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The mechanism of specific recruitment of the dosage compensation complex (DCC) to the X chromosome of male Drosophila remains an unresolved problem. Recently, a DNA-binding protein CLAMP, which binds to the primary chromosome entry sites (CESs) of the DCC, has been described as a factor involved in X chromosome recruiting of DCC. It has been shown that CESs can recruit DCC to autosomes. In this work, we studied the ability of a well-described CES to support distant interactions in model transgenic Drosophila lines. Negative results were obtained showing that the CES that is bound by CLAMP protein does not support distance interactions.

Polycomb group (PcG) proteins are chromatin-associated factors involved in the repression of gene transcription. In the present study, we characterized the interactome of the Sfmbt factor at the embryonic stage of development. For this, the Sfmbt protein complex was affinity purified from the nuclear extract, followed by highly specific peptide sequencing (IP/LC-MS). As a result, a number of previously uncharacterized Sfmbt interactions were discovered. In particular, Sfmbt top-interacting proteins include the DNA-binding protein Hangover and components of the SWI/SNF family of chromatin remodelers.

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., BMC Biology, 19 (1), ISSN:1741-7007

In Drosophila, the BEAF-32, Z4/putzig, and Chriz/Chromator proteins colocalize in the interbands of polytene chromosomes. It was assumed that these proteins can form a complex that affects the structure of chromatin. However, the mechanism of the formation of such a complex has not been studied. We have proved for the first time that the BEAF-32, Z4/putzig, and Chriz/Chromator proteins interact directly with each other and localized the protein domains that provide multiple protein-protein interactions. Based on the data obtained, we developed a model of the mechanism of the formation the BEAF/Z4/Chriz complex and its recruitment to chromatin.

Human CTCF (hCTCF) is a major architectural protein in mammals. In Drosophila, the CTCF homologue (dCTCF) interacts with the BTB domain of the CP190 protein, which is involved in the establishment of open chromatin and activity of insulators. Previously, it was shown that the BTB protein Kaiso interacts with hCTCF and regulates its activity. We have carried out a detailed study of the interaction between these proteins in the yeast two-hybrid assay. Surprisingly, Kaiso did not interact with hCTCF and its Drosophila homologue. On the other hand, CP190 interacted with the C-terminus of hCTCF. The results obtained demonstrate that the interaction between CTCF and CP190 proteins is highly conserved. It is likely that humans have other BTB proteins that perform the functions described for the Drosophila CP190.

PRC2 (Polycomb repressive complex 2) is a conserved protein complex in multicellular organisms that is required to maintain gene repression. The catalytic subunit of PRC2, the EZH2 protein, provides the methylation of histone H3K27 (H3K27me1/2/3). It was demonstrated that a number of human cancers were associated with overexpression of PRC2 subunits, as well as with mutations that enhanced the EZH2 catalytic activity. At the same time, a group of cancers correlate with mutations that inhibit PRC2. A number of small molecule inhibitors to the PRC2 subunits have been developed, primarily to EZH2. One of these, tazemetostat, received approval in January 2020 in the United States for the treatment of epithelioid sarcoma. This review focuses on the role of PRC2 in cancer development and summarizes information on the designed PRC2 inhibitors.

In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.

The mechanisms underlying long-range interactions between chromatin regions and the principles of chromosomal architecture formation are currently under extensive scrutiny. A special class of regulatory elements known as insulators is believed to be involved in the regulation of specific long-range interactions between enhancers and promoters. This review focuses on the insulators of Drosophila and mammals, and it also briefly characterizes the proteins responsible for their functional activity. It was initially believed that the main properties of insulators are blocking of enhancers and the formation of independent transcription domains. We present experimental data proving that the chromatin loops formed by insulators play only an auxiliary role in enhancer blocking. The review also discusses the mechanisms involved in the formation of topologically associating domains and their role in the formation of the chromosomal architecture and regulation of gene transcription.

Proteins MSL1 and MSL2 form the core of the Drosophila dosage compensation complex, which specifically binds to the X chromosome of males. Phosphorylation of certain amino acid residues was previously shown to regulate MSL1 activity. In the present work, transgenic lines of Drosophila expressing mutant variants of the MSL1 protein were obtained, in which amino acids undergoing phosphorylation were replaced. As a result, it was shown that inactivation of phosphorylation sites does not affect the efficiency of specific binding of the dosage compensation complex to the X chromosome of males and its functional activity.

