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Results for “chromatin loop formation”

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At least 19 recordsLinked to original sources

PubMed2006

Chromatin looping and the probability of transcription.

Recent studies of several multigene clusters have shown that gene activation by a remote enhancer is associated with chromatin loop formation. It is not fully understood how a chromatin loop forms in a nucleus or how it is involved in gene regulation. In this article, we propose that the major feature that determines loop formation is the flexibility of chromatin, and that this flexibility is modulated by histone acetylation (and other modifications). Thus, histone modifications will modulate distribution of the preferential looping site in chromatin, which, in turn, determines the probability of interaction between a remote enhancer and the cognate genes. This model can explain gene expression changes in the Hoxd gene cluster and the beta-globin locus.

Acetylation↗

PubMed2005

Association by guilt: identification of DLX5 as a target for MeCP2 provides a molecular link between genomic imprinting and Rett syndrome.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting almost exclusively girls. Although mutations in methyl-CpG-binding protein (MeCP2) are known to be associated with RTT, gene expression patterns are not significantly altered in MeCP2-deficient cells. A recent study1 identified MeCP2-mediated histone modification and formation of a higher-order chromatin loop structure specifically associated with silent chromatin at the Dlx5-Dlx6 locus in normal cells, and its absence thereof in RTT patients. This altered expression of Dlx5 through loss of silent chromatin loop formation provides a molecular mechanism underlying RTT and proposes a novel role for MeCP2 in chromatin organization and imprinting.

Animals↗

PubMed1996

Estrogen-mediated induction of rat prolactin gene transcription requires the formation of a chromatin loop between the distal enhancer and proximal promoter regions.

Rat PRL (rPRL) gene transcription is controlled by proteins that interact with two DNA elements located in the 5'-upstream regulatory region. These elements, the distal enhancer and the proximal promoter, are separated by nearly 1500 bp of DNA. Induction of rPRL transcription by the estrogen 17 beta-estradiol (E2) is conferred through the estrogen response element located in the distal enhancer. To activate transcription, the estrogen-estrogen receptor complex must "communicate" with RNA polymerase II located at the proximal promoter. Using a novel nuclear ligation assay, it is shown that estrogen treatment of rat pituitary GH3 cells stimulates the formation of chromatin loops between the distal enhancer and proximal promoter of the rPRL gene, juxtaposing these two transcriptional control regions. The formation of these chromatin loops was observed in both the endogenous rPRL gene and in a rPRL-Tn5 minichromosome. Induction of rPRL and rPRL-Tn5 expression by E2 was reduced by coincubation of cells with the antiestrogen 4-hydroxytamoxifen. Likewise, 4-hydroxytamoxifen was found to block the E2-induced formation of chromatin loops between the distal and proximal regions. Concurrent treatment with the synthetic glucocorticoid dexamethasone reduced the E2-induced transcription rate of the rPRL and rPRL-Tn5 gene to near basal levels. Similarly, dexamethasone treatment inhibited the ability of E2 to enhance the formation of the chromatin loops. These data suggest that the activation of rPRL gene transcription by E2 involves the stabilization of a chromatin loop that facilitates protein-protein interactions between transcription factors that are associated with the distal enhancer and the proximal promoter.

Animals↗

PubMed1997

The scaffold/matrix attachment region binding protein hnRNP-U (SAF-A) is directly bound to chromosomal DNA in vivo: a chemical cross-linking study.

The protein heterogeneous nuclear ribonucleoprotein U (hnRNP-U, also known as scaffold attachment factor A, SAF-A) is an abundant component of hnRNP particles and of the nuclear matrix. Previous experiments have demonstrated that, in vitro, hnRNP-U specifically binds to scaffold/matrix attachment (S/MAR) region DNA elements and could thus be involved in higher order chromatin structure. In this paper we report on the use of chemical cross-linking to investigate whether the protein is also bound to DNA in vivo, which is a prerequisite for its presumed function in chromatin loop formation. We have improved published methods for cross-linking proteins to DNA with the aim to minimize unspecific fixation and possible contamination with RNA binding proteins. Our protocol is based on a limited cross-linking of living human cells with formaldehyde, followed by the purification of DNA/protein complexes by two consecutive cesium chloride density gradient centrifugations. Analysis of the protein constituents of these complexes shows a specific subset of cross-linked proteins with the histones as major components. By western blotting, we demonstrate that hnRNP-U is efficiently cross-linked to DNA under experimental conditions that yield DNA/protein complexes with a buoyant density equivalent to that of native chromatin. Dimethylsulfate cross-linking and limited protease digestion of the complexes was used to establish that hnRNP-U is bound directly to DNA and not via cross-linking to other proteins. This is the first direct demonstration of the in vivo DNA binding of a S/MAR specific protein and suggests a structural role of hnRNP-U in chromatin organization.

