Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Chromatin loop”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed2001

Higher-order chromatin structure of human granulocytes.

The structural organisation of chromatin in eukaryotes plays an important role in a number of biological processes. Our results provide a comprehensive insight into the nuclear topography of human peripheral blood granulocytes, mainly neutrophils. The nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin DNA-containing filament. The segregation of chromosomes into the nuclear lobes was studied using fluorescence in situ hybridisation (FISH). We were able to distinguish different topographic types of granulocytes on the basis of the pattern of segregation. Five topographic types were detected using dual-colour FISH in two-lobed nuclei. The segregation of four sets of genetic structures could be studied with the aid of repeated FISH and a large number of topographic types were observed. In all these experiments a non-random distribution of chromosomes into nuclear lobes was found. The painting of a single type of chromosome in two-lobed nuclei showed the prevalence of symmetric topographic types (on average in 65.5% of cases) with significant variations among individual chromosomes. The results of analysis of five topographic types (defined by two chromosomes in two-lobed nuclei) showed that the symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. The frequency of symmetric topographic types for chromosome domains was systematically higher than for genes located on these chromosomes. It appears that the prevalence of symmetric segregation patterns is more probable for large objects such as chromosome domains than for genes located on chromatin loops extending outwards from the surface of the domain defined by specific chromosome paints. This means that one chromosome domain may occur in different lobes of granulocytic nuclei. This observation is supported by the fact that both genes and centromeres were observed on filaments joining different lobes. For all chromosomes, the distances between the membrane and fluorescence gravity centre of the chromosome were measured and correlated with the segregation patterns. A higher percentage of symmetric topographic types was found in those chromosomes that were located closer to the nuclear membrane. Nuclear positioning of all genetic elements in granulocytic nuclei was studied in two-dimensional projection; however, the results were verified using three-dimensional analysis.

Chromatin↗
PubMed2002

Higher order chromatin degradation: implications for neurodegeneration.

Higher order chromatin degradation (HOCD) is a hallmark of programmed cell death. HOCD is mediated by enzymatic digestion of the DNA backbone at matrix attachment regions, and ultimately results in the excision of chromatin loops and their oligomers from chromosomes. We have recently demonstrated that hydrogen peroxide (H2O2), the major mediator of oxidative stress, rapidly induces HOCD. This demonstration allowed us to characterize several kinetic features of HOCD. Moreover, H2O2-induced HOCD provides a mechanistic link between oxidative stress and the pathology of neurodegeneration. Thus, in acute neurodegenerative conditions, which feature severe oxidative stress, H2O2-induced HOCD efficiently dismantles the genome, and thus, irreversibly commits cells to death. In chronic neurodegenerative conditions, which feature sublethal but perennial oxidative stress, cells undergo only a partial fragmentation of the genome via H2O2-induced HOCD. If unrepaired of improperly repaired, such a partial fragmentation leads to the generation and accumulation of somatic mutations that are likely to play the key role in delayed degeneration and death of neural cells.

Animals↗
PubMed2006

[Nuclear organization and expression of milk protein genes].

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.

Animals↗
PubMed2004

Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance.

The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.

Animals↗
PubMed2005

Natural antisense LHCGR could make sense of hypogonadism, male-limited precocious puberty and pre-eclampsia.

The pleiotropic effects of human chorionic gonadotrophin (hCG), the key regulator of human pregnancy, are dependent upon cell surface expression of its functional cognate receptor LHCGR in the placental trophoblasts, corpus luteum, uterus, vascular endothelial and smooth muscle cells. Additionally, lutenizing hormone-mediated signalling failure has often been linked to activating/inactivating mutations in LHCGR. One of the intriguing aspects of these studies is that the mutations are most frequently located within C-terminal 200-350 residues of the receptor protein. In an attempt to reconcile the mechanistic basis of LHCGR regulation and mutations, we have carried out bioinformatic analyses to identify the CpG-rich regions and the major potential scaffold/matrix attachment sites (S/MARs) in LHCGR and neighbouring gene (ALF) at human chromosome 2p21. Based on these analyses, we propose a chromatin-loop model, which may explain the temporal regulation and susceptibility to mutation of the human LHCGR. One of the characteristic features of the model, is that the major potential S/MAR sequences of the human LHCGR gene (68 kb) are located at the 3' end of the gene, and unlike mouse, the transmembrane and C-terminal protein coding sequences at exon 11 are embedded in this S/MAR site. Moreover, this region is subject to antisense transcription from the neighbouring gene ALF, which is gonad-specific and is only activated in meiotic spermatocytes and oocytes. Together, these analyses suggest that exon 11 of human LHCGR could be more susceptible to mutation than the other 10 exons together and that activation of LHCGR, contingent to the somatic silencing of neighbouring ALF, could be linked to male-limited precocious puberty and pre-eclampsia.

