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At least 91 records · Page 5Linked to original sources
PubMed1998

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

Animals↗
PubMed2004

New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP.

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.

Animals↗
PubMed2004

Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes.

Targeted gene expression can be achieved through the use of cell-selective promoters. However, when the expression cassette is delivered by an adenovirus, "promoter interference," resulting in the loss of specificity, has been reported. To overcome this problem, insulator elements (the bovine growth hormone transcription stop signal or HS4 chromatin insulators of the chicken beta-globin locus) have been used. The present study examines the efficacy of these insulators elements, when two independent expression cassettes (one in which a strong, ubiquitous promoter drives the expression of the green fluorescent protein and another in which the "cancer-selective" ERBB2 promoter drives the expression of the herpes simplex virus thymidine kinase [HSVtk] gene) are incorporated into the same recombinant adenovirus. As expected, the presence of either insulator does not alter the expression of HSVtk in ERBB2-positive cells, measured through sensitization of the cells to ganciclovir. When ERBB2-negative cells were infected at a multiplicity of infection (MOI) of 10, the uninsulated virus sensitized ERBB2-negative cells to the same extent as it did for ERBB2-positive cells; partial sensitization was observed when transcriptional terminators were used, indicating a partial insulating effect; and complete insulation (no sensitization to ganciclovir) was observed when HS4 chromatin insulators were used. However, this complete insulation was lost when the MOI of virus was increased to 100. Our study demonstrates the possibility of insulating a conditionally expressed transgene in the vicinity of another expression cassette, but this insulating effect is lost when the multiplicity of infection is increased.

Adenoviridae↗
PubMed1999

Cohabitation of insulators and silencing elements in yeast subtelomeric regions.

In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements. We have investigated the influence of these sequences on telomeric silencing. The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene. These elements were named STARs, for subtelomeric anti-silencing regions. STARs can also counteract silencer-driven repression at the mating-type HML locus. When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene. In addition, an intervening STAR uncouples the silencing of neighboring genes. STARs thus display the hallmarks of insulators. Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs. In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing. They can promote silencing downstream of an insulated expressed domain. Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements.

Binding Sites↗
PubMed2002

Protecting against promiscuity: the regulatory role of insulators.

Eukaryotic genomes contain transcriptional regulatory elements that alter promoter activity through long-range interactions. Many control elements show a broad range of promoter interactions, suggesting that these elements are capable of inappropriate transcription. The identification of a novel class of directing regulatory elements, called insulators, has provided clues into mechanisms used in eukaryotic genomes to maintain transcription fidelity. Insulators contribute to the organization of independent domains of gene function by restricting enhancer and silencer function. This review describes the properties of insulators and related elements that have been isolated from several eukaryotic genomes. Two classes of models of insulator function are considered. These models provide insights into possible mechanisms used by these diverse elements to provide regulatory autonomy.

Animals↗
PubMed1983

Galvanic element produced by defective electrode insulation--a possible cause of abnormal pulse generator inhibition.

In patients with pacemaker, abnormal inhibition with prolongation of pacing intervals may cause alarming clinical symptoms. A case is described in which high current threshold in relation to voltage threshold indicated probability of an insulation defect with current leakage. Electrograms from the electrode disclosed false signals, which had appeared after replacement of a pulse generator six months earlier. A sharp bend of the wire in the tricuspid area was shown by X-ray and was accentuated by movements of the valve. Experimentally it was demonstrated that similar potentials, sufficient to inhibit a pulse generator, can be obtained from an electrode with defective insulation. A galvanic element forms between the metals of the electrode tip and the non-insulated cable, and potential variations are elicited by movement of the wire.

Arrhythmias, Cardiac↗
PubMed2000

Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo.

Recombinant adenoviruses (Ad) are attractive vectors for gene transfer in vitro and in vivo. However, the widely used E1-deleted vectors as well as newer generation vectors contain viral sequences, including transcriptional elements for viral gene expression. These viral regulatory elements can interfere with heterologous promoters used to drive transgene expression and may impair tissue-specific or inducible transgene expression. This study demonstrates that the activity of a metal-inducible promoter is affected by Ad sequences both upstream and downstream of the transgene cassette in both orientations. Interference with expression from the heterologous promoter was particularly strong by viral regulatory elements located within Ad sequences nucleotides 1-341. This region is present in all recombinant Ad vectors, including helper-dependent vectors. An insulator element derived from the chicken gamma-globin locus (HS-4) was employed to shield the inducible promoter from viral enhancers as tested after gene transfer with first-generation Ad vectors in vitro and in vivo. Optimal shielding was obtained when the transgene expression cassette was flanked on both sides by HS-4 elements, except for when the HS-4 element was placed in 3'-->5' orientation in front of the promoter. The insulators reduced basal expression to barely detectable levels in the non-induced stage, and allowed for induction factors of approximately 40 and approximately 230 in vitro and in vivo, respectively. Induction ratios from Ad vectors without insulators were approximately 40-fold lower in vitro and approximately 15-fold lower in vivo. This study proves the potential of insulators to improve inducible or tissue-specific gene expression from adenovirus vectors, which is important for studying gene functions as well as for gene therapy approaches. Furthermore, our data show that insulators exert enhancer-blocking effects in episomal DNA.

Adenoviridae↗
PubMed2004

Insulator dynamics and the setting of chromatin domains.

The early discovery of cis-regulatory elements able to promote transcription of genes over large distances led to the postulate that elements, termed insulators, should also exist that would limit the action of enhancers, LCRs and silencers to defined domains. Such insulators were indeed found during the past fifteen years in a wide range of organisms, from yeast to humans. Recent advances point to an important role of transcription factors in insulator activity and demonstrate that the operational observation of an insulator effect relies on a delicate balance between the "efficiency" of the insulator and that of the element to be counteracted. In addition, genuine insulator elements now appear less common than initially envisaged, and they are only found at loci displaying a high density of coding or regulatory information. Where this is not the case, chromatin domains of opposing properties are thought to confront each other at "fuzzy" boundaries. In this article, we propose models for both fixed and fuzzy boundaries that incorporate probabilistic and dynamic parameters.

Animals↗
PubMed2005

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication.

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

ATP-Binding Cassette Transporters↗
PubMed1998

Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster.

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.

ATP-Binding Cassette Transporters↗
PubMed2005

Insulators are fundamental components of the eukaryotic genomes.

The properties of cis-regulatory elements able to influence gene transcription over large distances have led to the hypothesis that elements called insulators should exist to limit the action of enhancers and silencers. During the last decades, insulators have been identified in many eukaryotes from yeast to human. Insulators possess two main properties: (i) they can block enhancer-promoter communication ('enhancer blocker activity'), and (ii) they can prevent the spread of repressive chromatin ('barrier activity'). This review focuses on recent studies designed to elucidate the molecular mechanisms of the insulator function, and gives an overview of the critical role of insulators in nuclear organization and functional identity of chromatin.

Alleles↗
PubMed2003

Chromatin insulation by a transcriptional activator.

In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression.

Cell Line↗
PubMed2003

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors.

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.

Adenoviridae↗
PubMed1999

UASrpg can function as a heterochromatin boundary element in yeast.

The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML alpha genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the alpha genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Binding Sites↗
PubMed2005

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.

Animals↗
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↗
PubMed2006

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

Animals↗