The Drosophila TTK protein is involved in the processes of cell differentiation and is represented by two isoforms, TTK69 and TTK88, which have a common N-terminal BTB domain and different C-terminal sequences. Earlier, it was shown that TTK69 represses the activity of enhancers and promoters by recruiting a conserved among higher eukaryotes NURD complex to chromatin. The Mep-1 protein was found in the NURD-complex of Drosophila, and this protein can interact with the C-terminal region of TTK69. In the present study, using the yeast two-hybrid system, we mapped the interacting regions of the TTK and Mep-1 proteins. We identified regions in the unique C-terminal regions of TTK isoforms that can interact simultaneously with two regions of the Mep-1 protein. The results show that, despite the low homology of the C-terminal regions, the TTK isoform retains the ability to interact with two conserved regions of the Mep-1 protein, which suggests the functional significance of this interaction.

Boundaries in the Drosophila bithorax complex (BX-C) enable the regulatory domains that drive parasegment-specific expression of the three Hox genes to function autonomously. The four regulatory domains (iab-5, iab-6, iab-7, and iab-8) that control the expression of the Abdominal-B (Abd-B) gene are located downstream of the transcription unit, and are delimited by the Mcp, Fab-6, Fab-7, and Fab-8 boundaries. These boundaries function to block cross talk between neighboring regulatory domains. In addition, three of the boundaries (Fab-6, Fab-7, and Fab-8) must also have bypass activity so that regulatory domains distal to the boundaries can contact the Abd-B promoter. In the studies reported here, we have undertaken a functional dissection of the Fab-8 boundary using a boundary-replacement strategy. Our studies indicate that the Fab-8 boundary has two separable subelements. The distal subelement blocks cross talk, but cannot support bypass. The proximal subelement has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC (large boundary complex), binds to sequences in the proximal subelement and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the Fab-7 boundary.

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.

The Abdominal-B (Abd-B) gene belongs to Bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold. Instead, we find that tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our findings indicate that the functioning of the iab-5 and iab-6 domains in development of the adult cuticle A5 and A6 in males fit better with an additive model much like that first envisioned by Ed Lewis.

Simple Summary PRC2 (Polycomb repressive complex 2) is a catalytic multi-subunit complex involved in transcriptional repression through the methylation of lysine 27 at histone 3 (H3K27me1/2/3). Dysregulation of PRC2 has been linked to tumor development and progression. Here, we performed a comprehensive analysis of data in the genomic and transcriptomic (cBioPortal, KMplot) database portals of clinical tumor samples and evaluated clinical correlations of EZH2, SUZ12, and EED. Next, we developed an original Python application enabling the identification of genes cooperating with PRC2 in oncogenic processes for the analysis of the DepMap CRISPR knockout database. Our study identified cancer types that are most likely to be responsive to PRC2 inhibitors. By analyzing co-dependencies with other genes, this analysis also provides indications of prognostic biomarkers and new therapeutic regimens. Abstract PRC2 (Polycomb repressive complex 2) is an evolutionarily conserved protein complex required to maintain transcriptional repression. The core PRC2 complex includes EZH2, SUZ12, and EED proteins and methylates histone H3K27. PRC2 is known to contribute to carcinogenesis and several small molecule inhibitors targeting PRC2 have been developed. The present study aimed to identify the cancer types in which PRC2 targeting drugs could be beneficial. We queried genomic and transcriptomic (cBioPortal, KMplot) database portals of clinical tumor samples to evaluate clinical correlations of PRC2 subunit genes. EZH2, SUZ12, and EED gene amplification was most frequently found in prostate cancer, whereas lymphoid malignancies (DLBCL) frequently showed EZH2 mutations. In both cases, PRC2 alterations were associated with poor prognosis. Moreover, higher expression of PRC2 subunits was correlated with poor survival in renal and liver cancers as well as gliomas. Finally, we generated a Python application to analyze the correlation of EZH2/SUZ12/EED gene knockouts by CRISPR with the alterations detected in the cancer cell lines using DepMap data. As a result, we were able to identify mutations that correlated significantly with tumor cell sensitivity to PRC2 knockout, including SWI/SNF, COMPASS/COMPASS-like subunits and BCL2, warranting the investigation of these genes as potential markers of sensitivity to PRC2-targeting drugs.

