SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
In eukaryotes, cis-regulatory sequences are often a long way away from the transcription start site, and interactions between regulatory elements can be blocked by 'insulator' sequences. A novel type of cis-regulatory element has now been found that selectively permits some interactions across insulators.
Locus control regions (LCR) were first defined by their theoretical ability to enhance the expression of linked genes in a tissue-specific, position-independent and copy-number dependent manner. In fact, few of the so-called LCR identified completely fulfil this definition. For example, the regulatory elements located in 5' (Emu) and 3' (HS3a; HS1,2; HS3b; HS4) of the IgH locus display some properties of a LCR but lack a copy-number dependence and sometimes display position effects in transgenes. In order to study whether addition of insulators would allow to overcome such problems in transgenes, we studied constructs harboring a V(H) promoter-green fluorescent protein reporter gene linked to the 3' and/or 5' IgH elements, surrounded or not with the chicken beta-globin 5'HS4 insulator. When flanked with insulators it appeared that either 3' IgH and 5' IgH regulatory elements now behave as true LCR elements and noticeably display copy-number dependence in transfected pre-B or B cell lines.
Hematopoietic stem cells (HSCs) are attractive targets for gene therapy because of their capacity for self renewal and the wide systemic distribution of their progeny. Sustained expression of transgenes at clinically relevant levels in the progeny of HSCs would provide novel and potentially curative treatments for a wide range of inherited and acquired blood diseases. Recent improvements in retroviral transduction protocols have resulted in the first successful amelioration of a human hematologic disease--a form of severe combined immunodeficiency--by HSC gene transfer. However, continued advances in gene transfer technology are necessary if the inherent promise of HSC gene therapy is to be fully realized. Ongoing efforts are focused on modifying oncoretroviral vector designs and pseudotyping with alternative envelope proteins. In addition, because of their ability to transduce non-divided cells, safety-modified human immunodeficiency virus-1-based lentiviral vectors have emerged as promising tools for gene modification of HSCs, which reside primarily in the G0/G1 phase of the cell cycle. Irrespective of these advances, accumulated data indicate that stably integrated transgenes are frequently subject to position-effect variegation and extinction of expression. Therefore, the extent to which genetic control elements such as chromatin domain insulators and scaffold/matrix attachment regions in conjunction with posttranscriptional regulatory elements will result in enhanced probability and level of transgene expression is under active investigation. Collectively, these developments increase the likelihood that HSC gene transfer will ultimately become an effective therapeutic strategy.
Chromatin boundaries and insulators are transcriptional regulatory elements that modulate interactions between enhancers and promoters and protect genes from silencing effects by the adjacent chromatin. Originally discovered in Drosophila, insulators have now been found in a variety of organisms, ranging from yeast to humans. They have been found interspersed with regulatory sequences in complex genes and at the boundaries between active and inactive chromatin. Insulators might modulate transcription by organizing the chromatin fiber within the nucleus through the establishment of higher-order domains of chromatin structure.
Insulators define chromosomal domains such that an enhancer in one domain cannot activate a promoter in a different domain. We show that the Drosophila gypsy insulator behaves as a cis-stimulatory element in the larval fat body. Transcriptional stimulation by the insulator is distance dependent, as expected for a promoter element as opposed to an enhancer. Stimulation of a test alcohol dehydrogenase promoter requires a binding site for a GATA transcription factor, suggesting that the insulator may be facilitating access of this DNA binding protein to the promoter. Short-range stimulation requires both the Suppressor of Hairy-wing protein and the Mod(mdg4)-62.7 protein encoded by the trithorax group gene mod(mdg4). In the absence of interaction with Mod(mdg4)-62.7, the insulator is converted into a short-range transcriptional repressor but retains some cis-stimulatory activity over longer distances. These results indicate that insulator and promoter sequences share important characteristics and are not entirely distinct. We propose that the gypsy insulator can function as a promoter element and may be analogous to promoter-proximal regulatory modules that integrate input from multiple distal enhancer sequences.
