Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.

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 are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs–scs, scs’–scs’, 1A2–1A2 and Wari–Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5–Su(Hw), dCTCF–Su(Hw), or dCTCF–Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer–promoter interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. Here we have shown that the ZAD domains of ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene promoter. However, no functional interaction between binding sites for different proteins has been revealed, suggesting that such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.

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.Eukaryotic genomes are highly organized into functional units containing individual genes or gene groups together with the corresponding regulatory elements. Regulatory elements, enhancers/silencers, may be separated from the promoters by dozens of thousands of base pairs (10,15,16,35,63). Most recent data (14,18,42,52,67) support the looping model (53), which postulates that enhancers and distant promoters are in physical contact with each other while the intervening sequences loop out. Accordingly, one of the key questions is how distant enhancers communicate with their target promoters. The complexity of higher eukaryotic regulatory systems, which contain many distantly located enhancers that nevertheless properly activate the target promoters, has prompted the hypothesis that the action of enhancers should be restricted by elements called insulators (6,32,64,66,68). Generally, insulators are defined by two properties: the enhancer-blocking activity, preventing communication between an enhancer and a promoter separated by the insulator, and the boundary function (barrier activity), preventing repressive chromatin spreading. In recent years, however, experimental evidences have been accumulated that insulator proteins may be involved in supporting long-distance interactions between regulatory elements located either within the same complex locus or in distantly located loci (12,13,31,40,42,43,57).One of the best model systems for studying the role of insulators in long-distance enhancer-promoter communication is the regulatory region of the homeotic Abdominal-B (Abd-B) gene of the bithorax complex (41,47,63). The three homeotic genes of the bithorax complex-Ultrabithorax (Ubx), abdominal-A (abd-A), and Abd-B-are responsible for s...

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.The discovery that eukaryotic transcriptional activators can operate over long distances in an orientation-independent manner posed several questions (3). One of these was how the long-range activation potential of eukaryotic enhancers could be restricted to the relevant target promoter. It has been proposed that eukaryotic chromatin is organized into functionally independent domains to prevent illegitimate enhancer-promoter communication. Recently, chromatin insulators, or boundary elements, have been identified. These DNA sequences are functionally characterized by two properties: they prevent enhancer-promoter interactions and buffer transgenes from the chromosomal-position effects of genomic sequences adjoining the transgene insertion site (11,14,22,23,36,42,43).An idea of a probable role of insulators in the cell can be gained from data on their distribution in the bithorax complex (36, 39). The three homeotic genes of the bithorax complex, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B), are responsible for specifying the identity of parasegments 5 to 14 (PS5 to PS14), which form the posterior half of the thorax and all abdominal segments of an adult fly (24, 32). The PS-specific expression patterns of Ubx, abd-A, and Abd-B are determined by a complex cis-regulatory region that spans a 300-kb DNA segment (32). For example, Abd-B expression in PS10, PS11, PS12, and PS13 is controlled by the iab-5, iab-6, iab-7, and iab-8 cis-regulatory domains, respectively (4, 9, 10, 12, 19, 24, 40). Each iab domain appears to contain at least one enhancer that initiates Abd-B expression in the early embryo, as well as a PRE (Polycomb response element) silencer element that maintains the expression pattern throughout development (2,5,6,17,18,29,31,33,44,45). It has been proposed that insulators flank each iab region and organize the Abd-B regulatory DNA into a series of separate chromatin loop dom...

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.In eukaryotic organisms, precise control of spatially and temporally regulated genes requires a large set of enhancers, which are often located at considerable distances from the regulated gene (4,6,15,19,20,21,39,66,72). Most of recent data (13,54,69) support the looping model (56), which postulates that enhancers and distant promoters are in physical contact, while the intervening sequences loop out. Accordingly, one of the key questions is how distant enhancers communicate with their target promoters. One of the best model systems for studying long-distance enhancer-promoter communication is the regulatory region of the homeotic Abdominal-B (Abd-B) gene of the bithorax complex (45, 66).The three homeotic genes of the bithorax complex-Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)-are responsible for specifying the identity of parasegment 5 (PS5) to PS14, which form the posterior half of the thorax and all abdominal segments of an adult fly (41,45,50,60). The PS-specific expression patterns of Ubx, abd-A, and Abd-B are determined by a complex cis-regulatory region that spans a 300-kb DNA segment (45, 66). For example, Abd-B expression in PS10, PS11, PS12, and PS13 is controlled by the iab-5, iab-6, iab-7, and iab-8 cis-regulatory domains, respectively (7, 14, 23, 34, 41, 51, 61). Each iab domain appears to contain at least one enhancer that initiates Abd-B expression in the early embryo, as well as a PRE silencer element that maintains the expression pattern throughout development (2,8,9,31,32,48,49,51,74,75). It has been proposed that insulators flank each iab region and organize the Abd-B regulatory DNA into a series of separate chromatin loop domains (25,30,50,51). The recent finding that iab-7 is flanked by two insulators, Fab-7 and Fab-8, is consistent with this model (2, 31, 74). MATERIALS AND METHODSPlasmid constructions. Details of transgene construction are available upon request. An 8-kb fragment containing the yellow gene was kindly provided by P. Geyer. The 5-kb BamHI-BglII fragment containing the coding region (yc) was subc...

BackgroundInsulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man.ResultsIn the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF–CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity.ConclusionsOur results indicate that dCTCF–CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF–dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0168-7) contains supplementary material, which is available to authorized users.