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Results for “chromatin loop formation”

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PubMed2001

DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules.

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.

Amino Acid Motifs↗

PubMed2026

Haplotype-resolved 3D genome maps reveal RNAPII-mediated allelic regulation in hybrid rice.

To understand how the two parental genomes coordinate transcription in hybrids, chromatin architecture must be resolved at the haplotype level. Here, using phased Bridge-Linker Hi-C, we reconstructed a haplotype-resolved three-dimensional (3D) genome of the elite hybrid rice (Oryza sativa) line Shanyou 63 (SY63). We identified extensive allele-specific chromatin conformations. Furthermore, we generated allele-resolved RNAPII ChIA-PET maps and phased transcriptomes to explore how chromatin interactions contribute to allelic regulation. Although maternal and paternal homologs share broadly similar chromatin features, we detected widespread haplotype-biased RNAPII binding and chromatin looping at high resolution. These allele-specific RNAPII-mediated contacts were significantly associated with biased expression. Stronger RNAPII binding on one haplotype promoted the formation of long-range regulatory loops with distal genes, thereby contributing to allele-biased transcription at a subset of loci, even when promoter-proximal RNAPII occupancy was comparable between alleles. These results demonstrate that subtle differences in RNAPII engagement and 3D regulatory wiring between parental haplotypes can reshape transcriptional output in hybrids, providing new insights into the mechanisms underlying the allelic regulation of gene expression.

Allele-specific chromatin interactions↗

PubMed2003

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Animals↗

PubMed1998

Mechanisms of radiation-induced chromatid breaks.

Chromatid breaks are thought to result from DNA double-strand breaks (dsb) but the mechanisms are not yet understood. The early (but still prevailing) 'breakage-first' hypothesis fails to explain the large size of chromatid breaks; many of which are estimated to represent the apparent loss of between 15 and 45 Mbp (up to 30% of an average chromatid). The alternative 'exchange' hypothesis of Revell has potential for explaining the large sizes of deletions, but assumes the interaction of two lesions which therefore predicts a quadratic dependence of chromatid breaks on radiation dose. The exchange hypothesis is not tenable for mammalian cells since chromatid breaks are observed to be induced linearly with dose in both human and rodent cells. An alternative 'signal' model of chromatid breaks is outlined whereby a single dsb, occurring within a large looped chromatin domain, is signalled (possibly by molecules such as DNAPK or ATM protein) and triggers the cell to undergo a recombinational exchange, either within a chromatid or between sister chromatids. If incomplete, such recombinational exchanges would appear as chromatid breaks at metaphase. It is suggested that the large looped chromatin domains could be equivalent to one or more likely several replication 'factories' in which the DNA processing enzymes required for exchange formation would be located.

Animals↗

PubMedDate not supplied

[Structure and function of nuclear matrix associated regions (S/MARs)].

Modern concepts on the chromatin loop-domain organization and the role of the DNA regions specifically binding the nuclear matrix (nuclear scaffold, or S/MARs) in its formation, maintenance, and regulation are discussed. Some S/MAR structural features, properties of binding the nuclear matrix, and probable mechanisms of their involvement in regulation of gene activity are considered. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Amino Acid Sequence↗

PubMed2005

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

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

DNA↗

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↗

PubMed1993

Histone H4 acetylation and transcription in amphibian chromatin.

