Drosophila tethering elements mediate long-range enhancer-promoter interactions and connect the promoters of distant paralogous genes. Micro-C maps identified 645 such loops in the Drosophila larval brain, spanning distances of 25 to 250 kb. Here, we demonstrate that the MADF-containing Vostok protein acts as a looping factor. It binds to GCAACA motifs that are overrepresented in brain tethering elements. There is a loss of 47 (7%) of the loops in Vostok mutants, resulting in diminished expression of associated genes. Vostok is largely independent of another looping factor, GAGA-associated factor (GAF). Only 9 loops are disrupted in both Vostok and GAF mutants, raising the possibility of a combinatorial code for tether-tether interactions. This is supported by the reliance of two previously identified meta-loops spanning 6 Mb on both GAF and Vostok. We discuss the prospects of using different combinations of looping factors to engineer 3D associations in animal genomes.

BACKGROUND:Steroid hormones drive the transcription of developmental genes by activating distinct sets of enhancers across tissues and developmental contexts, but the mechanisms that target specific enhancers remain incompletely understood, even in model organisms. It has been proposed that selective binding of nuclear receptors occurs at regulatory sites primed by other classes of DNA-binding transcription factors. However, direct studies of cooperation between nuclear receptors and different transcription factors remain limited. A previous study suggested that the GATA family factor SRP/dGATAb primes regulatory sites in S2 Schneider cells for activation by 20-hydroxyecdysone (20E), the principal steroid hormone in Drosophila development. Yet the genome-wide impact of SRP/dGATAb depletion on transcriptional responses to 20E has not been examined. RESULTS:We investigated the role of SRP/dGATAb in the response of S2 Schneider cells to 20E using SRP/dGATAb depletion via RNA interference. Combined RNA-Seq and ChIP-Seq analyses identified primary targets of SRP/dGATAb that (i) are transcriptionally induced by 20E and (ii) contain binding sites for both EcR and SRP/dGATAb. SRP/dGATAb depletion altered the transcription of different 20E-induced genes in opposite ways. For one subset of 20E-activated genes whose expression decreased upon depletion, SRP/dGATAb regulated active regulatory sites marked by H3K27Ac and enriched with SRP/dGATAb motifs. In these loci, SRP/dGATAb depletion reduced EcR binding at co-bound sites, demonstrating a priming role for this GATA family protein. In contrast, for a second subset of 20E-activated but SRP/dGATAb-suppressed genes (i.e., genes whose expression increased upon SRP/dGATAb depletion), SRP/dGATAb and EcR co-bound sites exhibited undisturbed EcR binding. Notably, the overall level of H3K27 acetylation at these loci increased upon SRP/dGATAb depletion. CONCLUSIONS:Our data indicate that SRP/dGATAb positively regulates 20E-inducible transcription in S2 Schneider cells for some genes, functioning as a priming factor that facilitates EcR recruitment and chromatin acetylation. In contrast, for another subset of 20E-inducible genes, SRP/dGATAb exerts a negative regulatory effect, restraining activity of the regulatory sites.

Telomeres ensure genome stability and the levels of telomeric RNA reflect the integrity of telomeric chromatin. The highly conserved RNA-binding protein Ars2 (Arsenite-resistance protein 2) plays an essential role in the RNA nuclear metabolism and negatively regulates the expression of telomeric transcripts in human cells and in Drosophila. We found that germline knockdown of Drosophila Ars2 does not affect small RNA abundance but causes overexpression of telomeric repeats and transposable elements (TEs), accompanied by chromatin decompaction of these regions. The expression of a transgene containing the HeT-A telomeric retrotransposon was also affected by Ars2 knockdown. The mutation of the G-rich region, which is prone to the formation of G-quadruplex structures, reduces the HeT-A transgene’s sensitivity to Ars2 depletion. Intriguingly, Ars2-regulated non-telomeric TEs are also enriched by G-quadruplex structures, implying their role in the Ars2 target recognition. Ars2 also prevents the formation of R-loops at telomeres, which are most likely caused by the accumulation of unreleased transcripts. Surprisingly, Ars2 is required for the expression of R1 retrotransposons, which are integrated in rRNA genes and essential for their amplification. Our findings point to a new mechanism for control of expression of telomeric repeats and TEs in the germline involving Ars2.

