of this process remain unknown. The mammalian sperm nuclear matrix serves as a distinctive model of DNA loop domain organization by the nuclear matrix since the integrity of the DNA structure can be measured by the ability of the paternal chromosomes to participate in embryogenesis. The structure of the nuclear matrix is known to be important for normal cellular functions such as transcriptional regulation and DNA replication. Even small aberrations in DNA structural organization in the sperm cell could have disastrous consequences for the embryo if they were essential for function. Recent work from our laboratory suggests that sperm nuclei with disrupted nuclear matrix structures but intact DNA cannot participate fully in embryogenesis, suggesting that the structural organization of DNA may provide important, heritable information that is necessary for development. We term the DNA sequence together with its three-dimensional organization the "genomic code." We suggest that the sperm nucleus is an ideal model for understanding the principles of the involvement of the three-dimensional structure of DNA in normal cellular function. Finally, the implications for cancer about what we can learn using sperm DNA as a model about the "genomic code" are discussed. J. Cell. Biochem. Suppl. 35:23-26, 2000.

The nuclear matrix prepared by amine modification.

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.