Lampbrush chromosomes from oocytes of the amphibian Triturus cristatus have been used to examine the role of histone acetylation in transcription by indirect immunofluorescence with antisera to H4 acetylated at specific lysine residues. Electrophoresis on acid-urea-Triton gels and Western blotting have confirmed the specificity of these antisera and defined the order in which particular lysine residues are acetylated in amphibian cells. As in mammals, lysine 16 is acetylated first, followed by 8 and/or 12 and then 5. With lampbrush chromosomes from immature (previtellogenic) oocytes, antisera to H4 acetylated at lysines 8, 12, and 16 labeled fluorescent foci at the bases of transcription loops. Antisera to H4 acetylated at lysine 5 labeled weakly (i.e., the tri- and tetraacetylated isoforms must be rare). Loops showed weak labeling of the chromatin axis but intense fluorescence at particular points, which probably represent incompletely decondensed chromatin. The RNP matrix of loops, including the RNP-rich sphere bodies and the dense matrix of "marker" loops, was not labeled. Treatment of immature oocytes with butyrate for 12 h to inhibit histone deacetylation did not affect immunolabeling, suggesting that turnover of H4 acetates is slow. In contrast, in chromosomes from mature oocytes, in which loops have retracted and transcription is low, butyrate caused an increase in labeling with all antisera, followed by the appearance of vestigial loops, weakly labeled, but with regions of intense fluorescence. These loops contain RNP and are presumably transcriptionally active. We conclude that H4 acetates turn over more rapidly in mature than immature oocytes and that histone hyperacetylation precedes, and possibly induces, loop formation and transcriptional activation.

Acetylation↗

PubMed1999

Histone octamer transfer by a chromatin-remodeling complex.

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

Animals↗

PubMed2003

Rationally designed insulator-like elements can block enhancer action in vitro.

Insulators are DNA sequences that are likely to be involved in formation of chromatin domains, functional units of gene expression in eukaryotes. Insulators can form domain boundaries and block inappropriate action of regulatory elements (such as transcriptional enhancers) in eukaryotic nuclei. Using an in vitro system supporting enhancer action over a large distance, the enhancer-blocking insulator activity has been recapitulated in a highly purified system. The insulator-like element was constructed using a sequence-specific DNA-binding protein making stable DNA loops (lac repressor). The insulation was entirely dependent on formation of a DNA loop that topologically isolates the enhancer from the promoter. This rationally designed, inducible insulator-like element recapitulates many key properties of eukaryotic insulators observed in vivo. The data suggest novel mechanisms of enhancer and insulator action.

Base Sequence↗

PubMedDate not supplied

[SLS--a new type of polynucleotide chain folding].

Short tandem repeats (5-8 base pairs) are not uncommon in the prokaryotic and eukaryotic DNA. Regions with such sequence motifs, when under superhelical stress, manifest unusual sensitivity to single-strand specific nuclease. To explain this, it has been suggested that one DNA thread should be shifted relatively to another, so that they could form two single-stranded loops protruding from the opposite chains and separated on the DNA helix by the length of a direct repeat. The structure was proposed to play a role in the regulation of transcription, organization of chromatin and in the recombination. Such type of folding could have been extra-stabilized by base pairing between the loops. This attractive possibility of the interloop minihelix formation requires a delicate stereochemical analysis and direct experimental support. Formation of the interloop minihelix in the Slipped Loop Structure (SLS) was tested by a chemical modification method at one nucleotide level resolution. The results show that bases located within the proposed interloop helix are well protected from the probes used. This fact encourages us to publish a 3-D model for the SLS-form DNA (and RNA). The SLS is characterized by a remarkable symmetry having three mutually perpendicular dyad axes. Scanning the bank of nucleotide sequences has revealed more than 500 sites, the transcripts of which are capable of folding into the SLS form, which allow us to regard the SLS form as a novel universal structural form. Remarkably, the abundance of SLS in intrones three times exceeds that of the coding sequences. This may reflect a functional role (or roles) of the SLS conformation.

Animals↗

PubMed2005

On a chromosome far, far away: LCRs and gene expression.

Transcription activation of a gene involves the ordered recruitment of components of the basal transcription machinery in concert with alterations in chromatin structure, including nucleosome remodeling and post-translational modification of histones. Enhancers and locus control regions (LCRs) that are remote from the genes they activate, recruit the complexes that carry out these alterations and, sometimes, recruit RNA polymerase II. The question of how these distant activators interact with their target genes has been of long-standing interest. Recent data indicate that LCRs mediate contact with their coordinate genes through the formation of domains of histone modification and of intra- and inter-chromosomal loops and that they might localize genes within nuclear regions that favor transcription.

Animals↗

PubMed2006

The regulation of HIV-1 transcription: molecular targets for chemotherapeutic intervention.