Animals↗
PubMed2003

Active poly(ADPribose) metabolism in DNAase- and salt-resistant rat testis chromatin with high transcriptional activity/competence.

A chromatin fraction, named pP fraction, was prepared from rat testis nuclei, which had been digested with nuclease in order to separate soluble and insoluble chromatin. This fraction resembled nuclear matrix as it was highly resistant to DNAase digestion, had a high content of proteins compared to the low DNA percentage, and a noticeable transcriptional activity. Moreover, poly(ADPribosyl)ation system (i.e., poly(ADPR)polymerase, poly(ADPribose), and acceptor proteins) was still present at high levels. In order to study whether it might be identified as the protein support surrounding chromatin loops, this pP fraction was further analyzed after 3 M NaCl extraction. The 3 M NaCl extract and the highly insoluble pellet, named Nuclear Matrix Pellet, were characterized as it regards DNA, newly synthesized RNA and proteins. Furthermore, poly(ADPribose) metabolism was analyzed by measuring both poly(ADPribose) polymerase and poly(ADPribose) glycohydrolase activities, poly(ADPribose) distribution and by identifying protein acceptors. The final pellet had features of nuclear matrix containing less than 10% DNA and high percentage of proteins; 28% of newly synthesized RNA was still associated with this fraction. Long and branched polyADPribose were found in the nuclear matrix-like pellet, although ADPribose acceptors (mainly H1 and core histones) appeared to be modified mostly with short ADPribose oligomers. Longest and branched polymers were retained on the top of protein gel, likely bound to automodified poly(ADPribose) polymerase.

Animals↗
PubMed1983

Architecture of metaphase chromosomes and chromosome scaffolds.

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.

Chromatin↗
PubMed2002

Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity.

It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 micro M CdSO(4) is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.

Apoptosis↗
PubMed2023

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Child↗
PubMed2000

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

Amino Acid Motifs↗
PubMed1989

Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions.

DNA elements termed scaffold-associated regions (SARs) are AT-rich stretches of several hundred base pairs which are known to bind specifically to nuclear or metaphase scaffolds and are proposed to specify the base of chromatin loops. SARs contain sequences homologous to the DNA topoisomerase II cleavage consensus and this enzyme is known to be the major structural component of the mitotic chromosome scaffold. We find that purified topoisomerase II preferentially binds and aggregates SAR-containing DNA. This interaction is highly cooperative and, with increasing concentrations of topoisomerase II, the protein titrates quantitatively first SAR-containing DNA and then non-SAR DNA. About one topoisomerase II dimer is bound per 200 bp of DNA. SARs exhibit a Circe effect; they promote in cis topoisomerase II-mediated double-strand cleavage in SAR-containing DNA fragments. The AT-rich SARs contain several oligo(dA).oligo(dT) tracts which determine their protein-binding specificity. Distamycin, which is known to interact highly selectively with runs of A.T base pairs, abolishes the specific interaction of SARs with topoisomerase II, and the homopolymer oligo(dA).oligo(dT) is, above a critical length of 240 bp, a highly specific artificial SAR. These results support the notion of an involvement of SARs and topoisomerase II in chromosome structure.

Base Composition↗
PubMed1993

Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

Animals↗
PubMed1994

Transcription factor binding sites in the matrix attachment region (MAR) of the chicken alpha-globin gene.