In this review, we look at the most recent studies of DNA elements that function over long genomic distances to regulate gene transcription and will discuss the mechanisms genes employ to overcome the positive and negative influences of their genomic neighbourhood in order to achieve accurate programmes of expression. Enhancer elements activate high levels of transcription of linked genes from distal locations. Recent technological advances have demonstrated chromatin loop interactions between enhancers and their target promoters. Moreover, there is increasing evidence that these dynamic interactions regulate the repositioning of genes to foci of active transcription within the nucleus. Enhancers have the potential to activate a number of neighbouring genes over a large chromosomal region, hence, their action must be restricted in order to prevent activation of non-target genes. This is achieved by specialized DNA sequences, termed enhancer blockers (or insulators), that interfere with an enhancer's ability to communicate with a target promoter when positioned between the two. Here, we summarize current models of enhancer blocking activity and discuss recent findings of how it can be dynamically regulated. It has become clear that enhancer blocking elements should not be considered only as structural elements on the periphery of gene loci, but as regulatory elements that are crucial to the outcome of gene expression. The transcription potential of a gene can also be susceptible to heterochromatic silencing originating from its chromatin environment. Insulator elements can act as barriers to the spread of heterochromatin. We discuss recent evidence supporting a number of non-exclusive mechanisms of barrier action, which mostly describe the modulation of chromatin structure or modification.
Gene therapy has emerged from the idea of inserting a wild-type copy of a gene in order to restore the proper expression and function of a damaged gene. Initial efforts have focused on finding the proper vector and delivery method to introduce a corrected gene to the affected tissue or cell type. Even though these first attempts are clearly promising, several problems remain unsolved. A major problem is the influence of chromatin structure on transgene expression. To overcome chromatin-dependent repressive transgenic states, researchers have begun to use chromatin regulatory elements to drive transgene expression. Insulators or chromatin boundaries are able to protect a transgene against chromatin position effects at their genomic integration sites, and they are able to maintain transgene expression for long periods of time. Therefore, these elements may be very useful tools in gene therapy applications for ensuring high-level and stable expression of transgenes.
Genes are recognized as undergoing genomic imprinting when they are capable of being expressed only from the paternal or only from the maternal chromosome. The process can occur coordinately within large physical domains in mammalian chromosomes. One interesting facet of the study of genomic imprinting is that it offers insight into the regulation of large chromosomal regions. Understanding this regulation involves elucidating the cis-acting regulators of gene expression and defining the elements that maintain chromatin insulation, both required for understanding more practically applicable areas of biological research, such as efficient transgene production. This review is focused on the regulation of the imprinted domain of human chromosome 11p15.5, responsible for Beckwith-Wiedemann syndrome (BWS). Recent findings indicate that the maintenance of imprinting within this domain is critically dependent on the stable maintenance of chromatin insulation.
The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.
Chromatin domain insulators are thought to insulate adjacent genes, including their regulatory elements, from each other by organizing chromatin into functionally independent domains. Thus insulators should play a global role in gene regulation by keeping regulatory domains separated. However, this has never been demonstrated. We previously designed and characterized a transgene that is under GAL4 UAS control and encodes a dominant-negative form of the Boundary Element-Associated Factors BEAF-32A and BEAF-32B. The BID transgene encodes the BEAF self-interaction domain but lacks a DNA binding domain. Expression of BID in eye imaginal discs leads to a rough eye phenotype. Here we screen for dominant mutations that modify this eye phenotype. This assay provides evidence for cross-talk between different classes of insulators, and for a broad role of the BEAF proteins in maintaining patterns of gene expression during eye development. Most identified genes encode other insulator binding proteins, transcription factors involved in head development, or general transcription factors. Because it is unlikely that insulator function is limited to eye development, the present results support the hypothesis that insulators play a widespread role in maintaining global transcription programs.
