Transient loss of Polycomb components induces an epigenetic cancer fate.

Although cancer initiation and progression are generally associated with the accumulation of somatic mutations1,2, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility3-6, suggesting that genetic mechanisms might not be the only drivers of malignant transformation7. However, whether purely non-genetic mechanisms are sufficient to initiate tumorigenesis irrespective of mutations has been unknown. Here, we show that a transient perturbation of transcriptional silencing mediated by Polycomb group proteins is sufficient to induce an irreversible switch to a cancer cell fate in Drosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including members of the JAK-STAT signalling pathway and zfh1, the fly homologue of the ZEB1 oncogene, whose aberrant activation is required for Polycomb perturbation-induced tumorigenesis. These data show that a reversible depletion of Polycomb proteins can induce cancer in the absence of driver mutations, suggesting that tumours can emerge through epigenetic dysregulation leading to inheritance of altered cell fates.

Recruitment to Chromatin of (GA)n-Associated Factors GAF and Psq in the Transgenic Model System Depends on the Presence of Architectural Protein Binding Sites.

Polycomb group (PcG) repressors and Trithorax group (TrxG) activators of transcription are essential for the proper development and maintenance of gene expression profiles in multicellular organisms. In Drosophila, PcG/TrxG proteins interact with DNA elements called PRE (Polycomb response elements). We have previously shown that the repressive activity of inactive PRE in transgenes can be induced by architectural protein-binding sites. It was shown that the induction of repression is associated with the recruitment of PcG/TrxG proteins, including the DNA-binding factors Pho and Combgap. In the present study, we tested the association of the two other PRE DNA-binding factors, GAF and Psq, with bxdPRE in the presence and absence of sites for architectural proteins. As a result, it was shown that both factors can be efficiently recruited to the bxdPRE only in the presence of adjacent binding sites for architectural proteins Su(Hw), CTCF, or Pita.

Sfmbt Co-purifies with Hangover and SWI/SNF-Remodelers in Drosophila melanogaster.

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.

A Novel PRE-Element from Drosophila virilis Genome as a Useful Model Silencer.

We tested the activity of the PRE element from the Drosophila virilis genome, which is the homologue of the known Drosophila melanogasterbxdPRE element from the regulatory region of the Ubx gene. It is easy to select unique primers to this element that do not occur in the Drosophila melanogaster genome. We showed that the studied PRE element causes a strong repression of the white marker gene upon insertion into the Drosophila melanogaster genome and interacts with the PcG proteins of the PRC1 and PhoRC complexes.

Effect of Transcription on the white Gene Enhancer Integrated into the Intron.

In this paper, we demonstrate that passing-through transcription suppresses the activity of the white gene enhancer integrated into the intron. At the same time, the SV40 transcription terminators flanking the transgene can completely remove the inhibitory effect of transcription.

Activation of Su(Hw)-Controlled Genes Is Associated with Increase in GAF Binding.

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.

[Control of the gene activity by polycomb and trithorax group proteins in Drosophila].

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.

PRE/TRE elements act as transcription activators in Drosophila S2 Cells.

The Drosophila PRE/TRE elements are DNA targets for Polycomb and Trithorax group proteins, which control repression and activation of gene transcription, respectively. In this study, we show that, in transiently transfected Drosophila S2 cells, bxdPRE activates transcription driven from different promoters. Using CG32795 gene promoter, we demonstrate that other PRE/TRE elements-Fab7, en, eve, and CycB-also act as transcription activators in this model system.

The bxdPRE/TRE element terminates passing through transcription.

The regulation of PRE/TRE activity is required for appropriate tissue and stage-specific gene expression. However, the molecular principles of PRE/TRE activity control remain unknown. Here we show that PRE/TRE element from Ubx regulatory region efficiently terminates passing through transcription.

[The Effect of Transcription on Enhancer Activity in Drosophila melanogaster].

In higher eukaryotes, the level of gene transcription is under the control of DNA regulatory elements, such as promoter, from which transcription is initiated with the participation of RNA polymerase II and general transcription factors, as well as the enhancer, which increase the rate of transcription with the involvement of activator proteins and cofactors. It was demonstrated that enhancers are often located in the transcribed regions of the genome. We showed earlier that transcription negatively affected the activity of enhancers in Drosophila in model transgenic systems. In this study, we tested the effect of the distance between the leading promoter, enhancer, and target promoter on the inhibitory effect of transcriptions of different strengths. It was demonstrated that the negative effect of transcription remained, but weakened with increased distance between the leading promoter and enhancer and with decreased distance between the enhancer and target promoter. Thus, transcription can modulate the activity of enhancers by controlling its maximum level.

[The Role of Su(Hw) Protein in Transcription Regulation in Drosophila melanogaster].

