The insulator BEAF32 controls the spatial-temporal expression profile of the telomeric retrotransposon TART in the Drosophila germline.

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.

Crol contributes to PRE-mediated repression and Polycomb group proteins 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.

CDK8 and CDK19: positive regulators of signal-induced transcription and negative regulators of Mediator complex proteins.

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.

Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap-, Zeste-, Psq-, and Adf1-associated proteins.

The Polycomb group (PcG) and Trithorax group (TrxG) proteins are key epigenetic regulators controlling the silenced and active states of genes in multicellular organisms, respectively. In Drosophila, PcG/TrxG proteins are recruited to the chromatin via binding to specific DNA sequences termed polycomb response elements (PREs). While precise mechanisms of the PcG/TrxG protein recruitment remain unknown, the important role is suggested to belong to sequence-specific DNA-binding factors. At the same time, it was demonstrated that the PRE DNA-binding proteins are not exclusively localized to PREs but can bind other DNA regulatory elements, including enhancers, promoters, and boundaries. To gain an insight into the PRE DNA-binding protein regulatory network, here, using ChIP-seq and immuno-affinity purification coupled to the high-throughput mass spectrometry, we searched for differences in abundance of the Combgap, Zeste, Psq, and Adf1 PRE DNA-binding proteins. While there were no conspicuous differences in co-localization of these proteins with other functional transcription factors, we show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste, and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription.

Polycomb Recruiters Inside and Outside of the Repressed Domains.

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.

Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment.

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.

GAGA factor: a multifunctional pioneering chromatin protein.

The Drosophila GAGA factor (GAF) is a multifunctional protein implicated in nucleosome organization and remodeling, activation and repression of gene expression, long distance enhancer-promoter communication, higher order chromosome structure, and mitosis. This broad range of activities poses questions about how a single protein can perform so many seemingly different and unrelated functions. Current studies argue that GAF acts as a "pioneer" factor, generating nucleosome-free regions of chromatin for different classes of regulatory elements. The removal of nucleosomes from regulatory elements in turn enables other factors to bind to these elements and carry out their specialized functions. Consistent with this view, GAF associates with a collection of chromatin remodelers and also interacts with proteins implicated in different regulatory functions. In this review, we summarize the known activities of GAF and the functions of its protein partners.

Boundaries potentiate polycomb response element-mediated silencing.

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.

RNA Polymerase II "Pause" Prepares Promoters for Upcoming Transcription during Drosophila Development.

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.

Boundaries of loop domains (insulators): Determinants of chromosome form and function in multicellular eukaryotes.

Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.

Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements.

In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched "on" and "off." When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. Here, we have directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, we show that transcription through the PRE is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. Our results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity.

Eukaryotic enhancers: common features, regulation, and participation in diseases.

Enhancers are positive DNA regulatory sequences controlling temporal and tissue-specific gene expression. These elements act independently of their orientation and distance relative to the promoters of target genes. Enhancers act through a variety of transcription factors that ensure their correct match with target promoters and consequent gene activation. There is a growing body of evidence on association of enhancers with transcription factors, co-activators, histone chromatin marks, and lncRNAs. Alterations in enhancers lead to misregulation of gene expression, causing a number of human diseases. In this review, we focus on the common characteristics of enhancers required for transcription stimulation.

Making connections: insulators organize eukaryotic chromosomes into independent cis-regulatory networks.

Insulators play a central role in subdividing the chromosome into a series of discrete topologically independent domains and in ensuring that enhancers and silencers contact their appropriate target genes. In this review we first discuss the general characteristics of insulator elements and their associated protein factors. A growing collection of insulator proteins have been identified including a family of proteins whose expression is developmentally regulated. We next consider several unexpected discoveries that require us to completely rethink how insulators function (and how they can best be assayed). These discoveries also require a reevaluation of how insulators might restrict or orchestrate (by preventing or promoting) interactions between regulatory elements and their target genes. We conclude by connecting these new insights into the mechanisms of insulator action to dynamic changes in the three-dimensional topology of the chromatin fiber and the generation of specific patterns of gene activity during development and differentiation.

Insulators form gene loops by interacting with promoters in Drosophila.

Chromatin insulators are regulatory elements involved in the modulation of enhancer-promoter communication. The 1A2 and Wari insulators are located immediately downstream of the Drosophila yellow and white genes, respectively. Using an assay based on the yeast GAL4 activator, we have found that both insulators are able to interact with their target promoters in transgenic lines, forming gene loops. The existence of an insulator-promoter loop is confirmed by the fact that insulator proteins could be detected on the promoter only in the presence of an insulator in the transgene. The upstream promoter regions, which are required for long-distance stimulation by enhancers, are not essential for promoter-insulator interactions. Both insulators support basal activity of the yellow and white promoters in eyes. Thus, the ability of insulators to interact with promoters might play an important role in the regulation of basal gene transcription.

Su(Hw) interacts with Combgap to establish long-range chromatin contacts.

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.

Context-dependent role of Pho binding sites in Polycomb complex recruitment in Drosophila.

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.

Analyses of Genes Critical to Tumor Survival Reveal Potential 'Supertargets': Focus on Transcription.

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.

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

Su(Hw) primes 66D and 7F Drosophila chorion genes loci for amplification through chromatin decondensation.

Suppressor of Hairy wing [Su(Hw)] is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. In previous studies we have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. Here, we describe the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. We show Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin impairs pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although we observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. We have discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and this binding is dependent on Su(Hw). We propose that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.

Clinical Correlations of Polycomb Repressive Complex 2 in Different Tumor Types.

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.