[The Drosophila CG9890 Protein is Involved in the Regulation of Ecdysone-Dependent Transcription].

Previously we showed that the CG9890 protein, which has zinc finger domains, interacts with ENY2-containing complexes and is localized mainly on the promoters of active genes. The CG9890 protein is involved in the regulation of the expression of some of the genes on the promoters of which it is located, and among these genes there are genes for the ecdysone cascade. In this work, the role of the CG9890 protein in the regulation of ecdysone-dependent inducible transcription was studied. For this, 12 ecdysone-dependent genes on the promoters of which the CG9890 protein is localized were identified. Their activation was studied after the addition of 20-hydroxyecdysone to cells, both in normal conditions and after RNA interference of CG9890. The expression of ecdysone-dependent genes is significantly increased in response to the treatment of cells with ecdysone, in contrast to the control genes. Moreover, in the cell line after RNA interference CG9890, the transcription of 8 out of 12 genes was significantly higher than in the control line. Thus, the CG9890 protein is involved in the regulation of transcription of ecdysone-dependent genes, and, in most cases, acts as a repressor.

MLE Helicase Is a New Participant in the Transcription Regulation of the ftz-f1 Gene Encoding Nuclear Receptor in Higher Eukaryotes.

MLE helicase is an evolutionarily conserved eukaryotic protein involved in a wide range of processes in the regulation of gene expression. Previously, we studied the properties of MLE on the Drosophila melanogaster model. In the present work we continue studying the functions of MLE and show that MLE interacts with the components of the SWI/SNF chromatin remodelling complex. To clarify the work of MLE, the profile of MLE binding to the regulatory elements of the SWI/SNF-dependent ftz-f1 gene was analyzed. The effect of MLE on the expression of this gene, the transcription of which occurs by the RNA polymerase II pausing mechanism, was investigated. The data obtained indicate the important role of MLE in ensuring timely activation and high level of expression of the ftz-f1 gene in vivo.

Drosophila Zinc Finger Protein CG9890 Is Colocalized with Chromatin Modifying and Remodeling Complexes on Gene Promoters and Involved in Transcription Regulation.

In this work, we conducted a genome-wide study of the zinc finger protein CG9890 and showed that it is localized mostly on the promoters of active genes. The CG9890 binding sites are low-nucleosome-density regions and are colocalized with the chromatin modifying and remodeling complexes SAGA and dSWI/SNF, as well as with the ORC replication complex. The CG9890 protein was shown to be involved in the regulation of the expression of some genes on the promoters of which it is located, with the ecdysone cascade genes accounting for a significant percentage of these genes. Thus, the CG9890 protein is a new member of the transcriptional network which is localized on active promoters, interacts with the main transcription and replication complexes, and is involved in the regulation of both basal and inducible transcription.

The Development of Reporter System for the Investigation of Molecular Mechanisms of Ecdysone Response.

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.

Multifunctional ENY2 Protein Interacts with RNA Helicase MLE.

ENY2 protein of Drosophila melanogaster was previously discovered and characterized in our laboratory [1, 2]. It was found that ENY2 is a subunit of several multiprotein complexes and connects various stages of gene expression [3-5]. This work is devoted to studying the interaction of ENY2 with RNA helicase MLE. This interaction was confirmed by independent methods. Data indicating that this interaction is conserved in evolution and is important for the functioning of MLE in both sexes were obtained.

Insulator Protein Su(Hw) Is Indispensable for Amplification of Part of Drosophila Amplicons in Follicular Cells (DAFCs) during Early Oogenesis.

We found that the Su(Hw) insulator protein binds to all known Drosophila amplicons in follicular cells (DAFCs). Su(Hw) binding is required for ORC replication complex positioning on part of DAFCs during early oogenesis. Our data allow us to expand the current view on the role of the Su(Hw) protein in Drosophila oogenesis as a transcription repressor. We assume that Su(Hw) is involved in the coordination of transcription and replication processes during oogenesis in Drosophila.

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila.

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

Studying a novel ecdysone-dependent enhancer.

Earlier [4], we developed a two-stage scheme of the activation of expression of the ecdysone cascade gene ftz-f1 in Drosophila melanogaster S2 cells. In the first intron of the ftz-f1 gene, we found a binding site for the transcription activator DHR3. In this work, we studied the properties of this genomic element: the change in the histone modification level at different stages of transcription activation were analyzed, and data indicating the interaction of the intron element with the promoter were obtained. Taken together, the results of this study indicate that the studied genomic element exhibits the properties of an enhancer and functions at the stage of active gene transcription.

Nuclear receptors EcR, Usp, E75, DHR3, and ERR regulate transcription of ecdysone cascade genes.

We found that an increase in the expression level of E75, DHR3, and ERR increases the degree of activation of dhr3 and hr4 genes in Drosophila S2 cells. We also detected a repressing effect of these nuclear receptors on the basal transcription level of these genes. This is the first study to show the ability of nuclear receptors E75, DHR3, and ERR to function as activators or repressors depending on external conditions. We also confirmed the existence of the interaction of all studied nuclear receptors with the promoters of dhr3 and hr4 genes of the ecdysone cascade in vivo.