Transcriptional activation by 20-hydroxyecdysone (20E) in Drosophila provides an excellent model for studying tissue-specific responses to steroids. An increase in the 20E concentration regulates the degradation of larval and the proliferation of adult tissues during metamorphosis. To study 20E-dependent transcription, we used the natural system for controlling the 20E concentration-the E23 membrane transporter-which exports 20E from the cell. We artificially expressed E23 in tissues to suppress the first wave of 20E-inducible transcription at metamorphosis. E23 expression revealed a plethora of 20E-dependent genes in salivary glands, while mildly affecting transcription in brain. We described the mechanisms controlling transcriptional activation by 20E in salivary glands. 20E depletion decreased the binding of Pol II and the TFIID subunit, TBP, to the promoters of primary targets, demonstrating the role of 20E in transcription initiation. At target loci, 20E depletion resulted in the malfunctioning of sites co-bound with EcR and CBP/Nejire and enriched for the H3K27Ac mark inherent to active enhancers. At these sites, the 20E concentration was found to control chromatin accessibility and acetylation. We suggest that the activity of these 'active' ecdysone-sensitive elements was responsible for the active status of 20E targets in the salivary glands of wandering larvae.

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.

Background Insulator-binding proteins (IBPs) play a critical role in genome architecture by forming and maintaining contact domains. While the involvement of several IBPs in organising chromatin architecture in Drosophila has been described, the specific contribution of the Suppressor of Hairy wings (Su(Hw)) insulator-binding protein to genome topology remains unclear.Results In this study, we provide evidence for the existence of long-range interactions between chromatin bound Su(Hw) and Combgap, which was first characterised as Polycomb response elements binding protein. Loss of Su(Hw) binding to chromatin results in the disappearance of Su(Hw)-Combgap long-range interactions and in a decrease in spatial self-interactions among a subset of Su(Hw)-bound genome sites. Our findings suggest that Su(Hw)-Combgap long-range interactions are associated with active chromatin rather than Polycomb-directed repression. Furthermore, we observe that the majority of transcription start sites that are down-regulated upon loss of Su(Hw) binding to chromatin are located within 2 kb of Combgap peaks and exhibit Su(Hw)-dependent changes in Combgap and transcriptional regulators' binding.Conclusions This study demonstrates that Su(Hw) insulator binding protein can form long-range interactions with Combgap, Polycomb response elements binding protein, and that these interactions are associated with active chromatin factors rather than with Polycomb dependent repression.

The establishment and stable inheritance of individual patterns of gene expression in different cell types are required for the development of multicellular organisms. The important epigenetic regulators are the Polycomb group (PcG) and Trithorax group (TrxG) proteins, which control the silenced and active states of genes, respectively. In Drosophila, the PcG/TrxG group proteins are recruited to the DNA regulatory sequences termed the Polycomb response elements (PREs). The PREs are composed of the binding sites for different DNA-binding proteins, the so-called PcG recruiters. Currently, the role of the PcG recruiters in the targeting of the PcG proteins to PREs is well documented. However, there are examples where the PcG recruiters are also implicated in the active transcription and in the TrxG function. In addition, there is increasing evidence that the genome-wide PcG recruiters interact with the chromatin outside of the PREs and overlap with the proteins of differing regulatory classes. Recent studies of the interactomes of the PcG recruiters significantly expanded our understanding that they have numerous interactors besides the PcG proteins and that their functions extend beyond the regulation of the PRE repressive activity. Here, we summarize current data about the functions of the PcG recruiters.

