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Results for “chromatin loop formation”

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PubMed2006

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation.

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.

Animals↗

PubMed2004

[The potential role of nuclear matrix attachment regions (MARs) in regulation of gene expression].

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.

Chromatin↗

PubMed2025

Barrier effects on the kinetics of cohesin-mediated loop extrusion.

Chromosome organization mediated by structural maintenance of chromosome complexes is crucial in many organisms. Cohesin extrudes chromatin into loops that are thought to lengthen until it is obstructed by CTCF proteins. In complex cellular environments, the loop extrusion machinery may encounter other chromatin-binding proteins. How these proteins interfere with the cohesin-meditated extrusion process is largely unexplored, but recent experiments have shown that some proteins serve as physical barriers that block cohesin translocation. Other proteins containing a cohesin-interaction motif serve as chemical barriers to induce cohesin pausing through interactions with it. Here, we develop an analytically solvable approach for the loop extrusion model incorporating barriers to investigate the effect of the barrier on the passive extrusion process. To further quantify the impact of barriers, we calculate the mean looping time it takes for cohesin to translocate to form a stable loop before dissociation. Our finding reveals that the physical barrier can accelerate the loop formation, and the degree of acceleration is closely related to the impedance strength of the physical barrier. In particular, the synergy of the cohesin loading site and the physical barrier site accelerates loop formation more significantly. The proximity of the cohesin loading site to the barrier site facilitates the rapid formation of stable loops in long genomes, which implies loop extrusion and chromatin-binding proteins might shape functional genomic organization. Conversely, chemical barriers consistently impede loop formation, with increasing impedance strength of the chemical barrier leading to longer loop formation time. Our study contributes to a more comprehensive understanding of the complexity of the loop extrusion process, providing a new perspective on the potential mechanisms of gene regulation.

Cohesins↗

PubMed1999

Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences.

Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.

Antigens, Nuclear↗

PubMed2025

Crosstalk between chromatin state and ATM signalling in DNA damage-induced transcription stress.

The DNA Damage Response (DDR) is a highly regulated process that safeguards genomic integrity against DNA lesions. Increasing evidence supports a reciprocal relationship between damaged chromatin architecture and the signalling pathways that coordinate the DDR. However, the mechanisms underlying this interplay in response to transcription-blocking DNA lesions remain largely unexplored. Here, we show that stalling of RNA polymerase II (RNAPII) at such lesions induces local chromatin acetylation, mediated primarily by the histone acetyltransferase p300. The resulting chromatin relaxation stimulates the dissociation of mature co-transcriptional spliceosomes from nascent RNA and promotes RNA:DNA hybrid (R-loop) formation, leading to ATM activation. In turn, activated ATM modulates chromatin conformation by phosphorylating histone H2A.X and triggering p38MAPK/MSK1-dependent histone H3S10 phosphorylation. Our findings highlight the cross-regulation between chromatin state and ATM signalling as a key component of the cellular response to transcription stress.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2026

Genome assembly of Astatotilapia latifasciata uncovers B chromosome-linked chromatin reorganization.

B chromosomes (Bs) are supernumerary genomic elements found in many eukaryotes, yet their full sequence composition, functional potential, and regulatory impact on the host genome remain unclear. Here, we present a chromosome-level genome assembly of the cichlid fish Astatotilapia latifasciata, integrating PacBio long reads, Illumina short reads, and Hi-C chromatin contact maps to resolve both A and B chromosomes. The 0.93 Gb assembly (N50 = 36.2 Mb) includes a 34 Mb B chromosome containing 789 predicted protein-coding genes and a markedly higher density of transposable elements (TEs), especially long terminal repeats (LTR) retrotransposons. Transcriptome profiling revealed that B-linked genes are predominantly transcriptionally repressed relative to their A chromosome paralogs. Hi-C-based chromatin modeling uncovered distinct 3D structural configurations associated with the B chromosome, including fewer topologically associating domains (TADs), reduced loop formation, and altered compartmentalization. These changes are linked to long-range chromatin interactions and genomic rearrangements, suggesting that the B chromosome reshapes the nuclear architecture of the host genome. Our study proposes a potential regulatory role of Bs in genome and provides a genomic resource for investigating chromosome evolution in cichlids.

Animals↗

PubMed1996

DNA segments sensitive to single-strand-specific nucleases are present in chromatin of mitotic cells.

It was observed before that DNA in situ in chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNA in situ in mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells' exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.

Bromodeoxyuridine↗

PubMed2005

DNA-loop formation on nucleosomes shown by in situ scanning force microscopy of supercoiled DNA.

The flexibility of the chromatin structure, necessary for the processing of the genomic DNA, is controlled by a number of factors where flexibility and mobility of the nucleosomes is essential. Here, the influence of DNA supercoiling on the structure of single nucleosomes is investigated. Circular supercoiled plasmid DNA sub-saturated with histones was visualized by scanning force microscopy (SFM) in aqueous solution. SFM-imaging compared with topological analysis indicates instability of nucleosomes when the salt concentration is raised from 10 mM to 100 mM NaCl. Nucleosomes were observed after the deposition to the used scanning surface, i.e. mica coated with polylysine. On the images, the nucleosomes appear with a high probability in end-loops near the apices of the superhelices. In 100 mM NaCl but not in 10 mM NaCl, a significant number of complexes present the nucleosomes on superhelical crossings mainly located adjacent to an end-loop. The morphology of these structures and statistical analysis suggest that DNA loops were formed on the histone octamers, where the loop size distribution shows a pronounced peak at 50 nm. Recently, the formation and diffusion of loops on octamers has been discussed as a mechanism of translocations of nucleosomes along DNA. The presented data likely confirm the occurrence of loops, which may be stabilized by supercoiling. Analysis of the structure of regular nucleosomes not located on crossings indicates that reducing the salt concentration leads to more conformations, where DNA is partially unwrapped from the distal ends of the octamer.