Nuclear matrix is a nuclear protein-DNA superstructure believed to be the exclusive site of DNA replication, transcription, repair, and recombination. The attachment regions of chromatin loops to the nuclear matrix, called MARs, nest origins of replication, have transcriptional enhancer activity, and via their interaction with protein transcription factors may govern gene switch during development and tissue-specific gene expression. In this study the 967 bp MAR of the chicken alpha-globin gene is analyzed for the presence of hexanucleotides from a number (83 in total) of vertebrate protein transcription factors and core origins of replication. A total number of 760 hexanucleotides from factor sites or origins of replication were used for this search. We found that: (1) The occurrence of protein transcription factor binding sites overall on the MAR fragment as well as on the enhancer and promoter regions of other genes is only about 1.2-1.5 times higher than in random DNA, something consistent for all MAR and enhancer sequences examined. However, a high concentration (up to 2.7 times over random sequences) of hexanucleotide factor sites is observed on small stretches of the alpha-globin gene MAR. (2) Some regulatory protein binding sites are underrepresented whereas others are overrepresented, giving to an MAR a particular transcription factor flavor. (3) The DNA curvature map of the MAR sequence and the potential sites of positioned nucleosomes suggest the sites where a competition between core histone octamers and protein transcription factors for DNA might be found. This approach might provide a novel technique to diagnose for the regulatory or nonregulatory function of a stretch of DNA. Furthermore, MARs are proposed to constitute important regulatory elements of genes in addition to enhancers, promoters, silencers, locus control regions, and origins of replication. Additional parameters such as interaction of a transcription factor with other transcription factors fixed at vicinal sites, DNA methylation, intrinsic DNA curvature torsional strain, and nucleosome positioning might also determine the high-affinity binding of a transcription factor to its functional sites and its exclusion from or low affinity binding to other nonregulatory regions.

Animals↗
PubMed1993

A new and rapid method for visualising DNA replication in spread DNA by immunofluorescence detection of incorporated 5-iododeoxyuridine.

We have developed a new and rapid immunofluorescent method for visualisation of replicated regions on fixed DNA fibers. Using this method we have found in 5-fluorodeoxyuridine (FrdU)-blocked human cells distinct replication units covering about 60 kb of DNA and corresponding in size to single replicons or chromatin loops. Our results also suggest that nonadjacent replicons within a replicon cluster may be activated after FrdU arrest and that the method may be adapted for localization on fibers of specific DNA sequences.

Chromosomes, Human↗
PubMed1998

Apoptosis, redifferentiation and arresting proliferation simultaneously triggered by oxidative stress in human hepatoma cells.

The effects of oxidative stress (ascorbic acid-ferrous system) on the proliferation, differentiation and apoptosis of the human hepatoma cell SMMC-7721 were studied. Oxidative stress significantly inhibited cell proliferation and induced morphological differentiation. Whatever the indices related with cell malignancy, such as alpha-fetoprotein and c-glutamyltranspeptidase or the index related with cell differentiation, such as tyrosine-alpha-ketoglutarate transaminase, all inclined evidently to normalization. The tumour's clonogenic potential decreased significantly. Moreover, together with differentiation, the phenomenon of apoptosis was found by the appearance of apoptotic bodies, detached cells, and apoptotic morphological feature. Although, their DNA was not degraded into oligonucleosomal fragmentation, the DNA was cut into larger fragments (about 21.2 kbp) of a size associated with chromatin loops. These findings indicated that oxidative stress can induce both differentiation and apoptosis simultaneously in tumour cells. All the results showed that oxidative stress may initiate the tumour cells reverse transformation. The possible mechanism of the differentiation and apoptosis induced by oxidative stress may be related to the lipid peroxidation of cell membrane.

Apoptosis↗
PubMed1993

DNA topoisomerase II is not detectable on lampbrush chromosomes but enriched in the amplified nucleoli of Xenopus oocytes.

In somatic cells DNA topoisomerase II (topo II) is thought to be involved in the domain organization of the genome by anchoring the basis of chromatin loops to a chromosomal scaffold. Lampbrush chromosomes of amphibian oocytes directly display this radial loop organization in cytological preparations. In order to find out whether topo II may play a role in the organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo II. Our results indicate that topo II is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomal loops. Topo II was, however, enriched in the amplified nucleoli of Xenopus oocytes.

Animals↗
PubMed1996

The topology of transcription by immobilized polymerases.

Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a polymerase immobilized in a large transcription factory. The evidence for immobilized polymerases is reviewed, and a model for transcription by such fixed enzymes is presented. According to the model, gene activation would involve reducing gene-factory distance and increasing the affinity of a promoter for a factory. Locus controlling regions and enhancers would attach to a factory and increase the chances that a promoter could bind to a polymerase; after transcriptional termination, the gene would detach from the factory. As some RNA processing occurs cotranscriptionally, processing sites are also likely to be associated with the factory.

DNA↗
PubMed1995

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis.

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.

Animals↗