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Results for “Chromatin long range interaction”

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PubMed2005

Long-range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3' boundary sequence spanning 46 kilobases.

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

3' Flanking Region↗

PubMed2004

Hypersensitive site 7 of the TH2 locus control region is essential for expressing TH2 cytokine genes and for long-range intrachromosomal interactions.

Several regulatory regions are important for the expression of genes encoding T helper type 2 (T(H)2) cytokines, including T(H)2-specific DNase I hypersensitivity sites in the T(H)2 cytokine locus control region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid T(H)2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in T(H)2 cell differentiation, we have generated RHS7-deficient mice. CD4(+) T cells and mast cells showed a notable reduction in T(H)2 cytokine expression in vitro and T(H)2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the locus control region and promoters of the T(H)2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of T(H)2 cytokine gene expression.

ATP-Binding Cassette Transporters↗

PubMed1976

Chromatic models. Interactions between DNA and polypeptides containing L-lysine L-valine: circular dichroism and thermal denaturation studies.

The interaction of calf thymus DNA with statistical copolymers of L-lysine and L-valine [poly(L-Lys100f-Lvalf)] and block copolymers [poly(L-Lys)100f-poly(L-Val)f] were investigated as a function of ionic strength using circular dichroism (CD) spectroscopy. It was found that valine suppresses the ability of the copolymer-DNA complexes to yield a psi-type CD spectra as found for poly(L-Lys)-DNA [Jordan, C.F., Lerman, L.S., and Venable, J.N. (1972), Nature (london), New Biol. 236, 67] and lowers the ionic strength at which CD distortion occurs. Thermal denaturation, simultaneously monitoring 280-nm ellipticity, [theta]280, and hyperchromicity, h280, was carried out on annealed complexes of poly(L-Lys)-DNA, poly(L-Lys84.5-L-Val15.5)-DNA, poly(L-Lys)87.2-poly(L-Val)12.8-DNA, and directly mixed complexes of poly(L-Lys)-DNA, IN 2.5 X 10(-4) MEDTA, pH 7.0 solution. The CD denaturation of uncomplexed DNA at several ionic strengths was also determined to examine pre-melting. Despite the inability of both statistical and block copolymers of L-Lys and L-Val to form psi-type complexes with DNA, they bind as well to DNA as does poly(L-Lys) and give rise to a thermal denaturation pattern showing bound peaks between 90 and 100 degrees C, seen clearly with CD denaturation. The thermal denaturation of mixed and annealed complexes of poly(L-Lys)-DNA shows similar patterns in hyperchromicity changes as a function of temperature but very different CD melts. From the CD melt of annealed poly(L-Lys)-DNA, it appears that aggregation and long-range order of the complex are significant in low salt (2.5 X 10(-4) MEDTA) as well as in 1.0 M NaCl. These studies further illustrate the importance of the nature of nonionic interactions (hydrophobic) between polypeptides and DNA in determining the behavior of their complexes, such as causing condensation into higher order asymmetric structures. In light of these observations, the possible significance to the CD melting of chromatin and the validity of identification of C-form DNA by CD spectroscopy are discussed.

Chromatin↗

PubMed1996

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

Base Sequence↗

PubMed2004

Rounding up active cis-elements in the triple C corral: combining conservation, cleavage and conformation capture for the analysis of regulatory gene domains.

Identification and functional analysis of potential cis-regulatory elements is a laborious process that often depends on removing putative elements from their natural context to study their activity. While such methods provide valuable information about the isolated element, they disregard the potential role of an element's interaction(s) with other regulatory sequences and the three-dimensional structure of an active gene locus. Here, two novel methods are discussed--chromosome conformation capture (3C) and RNA-TRAP--that can be used to detect interactions between distal regulatory sites and which thus indicate the chromosomal conformation that is adopted by a gene locus in various states of transcriptional activity. Combined with comparative genomics and traditional DNase I hypersensitive site mapping, these methods form a powerful approach for the study of the mechanisms of long-range transcriptional regulation.

Animals↗

PubMed2006

Activation of receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers.

RANKL is a tumor necrosis factor (TNF)-like factor secreted by mesenchymal cells, osteoblast derivatives, and T cells that is essential for osteoclastogenesis. In osteoblasts, RANKL expression is regulated by two major calcemic hormones, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and parathyroid hormone (PTH), as well as by several inflammatory/osteoclastogenic cytokines; the molecular mechanisms for this regulation are unclear. To identify such mechanisms, we screened a DNA microarray which tiled across the entire mouse RankL gene locus at a 50-bp resolution using chromatin immunoprecipitation (ChIP)-derived DNA precipitated with antibodies to the vitamin D receptor (VDR) and the retinoid X receptor (RXR). Five sites of dimer interaction were observed on the RankL gene centered at 16, 22, 60, 69, and 76 kb upstream of the TSS. These regions contained binding sites for not only VDR and RXR, but also the glucocorticoid receptor (GR). The most distant of these regions, termed the distal control region (RL-DCR), conferred both VDR-dependent 1,25(OH)(2)D(3) and GR-dependent glucocorticoid (GC) responses. We mapped these activities to an unusual but functionally active vitamin D response element and to several potential GC response elements located over a more extensive region within the RL-DCR. An evolutionarily conserved region within the human RANKL gene contained a similar vitamin D response element and exhibited an equivalent behavior. Importantly, hormonal activation of the RankL gene was also associated with chromatin modification and RNA polymerase II recruitment. Our studies demonstrate that regulation of RankL gene expression by 1,25(OH)(2)D(3) is complex and mediated by at least five distal regions, one of which contains a specific element capable of mediating direct transcriptional activation.

