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.     

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.     

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.     

Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.

A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.     

EAST affects the activity of Su(Hw) insulators by two different mechanisms in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.     

A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.     

Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on the activity of the enhancer in transiently transfected Drosophila S2R⁺ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.     

Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination

We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7⁺ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Δ cells are similar to those of meu13Δ cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Δ cells is not so conspicuous as meu13Δ cells, and no meiotic delay is observed in mcp7Δmeu13Δ cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Δ cells, whereas Meu13 becomes less stable in mcp7Δ cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination.     

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRβ-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.     

Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator

The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.     

Ibf1 and Ibf2 are novel CP190‐interacting proteins required for insulator function

Insulators are DNA‐protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190‐binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190‐interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190‐binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA‐binding proteins that form hetero‐oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer‐blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.     

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.     

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.