A task-specific exposure matrix was designed for workers installing building insulation materials. A priori, a matrix element was defined by type of task (installer or helper), type of work area (attic spaces or wall cavities) and type of insulation material (slabs from mineral wool, fiberglass or flax; loose-fill cellulosic material or perlite). In the laboratory a mock-up (full scale) of a one-family house was used for simulated installation of insulation materials (four replicates per matrix element). Personal exposure to dust and fibers was measured. The dust was analyzed for content of endotoxin and some trace elements (boron and aluminum) from fire-retardant or mold-resistant additives. Fibers were characterized as WHO fibers or non-WHO fibers. In support of the exposure matrix, the dustiness of all the materials was measured in a rotating drum tester. For installers in attic spaces, risk of exposure was low for inhalation of dust and WHO fibers from slab materials of mineral wool or fiberglass. Slab materials from flax may cause high risk of exposure to endotoxin. The risk of exposure by inhalation of dust from loose-fill materials was high for installers in attic spaces and for some of the materials risk of exposure was high for boron and aluminum. Exposure by inhalation of cellulosic WHO fibers was high but little is known about the health effects and a risk assessment is not possible. For the insulation of walls, the risk of installers' exposure by inhalation of dust and fibers was low for the slab materials, while a high risk was observed for loose-fill materials. The exposure to WHO fibers was positively correlated to the dust exposure. A dust level of 6.1 mg/m3 was shown to be useful as a proxy for screening exposure to WHO fibers in excess of 10(6) fibers/m3. In the rotating drum, slabs of insulation material from mineral wool or fiberglass were tested as not dusty. Cellulosic loose-fill materials were tested as very dusty, and perlite proved to be extremely dusty.
Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed approximately 300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, high-throughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.
The use of animal cells such as Chinese hamster ovary (CHO) cells for recombinant gene expression provides many advantageous features such as proper folding and post-translational modification of the recombinant protein. However, recombinant genes introduced into animal cells are often expressed at low levels mainly due to position effects from the neighboring chromatin context. The tedious and time-consuming selection and amplification procedure has been the major hurdle for using animal cell line such as CHO cells. To improve mammalian cell expression systems, we screened a variety of matrix/scaffold attachment region (MAR/SAR) elements for their ability to insulate transgene expression from the position effects in CHO cells. We found that the human beta-globin MAR element is particularly effective as the frequency of beta-Gal positive colonies was increased by up to 80%. The expression levels of these colonies were also enhanced about seven-fold. These improvements appear to be related to the increased copy numbers and a higher efficiency of expression of the integrated genes. When this element was used to express soluble TGF-beta type II receptor (sTbetaRII) through the gene amplification system, the frequency of colonies expressing detectable amounts of sTbetaRII was much higher than that of the control vector. We could also generate high sTbetaRII producers with uniform growth properties by a simple two-step amplification process involving two concentrations of methotrexate. This eliminates the need to isolate individual colonies followed by multi-step treatments of methotrexate and thereby greatly simplifies this mammalian expression system.
The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases.
Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.
Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.
We propose a protocol to create maximally entangled pairs, triplets, quartiles, and other clusters of Bose-condensed atoms starting from a condensate in the Mott insulator state. The essential element is to drive single atom Raman transitions using laser pulses. Our scheme is simple, efficient, and can be readily applied to the recent experimental system as reported by M. Greiner 413, 44 (2002)].
We previously reported the development and characterization of transgenic mice containing a large 160-kb P1 artificial chromosome (PAC) encompassing the renin (REN) locus from human chromosome 1. Here we demonstrate that PAC160 not only encodes REN, but also complete copies of the next upstream (KISS1) and downstream (FLJ10761) gene along human chromosome 1. Incomplete copies of the second upstream (PEPP3) and downstream (SOX13) genes are also present. The gene order PEPP3-KISS1-REN-FLJ10761-SOX13 is conserved in mice containing either one or two copies of the REN locus. Despite the close localization of KISS1, REN, and FLJ10761, they each exhibit distinct, yet overlapping tissue-specific expression profiles in humans. The tissue-specific expression patterns of REN and FLJ10761 were retained in transgenic mice containing PAC160. Expression of REN and FLJ10761 were also proportional to copy number. Expression of KISS1 in PAC160 mice showed both similarities and differences to humans. These data suggest that expression of gene blocks encoded on large genomic clones are retained when the clones are used to generate transgenic mice. Genomic elements which act to insulate genes from their neighbors are also apparently retained.