Binding Sites↗

PubMed2000

LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation.

Using a directional cloning strategy, DNA sequence information was obtained corresponding to the site of early radiation-induced apoptotic DNA fragmentation within the human lymphoblastoid cell line TK6. Data were obtained from 88 distinct clones comprising approximately 65 kbp of sequenced material. Analysis of all cloned material showed that sequences in the 10 bp immediately adjacent to the cleavage sites were enriched in short oligoT tracts. The proportion of repetitive DNA within the entire cloned material was found to be within the normal range. However the distribution of Alu and LINE repetitive DNA were biased to positions at or adjacent to the apoptotic cleavage site. In particular, a non-random distribution of five cleavage sites was found clustered within the second ORF of the LINE L1 that partially overlapped with two binding sites for the nuclear matrix-associated protein SATB1. Three other clones, containing alpha satellite elements, were also linked to a DNA matrix binding function. These data indicate that the site of chromatin loop formation at the nuclear matrix may be a specific target for early DNA fragmentation events during apoptosis.

Apoptosis↗

PubMed2006

DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.

Adenosine Triphosphate↗

PubMed2005

Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.

Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.

Animals↗

PubMed2006

Gene regulation and large-scale chromatin organization in the nucleus.

Regulation of gene expression involves a number of different levels of organization in the cell nucleus. The main agents of transcriptional control are the cis-acting sequences in the immediate vicinity of a gene, which combine to form the functional unit or domain. Contacts between these sequences through the formation of chromatin loops forms the most basic level of organization. The activity of functional domains is also influenced by higher order chromatin structures that impede or permit access of factors to the genes. Epigenetic modifications can maintain and propagate these active or repressive chromatin structures across large genomic regions or even entire chromosomes. There is also evidence that transcription is organized into structures called 'factories' and that this can lead to inter-chromosomal contacts between genes that have the potential to influence their regulation.

Animals↗

PubMed1995

The nuclear matrix as a site of anticancer drug action.

Many nuclear functions, including the organization of the chromatin within the nucleus, depend upon the presence of a nuclear matrix. Nuclear matrix proteins are involved in the formation of chromatin loops, control of DNA supercoiling, and regulation and coordination of transcriptional and replicational activities within individual loops. Various structural and functional components of the nuclear matrix represent potential targets for anticancer agents. Alkylating agents and ionizing radiation interact preferentially with nuclear matrix proteins and matrix-associated DNA. Other chemotherapeutic agents, such as fludarabine phosphate and topoisomerase II-active drugs, interact specifically with matrix-associated enzymes, such as DNA primase and the DNA topoisomerase II alpha isozyme. The interactions of these agents at the level of the nuclear matrix may compromise multiple nuclear functions and be relevant to their antitumor activities.

Animals↗

PubMed2004

Interaction between the GAGA factor and Mod(mdg4) proteins promotes insulator bypass in Drosophila.

Chromatin insulators or boundaries are proposed to structure the chromatin fiber into functionally independent domains by promoting the formation of chromatin loops. These elements can block the communication between an enhancer and a gene when placed between them. Interestingly, it has been previously observed that two tandem copies of the Drosophila Su(Hw) insulator abolish this enhancer-blocking activity, presumably through pairing. This bypass effect has not been described with other insulators, however. In this report, we show that the insertion of binding sites for the GAGA factor (GAF) between an enhancer and the Su(Hw) insulator allows bypassing of the insulator. This bypass relies on the activity of both the GAF protein and the Mod(mdg4)-67.2 protein, a factor required for Su(Hw) insulator activity. We show that these two proteins interact in vitro and in vivo, providing molecular evidence of pairing between the GAF sites and the Su(Hw) insulator. Finally, we show that placing the Mcp boundary together with the Su(Hw) insulator between an enhancer and a promoter leads to bypass, again in a GAF- and Mod(mdg4)-dependent manner. Our data provide direct evidence that heterologous insulators can be bypassed by distal enhancers and identify the interaction between GAF and Mod(mdg4) as a possible means to regulate insulator activity.