Polycomb and Trithorax group proteins (PcG and TrxG) are epigenetic factors responsible for the repression and activation of transcription, respectively. In Drosophila , PcG/TrxG proteins are recruited to specialized DNA elements called Polycomb response elements (PREs). Depending on the context, these elements may repress, activate, or be neutral with respect to the promoter of the target gene. Previously, in transgenic studies using PhiC31 site-specific integration system, we have demonstrated that sites for architectural proteins inserted next to PRE can induce the repressive activity of bxd PRE by stimulating the binding of PcG/TrxG factors to this element. However, this effect may depend on additional DNA elements present at the integration site after PhiC31-dependent transgene insertion. In the present study, using an alternative system of integration based on CRISPR/Cas9-catalyzed homology-directed repair, we have proved that the binding sites of the architectural protein Su(Hw) are indeed able to induce the repressive activity of bxd PRE and recruitment of PcG/TrxG proteins, regardless of the heterogeneous DNA-sequences present at the site of integration after PhiC31-dependent insertion of the transgenes.

Insulators are architectural elements implicated in the organization of higher-order chromatin structures and transcriptional regulation. However, it is still unknown how insulators contribute to Drosophila telomere maintenance. Although the Drosophila telomeric retrotransposons HeT-A and TART occupy a common genomic niche, they are regulated independently. TART elements are believed to provide reverse transcriptase activity, whereas HeT-A transcripts serve as a template for telomere elongation. Here, we report that insulator complexes associate with TART and contribute to its transcriptional regulation in the Drosophila germline. Chromatin immunoprecipitation revealed that the insulator complex containing BEAF32, Chriz, and DREF proteins occupy the TART promoter. BEAF32 depletion causes derepression and chromatin changes at TART in ovaries. Moreover, an expansion of TART copy number was observed in the genome of the BEAF32 mutant strain. BEAF32 localizes between the TART enhancer and promoter, suggesting that it blocks enhancer-promoter interactions. Our study found that TART repression is released in the germ cysts as a result of the normal reduction of BEAF32 expression at this developmental stage. We suggest that coordinated expression of telomeric repeats during development underlies telomere elongation control.

Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, "PcG recruiters," including Pho, Spps, Cg, and GAF. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here, we directly addressed the importance of Pho binding sites in 2 engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, 2 PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho. This supports the view that multiple mechanisms contribute to PcG recruitment in Drosophila.

The Polycomb group (PcG) proteins are fundamental epigenetic regulators that control the repressive state of target genes in multicellular organisms. One of the open questions is defining the mechanisms of PcG recruitment to chromatin. In Drosophila, the crucial role in PcG recruitment is thought to belong to DNA-binding proteins associated with Polycomb response elements (PREs). However, current data suggests that not all PRE-binding factors have been identified. Here, we report the identification of the transcription factor Crooked legs (Crol) as a novel PcG recruiter. Crol is a C2H2-type Zinc Finger protein that directly binds to poly(G)-rich DNA sequences. Mutation of Crol binding sites as well as crol CRISPR/Cas9 knockout diminish the repressive activity of PREs in transgenes. Like other PRE-DNA binding proteins, Crol co-localizes with PcG proteins inside and outside of H3K27me3 domains. Crol knockout impairs the recruitment of the PRC1 subunit Polyhomeotic and the PRE-binding protein Combgap at a subset of sites. The decreased binding of PcG proteins is accompanied by dysregulated transcription of target genes. Overall, our study identified Crol as a new important player in PcG recruitment and epigenetic regulation.

