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PubMedDate not supplied

[Variability of the highest levels of chromosome organization in vertebrates].

Biochemical assay of the length of the element of the highest level of compactization of deoxyribonucleoprotein fibril, i.e. the loop, in interphasic chromosomes of the liver of some animals (rat, cat, hen, pigeon, tortoise, frog) reveals significant variation of the magnitude of this value. The smallest length of the loop among the organisms investigated was found in tortoise chromosomes--5--6 X 10(3) nucleotide pairs. The length of the loop structures in mammalian chromosomes (rats, cats) is equal to 45-75 X 10(3) nucleotide pairs. In birds and amphibians, chromosomes exhibit an intermediate size of the loop element as compared to chromosomes from mammals and reptiles. Possible connection between the length of the loop structures and chromosomal diameter is discussed together with the "anomalous" size of chromosomal loops in the frog.

Animals↗
PubMed2000

Release of mitotic descendants by giant cells from irradiated Burkitt's lymphoma cell line..

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo-reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub-nuclear structure is apparent and within this, sub-nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10-20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid 'bouquets', where chromosome double-loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub-nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double-strand DNA breaks within tumour cells.

Apoptosis↗
PubMed1998

Centromere sequences localize to the nuclear halo of human spermatozoa.

Chromatin is organized into a series of discrete nuclear matrix-associated and non-nuclear matrix-associated domains. The non-matrix-associated domains consist of loops of DNA that are attached to the proteinaceous nuclear matrix by matrix-associated regions (MARs). Although this organization is well characterized in somatic cells, comparatively little is known of this mode of organizing the genome in the human sperm nucleus. To define this relationship, the interaction of human sperm chromatin with the nuclear matrix was assessed by fluorescence in situ hybridization using specific alpha satellite probes directed to the centromeric regions of chromosomes 13 plus 21 and 18. Hybridization of the centromeric sequences was visualized as segmented, bundled structures that extended from the nuclear core into the halo.

Cell Nucleus↗
PubMed2025

EWS::WT1 Isoform-Dependent Regulation of Neogenes in Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma characterized by the pathognomonic EWS::WT1 fusion protein (FP), an oncogenic chimeric transcription factor (OCTF) resulting from the t(11;22)(p13;q12) translocation. Recent studies have identified "neogenes" (NGs), genes normally silent in normal tissues but transcriptionally activated by OCTFs, as potential tumor-specific markers in fusion-driven cancers. In this study, we investigated the expression and regulation of DSRCT-specific NGs (DSRCT_NGs) using multimodal data across different cohorts of patients, PDX, and cell line data. We evaluated bulk and single-nucleus RNA sequencing of patient specimens from MD Anderson Cancer Center, revealing the robust ability for DSRCT_NGs to distinguish FP-positive DSRCT from samples failing detection of the EWS::WT1 FP. To elucidate the regulatory role of the EWS::WT1 FP in driving NG expression, we performed knockdown experiments in four DSRCT cell lines. This consistently resulted in a reduction of DSRCT_NG expression. Isoform-specific expression of EWS::WT1 in LP9 and MeT-5A mesothelial cells revealed that the E-KTS isoform of EWS::WT1 predominantly drives DSRCT_NG expression. Mechanistically, ATAC-seq and ChIP-seq analyses demonstrated that EWS::WT1 directly binds to accessible chromatin regions near NG transcription start sites, enriched for WT1 motifs and active histone marks. Integration of Hi-ChIP data further revealed that EWS::WT1 facilitates long-range enhancer-promoter looping at DSRCT_NG loci, promoting the expression of nearby genes. Collectively, these findings establish DSRCT_NGs as direct transcriptional outputs of the EWS::WT1 FP and implicate their loci as regulatory regions of the DSRCT transcriptome. Their fusion-dependent expression, chromatin accessibility, and promoter-enhancer connectivity underscore their potential utility as highly specific biomarkers and therapeutic targets in DSRCT.

DSRCT↗
PubMed2002

Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast.

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.

Blotting, Western↗
PubMed2003

Combinatorial control of DNase I-hypersensitive site formation and erasure by immunoglobulin heavy chain enhancer-binding proteins.

DNase I-hypersensitive sites in cellular chromatin are usually believed to be nucleosome-free regions generated by transcription factor binding. Using a cell-free system we show that hypersensitivity does not simply correlate with the number of DNA-bound proteins. Specifically, the leucine zipper containing basic helix-loop-helix protein TFE3 was sufficient to induce a DNase I-hypersensitive site at the immunoglobulin heavy chain micro enhancer in vitro. TFE3 enhanced binding of an ETS protein PU.1 to the enhancer. However, PU.1 binding erased the DNase I-hypersensitive site without abolishing TFE3 binding. Furthermore, TFE3 binding enhanced transcription in the presence and absence of a hypersensitive site, whereas endonuclease accessibility correlated strictly with DNase I hypersensitivity. We infer that chromatin constraints for transcription and nuclease sensitivity can differ.

