Nuclear Matrix DNA Research Articles

Transformation-induced changes in the DNA-nuclear matrix interface, revealed by high-throughput analysis of DNA halos

In higher eukaryotic nuclei, DNA is periodically anchored to an extraction-resistant protein structure, via matrix attachment regions. We describe a refined and accessible method to non-subjectively, rapidly and reproducibly measure both size and stability of the intervening chromatin loops, and use it to demonstrate that malignant transformation compromises the DNA-nuclear matrix interface.

The Set of Structural DNA-Nuclear Matrix Interactions in Neurons Is Cell-Type Specific and Rather Independent of Functional Constraints.

In metazoans, nuclear DNA is organized during the interphase in negatively supercoiled loops anchored to a compartment or substructure known as the nuclear matrix. The interactions between DNA and the nuclear matrix (NM) are of higher affinity than those between DNA and chromatin proteins since the last ones do not resist the procedures for extracting the NM. The structural interactions DNA-NM constitute a set of topological relationships that define a nuclear higher order structure (NHOS) although there are further higher order levels of organization within the nucleus. So far, the evidence derived from studies with primary hepatocytes and naïve B lymphocytes indicates that the NHOS is cell-type specific at the local and at the large-scale level, and so it has been suggested that such NHOS is primary determined by structural and thermodynamic constraints. We carried out a comparative characterization of the NHOS of postmitotic cortical neurons with that of hepatocytes and naïve B lymphocytes. Our results indicate that the NHOS of neurons is completely different at the large scale and at the local level from that one observed in hepatocytes or in naïve B lymphocytes, confirming on the one hand that the set of structural DNA-NM interactions is cell-type specific and supporting, on the other hand the notion that structural constraints that impinge on chromosomal DNA and the NM are more important for determining this NHOS than functional constraints related to replication and/or transcription. J. Cell. Biochem. 118: 2151-2160, 2017. © 2016 Wiley Periodicals, Inc.

Reorganization of the DNA–nuclear matrix interactions in a 210 kb genomic region centered on c‐myc after DNA replication in vivo

In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). DNA loops are operationally classified in structural and facultative. Varied evidence indicates that DNA replication occurs in replication foci organized upon the NM and that structural DNA loops may correspond to the replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration. Using this model we have previously determined that the DNA loops corresponding to a gene-rich genomic region move in a sequential fashion towards the NM during replication and then return to their original configuration in newly quiescent cells, once liver regeneration has been achieved. In the present work we determined the organization into structural DNA loops of a gene-poor region centered on c-myc and tracked-down its movement at the peak of S phase and after the return to cellular quiescence during and after liver regeneration. The results confirmed that looped DNA moves towards the NM during replication but in this case the configuration of the gene-poor region into DNA loops becomes reorganized and after replication only the loop containing c-myc resembles the original in the control G0 hepatocytes. Our results suggest that the local chromatin configuration around potentially active genes constraints the formation of specific structural DNA loops after DNA replication, while in non-coding regions the structural DNA loops are only loosely determined after DNA replication by structural constraints that modulate the DNA-NM interactions.

Abstract LB-442: The nuclear matrix anchor domain of the DNA replication factor Ciz1 is commonly disrupted in tumours and is a circulating biomarker for lung cancer

Abstract The DNA replication protein Ciz1 promotes initiation of mammalian DNA replication in cooperation with cyclin A-dependent kinase, most likely by delivering cyclin A to sites where cyclin E-dependent pre-replication complex assembly has taken place. Normally, Ciz1 is anchored within nuclear matrix-associated foci that co-localize with sites of DNA replication, but in the absence of anchor domain Ciz1 retains the ability to promote initiation of DNA replication in isolated nuclei so attachment to the nuclear matrix is not essential for function. Expression of DNA replication and nuclear matrix anchor domains of Ciz1 are uncoupled and uneven at the transcript level in a wide range of common solid tumours, including breast and lung. In cell-based assays, recombinant anchor domain protein interferes with attachment of endogenous Ciz1 to the nuclear matrix, revealing a dominant negative effect that also impacts on nuclear matrix-recruitment of key components of the pre-replication complex. This suggests that Ciz1 normally plays a role in localizing initiation of DNA replication to the nuclear matrix. These findings implicate spatially unconstrained DNA replication as a source of nuclear disorder in cancer cells. We identified a variant Ciz1 isoform with alterations in the nuclear matrix attachment domain and tumour-restricted expression. RNAi-mediated selective inhibition of variant Ciz1 expression is sufficient to restrain the growth of tumour cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation. We also present evidence that this form of Ciz1 is expressed in 34/35 lung tumours but not adjacent tissue, giving rise to stable protein quantifiable in less than a microlitre of patient plasma by western blot. Using two independent sets, with 170 and 160 samples, variant Ciz1 correctly identified stage 1 lung cancer patients with clinically useful accuracy. For set 1, mean variant Ciz1 level (+SD) in individuals without diagnosed tumours established a threshold that correctly classified 94% of small cell lung cancers (SCLC) and non-SCLC patients. Within set 2, comparison of stage 1 non-SCLC with asymptomatic age-matched smokers correctly classified 85% of patients, while comparison with individuals with benign lung nodules correctly classified 83% of patients. The data show that this cancer-specific, single marker is capable of indentifying early stage lung cancer within at-risk groups, without resort to invasive procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-442. doi:1538-7445.AM2012-LB-442