Acetylation↗

PubMed2001

Local action of long-range repressors in the Drosophila embryo.

Previous studies have identified two corepressors in the early Drosophila embryo: Groucho and dCTBP: Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence-specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long-range repression, while dCtBP directs short-range repression. The short-range Krüppel repressor was converted into a long-range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Krüppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long-range silencing within the hairy 5' regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho-dependent long-range repressors do not always cause the dominant silencing of linked enhancers within a complex cis-regulatory region. We suggest a "hot chromatin" model, whereby repressors require activators to bind DNA.

Alcohol Oxidoreductases↗

PubMed2004

A complex interaction of imprinted and maternal-effect genes modifies sex determination in Odd Sex (Ods) mice.

The transgenic insertional mouse mutation Odd Sex (Ods) represents a model for the long-range regulation of Sox9. The mutation causes complete female-to-male sex reversal by inducing a male-specific expression pattern of Sox9 in XX Ods/+ embryonic gonads. We previously described an A/J strain-specific suppressor of Ods termed Odsm1(A). Here we show that phenotypic sex depends on a complex interaction between the suppressor and the transgene. Suppression can be achieved only if the transgene is transmitted paternally. In addition, the suppressor itself exhibits a maternal effect, suggesting that it may act on chromatin in the early embryo.

Animals↗

PubMed2000

Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9.

Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. In this report we demonstrate that SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, we show that SUV39H1 is phosphorylated specifically at the G(1)/S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control.

3T3 Cells↗

PubMed2004

Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2.

How does an emerging transcriptional programme regulate individual genes as stem cells undergo lineage commitment, differentiation and maturation? To answer this, we have analysed the dynamic protein/DNA interactions across 130 kb of chromatin containing the mouse alpha-globin cluster in cells representing all stages of differentiation from stem cells to mature erythroblasts. The alpha-gene cluster appears to be inert in pluripotent cells, but priming of expression begins in multipotent haemopoietic progenitors via GATA-2. In committed erythroid progenitors, GATA-2 is replaced by GATA-1 and binding is extended to additional sites including the alpha-globin promoters. Both GATA-1 and GATA-2 nucleate the binding of various protein complexes including SCL/LMO2/E2A/Ldb-1 and NF-E2. Changes in protein/DNA binding are accompanied by sequential alterations in long-range histone acetylation and methylation. The recruitment of polymerase II, which ultimately leads to a rapid increase in alpha-globin transcription, occurs late in maturation. These studies provide detailed evidence for the more general hypothesis that commitment and differentiation are primarily driven by the sequential appearance of key transcriptional factors, which bind chromatin at specific, high-affinity sites.

Acetylation↗

PubMed1998

Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions.

BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters. The most common type of cooperativity is supported by direct protein-protein interactions. Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro. probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site. This mechanism does not require that the transcription factors have activation domains. We have examined whether this phenomenon occurs in vivo. RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances. In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains. CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates. These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

Bacterial Proteins↗

PubMed2000

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

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

Amino Acid Motifs↗

PubMed2006

Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.

The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

Animals↗

PubMed1995

Proximal and long-range alterations in chromatin structure surrounding the Chinese hamster dihydrofolate reductase promoter.

The chromatin structure of the dihydrofolate reductase (DHFR) gene was examined by DNA/protein cross-linking, chemical DNA methylation, and micrococcal nuclease digestion. The 5' promoter region of the gene displays two nucleosome-free zones (-550 to -300 and -150 to +100 bp relative to the ATG codon), each of which contains a number of micrococcal nuclease-hypersensitive sites. Regions upstream from the distal hypersensitive zone (-900 to -550 bp), downstream from the proximal hypersensitive zone (+100 to +400 bp), and between these two zones (-300 to -150 bp) appear either to be more than 80% histone-free or to contain histones whose globular domains have lost most of their contacts with DNA. Overall, a broad zone extending from -4300 to +4700 bp is altered relative to bulk chromatin, and within this region there are positioned nucleosomes and/or nucleosome-free zones in which the DNA appears to interact with a number of different non-histone proteins. By comparison, the chromatin in the 3' end of the gene (including the right end of the 5th intron and the 6th exon) contains randomly positioned nucleosomes, and its structure is intermediate between that of the 5' end of the gene and a downstream matrix attachment region that contains regularly organized chromatin. A 2.3 kb zone in the central part of the 5th intron reveals some features similar to the 5' end of the gene, suggesting a hitherto unrecognized functional role.

