1A2 Insulator can interact with promoter of hsp70 gene in D. melanogaster

Insulators are regulatory DNA elements that participate in the modulation of the interactions between enhancers and promoters. Depending on the situation, insulators can either stabilize or destroy the contacts between enhancers and promoters. A possible explanation for the activity of insulators is their ability to directly interact with gene promoters. In the present study, it was demonstrated that, in model systems, a 1A2 insulator could interact with the core sequence of an hsp70 promoter. In this case, the insulator protein CP190 is found on the hsp70 promoter, which depends on the presence of an insulator in the transgene. The data obtained are consistent with the model, which implies that direct contacts between insulators and promoters make a considerable contribution to the modulation of the interactions between insulators and promoters.     

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.     

Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene

Boundary elements have been found in the regulatory region of the Drosophila melanogaster Abdominal-B (Abd-B) gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, we have tested the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It has been found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. We have also shown that Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter.     

Genomic context modulates insulator activity through promoter competition.

Chromatin insulators regulate gene expression by preventing inappropriate enhancer-promoter interactions. Our previous study showed that insulators do not merely function as rigid blockers, rather their activities are quantitative and selective. We have investigated the factors and mechanisms that determine the effectiveness of the suHw insulator in transgenic Drosophila. We show that the suHw-mediated blockage of the AE1 enhancer from a downstream promoter depends on the ability of the promoter to compete for AE1. Promoters that are highly competitive for the enhancer are blocked less effectively. Moreover, blockage of AE1 from its cognate ftz promoter can range from virtually complete to non-detectable, depending on the property of the neighboring upstream promoter. A highly competitive neighboring promoter enhances the suHw-mediated blockage, whereas a less competitive promoter reduces the insulator effectiveness. The influence on insulator effectiveness by both the interacting and the neighboring competing promoters correlates with their ability to compete for the enhancer, which was previously shown to depend on core promoter sequences. Our findings suggest a mechanism at the level of gene organization that modulates insulator effectiveness through promoter competition. The dependence of insulator function on its cis contexts may provide it with more regulatory flexibility while imposing organizational restraints on eukaryotic gene complexes.     

Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome

The Suppressor of the Hairy wing [Su(Hw)] binding region within the gypsy retrotransposon is the best known chromatin insulator in Drosophila melanogaster. According to previous data, two copies of the gypsy insulator inserted between an enhancer and a promoter neutralize each other's actions, which is indicative of an interaction between the protein complexes bound to the insulators. We have investigated the role of pairing between the gypsy insulators located on homologous chromosomes in trans interaction between yellow enhancers and a promoter. It has been shown that trans activation of the yellow promoter strongly depends on the site of the transposon insertion, which is evidence for a role of surrounding chromatin in homologous pairing. The presence of the gypsy insulators in both homologous chromosomes even at a distance of 9 kb downstream from the promoter dramatically improves the trans activation of yellow. Moreover, the gypsy insulators have proved to stabilize trans activation between distantly located enhancers and a promoter. These data suggest that gypsy insulator pairing is involved in communication between loci in the Drosophila genome.     

Selective interactions of boundaries with upstream region of Abd-B promoter in Drosophila bithorax complex and role of dCTCF in this process

Expression of the genes Ubx, abd-A, and Abd-B of the bithorax complex depends on its cis-regulatory region, which is divided into discrete functional domains (iab). Boundary/insulator elements, named Mcp, Fab-6, Fab-7 and Fab-8 (PTS/F8), have been identified at the borders of the iab domains. Recently, binding sites for a Drosophila homolog of the vertebrate insulator protein CTCF have been identified in Mcp, Fab-6 and Fab-8 and also in several regions that correspond to predicted boundaries, Fab-3 and Fab-4 in particular. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when the activator and the promoter are separated by a 5-kb yellow gene, we have tested functional interactions between the boundaries. The results show that all dCTCF-containing boundaries interact with each other. However, inactivation of dCTCF binding sites in Mcp, Fab-6 and PTS/F8 only partially reduces their ability to interact, suggesting the presence of additional protein(s) supporting distant interactions between the boundaries. Interestingly, only Fab-6, Fab-7 (which contains no dCTCF binding sites) and PTS/F8 interact with the upstream region of the Abd-B promoter. Thus, the boundaries might be involved in supporting the specific interactions between iab enhancers and promoters of the bithorax complex.     

Enhancer-promoter communication at the yellow gene of Drosophila melanogaster: diverse promoters participate in and regulate trans interactions

The many reports of trans interactions between homologous as well as nonhomologous loci in a wide variety of organisms argue that such interactions play an important role in gene regulation. The yellow locus of Drosophila is especially useful for investigating the mechanisms of trans interactions due to its ability to support transvection and the relative ease with which it can be altered by targeted gene replacement. In this study, we exploit these aspects of yellow to further our understanding of cis as well as trans forms of enhancer-promoter communication. Through the analysis of yellow alleles whose promoters have been replaced with wild-type or altered promoters from other genes, we show that mutation of single core promoter elements of two of the three heterologous promoters tested can influence whether yellow enhancers act in cis or in trans. This finding parallels observations of the yellow promoter, suggesting that the manner in which trans interactions are controlled by core promoter elements describes a general mechanism. We further demonstrate that heterologous promoters themselves can be activated in trans as well as participate in pairing-mediated insulator bypass. These results highlight the potential of diverse promoters to partake in many forms of trans interactions.     

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3′ side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ^u1 mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.