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At least 19 recordsLinked to original sources
PubMed1997

The repeat organizer, a specialized insulator element within the intergenic spacer of the Xenopus rRNA genes.

We have identified a novel activity for the region of the intergenic spacer of the Xenopus laevis rRNA genes that contains the 35- and 100-bp repeats. We devised a new assay for this region by constructing DNA plasmids containing a tandem repeat of rRNA reporter genes that were separated by the 35- and 100-bp repeat region and a rRNA gene enhancer. When the 35- and 100-bp repeat region is present in its normal position and orientation at the 3' end of the rRNA reporter genes, the enhancer activates the adjacent downstream promoter but not the upstream rRNA promoter on the same plasmid. Because this element can restrict the range of an enhancer's activity in the context of tandem genes, we have named it the repeat organizer (RO). The ability to restrict enhancer action is a feature of insulator elements, but unlike previously described insulator elements the RO does not block enhancer action in a simple enhancer-blocking assay. Instead, the activity of the RO requires that it be in its normal position and orientation with respect to the other sequence elements of the rRNA genes. The enhancer-binding transcription factor xUBF also binds to the repetitive sequences of the RO in vitro, but these sequences do not activate transcription in vivo. We propose that the RO is a specialized insulator element that organizes the tandem array of rRNA genes into single-gene expression units by promoting activation of a promoter by its proximal enhancers.

Animals↗
PubMed2003

The c-myc insulator element and matrix attachment regions define the c-myc chromosomal domain.

Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

Animals↗
PubMed1997

The activity of the scs and scs' insulator elements is not dependent on chromosomal context.

Sequence elements that protect a reporter gene from chromosomal position effects or that block enhancer-activated transcription are called insulators. Using a plasmid-based microinjection assay with Xenopus laevis oocytes, we show that the heterologous Drosophila melanogaster scs and scs' insulator elements do not require chromosomal context to block enhancer-activated transcription. A single insulator element partially blocks enhancer-activated transcription, indicating that each element operates independently rather than as part of a pair. Deletion analysis of the 1.8-kb scs element identified a 220-bp fragment from one of the DNase I-hypersensitive regions that has full blocking activity in the oocyte assay. This fragment corresponds to the critical region of the scs mapped in previous studies with Drosophila. A time course of transcription shows that the scs blocks enhancer-activated transcription as early as transcription can be detected, about 30 min after injection. Complete assembly of the DNA template into nucleosomes requires 4 h. The scs and scs' sequences do not block site-specific recombination by FLP recombinase, implying that insulators do not operate by a general mechanism that physically sequesters the DNA. These data are most consistent with a model for insulator action in which direct interaction between the insulator and either the enhancer or promoter confers directionality to enhancer-activated transcription.

Animals↗
PubMed1996

Boundary and insulator elements in chromosomes.

Recent progress in understanding boundary and insulator elements has concentrated on the identification of their protein components. BEAF-32 is a protein present in the scs' element of Drosophila that is also localized to most interband regions and puffs of polytene chromosomes, suggesting a role in the organization of structural chromosomal domains. The suppressor of Hairy-wing and modifier of mdg4 proteins have been characterized as components of the gypsy insulator. The latter seems to play a crucial role in conferring on the insulator its ability to unidirectionally affect enhancer function.

Animals↗
PubMed1997

A transcriptional insulator element, the su(Hw) binding site, protects a chromosomal DNA replication origin from position effects.

Eukaryotic chromosomes are organized into domains of activity for both transcription and DNA replication. Transcriptional "border," or "insulator," elements have been implicated in mediating the organization of transcriptional domains. However, the DNA sequence elements which might demarcate domains of DNA replication activity are unknown. su(Hw) protein binding sites [su(Hw)BSs] are potent transcriptional insulator elements which can block enhancer action, as well as positive and negative chromosomal position effects. Here we report that flanking su(Hw)BSs can also create a chromosomal domain permissible for activity of the chorion gene DNA replication origin. During Drosophila oogenesis the chorion (eggshell) gene loci are amplified approximately 80-fold through repeated initiation of DNA replication. The cis-acting amplification control element, on the third chromosome (ACE3), is required for high levels of amplification initiating at the nearby major origin of replication, Ori-beta. A transgenic chorion locus construct containing ACE3 and Ori-beta was able to amplify but was extremely sensitive to position effects: only 7 of 21 independent insertions amplified >10-fold. The inclusion of flanking su(Hw)BSs in the construct dramatically protected DNA replication from position effects: 31 of 31 insertions now amplified >10-fold, and this protection was reduced in a su(Hw) mutant background. Amplification was equal on both sides of the su(Hw)BS, demonstrating that replication fork passage is not significantly impeded by these sites. Inclusion of only a single su(Hw)BS in the construct did not detectably protect the chorion gene DNA replication origin from position effects.

