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.     

New properties of Drosophila fab-7 insulator

In the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains, boundary elements have previously been detected, including the Fab-7 element providing for the autonomous functioning of the iab-6 and iab-7 cis-regulatory domains. Here, it has been shown that a single copy of the 860-bp Fab-7 insulator effectively blocks the yellow and white enhancers. The eye and testis enhancers can stimulate the white promoter across the pair of Fab-7, which is indicative of a functional interaction between the insulators. Unexpectedly, Fab-7 has proved to lose the enhancer-blocking activity when placed near the white promoter. It seems likely that Fab-7 strengthens the relatively weak white promoter, which leads to the efficient enhancer-promoter interaction and insulator bypass.     

Study of the functional interaction between Mcp insulators from the Drosophila bithorax complex: effects of insulator pairing on enhancer-promoter communication

Boundary elements have been found in the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains. Previously, a 340-bp insulator-like element, M(340), was identified in one such 755-bp Mcp fragment linked to the PcG-dependent silencer. In this study, we identified a 210-bp core that was sufficient for pairing of sequence-remote Mcp elements. In two-gene transgenic constructs with two Mcp insulators (or their cores) surrounding yellow, the upstream yeast GAL4 sites were able to activate the distal white only if the insulators were in the opposite orientations (head-to-head or tail-to-tail), which is consistent with the looping/bypass model. The same was true for the efficiency of the cognate eye enhancer, while yellow thus isolated in the loop from its enhancers was blocked more strongly. These results indicate that the relative placement and orientation of insulator-like elements can determine proper enhancer-promoter communication.     

Interacting insulators from the Drosophila melanogaster bithorax complex can form independent expression domains

Regulatory region of three bithorax complex genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) can be divided into nine iab domains, capable of directing expression of one of the genes in certain abdominal parasegment of Drosophila. In the Abd-B regulatory region, three insulators were identified, including Fab-7 and Fab-8, which flanked the iab-7domain, and Mcp, which separated the Abd-B and abd-A regulatory regions. It was suggested that boundary insulators formed a barrier between active and repressed chromatin. In the present study, using the yellow and white reporter genes and different combinations of known insulators, Mcp, Fab-7, and Fab-8, it was demonstrated that only specific interaction of two insulators was capable of isolation of active and repressed chromatin, i.e., the formation of independent expression domains.     

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.     

Study of the enhancer-blocking activities of new boundaries in the bithorax complex of Drosophila melanogaster

It was shown earlier that the Mcp, Fab-7, and Fab-8 boundaries of the bithorax complex contain insulators that effectively block the enhancers of the yellow and white genes. Other boundaries have not been studied so far. The recent mapping of binding sites for the insulator protein dCTCF in the regulatory regions of the bithorax complex genes permitted the Fab-3, Fab-4, and Fab-6 boundaries to be localized. Here, we showed despite the presence of dCTCF-binding sites fragments of the Fab-3, Fab-4, and Fab-6 boundaries do not exhibit the properties of insulators in the model system with the yellow and white genes. Moreover, in some regions of the genome the Fab-4 and Fab-6 boundaries display the properties of silencers.     

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

In complex genomes, insulators set up chromatin domain boundaries and protect promoters from inappropriate activation by enhancers from neighboring genes. The Drosophila Abdominal-B locus uses insulator elements to organize its large regulatory region into several body segment-specific chromatin domains. This organization leads to a problem in enhancer-promoter communication, that is, how do distal enhancers activate the Abd-B promoter when there are several insulators in between? This issue is partially resolved by the Promoter Targeting Sequence, which can overcome the enhancer blocking effect of an insulator. In this study, we describe a new Promoter Targeting Sequence, PTS-6, from the Abd-B 3' regulatory region. PTS-6, comprised of approximately 200 bp, was found to bypass both homologous Abdominal-B insulators, such as Fab-7 and Fab-8, and a heterologous insulator, suHw. Most importantly, it also overcomes a combination of two insulators such as Fab-7/Fab-8. Thus, PTS-6 could, in principle, target remote enhancers that are separated from the Abd-B promoter by multiple insulators. In addition, PTS-6 selectively targets the distal enhancer to only one transgenic promoter, and it strongly facilitates Abd-B enhancers. These results suggest that promoter targeting is necessary for long-range enhancer-promoter communication in Abd-B, and PTS elements could be a common occurrence in large, complex genetic loci.     

A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo

The Abd-B Hox gene contains an extended 3' cis-regulatory region that is subdivided into a series of separate lab domains. The lab-7 domain activates Abd-B in parasegment 12 (ps12), whereas lab-8 controls expression in ps13. iab-7 is flanked by two insulators, Fab-7 and Fab-8, which are thought to prevent regulatory factors, such as Polycomb silencers, from influencing neighboring iab domains. This organization poses a potential paradox, since insulator DNAs can work in a dominant fashion to block enhancer-promoter interactions over long distances. Here, we present evidence for a novel cis-regulatory sequence located within lab-7, the promoter targeting sequence (PTS), which permits distal enhancers to overcome the blocking effects of Fab-8 and the heterologous su(Hw) insulator. We propose that the PTS converts dominant, long-range insulators into local regulatory elements that separate neighboring lab domains.     

Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.     

Insulators: exploiting transcriptional and epigenetic mechanisms

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.     

Boundary swapping in the Drosophila Bithorax complex

Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, we tested whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, we exchanged the Fab-7 and Fab-8 boundaries within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, we find that the Fab-7 boundary can almost completely replace the Fab-8 boundary. Our results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries.     

Study of the enhancer-blocking activities of new boundaries in the <Emphasis Type="Italic">bithorax</Emphasis> complex of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

It was shown earlier that the Mcp, Fab-7, and Fab-8 boundaries of the bithorax complex contain insulators that effectively block the enhancers of the yellow and white genes. Other boundaries have not been studied so far. The recent mapping of binding sites for the insulator protein dCTCF in the regulatory regions of the bithorax complex genes permitted the Fab-3, Fab-4, and Fab-6 boundaries to be localized. Here, we showed that, despite the presence of dCTCF-binding sites, fragments of the Fab-3, Fab-4, and Fab-6 boundaries do not exhibit the properties of insulators in the model system with the yellow and white genes. Moreover, in some regions of the genome the Fab-4 and Fab-6 boundaries display the properties of silencers.