Insulators are regulatory DNA elements that modulate the effect of enhancers and silencers on gene transcription. However, the role of the insulator complex proteins in the expression of specific genes remains scarcely studied. In the present study, we examined the role of the Su(Hw)-dependent insulator complex in transcription of the rap, CG32810, and RpS15Aa genes. It was demonstrated that the interaction of Su(Hw) and Mod(mdg4)-67.2 insulator proteins with the terminator regions of the selected genes was dependent on the Su(Hw) factor. At the same time, the CP190 protein also interacts with the promoter and terminator regions in the absence of Su(Hw). In addition, the Su(Hw) protein does not affect the level of transcription and silencing efficiency of the gene models. A possible explanation for these results is the existence of another transcription factor, a protein that is able to recruit CP190 and functionally compensate for the lack of the Su(Hw) protein.

SV40 transcription terminators stabilize the activity of regulatory elements in Drosophila melanogaster.

To ensure stable operation of transgenic systems and maintain reporter gene expression at a certain level, it is necessary to search for the conditions that protect the activity of regulatory elements. In this study, we have shown that the SV40 transcription terminators flanking the transgene protect enhancers and silencers of Drosophila and ensure their efficient functioning.

[Interactions of 1A2 insulator with promoter of hsp 70 gene in Drosophila melanogaster].

Insulators are regulatory DNA elements that participate in the modulation of the interactions between enhancers and promoters. Depending on the situation, insulators can either stabilize or destroy the contacts between enhancers and promoters. A possible explanation for the activity of insulators is their ability to directly interact with gene promoters. In the present study, it was demonstrated that, in model systems, a 1A2 insulator could interact with the core sequence of an hsp70 promoter. In this case, the insulator protein CP190 is found on the hsp70 promoter, which depends on the presence of an insulator in the transgene. The data obtained are consistent with the model, which implies that direct contacts between insulators and promoters make a considerable contribution to the modulation of the interactions between insulators and promoters.

[Distant interactions between enhancers and promoters in Drosophila melanogaster are mediated by transgene-flanking Su(Hw) insulators].

Insulators are DNA elements modulating gene activation by enhancers. Interaction between insulators can result in either isolation, or activation of promoter by the enhancer. In the present study, it is demonstrated that in transgenic lines, the yellow enhancers are unable to activate promoter of the tagged gene through the mini-white gene. The mini-white promoter, which functions as an insulator, can block the enhancer activity. In case that the genes and regulatory elements in the construct are flanked by Su(Hw) insulators from retroposon MDG4, interaction between enhancers and the yellow promoter is restored. The data obtained are congruent with the model suggesting that promoters can function as insulators, and that interaction between the insulators facilitates the interaction between enhancers and promoters within transcriptional domain.

[Effects of functional interactions between nonhomologous insulators Wari and Su(Hw)].

Insulators are regulatory DNA elements restricting gene activation by enhancers. Interactions between insulators can lead to both insulation and activation of promoters by enhancers. In this work, we analyzed the effects of interaction of two Drosophila insulators, Wari and Su(Hw). The functional interaction between these insulators was found to enhance the activity of the Su(Hw) insulator only, but not of the Wari insulator. This suggests that the formation of a chromatin loop between interacting insulators is not a key factor for enhancement of insulation, which is in disagreement with the main idea of structural models. In addition, the effect of interaction between Wari and Su(Hw) depends on a distance between them and on the position in the system relative to other regulatory elements.

Polycomb and Trithorax Group Proteins: The Long Road from Mutations in Drosophila to Use in Medicine.

Polycomb group (PcG) and Trithorax group (TrxG) proteins are evolutionarily conserved factors responsible for the repression and activation of the transcription of multiple genes in Drosophila and mammals. Disruption of the PcG/TrxG expression is associated with many pathological conditions, including cancer, which makes them suitable targets for diagnosis and therapy in medicine. In this review, we focus on the major PcG and TrxG complexes, the mechanisms of PcG/TrxG action, and their recruitment to chromatin. We discuss the alterations associated with the dysfunction of a number of factors of these groups in oncology and the current strategies used to develop drugs based on small-molecule inhibitors.

[Insulators of higher eukaryotes: properties, mechanisms of action, and role in transcriptional regulation].

One of the dogmas of transcriptional regulation in higher eukaryotes suggests the existence of transcriptional domains with no promoter-enhancer interactions between them. Specific regulatory elements, known as insulators, block the interaction between an enhancer and a promoter. Insulators are believed to act as transcription domain boundaries. The review considers general properties of well-known insulators identified in Drosophila and vertebrates. The mechanism of action of insulators and their role in the regulation of gene expression are discussed on the basis of available information.

Role of Transcriptional Read-Through in PRE Activity in Drosophila melanogaster.

Maintenance of the individual patterns of gene expression in different cell types is required for the differentiation and development of multicellular organisms. Expression of many genes is controlled by Polycomb (PcG) and Trithorax (TrxG) group proteins that act through association with chromatin. PcG/TrxG are assembled on the DNA sequences termed PREs (Polycomb Response Elements), the activity of which can be modulated and switched from repression to activation. In this study, we analyzed the influence of transcriptional read-through on PRE activity switch mediated by the yeast activator GAL4. We show that a transcription terminator inserted between the promoter and PRE doesn't prevent switching of PRE activity from repression to activation. We demonstrate that, independently of PRE orientation, high levels of transcription fail to dislodge PcG/TrxG proteins from PRE in the absence of a terminator. Thus, transcription is not the main factor required for PRE activity switch.