Fragments of the Fab-3 and Fab-4 Boundaries of the Drosophila melanogaster Bithorax Complex That Include CTCF Sites Are not Effective Insulators.

The segment-specific regulatory domains of the Bithorax complex (BX-C), which consists of three homeotic genes Ubx, abd-A and Abd-B, are separated by boundaries that function as insulators. Most of the boundaries contain binding sites for the architectural protein CTCF, which is conserved for higher eukaryotes. As was shown previously, the CTCF sites determine the insulator activity of the boundaries of the Abd-B regulatory region. In this study, it was shown that fragments of the Fab-3 and Fab-4 boundaries of the abd-A regulatory region, containing CTCF binding sites, are not effective insulators.

The Variable CTCF Site from Drosophila melanogaster Ubx Gene is Redundant and Has no Insulator Activity.

CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin. One such variable CTCF site (vCTCF) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx.

The Piragua Gene Is not Essential for Drosophila Development.

Proteins with clusters of C2H2 zinc finger domains (C2H2-proteins) constitute the most abundant class of transcription factors in higher eukaryotes. N-terminal ZAD (zinc finger-associated domain) dimerization domain has been identified in a large group of C2H2-proteins mostly in insects. The piragua gene encodes one of these proteins, Fu2. We have generated CRISPR/Cas9-mediated deletion of the piragua gene that has no phenotype. We have used φC31-mediated attP/attB recombination to generate a transgenic line expressing Fu2 protein fused with HA epitope. This line will be useful for analysis of DNA binding profile and functions of Fu2 protein.

Study of dCTCF Insulator Activity in Drosophilamelanogaster Model Systems.

Using transgenic Drosophila model systems, we showed that four binding sites for the architectural protein dCTCF per se cannot form an effective insulator that blocks enhancers and protects against the Polycomb-dependent repression. These results suggest that, in the known Drosophila insulators, the dCTCF protein functions in cooperation with other architectural proteins.

[Insulators can disrupt weak transcription derived from the white gene enhancer in Drosophila transgenic lines].

Increasing evidence suggests that noncoding RNA transcribed from the enhancers play an important role in the regulation of gene transcription. Insulators are the regulatory elements that limit the activity of enhancers and form independent transcriptional domains. Using a transgenic lines, we show that the Fab-7 insulator of the bithorax complex and the MDG4 (gypsy) insulator are able to disrupt weak transcription derived from the enhancer regulating the white gene expression in the eyes. The ability of insulators to disrupt weak transcription may play a role in the enhancer-blocking activity.

[Induction of transcription through the scs insulator leads to abnormal development of Drosophila melanogaster].

A regulatory element named scs is one of the first insulators discovered in Drosophila, which was found on the boundary of the hsp70 domain. The 993-bp scs insulator contains two promoters at the ends and two polyadenylation signals located in the same orientation in the central part of the insulator. In the Drosophila transgenic lines, induction of a strong transcription through the scs insulator only in the direction that coincides with the direction of the two polyadenylation sites activity results in multiple phenotypic defects of the Drosophila development and embryonic lethality. A similar effect was not observed upon testing of other known Drosophila insulators.

[Recruiting of insulator protein ZIPIC of Drosophila melanogaster to minor binding sites in vivo depends on other DNA-binding transcription factors].

ZIPIC insulator protein of Drosophila has seven zinc finger domains at the C-terminus. Some of this zinc fingers are involved in binding of specific DNA sequence: CAGGGCTG. ZIPIC can interact only in vivo with minor form of this site (substitution of G to T at position 4). Possible explanation is interaction with additional transcription factors can help ZIPIC to bind minor form of consensus. On the other hand ZIPIC can efficiently bind in vitro other minor form of consensus (substitution of C to A at 6 position).

[The bithorax Complex of Drosophila melanogaster as a Model for Studying Specific Long-Distance Interactions between Enhancers and Promoters].

In higher eukaryotes regulation of the expression of genes responsible for the development of the organism is a complex process. Often, a single gene may be regulated in different cells and time intervals with the help of dozens of enhancers and promoters. It has recently become clear that enhancers directly interact with target promoters, and the intervening chromatin loops out. However, the mechanisms that ensure the specificity of long-distance interactions between enhancers and promoters remain unclear. One of the most convenient models for the study of remote interactions between the regulatory elements is the bithorax complex (BX-C) of Drosophila melanogaster. This review describes the regulation of the expression of the BX-C homeotic genes and discusses the existing models that explain the specificity of remote interactions between the enhancers and promoters at this locus.

[Drosophila Fab-7 insulator effectively blocks polycomb-mediated repression in transgenic lines].

The bithorax complex (BX-C) genes have a regulatory region containing nine independent parasegment-specific enhancers. Inactivation of the enhancers is a result of Pc-mediated repression, which leads to the appearance of active and repressed domains. Active domains are supposed to be separated from the repressed domains by insulators on the domain boundaries. Three insulators were found in the BX-C by the ability to block enhancers intransgenic model systems. It is shown that one of these three well studied insulators, Fab-7, is able to protect the yellow gene transcription against the most powerful Polycomb-dependent silencer from the bxd regulatory domain of the Ultra-bithorax gene (Ubx). The barrier activity requires a region containing GAF-binding sites. Interestingly, the silencer used in the model system (1.5 kb PRE from the regulatory region of Ubx) recruits a transgenic construct with a frequency of up to 47% into genomic regions that completely repress the yellow gene expression.

[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.

[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.

[Functional interaction between an insulator and a Pc-dependent silencer with the example of the Mcp boundary of the Drosophila melanogaster Abd-B gene].

Insulators are cis-regulatory elements that prevent improper gene activation and heterochromatin spreading. An insulator is contained in the Mcp boundary from the regulatory region of the Drosophila melanogaster Abd-B gene. The barrier function of the Mcp insulator was studied and proved to be due to two modules. One is responsible for long-distance interactions and the capability of blocking enhancers. The other is essential for blocking Pc-dependent repression. It was observed for the first time that an insulator increased the repressor activity of a neighbor silencer.

[Search for new regulatory elements of the Bithorax complex in Drosophila melanogaster]. / Poisk novykh reguliatornykh élementov Bithorax komplexa Drosophila melanogaster.

The Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B) homeotic genes of the Bithorax complex are responsible for the development of body segments in Drosophila melanogaster. Their regulatory region of approximately 300 kb harbors a series of specific enhancers, each regulating expression in a single parasegment. Molecular genetic analysis of the Abd-B gene revealed the regulatory modules MCP, Fab-7, and Fab-8, which contain insulators with adjacent silencers. The present work was aimed at searching for similar regulatory elements in the promoter regions of the Abd-A and Abd-B genes and new regulatory modules in the Abd-B gene region. Four DNA fragments, each containing numerous specific sequences characteristic of regulatory modules, were tested for the ability to suppress transcription of the yellow and white genes and for the interaction with MCP. The DNA fragment of the Abd-A promoter region proved to act as an effective silencer. The fragment of the iab4 domain showed insulator properties. The fragment of the Abd-B promoter region interacted with MCP in providing for communication between the enhancer and promoter of the white gene.