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At least 397 records · Page 22Linked to original sources
PubMed2002

Topological organization of DNA molecules in the macronucleus of hypotrichous ciliated protozoa.

The DNA in the macronucleus of a hypotrichous ciliate occurs as millions of short molecules packed into dense chromatin bodies 0.1-2 microm in diameter. We have studied by electron microscopy the organization of DNA molecules in these chromatin bodies of macronuclei lysed in water at pH 9. Proteinase K treatment of lysed macronuclei progressively releases from chromatin bodies many rosettes of DNA molecules bound at one or both ends to a central core of protein. With longer treatment with proteinase K, rosettes disappear, leaving individual free DNA molecules. We propose that, in the native state, both ends of DNA molecules are bound through telomere-binding protein to a central core to form rosettes. Many rosettes, with collapsed DNA loops, aggregate to form a chromatin body. Chromatin bodies are believed to dissociate into individual collapsed rosettes to form the granules in the forward zone of the replication band. In the rear zone of the band, the rosettes dissociate, presumably as a result of release of telomere-binding protein, which is preliminary to the replication of the DNA molecules.

Animals↗
PubMed1993

Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure.

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibers anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.

Calcimycin↗
PubMed1977

Chromatin ultrastructure of lower vertebrates.

Meiotic and mitotic chromosomes from amphibians and snakes were studied by electron microscopy. By using water spreading, preceded by a mild NaCl pretreatment, we showed: 1. 'Beads on a string' arrangement of the chromatin fibres; 2. The presence of loops at pachytene chromomeres as well as during metaphase of both mitosis and first meiosis; 3. Transcriptional activity for non-ribosomal RNA on peripheral loops during the middle pachytene.

Animals↗
PubMed2002

Cytochemical study of the distribution of RNA and DNA in the synaptonemal complex of guinea-pig and rat spermatocytes.

The distribution of DNA and RNA in the synaptonemal complex and related structures, was studied using high resolution cytochemical methods and in situ hybridization, in guinea pig and rat testis. Serial sectioning demonstrates that frequently the formation of the synaptonemal complex (SC) occurs without a previous development of isolated chromosomal axes. The lateral elements of the forming SC are in continuity with pairs of DNA-containing thin filaments. These chromatin filaments fold in numerous short loops just before incorporating to the lateral elements. Some of these loops are included in the ribbon-like structure of the lateral elements of the mature SC. We propose that these short loops contain the DNA attachment sequences associated with the proteins of the LE. During the formation of the SC one of the two chromatin filaments incorporates at the central surface of the forming lateral element (LE) and the other is located at the external side of the LE. This unexpected distribution does not correspond to the pair of thick filaments previously discerned in structure of the LE. The presence of RNA associated with the DNA-containing thin filaments, as well as with the axial chromatin elements of the forming SC, may be related with the transcription occurring during meiotic prophase, specially during zygotene stage. We propose that RNA is involved in a still uncharacterized process essential for pairing.

Animals↗
PubMed1998

Mutagenic properties of topoisomerase-targeted drugs.

Topoisomerases maintain DNA structure by relieving torsional stress occurring in DNA during transcription, replication and cell division. Topoisomerases are of two main types, causing transient breaks in one (type I) or both (type II) and strands of DNA, and a number of clinical anticancer drugs are thought to act by inhibiting religation of these transient breaks. Topoisomerase II appears to have a close association with the SMC (stable maintenance of chromosomes) family of proteins involved in organisation of the chromatin in a series of loops on the proteinaceous chromosomal scaffold. Inhibition of topoisomerase II function can result in deletions of such loops, probably mediated by reciprocal exchange of topoisomerase subunits. Disruption of topoisomerase I and/or II function during DNA replication results in smaller DNA deletions and other mutations, probably arising from non-homologous recombination. Inhibition of topoisomerase II action during mitosis and meiosis can cause incomplete separation of chromatids and chromosomes, with the consequent production of genomic mutations. Topoisomerase-mediated mutagenicity is important because it can lead not only to drug resistance but also to drug-induced secondary cancers. Mutagenicity of topoisomerase-directed agents has been underestimated in the past, since these drugs are not usually capable of reacting covalently with DNA and usually have low mutagenicity in microbial assays.

Antineoplastic Agents↗
PubMed2005

Long-range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3' boundary sequence spanning 46 kilobases.

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

3' Flanking Region↗
PubMed2005

The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription.

The expression of Myod is sufficient to convert a fibroblast to a skeletal muscle cell, and, as such, is a model system in developmental biology for studying how a single initiating event can orchestrate a highly complex and predictable response. Recent findings indicate that Myod functions in an instructive chromatin context and directly regulates genes that are expressed throughout the myogenic program, achieving promoter-specific regulation of its own binding and activity through a feed-forward mechanism. These studies are beginning to merge our understanding of how lineage-specific information is encoded in chromatin with how master regulatory factors drive programs of cell differentiation.

