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.

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.

[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.

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.

[The role of ATP-dependent chromatin remodeling complexes in regulation of genetic processes].

Compaction of the genomic DNA into the chromatin structure reduces the accessibility of DNAbinding protein sites and complicates the realization of replication and transcription. In the cell, the negative effects of DNA condensation into chromatin are overcome by recruiting the complexes that change the chromatin structure and are involved in the regulation of transcription and replication. The chromatin remodeling process includes the alteration of nucleosome position and chromatin density and changes in the histone composition of the nucleosomes. ATP-dependent chromatin remodeling is performed by enzymes­chromatin remodeling complexes. The united activity of these enzymes forms the dynamic properties of chromatin during different nuclear processes such as transcription, replication, DNA repair, homological recombination, and chromatin assembly. In this review, we summarize the currently available data on the structure of chromatin remodeling complexes of different families, the pathways of their recruitment to certain chromatin sites, and their functional activity.

THE COMPOSITION OF SWI/SNF CHROMATIN REMODELING COMPLEX IS STABLE DURING GENE TRANSCRIPTION.

SWI/SNF protein complex is a unique transcription activator, which takes part in almost all stages of transcription regulation including preinitiation complex formation, transition of RNA polymerase II to transcription elongation, enhancer and insulator action. Recently it was found that particular subunits of SWI/SNF become involved in gene transcription on distinct stages. In the current work we investigate subunit composition of complexes formed with Brahma protein (ATPase of SWI/SNF complex) on the sages of initiation and elongation of gene transcription. As the result we have demonstrated the composition stability of SWI/SNF complex during gene transcription. We suppose that differences in recruitment of particular SWI/SNF subunits, that were distinguished earlier, are due to formation of complexes distinct from SWI/SNF and not including Brahma ATPase. Functioning of non-enzymatic subunits independently of the main complex had not been shown earlier. This finding extend our knowledge concerning functional properties of the proteins, involved in chromatin remodeling process.

[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.

Chromatin Modifiers in Transcriptional Regulation: New Findings and Prospects.

Histone-modifying and remodeling complexes are considered the main coregulators that affect transcription by changing the chromatin structure. Coordinated action by these complexes is essential for the transcriptional activation of any eukaryotic gene. In this review, we discuss current trends in the study of histone modifiers and chromatin remodelers, including the functional impact of transcriptional proteins/ complexes i.e., "pioneers"; remodeling and modification of non-histone proteins by transcriptional complexes; the supplementary functions of the non-catalytic subunits of remodelers, and the participation of histone modifiers in the "pause" of RNA polymerase II. The review also includes a scheme illustrating the mechanisms of recruitment of the main classes of remodelers and chromatin modifiers to various sites in the genome and their functional activities.