Animals↗

PubMed2002

Spatial distribution and yield of DNA double-strand breaks induced by 3-7 MeV helium ions in human fibroblasts.

Accelerated helium ions with mean energies at the target location of 3-7 MeV were used to simulate alpha-particle radiation from radon daughters. The experimental setup and calibration procedure allowed determination of the helium-ion energy distribution and dose in the nuclei of irradiated cells. Using this system, the induction of DNA double-strand breaks and their spatial distributions along DNA were studied in irradiated human fibroblasts. It was found that the apparent number of double-strand breaks as measured by a standard pulsed-field gel assay (FAR assay) decreased with increasing LET in the range 67-120 keV/microm (corresponding to the energy of 7-3 MeV). On the other hand, the generation of small and intermediate-size DNA fragments (0.1-100 kbp) increased with LET, indicating an increased intratrack long-range clustering of breaks. The fragment size distribution was measured in several size classes down to the smallest class of 0.1-2 kbp. When the clustering was taken into account, the actual number of DNA double-strand breaks (separated by at least 0.1 kbp) could be calculated and was found to be in the range 0.010-0.012 breaks/Mbp Gy(-1). This is two- to threefold higher than the apparent yield obtained by the FAR assay. The measured yield of double-strand breaks as a function of LET is compared with theoretical Monte Carlo calculations that simulate the track structure of energy depositions from helium ions as they interact with the 30-nm chromatin fiber. When the calculation is performed to include fragments larger than 0.1 kbp (to correspond to the experimental measurements), there is good agreement between experiment and theory.

Cells, Cultured↗

PubMed1986

Activation of the Drosophila hsp27 promoter by heat shock and by ecdysone involves independent and remote regulatory sequences.

Transcription of the Drosophila hsp27 gene is induced both by heat shock and by the steroid hormone ecdysone. Deletion analysis of the promoter using a transient expression assay in Drosophila tissue culture cells reveals two separate and functionally independent regulatory regions, whose positions coincide with previously reported DNase I-hypersensitive sites in embryonic chromatin. Ecdysone induction is mediated by multiple elements between -579 and -455. Several matches to the consensus heat shock regulatory element (HSE) lie between -370 and -270, and deletion of these abolishes the response to heat shock. Heat inducibility can be restored by the addition of HSE sequences from the hsp70 promoter, even when these are placed at the 3' end of the gene. Furthermore, HSEs seperated by >2 kb can interact in a cooperative way to promote transcription. Deletion of sequences adjacent to the TATA box (-40 to -154) has little effect on induction by either heat shock or ecdysone. In this respect, activation of the hsp27 TATA box in Drosophila cells differs from the long-range activation of promoters by HSEs or viral enhancers in HeLa cells.

Journal Article↗

PubMed1974

Specific sites of interaction between histones and DNA in chromatin.

Staphylococcal nuclease digestion of purified chromatin from duck reticulocytes or calf thymus results in the production of a series of double-stranded DNA fragments of discrete molecular size, ranging from about 130 to 45 base pairs, which can be detected by polyacrylamide gel electrophoresis. Similar patterns of protected DNA fragments are obtained from limit digests of chromatin "reconstituted" from purified DNA and chromatin proteins. The results obtained with reconstituted material do not depend upon the origin of the DNA, which may be derived from a bacterial, viral, or homologous source. The specificity of the protective mechanism, therefore, resides in the structure of the bound histones, and probably not in any special nucleotide sequences present in the DNA. Removal of lysine-rich histones from chromatin before digestion results principally in disappearance from the digest of a DNA fragment about 130 base pairs long. Our preliminary results suggest that other elements of the digest pattern can be assigned uniquely to the remaining histone components. These results indicate that the binding of histones to DNA in chromatin involves a limited number of specific and very well defined contacts between protein and nucleic acid, which arise from structural properties of the histones.

Animals↗

PubMed2000

GT repeats are associated with recombination on human chromosome 22.

The rate of meiotic recombination is not a constant function of physical distance across chromosomes. This variation is manifested by recombination hot spots and cold spots, observed in all organisms ranging from bacteria to humans. It is generally believed that factors such as primary and secondary DNA sequence, as well as chromatin structure and associated proteins, influence the frequency of recombination within a specific region. Several such factors, for example repetitive sequences, gene promoters, or regions with the ability to adopt Z-DNA conformation, have been hypothesized to enhance recombination. However, apart from specific examples, no general trends of association between recombination rates and particular DNA sequence motifs have been reported. In this paper, we analyze the complete sequence data from human chromosome 22 and compare microsatellite repeat distributions with mitotic recombination patterns available from earlier genetic studies. We show significant correlation between long tandem GT repeats, which are known to form Z-DNA and interact with several components of the recombination machinery, and recombination hot spots on human chromosome 22.

Chromosomes, Human, Pair 22↗