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Results for “chromatin loop formation”

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PubMed1985

Differential formation of O6-ethylguanine in the DNA of rat brain chromatin fibers of different folding levels exposed to N-ethyl-N-nitrosourea in vitro.

Extended (histone H1-depleted), 11-nm-thick chromatin fibers and condensed 25- to 35-nm-thick chromatin fibers, representing the first and second level of DNA folding in chromatin, respectively, as well as nucleosome core particles, were isolated from fetal rat brain cells and briefly exposed to N-ethyl-N-nitrosourea (EtNU) in vitro. The O6-ethyl-2'-deoxyguanosine (O6-EtdGuo):2'-deoxyguanosine (dGuo) molar ration in DNA enzymatically hydrolyzed to 2'-deoxynucleosides was determined by competitive radioimmunoassay using an anti-O6-EtdGuo monoclonal antibody with an affinity constant for O6-EtdGuo of approximately 2 X 10(10) liters/mol. In comparison to naked DNA (O6-EtdGuo:dGuo relative value, 1.0) the O6 atom of guanine proved to be increasingly protected from ethylation by EtNU in the DNA of the histone H1-depleted chromatin fibers (relative value, approximately 0.6) and of the condensed chromatin fibers (relative value, approximately 0.4). From the O6-EtdGuo:dGuo relative values obtained for the DNA of nucleosome core particles (approximately 0.5) and of H1-depleted chromatin fibers (approximately 0.6), it follows that the accessibility of the O6 atom of guanine to the electrophilic ethyldiazonium ion generated from EtNU in internucleosomal DNA (protein free) is about twice that found in nucleosomal DNA. The overall O6-EtdGuo:dGuo molar ratio in the DNA of H5 rat hepatoma cells exposed to EtNU in vitro was similar to that of the DNA of the condensed 25- to 35-nm chromatin fibers. Since the bulk of genomic DNA is organized in the form of these chromatin fibers, the overall degree of intercellular O6-EtdGuo formation appears to be mainly determined by this folding level of DNA, and not, or only to a low degree, by the DNA folding levels of higher-order chromatin structures such as those found in large loops or domains of chromatin fibers within the cell nucleus.

Alkylation↗

PubMed1986

Factors controlling the size of DNA loops in frog embryos and Friend erythroleukemia cells.

Measurements were made of the average size of fluorescent halos of nucleoids of developing frog embryos, mouse fibroblasts (A9 cells) and Friend erythroleukemia cells cultured with various compounds. These halos are thought to represent relaxed lengths of loops of DNA attached to the nuclear matrix. Measurement of transcription of RNA from nuclei in vitro suggested that cells with more loops (smaller halos) had more initiation sites for transcription. DNA loop number decreases during development of the frog embryos, and red blood cells of adults have even fewer and larger loops. One or two days of culture of Friend cells in various compounds that induce differentiation resulted in more DNA loops than for control cells, but after 5 days of culture the differentiated erythrocytes had fewer DNA loops than control cells at stationary phase. Stationary phase cells have almost twice as many DNA loops as log phase cells. Various compounds that stall cells at the G1/S border induced more DNA loops, but subsequently the number of DNA loops decreased as the cells entered S phase. A number of compounds which slow the rate of cell division cause the formation of more DNA loops, perhaps due to longer G1 periods.

Animals↗

PubMed2005

Mot1-mediated control of transcription complex assembly and activity.

Mot1 is an essential Snf2/Swi2-related ATPase and TATA-binding protein (TBP)-associated factor (TAF). In vitro, Mot1 utilizes ATP hydrolysis to disrupt TBP-DNA complexes, but the relationship of this activity to Mot1's in vivo function is unclear. Chromatin immunoprecipitation was used to determine how Mot1 affects the assembly of preinitiation complexes (PICs) at Mot1-controlled promoters in vivo. We find that the Mot1-repressed HSP26 and INO1 promoters are both regulated by TBP recruitment; inactivation of Mot1 leads to increased PIC formation coincident with derepression of transcription. For the Mot1-activated genes BNA1 and URA1, inactivation of Mot1 also leads, remarkably, to increased TBP binding to the promoters, despite the fact that transcription of these genes is obliterated in mot1 cells. In contrast, levels of Taf1, TFIIB, and RNA polymerase II are reduced at Mot1-activated promoters in mot1 cells. These results suggest that Mot1-mediated displacement of TBP underlies its mechanism of repression and activation at these genes. We suggest that at activated promoters, Mot1 disassembles transcriptionally inactive TBP, thereby facilitating the formation of a TBP complex that supports functional PIC assembly.

Adenosine Triphosphatases↗

PubMed1999

cDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin.

The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.

Amino Acid Sequence↗

PubMed2005

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription.

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

Animals↗

PubMed1999

The homeodomain protein NK-3 recruits Groucho and a histone deacetylase complex to repress transcription.

