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PubMed2006

Chromatin insulators.

Active and silenced chromatin domains are often in close juxtaposition to one another, and enhancer and silencer elements operate over large distances to regulate the genes in these domains. The lack of promiscuity in the function of these elements suggests that active mechanisms exist to restrict their activity. Insulators are DNA elements that restrict the effects of long-range regulatory elements. Studies on different insulators from different organisms have identified common themes in their mode of action. Numerous insulators map to promoters of genes or have binding sites for transcription factors and like active chromatin hubs and silenced loci, insulators also cluster in the nucleus. These results bring into focus potential conserved mechanisms by which these elements might function in the nucleus.

Animals↗
PubMedDate not supplied

[Regulatory potential of S/MAR elements in transient expression].

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.

Animals↗
PubMed2002

The insulation of genes from external enhancers and silencing chromatin.

Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5' end of the chicken beta-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.

Animals↗
PubMed2006

Silicon-on-insulator irregular waveguide mode converters.

Irregular waveguide elements in a silicon-on-insulator platform that allow new device concepts are introduced. Specific designs are achieved using a multiresolution optimization strategy where the waveguide width is varied in stepwise increments. Simulation results are given for high-efficiency waveguide mode converters, power splitters, and waveguide transitions operating at 1.55 microm. The degrees of freedom available permit these functions and small dimensions, making these devices candidates for optical integrated circuits.

Journal Article↗
PubMed1998

Shaping animal body plans in development and evolution by modulation of Hox expression patterns.

Most animals exhibit distinctive and diverse morphological features on their anterior-posterior body axis. However, underneath the variation in design and developmental strategies lies a shared ancient structural blueprint that is based on the expression patterns of Hox genes. Both the establishment and maintenance of the spatial and temporal distribution of Hox transcripts play an important role in determining axial pattern. The study of many animal systems, both vertebrate and invertebrate, suggests that the mechanisms used to establish Hox transcription are nearly as diverse as the body plans they specify. The strategies for maintenance of Hox expression pattern seem more conserved among different phyla, and rely on the action of Pc and trx group genes as well as auto- and cross-regulation among Hox genes. In mice, the sharing of regulatory elements coupled with auto- and cross-regulation could explain the conservation of the clustered arrangement of Hox genes. In contrast, fly Hox genes seem to have evolved insulators or boundary elements to avoid sharing regulatory regions. Differences in Hox transcription patterns can be correlated with morphological modifications in different species, and it seems likely that evolutionary variation of Hox cis-regulatory elements has played a major role in the emergence of novel body plans in different taxa of the animal kingdom.

Animals↗
PubMed2004

Partition of distinct chromosomal regions: negotiable border and fixed border.

Chromosomes are partitioned into distinct functional regions. For example, heterochromatin regions consist of condensed chromatin and contain few transcriptionally active genes, whereas euchromatin regions are less condensed and majority of active genes reside in the euchromatin regions. Because distinct regions reside in each chromosome, borders are accordingly established between these regions. A prevailing view of the borders is that they are 'walls' that actively inhibit communication between distinct regions on chromosomes. Although little is known about the molecular bases of these walls, specific DNA elements are considered to recruit these walls to define the positions of the borders. We call the borders established with this mechanism as 'fixed borders'. Past studies have identified various insulators (boundary DNA elements) that have been suggested to recruit fixed borders to them. Another mechanism, which we introduce and focus on in this review, does not require walls recruited by specific DNA elements at the chromosomal borders. Instead, the borders are defined by a balance of opposing enzymatic activities located at the opposite sides of the resultant borders. We name these borders 'negotiable borders'. Here we review some of the recent progress in the field that offer valuable insight into mechanisms of establishing structural and functional borders on chromosomes.

Animals↗
PubMed1995

Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo.

Insulator DNAs, or boundary elements, functionally isolate neighbouring genes by blocking interactions between distal enhancers and inappropriate target promoters. The best-characterized insulators in Drosophila correspond to a 340-base-pair (bp) fragment from the gypsy retrotransposon, and the scs and scs' sequences flanking the 87A1 hsp70 locus. Here we demonstrate that both insulators block the interaction of defined even-skipped (eve) stripe enhancers when positioned between the enhancer and the target promoter. The simultaneous use of two stripe enhancers (eve stripes 2 and 3) provides evidence that enhancers lying distal to the insulator are selectively blocked. The insertion of stripe-insulator-stripe sequences between two divergently transcribed promoters indicates that enhancers barred from acting on one basal promoter are fully accessible to appropriate regulatory factors for activating the other promoter. These results suggest that insulators do not propagate changes in chromatin structure. Finally, we present evidence that the gypsy insulator does not block interactions between a silencer element and a basal promoter. Taken together, these results suggest that insulators might not be restricted to the functional isolation of neighbouring genetic loci. Rather, they might function as flexible regulatory elements that modulate enhancer-promoter interactions within complex promoters and complex genetic loci.

Animals↗
PubMed1998

A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence.

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between enhancer and promoter elements located apart. The insulator effect is dependent on the suppressor of Hairy-wing (su(Hw)) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, whereby it additionally acts as a transcriptional activator of gypsy. Here, we show that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves in addition as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a short "compacted" retroviral sequence three functional domains (insulator, enhancer, and the presently unraveled MAR/SAR) dispersed within more extended regions in other "boundary" domains, is discussed in relation to previously proposed models for insulation.

Animals↗
PubMed2006

Insulated piggyBac vectors for insect transgenesis.

BACKGROUND: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken beta-globin HS4 insulator function in both Drosophila and mammalian cells. RESULTS: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken beta-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. CONCLUSION: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.

Animals↗
PubMed2000

Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch.

The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.

