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PubMed2003

Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA.

In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken beta-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.

Animals↗
PubMed2002

Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains.

The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs). Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor). Here we focus on the characterization of a moderately repeated 1.2 kb DNA sequence that encompasses boundary element 28 (BE28). We show that it directionally blocks enhancer/promoter communication in transgenic flies. This sequence contains a BEAF-binding sequence juxtaposed to an AT-rich sequence that harbors a strong nuclease-hypersensitive site. Using a combination of DNA-protein and protein blotting techniques, we found that this region is recognized by the A+T-binding D1 non-histone chromosomal protein of D. melanogaster, and we provide evidence that D1 and BEAF physically interact. In addition, the multicopy BE28 element maps to pericentric regions of the D. melanogaster 2L, 2R and X chromosome arms to which D1 has been shown to localize. In yeast, BEs that mark the periphery of silenced chromosomal domains have recently been shown to block the spreading of heterochromatin assembly. We propose that the BE28 repeat clusters could fulfill a similar function, acting as a local boundary between hetero- and euchromatin in a process involving interactions between the BEAF and D1 proteins.

AT Rich Sequence↗
PubMed2002

Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis.

In transgenesis, the expression of two transgenes is often subject to mutual interference by each of the two expression cassettes when they are driven by different transcriptional regulatory elements in a single construct. To study this problem, we constructed vectors consisting of two expression units, one contains a strong ubiquitous promoter and the other contains a tissue-specific transcriptional element. The expression pattern of each transgene was examined in transfected cell lines and also in transgenic mice. In both cases, two expression units in a single construct were expressed in an independent manner and were controlled by their respective regulatory element only if we placed insulators at both ends of one expression unit. These results indicate that usage of insulators is a valuable tool for transfection of double gene constructs in transgenesis.

Animals↗
PubMed1997

Characterization of the chicken beta-globin insulator.

Insulators, first identified in Drosophila, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5' end of the chicken beta-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse beta-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5'HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.

Animals↗
PubMed2003

Communication over a large distance: enhancers and insulators.

Enhancers are regulatory DNA sequences that can work over a large distance. Efficient enhancer action over a distance clearly requires special mechanisms for facilitating communication between the enhancer and its target. While the chromatin looping model can explain the majority of the observations, some recent experimental findings suggest that a chromatin scanning mechanism is used to establish the loop. These new findings help to understand the mechanism of action of the elements that can prevent enhancer-promoter communication (insulators).

Animals↗
PubMed2002

Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.

The 1.2-kb DNA sequence element (5'HS4) at the 5' end of the chicken beta-globin locus has the two defining properties of an insulator: it prevents an "external" enhancer from acting on a promoter when placed between them ("enhancer blocking") and acts as a barrier to chromosomal position effect (CPE) when it surrounds a stably integrated reporter. We previously reported that a single CTCF-binding site in 5'HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp "core" element from within 5'HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5'HS4 is a compound element in which it is possible to separate enhancer blocking and barrier activities. We demonstrate that full protection against CPE is conferred by mutant 5'HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5'HS4 result in varying reductions in the ability to protect against CPE. We find that binding sites for CTCF are neither necessary nor sufficient for protection against CPE. Comparison of the properties of 5'HS4 with those of other CTCF-binding enhancer-blocking elements suggests that CPE protection is associated with maintenance of a high level of histone acetylation near the insulator, conferred by insulator binding-proteins other than CTCF.

Animals↗
PubMed1997

Stem cell gene therapy, position effects and chromatin insulators.

Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.

Animals↗
PubMed2005

Topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of Escherichia coli.

The double helical nature of DNA implies that progression of transcription machinery that cannot rotate easily around the DNA axis creates waves of positive supercoils ahead of it and negative supercoils behind it. Using topological reporters that detect local variations in DNA supercoiling, we have characterized the diffusion of transcription-induced (TI) positive supercoils in plasmids or in the chromosome of wild type Escherichia coli cells. Transcription-induced positive supercoils were able to diffuse and affect local supercoiling several kilobases away from the site of origin. By testing the effect of various DNA sequences, these reporters enabled us to identify elements that impede supercoil diffusion, i.e. behave as topological insulators. All the elements tested correspond to DNA gyrase catalytic targets. These results correlate the ability of a DNA sequence to be cleaved by DNA gyrase with topological insulator activity. Implications of the asymmetry in supercoil diffusion for the control of DNA topology are discussed.

Chromosomes, Bacterial↗
PubMed2005

A system for precise analysis of transcription-regulating elements of immunoglobulin genes.

BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.

Allergy and Immunology↗
PubMed2005

Organizing the genome: enhancers and insulators.

Enhancers can activate their target genes over large linear distances. Insulators can delimit the influence of an enhancer to an appropriate target. There are a number of intertwined mechanisms by which the regulatory functions of enhancers and insulators might be carried out at the level of the chromatin fiber. Recent evidence suggests that both enhancers and insulators participate in higher-order organization of chromatin in the nucleus and in localization of their regulated sequences to both subnuclear structures and compartments. Novel experimental approaches are helping to reveal the mechanisms underlying nuclear organization of developmentally regulated genes.