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates andDrosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in thedCTCFgene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new nulldCTCFmutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of theAbd-Bgene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the nulldCTCFmutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation inDrosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCFin vivo.HighlightsThe dCTCF 715–735 aa region interacts with the BTB and M domains of CP190Interaction with CP190 is not essential for dCTCF functionin vivoNulldCTCFmutants are viable but show severely reduced fertility

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila , CTCF is involved in the regulation of homeotic genes . Previously, it was found that null mutations in the dCTCF gene died as pharate adults, which failed to eclose from their pupal case, or shortly after hatching of adults. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears is limited mainly to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not important for the functional activity of dCTCF in vivo.

In mammals, most of the boundaries of topologically associating domains and all well-studied insulators are rich in binding sites for the CTCF protein. According to existing experimental data, CTCF is a key factor in the organization of the architecture of mammalian chromosomes. A characteristic feature of the CTCF is that the central part of the protein contains a cluster consisting of eleven domains of C2H2-type zinc fingers, five of which specifically bind to a long DNA sequence conserved in most animals. The class of transcription factors that carry a cluster of C2H2-type zinc fingers consisting of five or more domains (C2H2 proteins) is widely represented in all groups of animals. The functions of most C2H2 proteins still remain unknown. This review presents data on the structure and possible functions of these proteins, using the example of the vertebrate CTCF protein and several well- characterized C2H2 proteins in Drosophila and mammals.

The spatial organization of the genome is controlled by a special class of architectural proteins, in particular proteins containing ZAD domains that are able to form homo- and heterodimers. These domains are unique for arthropods and are present at the N-terminus of many proteins containing C2H2-type zinc fingers. Despite a growing interest in the characterization of architectural proteins, little is known about their structures (structures of their functional domains), which could shed light on the mechanisms of regulatory processes of the establishment and maintenance of the spatial organization of chromatin domains. Only two structures of ZAD domains are known. Here we report the crystallization and preliminary X-ray diffraction analysis of the previously unknown ZAD domain of the Serendipity-d protein from Drosophila melanogaster.

Using Drosophila transgenic model systems, we have shown that the four binding sites for the architectural protein dCTCF themselves have no insulator activity. They cannot block enhancers and protect from Polycomb-dependent repression. The results suggest that in the known Drosophila insulators, the dCTCF protein functions in cooperation with other architectural proteins.

The Su(Hw) protein was first identified as a DNA-binding component of an insulator complex in Drosophila. Insulators are regulatory elements that can block the enhancer-promoter communication and exhibit boundary activity. Some insulator complexes contribute to the higher-order organization of chromatin in topologically associated domains that are fundamental elements of the eukaryotic genomic structure. The Su(Hw)-dependent protein complex is a unique model for studying the insulator, since its basic structural components affecting its activity are already known. However, the mechanisms involving this complex in various regulatory processes and the precise interaction between the components of the Su(Hw) insulators remain poorly understood. Our recent studies reveal the fine mechanism of formation and function of the Su(Hw) insulator. Our results provide, for the first time, an example of a high complexity of interactions between the insulator proteins that are required to form the (Su(Hw)/Mod(mdg4)-67.2/CP190) complex. All interactions between the proteins are to a greater or lesser extent redundant, which increases the reliability of the complex formation. We conclude that both association with CP190 and Mod(mdg4)-67.2 partners and the proper organization of the DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators. In this review, we demonstrate the role of multiple interactions between the major components of the Su(Hw) insulator complex (Su(Hw)/Mod(mdg4)-67.2/CP190) in its activity. It was shown that Su(Hw) may regulate the enhancer–promoter communication via the newly described insulator neutralization mechanism. Moreover, Su(Hw) participates in direct regulation of activity of vicinity promoters. Finally, we demonstrate the mechanism of organization of “insulator bodies” and suggest a model describing their role in proper binding of the Su(Hw) complex to chromatin.

Expression of the reporter gene in transgenic animals depends on the surrounding chromatin environment. Recent genome-wide studies have shown that, in mammals, the entire genome is transcribed. Transcription through a transgene often has a negative effect on the expression of a reporter gene. Here, we compared the ability of well-studied chicken chromatin insulator HS4 and bidirectional transcription terminators from the human genome to support high-level expression of the firefly luciferase gene (Fluc) under control of the previously characterized goat β-casein gene promoter. The insertion of HS4 or either of the two transcription terminators upstream of the promoter resulted in tenfold enhancement of Fluc expression in the mammary glands of transgenic mice. These results suggest that transcriptional terminators, similar to the HS4 insulator, can be used to improve the reporter gene expression in transgenic animals.