The identification of mechanisms that underlie the biology of individual tumors is aimed at the development of personalized treatment strategies. Herein, we performed a comprehensive search of genes (termed Supertargets) vital for tumors of particular tissue origin. In so doing, we used the DepMap database portal that encompasses a broad panel of cell lines with individual genes knocked out by CRISPR/Cas9 technology. For each of the 27 tumor types, we revealed the top five genes whose deletion was lethal in the particular case, indicating both known and unknown Supertargets. Most importantly, the majority of Supertargets (41%) were represented by DNA-binding transcription factors. RNAseq data analysis demonstrated that a subset of Supertargets was deregulated in clinical tumor samples but not in the respective non-malignant tissues. These results point to transcriptional mechanisms as key regulators of cell survival in specific tumors. Targeted inactivation of these factors emerges as a straightforward approach to optimize therapeutic regimens.

We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.

Summary Cell fate depends on genetic, epigenetic and environmental inputs that are interconnected, making it difficult to disentangle their respective contributions to cell fate decisions 1-3 , and epigenetic reprogramming is a major contributor to tumor plasticity and adaptation 4-6 . Although cancer initiation and progression are generally associated with the accumulation of somatic mutations 7,8 , substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility 9-18 , suggesting that genetic mechanisms alone may not be sufficient to drive malignant transformations 19-23 . However, whether purely non-genetic reprogramming mechanisms are sufficient to initiate tumorigenesis irrespective of mutations is 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 JNK and JAK-STAT signalling pathways and zfh1 , the fly homolog of the ZEB1 oncogene, which we show to be a necessary driver of the cancer fate. These data show that a reversible perturbation of Polycomb-Group protein levels can induce cancer in the absence of driver mutations and suggest that this is achieved through epigenetic inheritance of altered cell fates.

Establishing precise gene expression patterns in different cell types is essential for the proper differentiation and development of multicellular organisms. The resulting level of transcription is determined both by the genetic nucleotide sequence of DNA and by epigenetic factors that modify chromatin. Epigenetic repressors of the Polycomb group (PcG) are regulatory proteins that repress gene transcription and maintain correct pattern of gene expression in multicellular organisms. PcG proteins form two main complexes: Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) that possess ubiquitin ligase and histone methyltransferase enzymatic activities, respectively. Several mechanisms have been suggested to account for the recruitment of Polycomb proteins in mammals, one of which involves interactions with specific DNA-binding factors. While deregulation main PcG genes in cancer have been well documented, the role of PcG DNA-binding partners in oncology remains elusive. In the present study, we analyzed genomic and transcriptomic databases of clinical tumor samples (cBioPortal, TNMplot, KMplot) to evaluate clinical correlations of Polycomb-associated DNA-binding proteins. We found that amplifications and higher expression of the ZNF281 gene are often found in pancreatic cancer and correlate with poor prognosis of overall survival.

According to previous studies, during Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II “paused” complexes associated with promoters at different developmental stages are described in detail. For this purpose, we performed ChIP-Seq analysis using antibodies for various modifications of RNA polymerase II (total, Pol II CTD Ser5P, and Pol II CTD Ser2P) as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the “paused” state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a “pause” state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the “paused” RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the “post-pause” state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the “pause” mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P.

CDK8 and CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex, have been implicated in development and diseases. CDK8 and CDK19 are differentially expressed but their roles in transcriptional regulation remained unclear. Here we show that CDK8 and CDK19 exert very similar transcriptomic and phosphoproteomic effects. While these effects are kinase-dependent, both proteins protect their binding partner cyclin C from proteolysis in a kinase-independent manner. Downregulation of a small number of genes is the immediate effect of Mediator kinase inhibition, whereas multiple genes are upregulated upon prolonged inhibition. CDK8/19 inhibition in cells exposed to serum or to NFκB or PKC agonists reduces the induction of the most responsive genes, indicating that Mediator kinases regulate transcriptional reprogramming by maximizing signal-induced transcription. Surprisingly, Mediator kinase inhibition increases protein levels of Mediator complex components at the post-transcriptional level, offering a potential new mechanism for transcriptional regulation by Mediator kinases.

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 bxd PRE 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 bxd PRE only in the presence of adjacent binding sites for architectural proteins Su(Hw), CTCF, or Pita.