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We simulate the effect that insulating or shielding a muscle may have on electromyographic signal propagation using the finite element method. The results suggest that the crosstalk between insulated or shielded muscles is small but that it increases with increasing subcutaneous fat. The findings may be useful in the control of multifunctional prostheses.
The sound transmission between adjacent rooms has been modeled using a finite-element method. Predicted sound-level difference gave good agreement with experimental data using a full-scale and a quarter-scale model. Results show that the sound insulation characteristics of a party wall at low frequencies strongly depend on the modal characteristics of the sound field of both rooms and of the partition. The effect of three edge conditions of the separating wall on the sound-level difference at low frequencies was examined: simply supported, clamped, and a combination of clamped and simply supported. It is demonstrated that a clamped partition provides greater sound-level difference at low frequencies than a simply supported. It also is confirmed that the sound-pressure level difference is lower in equal room than in unequal room configurations.
Insulators are genomic elements that define domains of transcriptional autonomy. Although a large number of insulators have been isolated, it is unclear whether these elements function by shared molecular mechanisms. Novel applications of FLP recombinase technology were used to dissect and compare the function of the Drosophila: gypsy and scs insulators. Inter actions between FLP monomers bound to chromosomally integrated FRT sites were unimpeded by either insulator, demonstrating that these insulators do not establish a chromosomal environment capable of disrupting all types of protein-protein interactions. The gypsy insulator blocked enhancer-activated transcription on FLP-generated extra-chromosomal episomes, whereas the scs insulator displayed silencing effects. These data indicate that these insulators differ in the mechanisms used to prevent enhancer function. That the gypsy insulator blocked enhancer-promoter communication within small episomes suggests that these effects may be accomplished without a global reorganization of chromatin structure. Instead, the gypsy insulator may disrupt enhancer-activated transcription by direct interference with transmission of the enhancer signal to the promoter.
Proper regulation of E-cadherin-mediated cell adhesion is important during early embryonic development and in organogenesis. In mice, E-cadherin is expressed from the fertilized egg onward and becomes down-regulated during gastrulation in mesoderm and its derivatives, but its expression is maintained in all epithelia. E-cadherin promoter analyses led to the identification of binding sites for two transcriptional repressors, Snail and SIP1, which are able to mediate down-regulation in vitro, but little is known about the regulatory elements that govern E-cadherin transcriptional activity in vivo. Here, we compared the developmentally regulated expression of a series of lacZ-reporter transgenes fused to different sequences of the murine E-cadherin gene between -6 kb, including the promoter, and +16 kb, covering one third of intron 2. Four different segments with distinct regulatory properties were identified. The promoter fragment from +0.1 to -1.5 kb remains inactive in most cases but occasionally induces ectopic expression in mesodermal tissues, although it contains binding sites for the repressors Snail and SIP1. This promoter fragment also lacks positive elements needed for the activation of transcription in ectoderm and endoderm. Sequences from -1.5 to -6 kb harbor regulatory elements for brain-specific expression and, in addition, insulator or silencer elements, because they are consistently inactive in the mesoderm. Only if sequences from +0.1 to +11 kb are combined with the promoter fragments is E-cadherin-specific transgene expression observed in endoderm and certain epithelia. Sequences between +11 and +16 kb contain cis-active elements that generally enhance transcription. Our analyses show that E-cadherin expression is governed by a complex interplay of multiple regulatory regions dispersed throughout large parts of the locus.
Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter.
Chromatin insulators are boundary elements between distinctly regulated, neighboring chromosomal domains, and they function by blocking the effects of nearby enhancers in a position-dependent manner. Here, we show that the SNF2-like chromodomain helicase protein CHD8 interacts with the insulator binding protein CTCF. Chromatin immunoprecipitation analysis revealed that CHD8 was present at known CTCF target sites, such as the differentially methylated region (DMR) of H19, the locus control region of beta-globin, and the promoter region of BRCA1 and c-myc genes. RNA interference-mediated knockdown of CHD8 significantly abolished the H19 DMR insulator activity that depends highly on CTCF, leading to reactivation of imprinted IGF2 from chromosome of maternal origin. Further, the lack of CHD8 affected CpG methylation and histone acetylation around the CTCF binding sites, adjacent to heterochromatin, of BRCA1 and c-myc genes. These findings provide insight into the role of CTCF-CHD8 complex in insulation and epigenetic regulation at active insulator sites.
Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs' insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs' are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions. This model was tested using transposons that carried a yellow gene to assess enhancer blocking and an hsp70-lacZ gene to examine the structure of a heat shock puff in the presence and absence of insulators. We found that although scs and scs' blocked enhancer function, these sequences did not prevent the spread of decondensation resulting from hsp70-lacZ transcription. Further analysis of the endogenous 87A locus demonstrated that scs and scs' reside within, not at, the borders of the puff. Taken together, our studies suggest that scs and scs' are not boundary elements that block the propagation of an altered chromatin state associated with puff formation. We propose that these insulators may have a direct role in limiting regulatory interactions in the gene-dense 87A region.