The regulation of transcription of the human immunodeficiency virus (HIV) is a complex event that requires the cooperative action of both viral and cellular components. In latently infected resting CD4(+) T cells HIV-1 transcription seems to be repressed by deacetylation events mediated by histone deacetylases (HDACs). Upon reactivation of HIV-1 from latency, HDACs are displaced in response to the recruitment of histone acetyltransferases (HATs) by NF-kappaB or the viral transcriptional activator Tat and result in multiple acetylation events. Following chromatin remodeling of the viral promoter region, transcription is initiated and leads to the formation of the TAR element. The complex of Tat with p-TEFb then binds the loop structures of TAR RNA thereby positioning CDK9 to phosphorylate the cellular RNA polymerase II. The Tat-TAR-dependent phosphorylation of RNA polymerase II plays an important role in transcriptional elongation as well as in other post-transcriptional events. As such, targeting of Tat protein (and/or cellular cofactors) provide an interesting perspective for therapeutic intervention in the HIV replicative cycle and may afford lifetime control of the HIV infection.

Animals↗

PubMed1998

Chromosome cores and chromatin at meiotic prophase.

We review the synaptonemal complex, SC, of the synapsed homologous chromosomes at meiotic prophase in insects and mammals in terms of its formation, and the association of specific chromatin elements with the synaptonemal complexes. The focus is: (1) The SC as visualized with a variety of techniques; (2) The nature of the chromatin loops where they are associated with the SCs--the bases of the loops may be instrumental in recombinant events judging from the presence of Rad51 protein and late recombination nodules at the SCs; (3) Differences in DNA content of similarly sized loops; (4) Requirements for chromatin attachment to the chromosome cores, requirements that are apparently lacking in foreign DNA inserts; (5) Regulation of loop size by the position along the chromosome; (6) The structural correlates of recombination at the SCs--these comments are based on studies of SC structure, DNA-core protein associations, fluorescent in situ hybridization to visualize specific DNA segments, and fluorescent immunocytology to visualize the chromosome core proteins.

Animals↗

PubMed2026

The dynamic pool of Rec8-cohesin is crucial for meiotic recombination and transcription regulation in the yeast Saccharomyces cerevisiae.

Cohesin is a ring-shaped protein complex that mediates sister-chromatid cohesion (SCC) to ensure accurate chromosome segregation during mitosis and meiosis. In Saccharomyces cerevisiae, cohesin consists of four core subunits-Smc1, Smc3, Scc1/Mcd1, and Scc3. During meiosis, the mitotic α-kleisin Scc1/Mcd1 is replaced by the meiosis-specific α-kleisin Rec8. Rec8-containing cohesin is essential for multiple meiotic processes, including chromosome morphogenesis, homologous recombination, axis and synaptonemal complex formation, SCC, and transcriptional regulation. While stable association of Rec8-cohesin with chromatin is required to maintain SCC from premeiotic S phase through anaphase II, dynamic chromatin association is thought to underlie its roles in recombination, chromosome architecture, and transcription via loop extrusion. Whether distinct stable and dynamic pools of Rec8-cohesin coexist during meiosis and how their functions are partitioned remained unclear. Here, we employed an anchor-away strategy to conditionally deplete only the dynamic pool of Rec8-cohesin from the nucleus while preserving the stable pool. Selective depletion reduced sporulation efficiency and spore viability without compromising SCC. Calibrated ChIP-seq revealed a genome-wide reduction in Rec8-cohesin levels rather than locus-specific loss. Functional analyses demonstrated that the dynamic pool of Rec8-cohesin is required for efficient meiotic recombination, establishment of meiosis-specific chromosome architecture and synaptonemal complex formation, and proper transcriptional regulation of key meiotic regulators. In contrast, the stable pool alone was sufficient to maintain spindle pole body cohesion. Together, our findings demonstrate the existence of two functionally distinct pools of Rec8-cohesin during yeast meiosis.

Saccharomyces cerevisiae↗

PubMed2003

Spatial organization of gene expression: the active chromatin hub.