In D. melanogaster, the nuclear matrix protein EAST functionally interacts with the Su(Hw)-dependent insulator complex. The y2 allele, in which the MDG4 retrotransposon is inserted between enhancers and the gene promoter, was previously described. The Su(Hw) insulator sequence is present in the regulatory region of MDG4. An increase in the EAST protein concentration or inactivation of the Mod(mdg4)-67.2 protein, which is part of the Su(Hw)-dependent complex, results in tissue-specific repression of the yellow gene in the y2 allele. In the present work, the degree of EAST-dependent and Su(Hw)-dependent repression in different regions of the genome was analyzed using transposition of transgenes containing long terminal repeat (LTR) sequences of the MDG4 retrotransposon and the Su(Hw) insulator. As a result of genetic experiments, it was shown for the first time that EAST-dependent repression of the yellow gene is observed in the same transgenic lines of D. melanogaster as Su(Hw)-dependent repression. The obtained data confirm our earlier suggestion that the regulatory complex, which is formed on the LTR sequence and functionally interacts with the EAST protein, transfers the transgene to the nucleus regions with a high concentration of factors that repress transcription.

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. Electronic supplementary material The online version of this article (10.1186/s13072-018-0249-2) contains supplementary material, which is available to authorized users.

Over the past decade, there has been an active study of the interactions between the population of transposable elements (TEs) and the rest of the genome. Many regulatory sequences of TEs have been used for different genes regulation and genome organization. These regulatory sequences can be transcription factor binding sites, enhancers, and insulators. TEs may contain promoters that can be domesticated during substitution of original gene promoters or during de novo formation of long noncoding RNAs. In addition, there are many examples of domestication of TE-encoded proteins, for example, transposases, proteases, and Gag proteins. This review highlights the role of TEs in the evolution of gene regulatory networks and the principles determining it.

A hallmark of aging is loss of differentiated cell identity. AgedDrosophilamidgut 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 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 that underly the specific distance interactions between enhancers and promoters remain unknown. This review describes the properties and activities of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.

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 an element that has PRE activity.

The architectural protein Pita is critical forDrosophilaembryogenesis and predominantly binds to gene promoters and insulators. In particular, Pita is involved in the organization of boundaries between regulatory domains that controlled the expression of threehoxgenes in the Bithorax complex (BX-C). The best-characterized partner for Pita is the BTB/POZ-domain containing protein CP190. Usingin vitropull-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 thewild-typeand mutant variants lacking CP190-interacting regions. The expression of the mutant protein completely complemented the nullpitamutation. ChIP-seq experiments withwild-typeand mutant embryos showed that the deletion of the CP190-interacting regions did not significantly affect the binding of the mutant Pita protein to most chromatin sites. However, the mutant Pita protein does not support the ability of multimerized Pita sites to prevent cross-talk between theiab-6andiab-7regulatory domains that activate the expression ofAbdominal-B(Abd-B), one of the genes in the BX-C. The recruitment of a chimeric protein consisting of the DNA-binding domain of GAL4 and CP190-interacting region of the Pita to the GAL4 binding sites on the polytene chromosomes of larvae induces the formation of a new interband, which is a consequence of the formation of open chromatin in this region. These results suggested that the interaction with CP190 is required for the primary Pita activities, but other architectural proteins may also recruit CP190 in flies expressing only the mutant Pita protein.Author SummaryPita 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. The CP190 protein is recruited to chromatin through interaction with the architectural proteins. Here we mapped two regions in Pita that are required for interaction with the CP190 protein. 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. At the same time, interaction with CP190 is not required for thein vivofunction 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. These results support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

SummaryA hallmark of aging is the inability of differentiated cells to maintain their identity. In the agedDrosophilamidgut differentiated enterocytes (ECs) lose their identity, and the integrity of the midgut tissue and its homeostasis are impaired. To discover regulators of EC identity relevant to aging we performed an RNAi screen targeting 453 ubiquitin-related genes in fully differentiated ECs. Seventeen genes were identified, including the de-ubiquitinase Non-stop (Not/dUSP22; CG4166). Acute loss of Non-stop in young ECs phenotypically resembled aged ECs. Lineage tracing experiments established that Non-stop-deficient young ECs as well as wild-type aged ECs are no longer differentiated. Aging or acute loss of Non-stop also resulted in progenitor cell hyperproliferation and mis-differentiation, loss of gut integrity, and reduced organismal survival. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) that contains E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Transcriptionally, Non-stop ensured chromatin accessibility at EC genes, maintained an EC-specific gene expression signature, and silenced non-EC-relevant transcriptional programs. Within the NIC, Non-stop was required for stabilizing of NIC subunits. Upon aging, the levels of Non-stop and NIC subunits declined, and the large-scale organization of the nucleus was distorted. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguarded the protein level of NIC subunits, restored the large-scale organization of the differentiated nucleus, and suppressed aging phenotypes and tissue integrity. Thus, the isopeptidase Non-stop, and NIC, supervise EC identity and protects from premature aging.