Animals↗
PubMed2002

An evolutionarily conserved putative insulator element near the 3' boundary of the imprinted Igf2/H19 domain.

Igf2 and H19 are closely linked imprinted genes lying at the centromeric end of a 1 Mb imprinted domain on mouse chromosome 7. L23mrp and other genes located 3' (more centromeric) to H19 are not imprinted and do not interact with the enhancers shared by Igf2 and H19. It is therefore suggested that the intergenic region between H19 and L23mrp contains a boundary or an insulator element. We have identified a binding site for CTCF, a nuclear factor that mediates insulator activity in vertebrates, in the intergenic region. This site is conserved between human and mouse, associated with a major DNase I-hypersensitive site, and bound by CTCF in vivo. Functional assays using reporter constructs demonstrated that this element functions as an insulator in transfected cells. The findings suggest that this CTCF site contributes to the 3' boundary of this imprinted domain. Together with the findings on the differentially methylated CTCF sites 5' to H19, CTCF-dependent insulators may not only regulate but also delimit the imprinted domain.

3' Flanking Region↗
PubMed2003

Variegated expression from the murine band 3 (AE1) promoter in transgenic mice is associated with mRNA transcript initiation at upstream start sites and can be suppressed by the addition of the chicken beta-globin 5' HS4 insulator element.

The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or beta-spectrin in mouse fetal liver cells. When linked to a human gamma-globin gene, two different AE1 promoters directed erythroid-specific expression of gamma-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of gamma-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of gamma-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5'HS4 of the chicken beta-globin cluster to the AE1/gamma-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/gamma-globin transgene mapped to the start site of the endogenous AE1 mRNA, and gamma-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken beta-globin 5'HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.

Animals↗
PubMed2002

The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain.

Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin insulator element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.

Animals↗
PubMed2000

Polarity of transcriptional enhancement revealed by an insulator element.

Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their normal chromosomal context. They stimulate transcription in an orientation-independent manner when assayed on circular plasmids. We describe a transient transformation system to evaluate the orientation preference of transcriptional enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to block bidirectional action of an enhancer on circular plasmids. In this system, as in the chromosome, blocking of enhancer activity requires wild-type levels of the su(Hw) protein. We evaluated the orientation preference for the relatively large (4.4 kb) Adh larval enhancer from Drosophila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revealed by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promoter was weakened by removing binding sites for critical transcription factors, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The orientation preference, as much as 100-fold, is a property of the enhancer itself because it is displayed by gene constructions introduced into the chromosome regardless of the presence of the insulator in a distal location. These findings are most easily reconciled with a facilitated tracking mechanism for enhancer function in a native chromosomal environment.

Animals↗
PubMed2003

Putative FLJ20436 gene characterisation in goat. Observed ubiquitous expression in goat and transgenic mice allowed to restrict the location of an hypothesised insulator element.

Eukaryotic genomes are organised into independent domains through the establishment of boundaries which allow to have distinct pattern of gene expression both during development and in differentiated cells. The previously reported site independent expression of the mammary-specific goat alpha-lactalbumin gene in transgenic mice suggested the existence of cis-regulatory elements located upstream of this gene. The nearby presence of a second ubiquitously expressed gene (the cyclin T1 gene) allowed to define two chromatin domains putatively separated by a boundary insulator-like element. In this study, the characterisation of a third putative gene (FLJ20436) present between the alpha-lactalbumin and the cyclin T1 loci is reported. It was found to be a functional gene, ubiquitously expressed both in goat and transgenic mice. A complex pattern of alternative splicing events was observed in several analysed tissues leading to various mRNAs and putative FLJ20436 proteins, as suggested in human and mouse species. This allowed us to assign this gene to one of the two hypothesised chromatin domains, refining the location of the potential insulator element.

Alternative Splicing↗
PubMed1997

The role of insulator elements in defining domains of gene expression.