DNA, Superhelical↗

PubMed1995

A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes.

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

Animals↗

PubMed2006

Chromatin remodeling: RSC Motors along the DNA.

Single-molecule experiments show that the chromatin-remodeling complex RSC, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation.

Adenosine Triphosphatases↗

PubMed1995

DNA looping by Saccharomyces cerevisiae high mobility group proteins NHP6A/B. Consequences for nucleoprotein complex assembly and chromatin condensation.

The formation of higher order protein.DNA structures often requires bending of DNA strands between specific sites, a process that can be facilitated by the action of nonspecific DNA-binding proteins which serve as assembly factors. A model for this activity is the formation of the invertasome, an intermediate structure created in the Hin-mediated site-specific DNA inversion reaction, which is stimulated by the prokaryotic nucleoid-associated protein HU. Previously, we have shown that the mammalian HMG1/2 proteins substitute for HU in this system and display efficient DNA wrapping activity in vitro. In the present work, we isolate the primary sources of assembly factor activity in Saccharomyces cerevisiae, as measured by the ability to stimulate invertasome formation, and show that these are the previously identified NHP6A/B proteins. NHP6A/B have comparable or greater activity in DNA binding, bending, and supercoiling with respect to HU and HMG1 and appear to form more stable protein.DNA complexes. In addition, expression of NHP6A in mutant Escherichia coli cells lacking HU and Fis restores normal morphological appearance to these cells, specifically in nucleoid condensation and segregation. From these data we predict diverse architectural roles for NHP6A/B in manipulating chromosome structure and promoting the assembly of multicomponent protein.DNA complexes.

Base Sequence↗

PubMedDate not supplied

Sperm chromatin.

Available data on dry and hydrated nuclear volume of mammalian spermatozoa indicate that available volume is clearly insufficient to contain sperm chromatin packed in nucleosome-like structures. Therefore, sperm DNA-protein complexes must be packed differently than somatic DNA-protein complexes. Packing of DNA in fixed, dehydrated mammalian sperm approaches the physical limits of molecular compaction, making mammalian sperm chromatin the most condensed eukaryotic DNA known. The fundamental packaging unit of sperm chromatin is a toroid approximately 900-A outer diameter. 200-A thickness, and 150-A diameter hole. Each toroid contains 60 kilobases of DNA and is linked to other toroids by uncoiled DNA stretches. The factors that contribute to mammalian chromatin structuration are still under study. The role of protamines in sperm chromatin condensation and nuclear shaping has been overstressed to the exclusion of other possible factors. Chromatin organization in sperm nuclei is maintained during sperm condensation by tight interactions with the nuclear matrix at fixed sites, inducing the formation of individual toroid-shaped DNA loop stuctures. Observations that abnormal manchettes affect sperm head shape and chromatin organization inducing sterility speak about manchette importance during chromatin organization. The presence in sperm chromatin of regions packaged in specific ways with several types of protamines or even with histones, indicates that nuclear shaping and chromatin organization must be under DNA control. The structural properties that distinguish sperm DNA from somatic DNA may play the most important role in chromatin organization.

Animals↗

PubMed2003

Role of the M-loop and reactive center loop domains in the folding and bridging of nucleosome arrays by MENT.

MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL.

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↗

PubMed1985

Distribution of initial and persistent 2-acetylaminofluorene-induced DNA adducts within DNA loops.

The intranuclear distribution of initial and persistent DNA adducts induced in vivo after four weekly injections of the hepatocarcinogen 2-acetylaminofluorene was examined in rat liver by using a protocol that fractionates chromatin from various regions of each of the multiple nuclear DNA loops. Ten hours after the initial dose, two acetylated [(N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene] and one deacetylated [N-(deoxyguanosin-8-yl)-2-aminofluorene] adduct were detected by a 32P-labeling assay and were found to have a random genomic distribution, as evident by their relative concentrations in various chromatin fractions. These data suggest that all regions of the DNA loops are equally susceptible to adduct formation. A nonrandom persistence of the deacetylated adduct in the regions where the DNA loops are constrained by the nuclear matrix was evident by 6 days after the last dose and was markedly apparent by 60 days. In contrast, all chromatin fractions had equally inefficient removal of the N2-acetylated adduct by 6 days as well as 60 days but had complete removal of the C8-acetylated adduct. These findings suggest that pronounced regional differences in adduct repair along the DNA loops may play a role in chemically induced hepatocarcinogenesis.

2-Acetylaminofluorene↗

PubMed1996

The unique, complex organization of the transcriptionally silent sperm chromatin.

The sperm nucleus contains one haploid copy of the genome that is completely transcriptionally silent and is not being replicated. Recent evidence has revealed that this "silent" chromatin nevertheless contains a complex organization at all levels. This includes DNA loop domain formation by the sperm nuclear matrix that is gene specific and highly ordered folding patterns of the chromosomes, particularly with respect to centromere and telomere positioning. Such specificity in the sperm DNA organization suggests functional requirements for their existence. As these begin to emerge, the sperm nucleus is becoming an important model for the study of the eukaryotic genome.

Animals↗

PubMed2004

Taz1 binding to a fission yeast model telomere: formation of telomeric loops and higher order structures.

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.

DNA↗

PubMed2002

Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation.

Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

Cell Cycle Proteins↗