Abstract 3022: Disruption of the nuclear matrix attachment domain of the DNA replication factor Ciz1 in common solid tumors and its potential as a biomarker

Abstract The DNA replication protein Ciz1 promotes initiation of mammalian DNA replication in cooperation with cyclin A-dependent kinase, most likely by delivery cyclin A to sites where cyclin E-dependent pre-replication complex assembly has taken place. Normally, Ciz1 is anchored within nuclear matrix-associated foci that co-localize with sites of DNA replication, but in the absence of anchor domain Ciz1 retains the ability to promote initiation of DNA replication in isolated nuclei so attachment to the nuclear matrix is not essential for function. Here, we show that expression of DNA replication and nuclear matrix anchor domains of Ciz1 are uncoupled and uneven at the transcript level in a panel of common cancer cell lines, giving rise to excess DNA replication domain protein that is not attached to the nuclear matrix. Moreover Ciz1 domain expression is also uncoupled in a wide range of common solid tumours, including breast and lung, so that anchor domain transcript is elevated over control tissues, and exceeds replication domain in all primary stage I, II and III tumours tested. Notably, more than half of all stage IV tumours tested resemble established cell lines, having more replication domain than anchor domain. In cell based assays, recombinant anchor domain protein interferes with attachment of endogenous Ciz1 to the nuclear matrix, revealing a dominant negative effect that also impacts on nuclear matrix-recruitment of a key component of the pre-replication complex, Cdc6. This shows that Ciz1 normally plays a role in localizing Cdc6 to the nuclear matrix and suggests that cancer-associated uncoupled expression influences recruitment of mammalian pre-replication complexes to the nuclear matrix. These findings implicate spatially unconstrained DNA replication as a source of nuclear disorder in cancer cells. We identified a variant Ciz1 isoform with alterations in nuclear matrix attachment domain and tumour-restricted expression. Variant Ciz1 is elevated at the transcript and protein level with high frequency in small cell lung tumours, and with lower frequency in a range of other tumour types. Selective suppression of variant Ciz1 expression using inducible shRNA restricts proliferation of lung cancer cells that express it in vitro and in vivo, identifying a novel, exploitable therapeutic target with potential application in the treatment of lung cancer. Recent evidence will also be presented on the application of variant Ciz1 as a serum biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3022. doi:10.1158/1538-7445.AM2011-3022

Chromatin supraorganization and extensibility in mouse hepatocytes with development and aging

Chromatin supraorganization and extensibility, which lead to the formation of extended chromatin fibers (ECF), are affected by starvation and refeeding in adult mouse hepatocytes. It is expected that they could also change with mouse development and aging. Methods used involved topochemistry, image analysis, microspectrophotometry, gravity action, and polarization microscopy. Increased nuclear areas and Feulgen-DNA amounts with advancing hepatocyte polyploidy were found with development and aging. A slightly less packed chromatin with more heterogeneously distributed condensation levels was detected in young and old mice. Con-A responsiveness was almost absent in young mice but very deep in aged mice. ECFs formed from nuclei of adult and aged mice but not from nuclei of young mice. The frequency of ECF formation with the long lysis protocol increased with aging. In young mice, a less packed chromatin state may be associated with more intense gene activity, thus increasing the DNA-nuclear matrix interactions, and inhibiting ECF formation. Reduced DNA-nuclear matrix interactions besides defects in heterochromatin formation may induce higher ECF formation and chromatin unpackaging in old mice. We suggest that differences in Con-A staining relate to different gene activity with advancing development and aging.

Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

Natural ageing in the rat liver correlates with progressive stabilisation of DNA–nuclear matrix interactions and withdrawal of genes from the nuclear substructure

In the interphase nucleus, the DNA of higher eukaryotes is organised in supercoiled loops anchored to a nuclear matrix (NM). Replication, transcription and splicing seem to occur at macromolecular complexes organised upon the NM. Thus, the topological relationship between genes located in the loops and the NM appears to be very important for nuclear physiology. Here, we report that natural ageing in the rat liver correlates with a progressive strengthening of the NM framework and the stabilisation of the DNA loop–NM interactions, as well as with a progressive increase in the relative distance of genes to the NM. Both phenomena correlate with the gradual loss of proliferating potential and progression towards terminal differentiation in the hepatocytes, suggesting that wholesale modifications in the topological relationships within the cell nucleus are markers of tissue ageing and senescence, at least in the mammalian liver. We discuss the possible functional implications of such structural modifications that may underlie both terminal hepatocyte differentiation and their eventual replicative senescence.

Visualization of DNA Loops and Nuclear Matrix Anchorage Regions in the Human Dystrophin Gene

Visualization of DNA Loops and Nuclear Matrix Anchorage Regions in the Human Dystrophin Gene

Heat induced ‘masking’ of redox sensitive component(s) of the DNA-nuclear matrix anchoring complex

The ‘masking effect’ is the observation that heat shock reduces or masks the apparent expression of ionizing radiation (IR) damage to DNA. The mechanism of this effect is thought to involve the aggregation of proteins to the nuclear matrix or chromatin, thereby stabilizing these structures and masking actual DNA damage from assays and presumably from DNA repair complexes. Previously, using the ‘halo assay’, it has been shown that nucleoids treated with 1 mm dithiothreitol (DTT) and/or IR inhibited the rewinding of DNA supercoils and that this effect was masked in nucloids isolated from heated cells. Here it is reported that treatment of living cells with reducing agents diminishes the interaction between DNA and Protein Disulphide Isomerase (PDI) and that hyperthermia restored the PDI-DNA interaction, indicating that the masking effect occurred in vivo. PDI is a nuclear matrix protein which binds MAR DNA sequences and may be involved in regulating the degree of DNA supercoiling. It is hypothesized that heat-induced stabilization of PDI-DNA interaction will mask changes in supercoiling observed with reducing reagents and also IR. This stabilization may be affected through either the heat-induced association or enhancement of the binding of proteins to MAR DNA at the NM. Several proteins, including B23 and Hsp60, have been identified whose interaction with DNA increased following heat shock. Further work will be needed to determine if these proteins do, in fact, play a role in the masking effect.

Hydrogen Peroxide Induced Apoptosis in Amnion-derived WISH Cells is not Inhibited by Vitamin C

Increased reactive oxygen species (ROS) have been identified as a potential cause of remodelling and apoptotic change in fetal membrane. Vitamin C has been suggested as a therapeutic agent to prevent ROS induced chorio-amnion apoptosis. The purpose of this study was to determine whether hydrogen peroxide (HP), a ROS, initiates apoptosis in the WISH cell model and whether vitamin C would inhibit HP induced apoptosis. HP induced apoptosis in WISH cells; as assessed by cytochrome-c release from mitochondria, Poly-(ADP-ribose)-Polymerase (PARP) cleavage, nuclear matrix protein (NMP) release and DNA fragmentation analysis. HP induced dose dependent release of cytochrome-c, PARP cleavage, NMP release, and DNA fragmentation. HP also increased PGE 2release in parallel with apoptosis in WISH cells, in a manner similar to that reported with other apoptotic agents. Vitamin C pre-incubation caused cytochrome-c release earlier, and at lower HP doses, than HP alone. It had no effect on HP induced PARP cleavage, but enhanced DNA fragmentation, and induced NMP release on its own. Vitamin C partially suppressed dose dependent HP induced PGE 2release. We conclude that HP causes apoptosis in WISH cells and vitamin C pre-incubation does not inhibit, and may accelerate and exacerbate, HP induced apoptosis.

Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.

DNA-nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative-reductive environment can affect DNA-NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA-NM anchoring. First, the relative stringency of the DNA-NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from gamma-irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and gamma-rays alone. Nucleoids isolated from heat-shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT-treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA-NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4 degrees C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT-treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT-induced change in DNA association, but not its NM association. These results also imply that DNA-NM anchoring involves the redox state of protein sulfhydryl groups.