Insulators are naturally occurring DNA sequences that protect transgenes from genomic position effects, thereby establishing independent functional domains within the chromosome. Recent studies have focused on the identification of the cis and trans requirements for insulator activity. These experiments demonstrate that insulators contain multiple components that cooperate to confer their unique properties. Additionally, they suggest that the mechanism of insulation is related to that of enhancer function. Two models of insulator can be considered: a domain boundary and a transcriptional decoy model.

Animals↗
PubMed1997

Chromatin insulator elements block the silencing of a target gene by the Drosophila polycomb response element (PRE) but allow trans interactions between PREs on different chromosomes.

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. We have tested the ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ubx PRE. The gypsy element and the scs element interposed between PRE and miniwhite gene protect it against silencing but the scs' is only weakly effective. When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, it can still establish efficient silencing in some lines but not others. We show that this can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action.

Animals↗
PubMed2000

Deletion of an insulator element by the mutation facet-strawberry in Drosophila melanogaster.

Eukaryotic chromosomes are thought to be subdivided into a series of structurally and functionally independent units. Critical to this hypothesis is the identification of insulator or boundary elements that delimit chromosomal domains. The properties of a Notch mutation, facet-strawberry (fa(swb)), suggest that this small deletion disrupts such a boundary element. fa(swb) is located in the interband separating polytene band 3C7, which contains Notch, from the distal band 3C6. The fa(swb) mutation alters the structural organization of the chromosome by deleting the interband and fusing 3C7 with 3C6. Genetic studies also suggest that fa(swb) compromises the functional autonomy of Notch by allowing the locus to become sensitive to chromosomal position effects emanating from distal sequences. In the studies reported here, we show that a DNA fragment spanning the fa(swb) region can insulate reporter transgenes against chromosomal position effects and can block enhancer-promoter interactions. Moreover, we find that insulating activity is dependent on sequences deleted in fa(swb). These results provide evidence that the element defined by the fa(swb) mutation corresponds to an insulator.

5' Untranslated Regions↗
PubMed1998

The scs and scs' insulator elements impart a cis requirement on enhancer-promoter interactions.

The Xenopus rRNA enhancer activates its cognate promoter when the two elements are placed on opposite rings of dimeric catenanes. Here we show that when scs elements flank either the enhancer or promoter in catenanes, the enhancer cannot activate the promoter on the ring in trans. A series of catenanes containing different permutations of the insulators, enhancer, and promoters shows that when insulators are present, the enhancer is permitted to a activate the promoter only when both elements are on the same piece of DNA with no intervening insulator. These results suggest that insulators have the potential to block enhancer-promoter interactions between chromosomes and between independent topological domains within a chromosome.

Animals↗
PubMed2001

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Animals↗
PubMed2002

Conserved CTCF insulator elements flank the mouse and human beta-globin loci.

A binding site for the transcription factor CTCF is responsible for enhancer-blocking activity in a variety of vertebrate insulators, including the insulators at the 5' and 3' chromatin boundaries of the chicken beta-globin locus. To date, no functional domain boundaries have been defined at mammalian beta-globin loci, which are embedded within arrays of functional olfactory receptor genes. In an attempt to define boundary elements that could separate these gene clusters, CTCF-binding sites were searched for at the most distal DNase I-hypersensitive sites (HSs) of the mouse and human beta-globin loci. Conserved CTCF sites were found at 5'HS5 and 3'HS1 of both loci. All of these sites could bind to CTCF in vitro. The sites also functioned as insulators in enhancer-blocking assays at levels correlating with CTCF-binding affinity, although enhancer-blocking activity was weak with the mouse 5'HS5 site. These results show that with respect to enhancer-blocking elements, the architecture of the mouse and human beta-globin loci is similar to that found previously for the chicken beta-globin locus. Unlike the chicken locus, the mouse and human beta-globin loci do not have nearby transitions in chromatin structure but the data suggest that 3'HS1 and 5'HS5 may function as insulators that prevent inappropriate interactions between beta-globin regulatory elements and those of neighboring domains or subdomains, many of which possess strong enhancers.

Animals↗
PubMed1999

An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene.

We have identified a folate receptor gene upstream of the chicken beta-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of beta-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the beta-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the beta-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3' boundary coincides with the 5' beta-globin insulator. We speculate that the 5' beta-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.

Amino Acid Sequence↗