Search

Abstract Background CTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer–promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif. Results Previously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCF attP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein. Conclusions Our findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, male absent on the first (MOF), and maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. Here, we found that MLE's unstructured C-terminal region interacts with the sixth zinc-finger domain of CLAMP. In vitro, 4–5 zinc fingers are critical for the specific DNA-binding of CLAMP with GA repeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome.

The reviewed article examines the results of Reflectance Transformation Imaging (RTI) and 3D Laser Scanning documentation recently carried out by E. Komatarova-Balinova, P. Charalampakis and M. Raykovska on a well-known stone block with carvings and inscriptions discovered during the 1948 excavations in Pliska. Reviewing the data about the place of its discovery, its manufacture, as well as the presence of two “signs” with the character of letter combinations, the author of this paper confirms the conclusions of St. Stanchev (Vaklinov) from 1955 that the stone block with the graffiti originated from the palatial complex, most likely from the pagan temple in the Citadel, from which the stone block was removed for repeated subsequent use as early as the middle of the 9th c. The current review also takes into account older photographs of the monument that are compared to the RTI and 3D Laser Scanning documentation to achieve a more substantial reconstruction of the details in the carvings and inscriptions. Compared to the new documentation, they offer opportunities for a more objective consideration of the controversial issues related to the understanding of these graffiti. Regarding the images, the author maintains the opinion that they occupy the entire obverse of the stone and that, regardless of the different qualities of execution, they form a single composition dedicated to an important event – a religious ceremony held most likely at a Bulgarian ruler’s residence.

Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer–promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.

Expression of Abdominal-B (Abd-B) in abdominal segments A5–A8 is controlled by four regulatory domains, iab-5–iab-8. Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements (Mcp, Fab-7, Fab-7 and Fab-8). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can ‘jump over’ intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation.

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.

Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. In this study, we show that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein–protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, our results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein.

In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether readthrough transcription (RT) can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters, and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.

Abstract Background Epigenetic memory plays a critical role in the establishment and maintenance of cell identities in multicellular organisms. Polycomb and trithorax group (PcG and TrxG) proteins are responsible for epigenetic memory, and in flies, they are recruited to specialized DNA regulatory elements termed polycomb response elements (PREs). Previous transgene studies have shown that PREs can silence reporter genes outside of their normal context, often by pairing sensitive (PSS) mechanism; however, their silencing activity is non-autonomous and depends upon the surrounding chromatin context. It is not known why PRE activity depends on the local environment or what outside factors can induce silencing. Results Using an attP system in Drosophila, we find that the so-called neutral chromatin environments vary substantially in their ability to support the silencing activity of the well-characterized bxdPRE. In refractory chromosomal contexts, factors required for PcG-silencing are unable to gain access to the PRE. Silencing activity can be rescued by linking the bxdPRE to a boundary element (insulator). When placed next to the PRE, the boundaries induce an alteration in chromatin structure enabling factors critical for PcG silencing to gain access to the bxdPRE. When placed at a distance from the bxdPRE, boundaries induce PSS by bringing the bxdPREs on each homolog in close proximity. Conclusion This proof-of-concept study demonstrates that the repressing activity of PREs can be induced or enhanced by nearby boundary elements. Graphical abstract

We aim to present diagnostic approach and results from surgical treatment in avulsion fractures of the apophysis of Spina Iliaca Anterior Inferior (SIAI). Material and methods. During the period 2011 – 2021, seven(7) children with acute traumatic apophysiolysis of SIAI, were operated on, 61 A1.2 fracture by AO/OTA classification. All of the patients are boys with average age 14,1±1,35 years. In all cases the right SIAI is affected and the mechanism of the injury is sports trauma. The diagnostic methods used by us are complete orthopedic clinical examination, AP radiographs of the pelvis and lateral radiographic projections of the hip joint by modified Dunlap technique and also CT scan with 3D reconstruction of the pelvic bones (3cases). In four (4) cases the apophysiolyses are Salter-Harris type I and in three cases – SH type II. The average size of the avulsed bone fragment is 3cm/1cm measured on CT scan and 29,66±3,14 mm on AP radiographic view. Dislocation of the fractured fragment is greater than 1,5cm. In four children the diagnosis from the outpatient care is neglected or primary wrong. This fact procrastinated the treatment for between 15 and 30 days. In the rest of the cases the treatment is prompt in an urgent matter. For all patients open reduction and internal fixation with screw osteosynthesis is performed by using one (4 cases) or two (2 cases) 3,5mm cannulated titanium screws. Consolidation of the fracture and the functional condition by Lower Extremity Functional Scale (LEFS) have been followed.