It is shown that mutations in two GC-rich sequences (GC-boxes) from the promoter region of the yellow gene during enhancer-dependent transcription activation do not affect the basal level of the yellow gene transcription but destabilize the interaction between the enhancers and the promoter.

Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3′ variable exons generated from alternative promoters. To map the regions required for trans-splicing, we have developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. We have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5′ splice site in the last common exon with the 3′ splice site in one of the alternative exons.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.

The EAST protein is a component of nuclear matrix. In the interphase cells, EAST is localized in the extrachromosomal nuclear domain. Increased EAST concentration leads to a change in spatial chromatin structure. However, previously, it was not known whether this protein played any role in the regulation of transcription. This study examines the mechanism of the EAST protein effect on the yellow gene transcription. It was demonstrated that the EAST-dependent repression observed in a model system of the yellow gene could occur not only in the presence of the Su(Hw) insulator sequence but also in the presence of the long terminal repeat sequence of the gypsy retrotransposon. In the LTR, a 92-bp sequence involved in the mechanism of EAST-dependent repression was detected. These results suggest that the found DNA motif is the place of the assembly of a protein complex, which functionally interacts with the EAST protein. This complex either independently suppresses the yellow gene expression in bristles or moves the transgene in the nuclear region with a high concentration of transcription repression factors.

The emergence of whole-genome assays has initiated numerous genome-wide studies of transcription factor localizations at genomic regulatory elements (enhancers, promoters, silencers, and insulators), as well as facilitated the uncovering of some of the key principles of chromosomal organization. However, the proteins involved in the formation and maintenance of the chromosomal architecture and the organization of regulatory domains remain insufficiently studied. This review attempts to collate the available data on the abundant but still poorly understood family of proteins with clusters of the C2H2 zinc finger domains. One of the best known proteins of this family is a well conserved protein known as CTCF, which plays a key role in the establishment of the chromosomal architecture in vertebrates. The distinctive features of C2H2 zinc finger proteins include strong and specific binding to a long and unique DNA recognition target sequence and rapid expansion within various animal taxa during evolution. The reviewed data support a proposed model according to which many of the C2H2 proteins have functions that are similar to those of the CTCF in the organization of the chromatin architecture.

One of the features of transcriptional regulation in higher eukaryotes is the ability of enhancers to activate gene transcription, being at a distance, sometimes reaching several hundred kilobases away from the promoter. For several genes, it was demonstrated that cis-regulatory DNA sequences located near the core promoter sequences play an important role in the enhancer–promoter communication. In this study, we first showed that the sequence from–890 to–1620 bp relative to the yellow transcription start site, called a communicator, is required for the long-distance activation of the yellow promoter by the body and wing enhancers, as well as for their bypass of the Su(Hw)-dependent insulation. The communicator is a functional partner of a previously described regulatory element located between–69 and–100 bp in the upstream promoter region of the yellow gene. The results of this study demonstrate that specific regulatory elements in the promoter and enhancer regions may be involved in providing long-distance interactions between them.

Earlier, we showed in a model system of the yellow gene of D. melanogaster that an increase in the EAST protein concentration leads to repression in bristles, the mechanism of which remained obscure. In this study, an inverted repeat was localized by genetic methods in the long terminal repeat (LTR) sequence of the MDG4 retrotransposon. This repeat includes the binding site for the Mad protein-the key component of the of TGF-β/BMP signaling cascade. The results of this work suggest that the Mad protein recruits to chromatin a regulatory complex that functionally interacts with the EAST protein. This complex either itself suppresses the yellow gene expression in bristles or moves the transgene to the nuclear regions with a high concentration of transcription repression factors.

Combinatorial expression of the genes in multicellular organisms leads to the development of different cell types. The important epigenetic regulators of higher eukaryotes are the Polycomb group (PcG) and Trithorax group (TrxG) proteins. These factors control the transcription of a large number of genes involved in various cellular processes. Dysregulation of PcG and TrxG systems leads to developmental abnormalities and cancer. This review focuses on the main characteristics and properties of the Drosophila PRE elements. Furthermore, we summarize the information on the protein components of the PcG and TrxG groups and their functional activities and discuss the main aspects of competition between the proteins of these classes as well as their possible mechanisms of action.