Animals↗

PubMed2025

MIA-Jet: Multi-scale Identification Algorithm of Chromatin Jets.

The mammalian genome is organized into large-scale chromosome territories, compartments, domains, and at the smallest scale, chromatin loops and stripes. The newest element is a chromatin jet, a diffused line perpendicular to the main diagonal in the Hi-C contact map, which was reported in quiescent mammalian lymphocytes supporting a two-sided symmetric cohesin loop extrusion model. A similar structure is observed in Repli-HiC data, where relatively thin and straight chromatin fountains indicate coupling of DNA replication forks. However, the precise biological implications of these jet-like structures are unknown due to the limitations in computational methods. We developed MIA-Jet, a multi-scale ridge detection algorithm that can accurately detect jets of variable lengths, widths, and angles. When tested on Hi-C, Repli-HiC, ChIA-PET, ChIA-Drop, and Micro-C data in mouse, human, roundworm, and zebrafish cells, MIA-Jet outperformed existing methods. In human cells, jets were enriched in cohesin loading sites and early replication initiation zones. Applying MIA-Jet to Hi-C data generated from protein-degraded cells revealed that jets are dependent on cohesin but not YY1, and jet signals are strengthened after depleting WAPL. We envision MIA-Jet to be broadly applicable to any 3D genome mapping data, thereby providing new insights into the functional roles of chromatin jets.

3D genome mapping↗

PubMed2001

Effects of cis arrangement of chromatin insulators on enhancer-blocking activity.

Chromatin boundary elements or insulators are believed to regulate gene activity in complex genetic loci by organizing specialized chromatin structures. Here, we report that the enhancer-blocking activity of the Drosophila suHw insulator is sensitive to insulator copy number and position. Two tandem copies of suHw were ineffective in blocking various enhancers from a downstream promoter. Moreover, an enhancer was blocked more effectively from a promoter by two flanking suHw insulators than by a single intervening one. Thus, insulators may modulate enhancer-promoter interactions by interacting with each other and facilitating the formation of chromatin loop domains.

Animals↗

PubMed1999

The role of the nuclear matrix in cancer chemotherapy.

The nuclear matrix is the site of many nuclear functions including transcription, replication, formation of chromatin loops, and control of DNA supercoiling. It contains various structural and functional components that represent targets for antineoplastic agents. Antimetabolites and topoisomerase II inhibitors interact specifically with matrix-associated enzymes, DNA primase, and DNA topoisomerase II, respectively. Alkylating agents and ionizing radiation interact with nuclear matrix proteins and matrix-associated DNA. Many nuclear functions, including multidrug resistance, and others which lead to cell death, have been shown to be compromised when these anticancer agents interact with the nuclear matrix.

Antineoplastic Agents↗

PubMed2004

Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome.

Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize gamma-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, approximately 1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation-combined loop assays showed that Mecp2 mediated the silent chromatin-derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin-associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.

Animals↗

PubMed2025

Proteins driving liquid-liquid phase separation and histone modifications cooperatively associate with chromatin looping and transcriptional regulation.

BACKGROUND: Although liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks. RESULTS: We identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors. CONCLUSIONS: Our study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.

Humans↗

PubMed2002

Chromatin: a connection between loops and barriers?

A genetic screen for proteins that can block the spread of silenced heterochromatin has identified components of the nuclear pores with potential barrier activity. These results suggest that formation of loops of chromatin anchored to the pore could be one mechanism of barrier function.

Chromatin↗

PubMed2006

Chromatin structure can strongly facilitate enhancer action over a distance.

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer-promoter E-P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E-P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E-P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Animals↗

PubMed1997

In vitro genotoxic effects of hard metal particles assessed by alkaline single cell gel and elution assays.

Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers.

Cobalt↗