Animals↗
PubMed1985

Frame fungi on insulated windows.

Insulated windows (fixed triple glass window elements), became common in Finland during and after the energy crisis. Moisture tends to condense along the edges causing mold growth on the frames. Nine houses with insulated glass windows and eight with other types of window were studied. Fungus samples were taken from the window edges and from the inside air and cultivated on malt extract agar. In five of the houses with insulated windows there was visible mold growth on the window edges. The predominant fungus was Aureobasidium pullulans. With other types of windows there was only one case of visible mold. The concentrations of fungi were about the same in the air of houses with both types of windows. Aureobasidium was rare in the air samples. Thus, window frame molds seem to contribute very little to airborne fungus concentrations. In situ trials were done to control the mold growth with disinfectants. Repeated spraying with aldehydes or quaternary ammonium chlorides in alcohols proved to be successful.

Air Microbiology↗
PubMed2001

The sea urchin sns insulator blocks CMV enhancer following integration in human cells.

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter upstream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells.

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↗
PubMed2002

Description and evaluation of a sampling system for long-time monitoring of PAHs wet deposition.

In this paper a new electronically controlled year-round wet-only sampler for wet deposition of trace organic compounds (e.g. airborne PAHs) is described. The sampler provides in situ filtration of the precipitation as well as preconcentration of nonpolar organic compounds by means of a C18-PAH modified silica gel cartridge. The whole assembly is insulated and equipped with heating elements which permit collection of wet deposition as ice or snow and insure correct function of the sampling system even during cold weather. Concurrent chemical analysis of both the particulate and the dissolved phases is performed by high resolution gas chromatography with flame ionization detection or HPLC with fluorescence detection. The reliability of the method was proved by analyzing PAH spiked water (simulated rain) and using NIST SRM 1649 ('urban dust') as certified material for particle-bound PAHs in precipitation. This study proved satisfactorily recoveries of as both particle-bound and unbound aqueous PAH, with only small losses to collector surfaces. It was proved that this new wet-only precipitation sampler can successfully be used for long-time monitoring of PAH in wet depositions in urban areas.

Air Pollutants↗
PubMed2005

All-diamond microelectrode array device.

We report the development of all-diamond microelectrochemical devices, namely, a microelectrode array (MEA), in which a periodic array structure with well-defined diameters, distance, and hexagonal unit cell pattern is micromachined using a combination of state-of-the-art microwave-induced plasma growth and laser ablation shaping techniques to prepare and coat a patterned boron-doped diamond (BDD) substrate with an intrinsic diamond insulating layer. The active BDD element can be tuned to between 10 and 50 microm in diameter with a 10 times diameter center-to-center distance between two adjacent conducting elements, which are exactly coplanar to the dielectric surroundings. This type of device should enable applications in harsh conditions such as high temperature, high pressure, and resistive media under dynamic flow regimes.

Journal Article↗
PubMed2005

Metal to insulator transitions in clusters.

The strict criterion for metallicity, a finite density of states (DOS) at the Fermi energy (E(F)), cannot be applied to clusters because energy levels are always discrete in a system of finite size. We propose an alternative definition whereby clusters can be considered metallic when the gap between occupied and unoccupied states at E(F) is consistently smaller than or equal to the Kubo band gap delta. We use the experimental findings of photoelectron spectroscopy of anionic clusters to analyze band gaps of various cluster families. Monovalent clusters (alkali and noble metals) grossly follow the shell structure pattern, producing band gaps smaller than delta for most cluster sizes, with some exceptional sizes exhibiting electronic shell closure or symmetry-induced band gaps. Among the bivalent metals, only mercury shows consistent band gap closure with increasing cluster size, that is a simple insulator-metal transition. Other bivalent elements such as Zn and Mg exhibit a much more complicated behavior. We also briefly discuss complex cluster families such as aluminum and transition metals.

Aluminum↗
PubMed2006

The H19 gene: regulation and function of a non-coding RNA.

The H19 gene encodes a 2.3-kb non-coding mRNA which is strongly expressed during embryogenesis. This gene belongs to an imprinted cluster, conserved on mouse chromosome 7 and human chromosome 11p15. H19 is maternally expressed and the neighbouring Igf2 gene is transcribed from the paternal allele. These two genes are co-expressed in endoderm- and mesoderm-derived tissues during embryonic development, which suggests a common mechanism of regulation. The regulatory elements (imprinted control region, CTCF insulation, different enhancer sequences, promoters of the two genes, matrix attachment regions) confer a differential chromatin architecture to the two parental alleles leading to reciprocal expression. The role of the H19 gene is unclear but different aspects have been proposed. H19 influences growth by way of a cis control on Igf2 expression. Although H19(-/-) mice are viable, a role for this gene during development has been suggested by viable H19(-/-) parthenogenetic mice. Finally it has been described as a putative tumour suppressor gene. H19 has been studied by numerous laboratories over the last fifteen years, nevertheless the function of this non-coding RNA remains to be elucidated.

Animals↗