The experiments in a yeast two-hybrid system, on co-immunoprecipitation of proteins from the D. melanogaster embryonic cell lysates, and on cell immunostaining have shown for the first time that the product of mod(mdg4) locus, Mod (mdg4)-58.0 (isoform C), directly interacts with Kermit protein (dGIPC). Kermit protein plays an important role in locomotor activity, polarization of the cells within the plane of the epithelium at the early stages of development, transfer of intercellular signals, and determining the longevity of Drosophila. These data expand the understanding of the role of Mod(mdg4)-58.0 protein in the eukaryotic cell activity.

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic animals.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

A large class of arthropod transcription factors is formed by proteins with a characteristic N-terminal Zinc-finger-Associated Domain (ZAD) which contains four cysteine residues that coordinate a zinc ion. The number of putative proteins with ZAD in the Drosophila genome exceeds 90, and the degree of sequence similarity between these domains can be as low as 23%. Efficient binding of ZADs from the proteins Grau, ZIPIC, and Zw5 to the translation elongation factor EF1α1 in nuclear and cytoplasmic extracts has been demonstrated. EF1α1 is probably involved in the regulation of the activity of ZAD-containing transcription factors.

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.

Proteins Z4/putzig and Chriz/Chromator are involved in the chromatin organization on the promoters of the majority of Drosophila genes. It was shown that the Chriz protein region from aa 273 to 503 is required for the interaction with the Z4 protein. Deletion of this sequence leads to derepression of a number of STAT-dependent genes and development of melanotic tumors in flies. The results of this study suggest that the Chriz protein promotes the recruitment of the Z4 protein to chromatin.

CTCF is the main architectural protein found in most of the examined bilaterian organisms. The cluster of the C2H2 zinc-finger domains involved in recognition of long DNA-binding motif is only part of the protein that is evolutionarily conserved, while the N-terminal domain (NTD) has different sequences. Here, we performed biophysical characterization of CTCF NTDs from various species representing all major phylogenetic clades of higher metazoans. With the exception of Drosophilides, the N-terminal domains of CTCFs show an unstructured organization and absence of folded regions in vitro. In contrast, NTDs of Drosophila melanogaster and virilis CTCFs contain unstructured folded regions that form tetramers and dimers correspondingly in vitro. Unexpectedly, most NTDs are able to self-associate in the yeast two-hybrid and co-immunoprecipitation assays. These results suggest that NTDs of CTCFs might contribute to the organization of CTCF-mediated long-distance interactions and chromosomal architecture.

The interaction of the GAF protein with the promoters of neuron-specific genes during activation and repression of transcription was studied. We showed that, while the Su(Hw) protein remains stably associated with the promoters of these genes at different transcriptional state, the GAF protein level is significantly higher when transcription is activated.

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.

TRF2 protein (TBP-related factor 2) can substitute for TBP forming alternative transcription initiation complexes on TATA-less promoters, including the promoters of histone H1 and piRNA clusters required for transposon repression. The Drosophilatrf2 gene codes for two isoforms: a “short” and a “long” one, in which the same short TRF2 sequence is preceded by a long N-terminal domain. Here, we demonstrated that the long TFR2 isoform has a greater functional activity than the short isoform by expressing each of them at a reduced rate under the endogenous promoters. Expression of the long isoform alone affects neither the flies’ viability nor the sex ratio. Expression of the short isoform alone leads to the phenotype described for the trf2 gene insufficiency and derepression of transposable elements, that is, decreased viability, disturbance of homologous chromosome pairing and segregation, and apparent female-biased sex ratio.

The binding of the Drosophila male-specific lethal dosage compensation complex (DCC) exclusively to male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment in vitro. Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc finger domain of CLAMP. Here, we found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in Drosophila males.

To study the mechanisms of transcriptional regulation, a convenient experimental approach is to use the artificial chimeric constructs carrying the regulatory elements of interest. In the present work, we describe the creation and characterization of a novel genetic construct that makes it possible to study the transcriptional regulation of the early-late gene of the ecdysone cascade. Using the data of genome-wide experiments, we have isolated the main regulatory region of the hr4 gene, which was successfully used to create a chimeric reporter construct expressing a fluorescent protein upon the treatment with the ecdysone hormone. This reporter system can be used to study the mechanisms of the ecdysone response, both in cell culture and in tissues, at various stages of the Drosophila development.

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.