Proximal femoral osteotomies are one of the most commonly used surgical techniques and play an important role in the spatial correction of the hip joint in a number of pediatric orthopedic disorders such as Legg-Calve-Perthes disease, Slipped capital femoral epiphysis, Developmental dysplasia of the hip, osteoarthritis, etc.14,21,23,28,30,31 The development of orthopaedic osteosynthesis for fixation of bone fragments includes the use of Kirschner wires, intraosseous nail plates, AO-plates with fixed angle, external fixators, etc. Over time, many of the techniques and osteosynthesis materials have fallen into disuse and have remained only of historical significance. The purpose of this study is to follow how osteosynthesis fixation devices for varus osteotomies in the proximal femur have historically evolved and developed from the beginning to the present.

Abstract Background Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins. Results Using in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of the wild-type and mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions. Conclusions The results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

Abstract The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary we generated deletions of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary – Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.

In higher eukaryotes, the regulation of developmental gene expression is determined by enhancers, which are often located at a large distance from the promoters they regulate. Therefore, the architecture of chromosomes and the mechanisms that determine the functional interaction between enhancers and promoters are of decisive importance in the development of organisms. Mammals and the model animal Drosophila have homologous key architectural proteins and similar mechanisms in the organization of chromosome architecture. This review describes the current progress in understanding the mechanisms of the formation and regulation of long-range interactions between enhancers and promoters at three well-studied key regulatory loci in Drosophila.

The article aims to briefly overview the three types of inscriptions on the vessels of the Nagyszentmiklós treasure (NSM) as an uniform evidence of the existence of specific religious beliefs in the Middle Danube area in the 8th – 9th c. The emphasis of the study is on deciphering the runiform inscriptions, which the author considers as cryptography of sacred concepts and names of a Christian community with Gnostic conceptions and ideas. At the basis of their decoding he placed the decipherment of individual runiform graphemes and short texts in related written monuments from the Lower Danube area (Pliska, Murfatlar, etc.). The concepts and names decoded (Tetragrammatôn, Hexagrammatôn, Eôn, Five/the Fifth Eon, Hiueh, Eueh and others) allow establishing that they are cryptograms, attested in the ideas about the God of Gnostic Christians in the first centuries AD in the Middle East. This is evidenced by the use of specific Semitic and Greek graphemes in the three types of inscriptions from the treasure. Their use on vessels of precious metal in the Early Middle Ages is part of these beliefs and is evidence of their stability and transfer to the Middle and Lower Danube area by adherents of Christian Gnosis, most likely the doctrine of the socalled Valentinians. The link of repetitive or similar cryptograms and Greek-language texts to vessels of identical or similarshape and similar use allows those marked with them to be distributed in “sets” that met the cult and ritualneeds of different categories of believers in Christian Gnosis (“pneumatikoi”, “psyxikoi” and “hylics”). Finally, the author tries to highlight cultural and historical traces of the presence of a population with similar ideas in the region of the Middle Danube. Although fragmentary and hypothetical, they allow us assuming that the so-called Danube runic script was in fact a relic of the life of Gnostic-Christian sects in Central and Southeastern Europe in the Early Middle Ages.

Kaiso is a methyl-DNA-binding protein containing three C2H2 zinc fingers with a C-terminal extension that participates in DNA binding. The linker between the last zinc finger and the DNA-binding portion of the extension contains two prolines that are highly conserved in vertebrates and in cognate ZBTB4 and ZBTB38 proteins. Prolines provide chain rigidity and can exist in cis and trans conformations that can be switched by proline isomerases, affecting protein function. We found that substitution of the conserved proline P588, but not of P577, to alanine, negatively affected KaisoDNA-binding according to molecular dynamics simulation and in vitro DNA-binding assays. Molecular dynamics simulations of the Kaiso DNA-binding domain with P588 either substituted to alanine or switched to the cis-conformation revealed similar alterations in the H-bonding network and uncovered allosteric effects leading to structural rearrangements in the entire domain that resulted in the weakening of DNA-binding affinity. The substitution of proline with a large hydrophobic residue led to the same negative effects despite its ability to partially rescue the intrinsic DNA-binding activity of the C-terminal loop. Thus, the presence of the C-terminal extension and cis-conformation of proline residues are essential for efficient Kaiso–DNA binding, which likely involves intramolecular tension squeezing the DNA chain.