[Coactivator complexes participate in different stages of the Drosophila melanogaster hsp70 gene transcription].

The objective of this study was to identify transcriptional coactivators participating in transcription elongation of the hsp70 gene induced by heat shock. We found that all investigated coactivator complexes participate in transcription of this gene, as significant level of them were present at the gene promoter in its active state. For most of the coactivators (except for p300/CBP, Set2, and Mediator complex), we also observed a considerable increase of their binding level at the coding region of the gene after activation of its transcription by heat shock. We assume that coactivators CHD1, ISWI, Brm, Kismet-L, INO80, Mi-2, Gcn5, Lid/KDM5, Set1, DART1, DART4, SSRP1, PAF1, and Fs(1)h/Brd4 bind to the promoter of the active hsp70 gene and migrate to its coding region together with elongating RNA polymerase II. It can be suggested that some of these coactivators play an important role in stimulating the transition of the RNA polymerase II complex from transcription initiation to elongation stage.

[SWI/SNF Protein Complexes Participate in the Initiation and Elongation Stages of Drosophila hsp70 Gene Transcription].

The participation of the SWI/SNF chromatin remodeling complex in the stimulation of the RNA polymerase II binding to gene promotors was demonstrated in all model eukaryotic organisms. It was shown eight years ago that the SWI/SNF complex influence on transcription is not limited to its role in initiation but also includes participation in elongation and alternative splicing. In the current work, we describe the subunit composition of the SWI/SNF complexes participating in initiation, preparing for the elongation and elongation of hsp70 gene transcription in Drosophila melanogaster. The data reveal the high mobility of the SWI/SNF complex composition during the hsp 70 gene transcription process. We suggest a model describing the process of sequential SWI/SNF complex formation during heat-shock induced transcription of the hsp 70 gene.

[Ability of Su(Hw) to create a platform for ORC binding does not depend on the type of surrounding chromatin].

DNA replication begins from multiple sites distributed thoughout the genome and named replication origins. Despite the increasing amount of data on the properties of replication origins, it is still unknown what factors(s) is the primary determinant of ORC localization. Su(Hw) is a zinc-finger protein that is responsible for the activity of the best-studied Drosophila insulators. Here, we show that insulator protein Su(Hw) recruits histone acetyltransferase complex SAGA and chromatin remodeler dSWI/SNF to Su(Hw)-dependent insulators and creates platform for ORC binding. We have found that Su(Hw) is necessary for chromatin remodeling and ORC recruitment regardless of type of surrounding chromatin. Thus, global chromatin state does not influence molecular mechanism underlying ORC positioning in the genome, rather DNA-binding proteins are key determinants that create proper chromatin structure for ORC binding. Su(Hw) is the first example of such a protein.

[SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation].

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome.

Production of human lactoferrin in animal milk.

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

[Multifunctional factor ENY2 integrates the various stages of gene expression].

The ENY2/Sus1 is a multifunctional transcription factor which couples transcription with mRNA export. It is the component of the SAGA/TFTC and TREX-2/AMEX protein complexes. Recently, we described the interaction of ENY2 with one more protein complex, THO. Moreover, our data indicate that ENY2 associates with other nuclear and cytoplasmic factors. Thus, being the component of a number of protein complexes, ENY2 plays the role of an adaptor molecule, involved into the integration of different cellular processes, specifically, subsequent stages of gene expression.

[Conservative E(y)2/Sus1 protein is the member of SAGA complex and new nuclear pore-associated complex in Drosophila].

The SGA/TFTC complex plays an important role in the regulation of transcription. We have examined the significance of the gene positioning in the nucleus for its transcription and subsequent export of nascent mRNA. It was demonstrated that E(y)2 protein was a subunit of the SAGA/TFTC histone acetyl transferase complex in Drosophila and that E(y)2 concentrated at the nuclear periphery. An interaction between E(y)2 and the nuclear pore complex (NPC) was demonstrated, as well as that SAGA/TFTC also contacted the NPC at nuclear periphery. In addition, it was shown that E(y)2 formed complex with the Xmas-2 protein (X-linked male sterile 2) both in normal conditions and after heat shock. Importantly, the E(y)2 and Xmas-2 knockdown decreased the contact between the heat-shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation, and interfered with the transcription. Thus, E(y)2 and Xmas-2 together with SAGA/TFTC functioned in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

[Conservative E(y)2/Sus1 protein interacts with the Su(Hw)-dependent insulators in Drosophila].

Insulators are regulatory elements having two properties. First, they are able to disturb the interaction between promoters and enhancers/silencers. Second, they are able to block distribution of the heterochromatin. The best-studied are the Su(Hw)-dependent insulators of Drosophila melanogaster, activity of which is determined by the Su(Hw) protein. In this study it was demonstrated that novel, evolutionary conservative transcription factor E(y)2/Sus1 interacted with the Su(Hw) zinc-finger domain and was present in the protein complex, associated with the Su(Hw)-dependent insulators.