Animals↗
PubMed1988

[Ultrastructure of the mitotic chromosomes in pig embryonic kidney cells during their reversible artificial decondensation in vivo].

Using methods of in vivo observation and ultrathin sectioning, it is shown that chromosomes of metaphase PE cells, previously treated with diluted Henk's solutions (70, 30 and 15%), undergo some structural transitions resulting in the formation of micronuclei. At the early stages of hypotonic treatment chromosomes are seen considerably swollen and losing the higher levels of organization, including the chromonema and chromomeres. The chromosomal bodies are formed by DNP fibers 10-25 nm in diameter making loops radiating from the central part of the chromatids. Chromosomes are capable of recondensing from this state by consecutive reconstitution of G-bands, chromomeres and the chromonema. The subsequent secondary decondensation of chromosomes is analogous to telophase decondensation at the normal mitosis, but it results in the formation of a great number of small nuclei (micronuclei). The chromatin structure in micronuclei as well as their ability to synthesize RNA and to replicate DNA show these effects to be reversible. It has been suggested that the loop organization of DNP may be essential for sustaining the structural integrity of the mitotic chromosome.

Animals↗
PubMed1975

Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-1-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core.

Animals↗
PubMed2000

Gene expression array of HTLV type 1-infected T cells: Up-regulation of transcription factors and cell cycle genes.

By utilizing a human cDNA expression array blot (588 genes), we have observed overexpression of various transcription factors, cell cycle regulated kinases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibitor, p21/waf1. The p21/waf1 transcription and protein is overexpressed in all HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samples. While p21/waf1 has been shown to display a selectivity for G(1)/S cyclin/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/cdk2. Functionally, the association of p21/cyclin A/cdk2 decreased the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, as well as affecting other substrates such as the C-terminus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p21/waf1 promoter in a p53-independent manner. We found that the minimal p21/waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal promoter contained two E2A transcription factor binding sites located between the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1-infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA binding site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p21/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1-infected cells.

Basic Helix-Loop-Helix Proteins↗
PubMed2001

Multiple modes of transcriptional regulation by the HIV-1 Tat transactivator.

Regulation of HIV-1 gene expression by the viral Tat transactivator is a critical step in the viral life cycle. Tat acts as a highly unusual transcription factor that interacts with a stem-loop RNA structure (TAR) found at the 5' end of all viral transcripts. There, it induces a modification of chromatin at the HIV-1 long terminal repeat (LTR) promoter and stimulates the recruitment of elongation-competent RNA polymerase II complexes capable of processive transcription. Increase of transcriptional elongation is the consequence of the interaction of Tat with cyclin T1, the cyclin component of CDK9, which phosphorylates the carboxy-terminal domain of RNA polymerase II to enhance its processivity. Tat-induced transcriptional activation of the LTR promoter is concomitant with recruitment of the transcriptional coactivators p300 and the highly homologue cAMP-responsive transcription factor binding protein (CBP). These large proteins act at the level of transcriptional initiation by bridging the basal transcription machinery with specific transcriptional activators. Furthermore, p300/CBP are histone acetyl-transferases capable of modulating the interaction of nucleosomes with DNA and with chromatin remodeling complexes. Besides histones, Tat itself is a substrate for the enzymatic activity of p300/CBP and of the associated factor P/CAF, suggesting a regulatory role of acetylation on the protein itself. Devising a unifying model for LTR activation that includes activities of Tat at the levels of both transcriptional initiation and transcriptional elongation is a challenging task at this moment. Nevertheless, protein localization studies indicate that both cyclin T1 and p300/CBP co-localize in specific subnuclear compartments, thus suggesting participation of both proteins in the formation of multimolecular complexes governing coordinated steps of transcriptional activation.

Acetyltransferases↗
PubMed2005

Thermodynamics of DNA loops with long-range correlated structural disorder.

We study the influence of a structural disorder on the thermodynamical properties of 2D-elastic chains submitted to mechanical/topological constraint as loops. The disorder is introduced via a spontaneous curvature whose distribution along the chain presents either no correlation or long-range correlations (LRC). The equilibrium properties of the one-loop system are derived numerically and analytically for weak disorder. LRC are shown to favor the formation of small loop, larger the LRC, smaller the loop size. We use the mean first passage time formalism to show that the typical short time loop dynamics is superdiffusive in the presence of LRC. Potential biological implications on nucleosome positioning and dynamics in eukaryotic chromatin are discussed.

Chromatin↗
PubMed1984

Modeling Balbiani Ring gene transcription with electron microscope tomography.

Cross-sectional views of mature regions of Balbiani Ring (BR) transcription loops were reconstructed by electron microscope tomography. Balsa wood models were built based upon the resulting tomograms. Coordinates of the centers of the ribonucleoprotein BR granules were estimated and employed to calculate the approximate orientation of the BR transcription unit axis in the plastic section. The density of BR granules per micron of transcription unit axis was estimated. Employing structural parameters for mature regions of BR transcription loops derived from present and previous studies, theoretical calculations were performed to examine potential steric restrictions around the central chromatin axis. Tomograms were also observed and photographed on a varifocal (vibrating) mirror to illustrate the utility of a rapid user-interactive 3-D display.