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus.

Abstract Background Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. Results Here, we have used a structure–function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. Conclusions The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.

A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, we performed an RNAi screen targeting ubiquitin-related genes in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging.

In Drosophila melanogaster, CLAMP is an essential zinc-finger transcription factor that is involved in chromosome architecture and functions as an adaptor for the dosage compensation complex. Most of the known Drosophila architectural proteins have structural N-terminal homodimerization domains that facilitate distance interactions. Because CLAMP performs architectural functions, we tested its N-terminal region for the presence of a homodimerization domain. We used a yeast two-hybrid assay and biochemical studies to demonstrate that the adjacent N-terminal region between 46 and 86 amino acids is capable of forming homodimers. This region is conserved in CLAMP orthologs from most insects, except Hymenopterans. Biophysical techniques, including nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS), suggested that this domain lacks secondary structure and has features of intrinsically disordered regions despite the fact that the protein structure prediction algorithms suggested the presence of beta-sheets. The dimerization domain is essential for CLAMP functions in vivo because its deletion results in lethality. Thus, CLAMP is the second architectural protein after CTCF that contains an unstructured N-terminal dimerization domain.

In this article the author reviews the data about a section of the medieval fortress wall excavated in 2005 in the oldest and highest part of the Ancient Odessos. Based on the published geodetic plan, as well as on unpublished photographs made after the wall was exposed and during its partial dismantlement, he makes new observations and conclusions regarding its structure and relation to previous and contemporary constructions such as street pavements, channels, water pipes, etc. A conclusion is made that several entrances existed consecutively along the 25 m stretch of the uncovered wall. The entrances, as well as several of the pavements and the channels passing through them or located nearby, are situated along a Late Antique cardo and are part of a street that served as a link to large and durable ancient constructions and to the Large Roman Baths. Outside of the limits of the medieval city, the street passed through abandoned neighbourhoods and headed to an Old-Christian urban basilica and from there further north and north-west. The structure of the wall and particularly the lack of towers at the entrances shows that it was not part of a separate fortress, as currently presumed, but a relatively low (up to 4 m high) barrier. Most probably the wall followed the highest possible points of the terrain and her both ends headed towards the ancient fortification located nearby. Judging by earlier and contemporary evidence, at the seaside the ancient fortress wall existed until the 15th – 16th c. Taking into account three main factors – the relief of the terrain, the presence of well preserved Late Antique buildings, and the nearby key points of the Romano-Byzantine fortification, the author presumes that the medieval fortress occupied the entire cape part of Odessos and enclosed an area of 10 ha. With the construction of a new defensive line from north and north-west only, the fortified area of Varna comprised no more than one third of the total fortified area of Odessos. From this point of view this can be considered as a consequence of the reduction of the size of the ancient cities, a practice adopted in Byzantium in the 5th – 6th c., but also applied in the later centuries. It has to be noted that Varna’s transformation from an ancient to a medieval fortress happened relatively late. If we assume that the fortress wall was constructed in the first two decades of the 11th c., as stated by V. Pletniov, this transformation should have resulted from an imperial initiative for restoration of the city and integration of its citizens after the decline of the First Bulgarian Kingdom. Integrating his own conclusions among the data about the defensive system of Varna collected so far, the author points out that the fortress under consideration is a “hybrid“ between the Early and Middle Byzantine defensive system of the city and represents only its outer belt. The inner fortress, as mainly established by Al. Kuzev, was the fortress “Kale ichi” (literally from Turkish – Inner fortress), which existed until 1830. After commenting upon well-known Byzantine literary sources, the author concludes that the fortress (or a part of it) was used as a citadel at least since the late 12th c. and not after the mid – 13th c. It is possible that some early construction phases were contemporary to the construction of the outer defensive ring, or were immediately subsequent. In this regard, the author reminds about the rejected hypothesis of the Shkorpil brothers, that the Roman Baths or parts of them, located at the highest point of Odessos, could have been used as an “acropolis” and as an “inner fortification” since the Late Antiquity and the Early Middle Ages.

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must "jump over" the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C-they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.

In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms underlying specific distance interactions between enhancers and promoters remain poorly understood. This review briefly describes the properties of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.