Animals↗
PubMed1999

Enhancer-blocking activity within the DNase I hypersensitive site 2 to 6 region between the TCR alpha and Dad1 genes.

Although tightly linked, the TCR alpha and delta genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR alpha and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR alpha enhancer (Ealpha) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2-6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2-6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2-6. We show that HS2-6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2-6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR alpha and Dad1 genes.

Animals↗
PubMed1993

A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila.

We have characterized an element near the 5' boundary of the chicken beta-globin domain that insulates a reporter gene from the activating effects of a nearby beta-globin locus control region (5'HS2) when assayed in the human erythroid cell line K562. We show that the insulation mechanism is directional, that it operates at the level of transcription, and that it involves the alteration of chromatin structure over the promoter of the gene. The insulator has no significant stimulatory or inhibitory effects of its own. In transgenic Drosophila, the insulator protects the white minigene from position effects. The action of the insulator thus is not restricted to erythroid or mammalian cells, suggesting that such elements may serve an important and widely distributed function in the organization of chromatin structure.

Animals↗
PubMed1999

Transcriptional and structural study of a region of two convergent overlapping yeast genes.

The exceptionally close packing of many yeast genes and other chromosomal elements raises the question of how those elements are functionally insulated. All published work shows that natural insulators are very effective, but transcriptional interference (TI) occurs if they are mutated or if their natural context is altered. Mechanisms to avoid TI are poorly understood, but are thought to involve an interplay of cis sequences and trans factors in a chromatin context. We have studied the case of two convergent closely packed ORFs (56 bp of separation) in chromosome IX of Saccharomyces cerevisiae. mRNAs from POT1 and YIL161w overlap by up to 115 nt. Convergent transcription causes a small but noticeable negative effect on the level of POT1 mRNA and nucleosome displacement in the intergenic region. This suggests for the first time that some TI could occur in convergently transcribed yeast genes, even in a natural chromosomal context.

Fungal Proteins↗
PubMed1998

A role for RNA processing in regulating expression from transfected genes.

We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.

Animals↗
PubMed2006

Subtelomeric factors antagonize telomere anchoring and Tel1-independent telomere length regulation.

Yeast telomeres are anchored at the nuclear envelope (NE) through redundant pathways that require the telomere-binding factors yKu and Sir4. Significant variation is observed in the efficiency with which different telomeres are anchored, however, suggesting that other forces influence this interaction. Here, we show that subtelomeric elements and the insulator factors that bind them antagonize the association of telomeres with the NE. This is detectable when the redundancy in anchoring pathways is compromised. Remarkably, these same conditions lead to a reduction in steady-state telomere length in the absence of the ATM-kinase homologue Tel1. Both the delocalization of telomeres and reduction in telomere length can be induced by targeting of Tbf1 or Reb1, or the viral transactivator VP16, to a site 23 kb away from the TG repeat. This correlation suggests that telomere anchoring and a Tel1-independent pathway of telomere length regulation are linked, lending a functional significance to the association of yeast telomeres with the NE.

Chromosomes, Fungal↗
PubMed1975

The evolutionary origin of feathers.

Previous theories relating the origin of feathers to flight or to heat conservation are considered to be inadequate. There is need for a model of feather evolution that gives attention to the function and adaptive advantage of intermediate structures. The present model attempts to reveal and to deal with, the spectrum of complex questions that must be considered. In several genera of modern lizards, scales are elongated in warm climates. It is argued that these scales act as small shields to solar radiation. Experiments are reported that tend to confirm this. Using lizards as a conceptual model, it is argued that feathers likewise arose as adaptations to intense solar radiation. Elongated scales are assumed to have subdivided into finely branched structures that produced a heat-shield, flexible as well as long and broad. Associated muscles had the function of allowing the organism fine control over rates of heat gain and loss: the specialized scales or early feathers could be moved to allow basking in cool weather or protection in hot weather. Subdivision of the scales also allowed a close fit between the elements of the insulative integument. There would have been mechanical and thermal advantages to having branches that interlocked into a pennaceous structure early in evolution, so the first feathers may have been pennaceous. A versatile insulation of movable, branched scales would have been a preadaptation for endothermy. As birds took to the air they faced cooling problems despite their insulative covering because of high convective heat loss. Short glides may have initially been advantageous in cooling an animal under heat stress, but at some point the problem may have shifted from one of heat exclusion to one of heat retention. Endothermy probably evolved in conjunction with flight. If so, it is an unnecessary assumption to postulate that the climate cooled and made endothermy advantageous. The development of feathers is complex and a model is proposed that gives attention to the fundamental problems of deriving a branched structure with a cylindrical base from an elongated scale.

Animals↗
PubMed2002

Gene transfer strategies in animal transgenesis.

Position effects in animal transgenesis have prevented the reproducible success and limited the initial expectations of this technique in many biotechnological projects. Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression domain. An expression domain is thought to contain all regulatory elements that are needed to specifically control the expression of a given gene in time and space. The lack of profound knowledge on the chromatin structure of expression domains of biotechnological interest, such as mammary gland-specific genes, explains why most standard expression vectors have failed to drive high-level, position-independent, and copy-number-dependent expression of transgenes in a reproducible manner. In contrast, the application of artificial chromosome-type constructs to animal transgenesis usually ensures optimal expression levels. YACs, BACs, and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant key regulatory sequences, previously unknown, that are characteristic for each expression domain. These elements contribute to insulating the artificial chromosome-type constructs from chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes.

Animals↗
PubMed1999

Upstream element of the sea urchin arylsulfatase gene serves as an insulator.

Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we report that a DNA fragment located in the upstream region of sea urchin, H. pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner. The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation mechanism operates also unidirectionally in Drosophila. We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution.

Animals↗