Animals↗
PubMed2004

Hairy transcriptional repression targets and cofactor recruitment in Drosophila.

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2-6 h) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for Hairy in segmentation, we find groups of targets suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find here that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development.

Adenine↗
PubMed1981

Chromatin fine structure of active and repressed genes.

Study of the structural organization of chromatin during transcription and replication may reveal important aspects of these processes. At the lowest level of organization, chromatin consists of a repeating subunit, the nucleosome (for reviews see refs 1-3). Electron microscopy indicates that the nucleosomes are arranged helically or form discrete superbeads, generating the familiar 250 A-300-A fibre. It has been suggested that this fibre is further folded into loops containing up to several hundred nucleosomes. Despite extensive study, the significance and fate of these nucleosomes remain obscure. We have used here micrococcal nuclease digestion to compare the structures of actively transcribing and inert chromatin of the genes coding for the major heat-shock protein of Drosophila melanogaster. The repressed hsp 70 genes were considerably more resistant to cleavage by micrococcal nuclease than their flanking regions and the bulk of chromatin. The active genes, previously shown to be more sensitive than the repressed genes, are also more susceptible to the nuclease than their 3'-flanking regions and bulk chromatin.

Animals↗
PubMed1985

Stepwise unfolding of chromatin by urea. A flow linear dichroism and photoaffinity labeling study.

The unfolding of chromatin by urea (0-7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1-2 M urea and 6-8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2-6 M urea), and finally resulting in an almost total stretching of the DNA (greater than 6 M urea).

Affinity Labels↗
PubMed2006

Cooperativity between Rad51 and C/EBP family transcription factors modulates basal and Tat-induced activation of the HIV-1 LTR in astrocytes.

Transcription of the HIV-1 genome is a complex event that requires functional and physical communication of cellular proteins that recognize the LTR sequence with viral proteins, most notably, Tat. Moreover, studies have revealed the ability of Tat to induce transcription of a variety of cellular genes whose products can affect the status of cells, thus contributing to the pathogenesis of AIDS. Recently, we demonstrated that expression of Tat in astrocytes and other neural cells leads to upregulation of Rad51, a major component of DNA repair via homologous recombination. The unscheduled upregulation of Rad51, in turn, has an impact upon the extent of chromosomal abnormalities that are seen in Tat-producing cells. Here, we asked whether an elevation in Rad51 levels influences the extent of viral gene transcription in astrocytic cells. Our results demonstrate that ectopic expression of Rad51 enhances the basal- and the Tat-induced transcription of the LTR promoter. This event requires cooperativity from the C/EBP family of transcription factors including C/EBPbeta and C/EBPbeta homologous protein (CHOP). Similar to Tat, we showed that Rad51 interacts with C/EBPbeta and augments its interaction with the DNA motif spanning nucleotides -120 to -94 of the LTR. Interestingly, Tat exhibited the capacity to augment the synergism between Rad51 and C/EBPbeta. Our results also demonstrate that the level of activation of the LTR by CHOP and Tat, either alone or together, is elevated in the presence of the SW1/SNF1 chromatin remodeling complex. These observations unravel a new pathway for Tat activation of the LTR that includes the positive feedback loop involving Rad51 and C/EBPbeta family proteins.

Astrocytes↗
PubMed1989

SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size.

By in situ hybridization, short interspersed repeated DNA elements (SINEs), exemplified by Alu repeats, are located principally in Giemsa-light human metaphase chromosome bands. In contrast, the L1 family of long interspersed repeats (LINEs) preferentially cluster in Giemsa-dark bands. These SINE/LINE patterns also generally correspond to early and later replication band patterns. In order to provide a molecular link between structurally visible chromosome bands and a framework of interspersed repeats, we investigated patterns of SINE and LINE hybridization using pulse-field gel electrophoresis (PFGE). Interspersed SINEs and LINEs hybridize with high intensity to specific size fragments of 0.2-3 megabase pairs (Mb). Using appropriate restriction enzymes and pulse-field conditions, a number of fragments were delineated that were either SINE or LINE rich, and were mutually exclusive. Control studies with a human endogenous retroviral repeat that is related in sequence to the major LINE family, delineated a subset of fragments of 0.07-0.4 Mb with unequal intensity. Thus these less numerous repeats also appear to cluster selectively in DNA domains that are larger than a chromosome loop (60-120 kb). In summary, PFGE studies independently confirm the clustering of interspersed repeats on contiguous DNA loops. Selective clustering of repeat motifs may contribute to special structural or functional properties of large chromosome domains, such as chromatin extension/condensation or replication characteristics. In some cases the DNA fragments defined by these repeats approach the size of tandem satellite arrays.

Animals↗