Transcriptional repression by sequence-specific DNA binding factors is mediated by the recruitment of a corepressor complex to the promoter region. The NK-3 homeodomain protein is a transcriptional repressor that recruits the nuclear protein kinase, homeodomain interacting protein kinase 2 (HIPK2). Here we show that HIPK2 is a component of a corepressor complex containing Groucho and a histone deacetylase complex. Groucho, like HIPK2, acts as a corepressor for NK-3 and binds to NK-3 and HIPK2. Moreover, HIPK2 appears to regulate the corepressor activity of Groucho. Transcriptional repression by NK-3 and Groucho is relieved by the histone deacetylase inhibitor trichostatin A, and both NK-3 and Groucho directly interact with the histone deacetylase HDAC1 that is associated with mSin3A in vivo. Recruitment of the histone deacetylase complex by NK-3 decreases the acetylated histones that are associated with the target gene promoter. These results indicate that NK-3 represses transcription by recruiting a complex containing Groucho and a histone deacetylase complex that leads to histone modification on chromatin and suggest that HIPK2 may play a regulatory role in the corepressor complex formation.

Base Sequence↗

PubMed2003

Structural and dynamic functions establish chromatin domains.

Drosophila and mammalian proteins protect genes from heterochromatic repression in Saccharomyces cerevisiae by two different mechanisms. Factors termed genuine boundary activities (BAs) establish a structural, unidirectional bulwark against heterochromatin. In contrast, factors termed desilencing activities (DAs) act by the formation of a bidirectional, euchromatic island that blocks spreading of heterochromatin. The Drosophila boundary protein BEAF and, unexpectedly, the mammalian factor Sp1 exhibited a robust BA in yeast. In contrast, mammalian CTCF, Drosophila GAGA factor, yeast Gcn5p, and many mammalian transcription factors, although inactive as upregulators of nonsilenced genes, work as DAs. DAs but not BAs protect telomere-linked genes from silencing, presumably due to looping of telomeres and ensuing multidirectional silencing. The data demonstrate that "genetic autonomy" of chromatin domains is established by both passive and active mechanisms.

Animals↗

PubMed1981

Correlation of hnRNP structure and nascent transcript cleavage.

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (approximately 50 A wide) and retained segments have a thicker particulate morphology (approximately 250 A diameter). A characteristic secondary structure formation also accompanies cleavage--that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length from 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3-3 min after transcription of the cleavage site.

Animals↗

PubMed1999

Chromatin condensation is confined to the loop and involves an all-or-none structural change.

Using differential scanning calorimetry in combination with pulsed field gel electrophoresis, we relate here the changes in the thermal profile of rat liver nuclei induced by very mild digestion of chromatin by endogenous nuclease with the chain length distribution of the DNA fragments. The enthalpy of the endotherm at 106 degrees C, which reflects the denaturation of the heterochromatic domains, decreases dramatically after the induction of a very small number of double-strand breaks per chromosome; the thermal transition disappears when the loops have undergone on average one DNA chain scission event. Quantitative analysis of the experimental data shows that the loop behaves like a topologically isolated domain. Also discussed is the process of heterochromatin formation, which occurs according to an all-or-none mechanism. In the presence of spermine, a strong condensation agent, only the loops that have undergone one break are able to refold, in confirmation of the extremely cooperative nature of the transition. Furthermore, our results suggest a relationship between the states that give rise to the endotherms at 90 degrees C and 106 degrees C and the morphologies referred to as class II and class III in a previous physicochemical study of the folding of chromatin fragments (Widom, 1986. J. Mol. Biol. 190:411-424) and support the view that the overall process of condensation follows a sequential (two-step) pathway.

Animals↗

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↗

PubMed2006

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome.

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.

Active Transport, Cell Nucleus↗

PubMed1981

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus.

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.

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↗

PubMed1997

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Antigens, Nuclear↗

PubMed1996

[Ultrastructural and immunocytochemical analysis of the synaptonemal complex at the initiation of synapsis].

The synaptonemal complexes (SCs) are nuclear structures specific for meiosis. They have a central role in homolog chromosomes coupling; they are essential in crossing over events and chromosomic segregation during the first meiotic division. When its joining ends in pakiteno stage, each synaptonemal extends along the bivalent joining the ends to nuclear wrapping. The SCs are characterized by the presence of two lateral elements and a central region. The lateral elements are parallel and equidistant. The chromatine of homolog chromosomes fixes in a series of loops to these elements. The central region is between the lateral elements. It is formed by the latero-medial fibers and the medial element. The first ones are perpendicularly oriented to the longitudinal axis of CS and connect lateral elements with the medial element. The recombination modules have an active role in recombination processes and quiasma formation, they are associated, at intervals, with the central region among the homolog chromosomes. The localization and function of nucleic acids in formation and coupling of synaptonemal complex is little known, so methodologic alternatives are looked for to resolve this type of problems. In this work, ADN distribution in chicken ovocytes in cigotene, using techniques for electronic microscopy of immuno-oro, were studied. Besides, cytochemical techniques, were used as preferential contrast for ADN or preferential for ribonucleoproteins (RNPs). The combination of preferential tincture for RNPs and immunolocalization of ADN show that chromatin accumulates jointly with ribonucleoproteins in nor coupled lateral elements and the presence of numerous RNPs fibers distributed around lateral elements. Recombination nodules were found among lateral elements during the coupling, these nodules are PTA positives, which means ADN presence, and so, ADN presence among lateral elements. THe presence of a bridge of marked fibers with coloidal gold (ADN) uniting not coupled lateral elements, suggests ADN as a sort of macromollecule forming synapsis sites.