A complete study of the assemblage of four mounds near the earthen fortification facility in Kabayuk excavated by R. Rashev was published in 2014. The researchers’ attention focused specifically on a burial under one of the barrows. It is a burial of a young aristocrat buried with an abundance of luxurious grave goods in accordance with the pagan ritual. The authors suggest that the burial dates back to the very late 7th or very early 8th century, and the barrow dates back to the early 9th century. Recently, St. Stanilov has argued that these results provide strong evidence supporting the argument about the functioning of a royal residence at this location form the late 7th and the early 8th century. This allowed him to defends the traditional view that Pliska, which is situated ca. 15 km away from it, has emerged as the center of the First Bulgarian Kingdom during the reign of Khan Asparukh (in the years after 681). This article reports the results of a critical comparison of the burial and barrow assemblage, which concludes that there is no clear chronological difference between the two archaeological structures. The burial preceded the barrow assemblage, and its location was carefully hidden. The four barrows were accumulated immediately after that in two stages. The barrows were smaller during the first stage, and barrow 4 covered the burial whose grave pit was cut into the bedrock. Pits and ovens were made at the periphery of the barrows. They were used for preparing ritual meals and grave goods during commemoration ceremonies. Following the completion of the funerary and commemorative ceremonies, the covers of the barrows were finalized. The author believes that the assemblage cannot be dated earlier than the beginning of the 9th century. A parallel can be made with similar assemblages in Pliska, the Fortification of Khan Omurtag on the Ticha River among others, all dating back to the first half of the 9th century. He believes that the four barrows in Kabayuk have important characteristics, but they cannot be linked to a ruler of the capital Pliska; it is more reasonable to link them to an inhabitant of a nearby earthen fortification. The author also reports the results of stratigraphic and comparative analyses used to establish the chronology of the grave good yielded by the burial under the barrow. R. Rashev’s conclusions that the barrows represent elements of certain activities form the early 9th century have been taken into consideration. Commenting on Rashev’s thesis and recent studies on the so-called Vrap–Velino group dated back to the 760s, the author suggests that the artifacts yielded by the burial are similar to others yielded by several individual burials excavated in the Pliska Field. The author believes that the earliest burials (such as burial 5 under barrow III, situated between Pliska and Madara ritual center) can be linked to the two important crossroads (crosswise and parallel) in the Pliska field. The field is situated between two territories: the Black Sea Coast and Dobrogea and the territory to the north of the Stara Planina Mountain, both densely populated with Bulgars in the 8th and 9th century. These finding allow to conclude that the currently available evidence does not support the argument of the existence of Pliska as an administrative and political center of the First Bulgarian Kingdom before the end of 8th century.

Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.

The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG) proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs). A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

Studies of regulatory systems in transgenic Drosophila are often compromised by possible genomic position effects on gene expression. As a result, it is desirable to be able to manipulate multiple regulatory elements in a single transgene construct. We developed an I-SceI endonuclease-based method to efficiently delete preassigned sequences from transgenes with the use of direct repeat sequences of just 126 nucleotides. This system can be used in combination with the existing cre-lox and FLP-FRT recombinational mechanisms in order to modify up to three regulatory regions in a given transgene. We validated the utility of our combination approach by demonstrating new properties of the Fab-7 insulator.

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. The first insulators to be identified were scs and scs', which flank the domain including two heat shock 70 genes. Zw5 and BEAF bind to scs and scs', respectively, and are responsible for the interaction between these insulators. Using the regulatory regions of yellow and white reporter genes, we have found that the interaction between scs and scs' improves the enhancer-blocking activity of the weak scs' insulator. The sequences of scs and scs' insulators include the promoters of genes that are strongly active in S2 cells but not in the eyes, in which the enhancer-blocking activity of these insulators has been extensively examined. Only the promoter of the Cad87A gene located at the end of the scs insulator drives white expression in the eyes, and the white enhancer can slightly stimulate this promoter. The scs insulator contains polyadenylation signals that may be important for preventing transcription through the insulator. As shown previously, scs and scs' can insulate transcription of the white transgene from the enhancing effects of the surrounding genome, a phenomenon known as the chromosomal position effect (CPE). After analyzing many independent transgenic lines, we have concluded that transgenes carrying the scs insulator are rarely inserted into genomic regions that stimulate the white reporter expression in the eyes.

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer-white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer-promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer-promoter pair.