Topoisomerase II cleavable complex formation within DNA loop domains

The distribution of VM-26 (Teniposide)-stabilized cleavable complexes within DNA loops bound to the nuclear matrix was determined to provide further insights into the mode of DNA synthesis inhibition by VM-26. Covalent binding of [ 3H]VM-26 was 9-fold greater per milligram of nuclear matrix protein compared with high salt-soluble nonmatrix protein of CEM cells. The ratio declined from 9-fold in CEM cells to 4-fold in drug-resistant VM-1/C2 cells, which have decreased nuclear matrix DNA topoisomerase IIα. VM-26 induced a concentration-dependent increase in the frequency of cleavable complex formation with actively replicating matrix DNA. At 25 μM VM-26, the frequency was 32 ± 2 (SEM) complexes per 10 6 bp of replicating matrix DNA compared with 13 ± 2 (SEM) complexes per 10 6 bp of nonreplicating DNA in the matrix fraction. VM-26 at concentrations as high as 25 μM stabilized less than 3 complexes per 10 6 bp in the various nonmatrix DNA domains, since the nonmatrix DNA comprises the DNA loop domains that are distal to the matrix-bound replication sites. A negligible frequency of cleavable complex formation was detected in both the matrix and nonmatrix DNA domains of drug-resistant VM-1/C2 cells. Compared with untreated control cells, VM-26 induced an accumulation of nascent DNA in the nuclear matrix fraction of CEM cells but decreased the amount of nascent DNA in the nonmatrix fraction. The extensive cleavable complex formation on matrix replicating DNA stalled most of the replication forks within 1 kb of the replication sites on the nuclear matrix. The results provide evidence that nascent DNA bound to the nuclear matrix is an important site of VM-26 cleavable complex formation, and that these complexes inhibit DNA synthesis by blocking the movement of nascent DNA away from replication sites on the nuclear matrix.

Differentiation-Specific Nuclear Matrix Proteins Cross-linked to DNA bycis-Diammine Dichloroplatinum

DNA–protein cross-linkages were performed in intact undifferentiated and differentiated HL60 cells by the action ofcis-diammine dichloroplatinum. Total nuclear matrix proteins and DNA cross-linked nuclear matrix proteins were resolved by two-dimensional gel electrophoresis. The comparison of the electrophoretic patterns allowed the identification of a set of differentiation-induced nuclear matrix proteins cross-linked to DNA. One of these proteins binds cloned histone SAR sequences. Our results outline an experimental strategy for isolating and characterizing nuclear matrix components that may play a fundamental role in the overall control and coordination of gene expression during differentiation.

Differential Effects of 1,25-Dihydroxyvitamin D3 and Tetradecanoylphorbol Acetate on Cell Cycle and Apoptosis of MCF-7 Cells and a Vitamin D3-Resistant Variant

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, and tetradecanoylphorbol acetate (TPA) are potent negative growth regulators of breast cancer cells. In this study, we compared the mechanism of action of these two compounds in MCF-7 cells and a vitamin D3-resistant variant (MCF-7D3Res). In parental MCF-7 cells, 1,25-(OH)2D3 induced morphological and biochemical markers of apoptosis (chromatin and nuclear matrix condensation and DNA fragmentation), whereas TPA induced growth arrest without apoptosis. Both 1,25-(OH)2D3 and TPA independently up-regulated the vitamin D receptor, p21, and the hypophosphorylated form of retinoblastoma (Rb) protein. The growth regulatory effects of 1,25-(OH)2D3 and TPA did not correlate with induction of p53 protein expression. When both compounds were added simultaneously, synergistic effects on MCF-7 cell number were observed, and cell cycle regulatory proteins were down-regulated. The MCF-7D3Res cells, which are not sensitive to 1,25-(OH)2D3, were growth inhibited by TPA, and TPA partially sensitized MCF-7D3Res cells to the growth inhibitory effects of 1,25-(OH)2D3. In MCF-7D3Res cells, 1,25-(OH)2D3 treatment had minimal effects on p21 or Rb protein expression, whereas TPA down-regulated Rb protein and transiently up-regulated p21. These studies indicate dissociation between the pathways triggered by 1,25-(OH)2D3 and TPA, which mediate growth regulation in MCF-7 cells. Because both compounds induce growth arrest, but only 1,25-(OH)2D3 mediates apoptosis, we conclude that cell cycle arrest is not sufficient to trigger cell death of MCF-7 cells, and that 1,25-(OH)2D3 generates distinct signals which lead to induction of apoptosis in breast cancer cells.