Animals↗
PubMed1979

Injected histone antibodies interfere with transcription of lampbrush chromosome loops in oocytes of Pleurodeles.

Antibodies to calf thymus histone H2B were purified by chromatography on DEAE-cellulose and injected into oocyte nuclei of Pleurodeles waltlii. As shown by indirect immunofluorescence these antibodies cross-reacted strongly with corresponding histones associated with lampbrush chromosomes. Shortly after injection the lateral loops of the chromosomes retracted into the chromomeres and by 3 h postinjection the 'lampbrush' appearance was completely lost and the chromosomes appeared in light-microscopic preparations as rod-like structures consisting of longitudinally coalesced chromomeres. In control oocytes injected with non-immune immunoglobulins or antibodies against a ubiquitous transcript-associated protein no morphological alterations of the lampbrush chromosomes could be observed. Electron microscopic spreads of chromosomes prepared at various times after injection of anti-H2B revealed a progressive loss of transcriptional complexes from the loop axes. Finally, higher-order chromatin configurations, like supranucleosomal globules ('superbeads') or cable-like chromatin strands 50-60 nm thick predominated, indicating complete transcriptional inactivation of all chromosomal regions. The results indicate that H2B antibodies react specifically with histones associated with the transcribed DNA of lateral loops in their native state. The resulting antigen-antibody complexes seem to inhibit progression of the RNA polymerases along the template, thus causing the premature release of transcripts, a process analogous to the stripping effect of actinomycin D. The demonstration of histones associated with heavily transcribed regions, which are not compacted into nucleosomes but largely extended, supports the current concept that unfolding of nucleosomes to allow transcription of the DNA does not involve dissociation of histones. In contrast, amplified ribosomal RNA genes are unaffected by injected H2B antibodies. This does not necessarily indicate absence of histones from nucleolar chromatin, since we do not know whether it is accessible in vivo to antibodies or whether the histone antigenic determinants are masked by the presence of other proteins. The technique of injecting specific antibodies should be widely applicable when analysing the in vivo distribution of chromosomal components at the electron-microscopic level and when studying complex metabolic processes, like the cleavage and modification of RNA, by selective inhibition of defined enzymic steps.

Animals↗
PubMed2026

Bridging-driven condensation by eukaryotic SMC complexes is a conserved feature of genome organization.

The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin-DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.

Chromosomal Proteins, Non-Histone↗
PubMed2000

Fatal connections: when DNA ends meet on the nuclear matrix.

A damaged nucleus has long been regarded simply as a "bag of broken chromosomes," with the DNA free ends moving around and forming connections with randomly encountered partners. Recent evidence shows this picture to be fundamentally wrong. Chromosomes occupy specific nuclear domains within which only limited movement is possible. In a human diploid nucleus, 6.6 x 10(9) base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair. Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so-called nuclear scaffold or matrix. Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur. This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair. Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies. The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes. In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved. Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis. J. Cell. Biochem. Suppl. 35:3-22, 2000.

Animals↗
PubMed2006

Assembly of nuclear matrix-bound protein complexes involved in non-homologous end joining is induced by inhibition of DNA topoisomerase II.

Topoisomerases maintain the DNA structure by relieving the torsional stress and alleviating other topological problems occurring in DNA during transcription and replication. Topoisomerase II appears to have a close association with the family of proteins involved in the organization of chromatin in a series of loops on the proteinaceous chromosomal matrix. Beyond its physiological functions, topoisomerase II is the target for some of the most active anticancer drugs. Inhibition of the topoisomerase II function can result in DNA double-strand breaks (DSBs) and, thus, lead to chromosomal translocations. The earliest event during DSB repair is phosphorylation of histone H2AX at S139 (so-called gammaH2AX) which is believed to serve as a focal point for the assembly of repair proteins at the DSB. In this work, we have demonstrated the formation of gammaH2AX foci in two human cell lines--K562 and HeLa--after suppression of topoisomerase II activity with etoposide. Furthermore, these foci remained visible at nuclear matrices and colocalized with the major components of non-homologous end joining (NHEJ) system of DSBs repair. Thus, inhibition of topoisomerase II activity triggers assembly of NHEJ complexes at the nuclear matrix.

Antigens, Nuclear↗
PubMed2005

Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation.

It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribed RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine.

DNA↗
PubMed1995

Poly(ADP-ribosyl)ation of proteins associated with nuclear matrix in rat testis.

We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.

Animals↗
PubMed1996

[Studies on prematurely condensed chromosomes and induced condensed chromosomes with SEM].