The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering our studies of the role of insulators in the control of gene expression, here we present a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. We demonstrate that their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs.
Insulators are DNA sequence elements that prevent inappropriate interactions between adjacent chromatin domains. One type of insulator establishes domains that separate enhancers and promoters to block their interaction, whereas a second type creates a barrier against the spread of heterochromatin. Recent studies have provided important advances in our understanding of the modes of action of both types of insulator. These new insights also suggest that the mechanisms of action of both enhancer blockers and barriers might not be unique to these types of element, but instead are adaptations of other gene-regulatory mechanisms.
Matrix attachment regions (MARs) have been shown to participate in the insulation of transcription elements from surrounding chromatin in tissue culture cells and transgenic mice. A whey acidic protein (WAP) transgene containing 1 kb promoter sequence was active in mammary tissue from 1 out of 17 lines of mice, demonstrating that the transcription elements were highly susceptible to position effects. To test whether MARs could insulate this WAP gene promoter and thereby restore transcription, we ligated MARs from the chicken lysozyme gene to the WAP transgene. Seven of the nine lines generated exhibited WAP transgene activity, expression was confined to mammary tissue, and correct regulation was observed in three of the four lines analyzed. This study provides strong additional evidence that the MAR fragments from the chicken lysozyme gene have the capacity to insulate transgenes from severe position effects.
Dosage compensation (equalization of X-linked gene products) occurs in Drosophila melanogaster by a two-fold transcriptional increase of X-linked gene expression in the male. The cis-acting X-linked DNA sequences required for dosage compensation (called DCREs) remain elusive, despite numerous attempts to identify them. We have developed an insulated reporter system to minimise problems previously encountered with identifying these elements. The system consists of the constitutive autosomal armadillo promoter fused to the lacZ reporter gene (called arm-lacZ) which was flanked by SCS insulator elements to block potential repressive effects of an autosomal chromatin environment. Seven X-linked DNA fragments, totaling 62.7 kb, were each inserted between the SCS element and the armadillo promoter. If an X-linked fragment contains a DCRE, then transgenic males carrying an autosomal insert of the construct should produce twice the beta-galactosidase activity of females. However, in all cases, males and females expressed the same level of lacZ. Thus, it's likely that none of the X-linked fragments contained a DCRE, suggesting these elements may be rarer than previously thought. The insulated reporter system was also used to test the hypothesis that some genes may be dosage compensated due to repression by Sex lethal (Sxl) in females. A fragment from the runt gene containing three Sxl binding sites was inserted into the 3' untranslated region of arm-lacZ. Transgenic males carrying an autosomal insert of the construct had on average 1.31-1.46 times the level of beta-galactosidase than females, suggesting that some genes could be compensated, at least partially, by Sxl repression in females.
Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox.
Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2.
Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.
The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function.
BACKGROUND: One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes. RESULTS: We have identified and characterized putative CTCF homologs in the medically-important mosquitoes, Aedes aegypti and Anopheles gambiae. These genes encode polypeptides with eleven C2H2 zinc fingers that show significant similarity to those of vertebrate CTCFs, despite at least 500 million years of divergence. The mosquito CTCFs are constitutively expressed and are upregulated in early embryos and in the ovaries of blood-fed females. We have uncovered significant bioinformatics evidence that CTCF is widespread, at least among Drosophila species. Finally, we show that the An. gambiae CTCF binds two known insulator sequences. CONCLUSION: Mosquito CTCFs are likely orthologous to the widely-characterized vertebrate CTCFs and potentially also serve an insulating function. As such, CTCF may provide a powerful tool for improving transgene expression in these mosquitoes through the identification of endogenous binding sites.
Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking-Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.
Insulators are a class of elements that define independent domains of gene function. The Drosophila gypsy insulator is proposed to establish regulatory isolation by forming loop domains that constrain interactions between transcriptional control elements. This supposition is based upon the observation that insertion of a single gypsy insulator between an enhancer and promoter blocks enhancer function, while insertion of two gypsy insulators promotes enhancer bypass and activation of transcription. To investigate this model, we determined whether non-gypsy insulators interacted with each other and with the gypsy insulator. Pairs of scs or scs' insulators blocked enhancer function. Further, an intervening scs insulator did not block gypsy insulator interactions. Taken together, these data suggest that not all Drosophila insulators interact, with this property restricted to some insulators, such as gypsy. Three gypsy insulators inserted between an enhancer and promoter blocked enhancer function, indicating that gypsy insulator interactions may be restricted to pairs. Our studies imply that formation of loop domains may represent one of many mechanisms used by insulators to impart regulatory isolation.