Developmental and tissue-specific expression of higher eukaryotic genes involves activation of transcription at the appropriate time and place and keeping it silent otherwise. Unlike housekeeping genes, tissue-specific genes generally do not cluster on the chromosomes. They can be found in gene-dense regions of chromosomes as well as in regions of repressive chromatin. Depending on the location, shielding against positive or negative regulatory effects from neighboring chromatin may be required and hence insulator and boundary models were proposed. They postulate that chromosomes are partitioned into physically distinct expression domains, each containing a gene or gene cluster with its cis-regulatory elements. Specialized elements at the borders of such domains are proposed to prevent cross-talk between domains, and thus to be crucial in establishing independent expression domains. However, genes and associated cis-acting sequences often do not occupy physically distinct domains on the chromosomes. Rather, genes can overlap and cis-acting sequences can be found tens or hundreds of kilobases away from the target gene, sometimes with unrelated genes in between. Therefore the ability of a gene to communicate with positive cis-regulatory elements rather than the presence of specialized boundary elements appears to be key to establishing an independent expression profile. Our recent finding that active beta-globin genes physically interact in the nuclear space with multiple cis-regulatory elements, with inactive genes looping out, has provided a potential mechanistic framework for this model. We refer to such a spatial unit of regulatory DNA elements as an active chromatin hub (ACH). We propose that productive ACH formation underlies correct gene expression, requiring the presence of protein factors with the appropriate affinities for each other bound to their cognate DNA sequences. Proximity and specificity determines which cis-acting sequences and promoter(s) form an ACH, and thus which gene will be expressed. Other regulatory sequences can interfere with transcription by blocking the appropriate physical interaction between an enhancer and promoter in the ACH. Possible mechanisms by which distal DNA elements encounter each other in the 3D nuclear space will be discussed.

Animals↗

PubMed2006

Formation and positioning of nucleosomes: effect of sequence-dependent long-range correlated structural disorder.

The understanding of the long-range correlations (LRC) observed in DNA sequences is still an open and very challenging problem. In this paper, we start reviewing recent results obtained when exploring the scaling properties of eucaryotic, eubacterial and archaeal genomic sequences using the space-scale decomposition provided by the wavelet transform (WT). These results suggest that the existence of LRC up to distances approximately 20-30 kbp is the signature of the nucleosomal structure and dynamics of the chromatin fiber. Actually the LRC are mainly observed in the DNA bending profiles obtained when using some structural coding of the DNA sequences that accounts for the fluctuations of the local double-helix curvature within the nucleosome complex. Because of the approximate planarity of nucleosomal DNA loops, we then study the influence of the LRC structural disorder on the thermodynamical properties of 2D elastic chains submitted locally to mechanical/topological constraint as loops. The equilibrium properties of the one-loop system are derived numerically and analytically in the quite realistic weak-disorder limit. The LRC are shown to favor the spontaneous formation of small loops, the larger the LRC, the smaller the size of the loop. We further investigate the dynamical behavior of such a loop using the mean first passage time (MFPT) formalism. We show that the typical short-time loop dynamics is superdiffusive in the presence of LRC. For displacements larger than the loop size, we use large-deviation theory to derive a LRC-dependent anomalous-diffusion rule that accounts for the lack of disorder self-averaging. Potential biological implications on DNA loops involved in nucleosome positioning and dynamics in eucaryotic chromatin are discussed.

Base Sequence↗

PubMed1991

The remote control of transcription, DNA looping and DNA compaction.

mRNA synthesis can be controlled at some distance from the start of transcription in eukaryotes and prokaryotes. It is generally assumed that the distal elements of the transcriptional machinery directly interact with the proximal elements, forcing the DNA to bend in a loop. DNA loop formation and transcription can be affected by the distance between the sites, their helical positioning, their orientation, their concentration (responsible for a cis- or a trans-effect of the DNA sequences), and DNA compaction in chromatin. The corresponding in vitro and in vivo situations have been critically examined for a number of systems, mostly prokaryotic.

Binding Sites↗