Differential effects of 1,25-dihydroxyvitamin D3 and tetradecanoylphorbol acetate on cell cycle and apoptosis of MCF-7 cells and a vitamin D3-resistant variant.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, and tetradecanoylphorbol acetate (TPA) are potent negative growth regulators of breast cancer cells. In this study, we compared the mechanism of action of these two compounds in MCF-7 cells and a vitamin D3-resistant variant (MCF-7D3Res). In parental MCF-7 cells, 1,25-(OH)2D3 induced morphological and biochemical markers of apoptosis (chromatin and nuclear matrix condensation and DNA fragmentation), whereas TPA induced growth arrest without apoptosis. Both 1,25-(OH)2D3 and TPA independently up-regulated the vitamin D receptor, p21, and the hypophosphorylated form of retinoblastoma (Rb) protein. The growth regulatory effects of 1,25-(OH)2D3 and TPA did not correlate with induction of p53 protein expression. When both compounds were added simultaneously, synergistic effects on MCF-7 cell number were observed, and cell cycle regulatory proteins were down-regulated. The MCF-7D3Res cells, which are not sensitive to 1,25-(OH)2D3, were growth inhibited by TPA, and TPA partially sensitized MCF-7D3Res cells to the growth inhibitory effects of 1,25-(OH)2D3. In MCF-7D3Res cells, 1,25-(OH)2D3 treatment had minimal effects on p21 or Rb protein expression, whereas TPA down-regulated Rb protein and transiently up-regulated p21. These studies indicate dissociation between the pathways triggered by 1,25-(OH)2D3 and TPA, which mediate growth regulation in MCF-7 cells. Because both compounds induce growth arrest, but only 1,25-(OH)2D3 mediates apoptosis, we conclude that cell cycle arrest is not sufficient to trigger cell death of MCF-7 cells, and that 1,25-(OH)2D3 generates distinct signals which lead to induction of apoptosis in breast cancer cells.

A DNA-binding Element for a Steroid Receptor-binding Factor Is Flanked by Dual Nuclear Matrix DNA Attachment Sites in the c-myc Gene Promoter

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a beta-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.

Radiation-Induced Oxidative DNA Base Damage and Its Repair in Nuclear Matrix-Associated DNA and in Bulk DNA in Hepatic Chromatin of Rat Upon Whole-Body γ-Irradiation

In the present work, we examined the formation and repair of DNA base damages induced by γ-irradiation in different fractions of rat hepatic chromatin. Animals were exposed to radiation with the dose of 10 Gy. Nuclear matrix DNA and whole chromatin were isolated from the liver of rats killed before and in different time after irradiation. In those samples the pyrimidine-derived and the purine-derived modified DNA bases were identified and quantitated by gas chromatography/isotope-dilution mass spectrometry with selected-ion monitoring. We found elevated levels of modified DNA bases over control values after whole body irradiation in both matrix DNA and bulk chromatin samples. Our results suggest that modified bases are preferentially removed from matrix DNA then bulk chromatin. Copyright © 1996 Elsevier Science Inc.

The non-random distribution of UV-induced photoproducts in the nuclear matrix and non-matrix DNA fractions.

The formation of UV-induced photoproducts in the chromatin fractions of human lymphocytes was studied by 32P-post-labeling. A higher level of DNA lesions was found in the matrix-attached DNA fraction as compared to non-matrix DNA of irradiated cells (about 150 and 110 adducts per 10(6) nucleotides, respectively, at a 500 J/m2 254 nm-UV dose). Formation of photoproducts in a MAR (matrix attached region) sequence from the mouse kappa immunoglobulin gene irradiated in vitro was examined as well. The MAR sequence showed a two-fold higher level of adducts as compared to non-MAR DNA. The effect of photoproducts on complex-formation between MAR DNA and proteins of the nuclear matrix was studied in vitro. The amount of UV-induced adducts was 1.5-fold higher in matrix-bound fraction as compared to non-fractionated DNA (and five-fold higher as compared to unbound fraction), which possibly resulted from preferential binding of lesion-containing DNA fragments to the nuclear matrix proteins.

Comparison of DNA‐protein interactions in intact nuclei from avian liver and erythrocytes: A cross‐linking study

DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei. © 1996 Wiley-Liss, Inc.