The ultrastructure of prematurely condensed chromosomes (PCC) in BK cells and the CHO metaphase chromosomes (the PCC inducer) were studied with cell fusion technique and SEM. The methods for preparing complete specimens for SEM, were used to eliminate artifact as complete as possible, so the natural states of PCC ultrastructure, especially the ultrastructure of the early, mid, and late S-PCC, were observed for the first time. In the three phases of S-PCC multiple double strand helical structure were revealed, on the chromosome segments of BK cells. On the surface of CHO metaphase chromosomes which may induce PCC may regularly arrayed short hair-like loop-protrusions consisting of helical chromatin fibers were observed.

Animals↗
PubMed2026

Reduced R-loop abundance at proinflammatory loci: a shared epigenetic mechanism in inflammatory and metabolic diseases.

INTRODUCTION: R-loops, RNA-DNA hybrid structures with a displaced single-stranded DNA loop, are key regulators of transcriptional control, chromatin architecture, and genome stability and have emerging roles in inflammatory signaling. However, the relationship between R-loop abundance and strongly modulated inflammatory effector genes in metabolic inflammation and influenza virus infection remains underexplored. METHODS: We performed a locus-centric integrative analysis combining robust differentially expressed genes (DEGs) from multiple inflammatory and infection-related murine and human transcriptomic disease models with experimentally validated multi-cell R-loop annotations from the reference atlas RLoopBase. Our correlation framework evaluated the directional relationship between R-loop abundance and inflammatory gene expression rather than assuming disease-sample-matched R-loop measurements. We further analyzed R-loop regulatory proteins, NRF2-associated R-loop regulators, and overlaps between R-loop regulators and CRISPRi-identified mitochondrial and cellular reactive oxygen species (ROS) regulators. RESULTS: In angiotensin II-infused apolipoprotein E-deficient (ApoE-/-) mice, a model of abdominal aortic aneurysm (AAA), genomic regions encoding the top significantly upregulated genes exhibited significantly fewer R-loops than those encoding downregulated genes at days 14 and 28. Similarly, in atherosclerotic ApoE-/- mice fed a high-fat diet for 32 and 78 weeks, upregulated genes were associated with fewer R-loops than downregulated genes. Reduced R-loop abundance was also observed in genomic regions encoding the top significantly upregulated genes in liver tissues from patients with non-alcoholic steatohepatitis (NASH), as well as in monosodium urate (MSU)-stimulated lymphatic endothelial cells (LECs) and influenza virus-infected human umbilical vein endothelial cells (HUVECs). R-loop regulatory proteins upregulated during metabolic inflammation were enriched in immune and inflammatory pathways. NRF2 was identified as a regulator of 27 R-loop regulatory proteins, including 10 positively and 17 negatively regulated proteins. Furthermore, 54 R-loop regulatory proteins overlapped with CRISPRi-identified mitochondrial and cellular ROS regulators, suggesting potential reciprocal regulation between R-loop homeostasis and ROS signaling. Disease-associated changes in pro-ROS and anti-ROS R-loop regulatory proteins further linked R-loop regulation to inflammatory and oxidative stress pathways. DISCUSSION: These findings identify reduced R-loop abundance at genomic regions encoding strongly upregulated inflammatory genes as a shared feature across multiple models of metabolic inflammation and influenza virus infection. The results further suggest that immune-associated R-loop regulatory proteins and the NRF2-ROS axis may contribute to R-loop remodeling during inflammatory disease. This integrative framework provides new insight into the potential role of R-loops and ROS-sensitive R-loop regulators in inflammatory and metabolic diseases and identifies candidate pathways for future mechanistic investigation and therapeutic targeting.

R-loop regulatory proteins↗
PubMed1991

Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high level gene expression. The result of these experiments will have important consequences for random gene transfer with predictable and reproducible expression of transgenes.

Animals↗
PubMed1983

Specific DNA sequences associated with the nuclear matrix in synchronized mouse 3T3 cells.

Eukaryotic chromatin appears to be organized into arrays of supercoiled loops anchored to the scaffolding structure of the mitotic chromosome core or to the nuclear matrix of interphase nuclei. To reveal whether specific DNA sequences are involved in this level of chromatin organization, we isolated and cloned a population of DNA molecules [average length of 150 base pairs (bp)] closely associated with the nuclear matrix after exhaustive DNase digestion and subsequent extensive protease digestion. The nuclear matrix was obtained from murine BALB/c 3T3 cells synchronized at the G1/S border of the cell cycle. We report the structure of two sequences, designated G4 and G5, which are highly enriched in the matrix DNA. Sequence G4, of 152 bp, contains three 31-bp direct head-to-tail repeats. An 11-bp sequence at the end of each repeat is homologous to the first large tumor antigen recognition site of human papova virus. Sequence G5, of 135 bp, consists of two well-defined domains, in which the first domain is a fragment of the B1 repetitive sequence. The results suggest the possibility that the loops of histone-depleted chromatin are connected to the scaffold of the nuclear matrix, with specific DNA sequences at the anchorage sites.

Animals↗