Animals↗

PubMed1980

Ribonucleoprotein structure in nascent hnRNA is nonrandom and sequence-dependent.

We have studied the ribonucleoprotein morphology of nascent hnRNA using chromatin spreading methods. This approach allows visualization of multiple transcripts of the same DNA sequence. Thus the RNP structure of hnRNA molecules with the same nucleotide sequence can be compared. We find that RNP particles averaging 240 A in diameter occur on the majority of hnRNA transcription units, but that RNA fibrils are generally not completely covered with particles. Furthermore, the RNP particle location is nonrandom for the transcripts of a given gene. Particle number and arrangement on RNP fibrils vary from one transcription unit to the next, and are not obviously related to transcription unit size or activity. Analysis of particle location with respect to the 5' RNA terminus allows RNP particle localization to homologous sequences of less than 500 ribonucleotides on the different fibrils of a single transcription unit. RNP particles assemble very soon after synthesis of the particular sequence. Sites of particle formation are distinct from sites of double-stranded hairpin loops on the RNA fibrils. A differential, or differentially stable, RNP structure with respect to RNA sequence may bear on mechanisms of specific hnRNA processing.

Animals↗

PubMed1992

Chromatin compaction and the efficiency of formation of DNA-protein crosslinks in gamma-irradiated mammalian cells.

Chromatin has been prepared from Chinese hamster V79 cell nuclei by successive suspension and sedimentation in buffers of decreasing ionic strength. For buffer concentrations from 50 to 1 mM, the resultant chromatin maintained a normal histone content, nucleosomal organization, and attachment to the nuclear matrix; however, as the buffer concentration was reduced from 50 to 10 and 1 mM, the higher-order chromatin structures became increasingly relaxed. Fully expanded chromatin is 5- to 10-fold more susceptible to the induction of DNA-protein crosslinks (DPCs) by gamma radiation than is chromatin residing in living interphase cells. As much as 60-70% of expanded chromatin can be induced to form DPCs as compared to a maximum of about 20% of cellular DNA. For expanded chromatin, the maximum level of induced DPCs is two to three times higher than would be expected if only matrix-associated DNA were induced to form DPCs. Therefore, DNA in distal regions of chromatin loops must also be induced to form DPCs with histones or other nonhistone chromosomal proteins. The hypersensitivity of isolated chromatin to radiation-induced production of DPCs appears to be related to the expansion of chromatin conformation rather than to the removal of intracellular radical scavengers for the following reasons: (a) there is an inverse relationship between the buffer concentration in which the chromatin is suspended and DPC formation, and (b) the induction of a more compact 30-nm chromatin fiber from the expanded 10-nm chromatin fiber in the presence of a low concentration of MgCl2 results in a marked reduction in DPC formation. The formation of radiation-induced DPC seems to occur at maximum efficiency in fully expanded chromatin, since DPC formation cannot be further stimulated by the addition of Cu2+, which can catalyze the production of OH by Fenton chemistry. It is concluded that radiation-induced DNA damage production is greatly influenced by chromatin conformation, and that chromatin as it exists in the cell is a relatively poor substrate for DNA-protein crosslinking in comparison to completely expanded chromatin.

Animals↗

PubMed1987

The higher order structure of the centromere.

The architecture of the centromere region of mouse chromosomes has been studied in cells grown in the presence of 5-azacytidine. This drug interferes with normal condensation producing elongated centromere regions. It has been found that this effect is reversible in the presence of the drug, allowing the observation of the repackaging of the extended centromere into a structure exhibiting native centromere morphology. Light microscopy as well as transmission and scanning electron microscopy of this condensation process suggests that the native centromere is formed by the helical folding of a subfiber with an approximate diameter of 100 nm. This fiber is in turn composed of loops of the 30-nm fiber class. The boundary between successive gyres of the subfiber are obscured at the completion of condensation resulting in the formation of a homogenous 250- to 300-nm fiber that is the native centromere. These observations provide evidence for an additional level of chromatin organization within the metaphase chromosome.

Animals↗