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Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer–promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model. [ABSTRACT FROM AUTHOR]

The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190. [Display omitted] • ZFH-2 is expressed in both the germline and somatic cells of the ovary. • ZFH-2 has a role in early oogenesis in the development of functional egg chambers. • ZFH-2 interacts with a springer element insertion to disrupt daughterless expression. • Zfh-2 binds preferentially at the band/interband boundaries of polytene chromosomes. [ABSTRACT FROM AUTHOR]

In Drosophila, a large group of actively transcribed genes is located in pericentromeric heterochromatin. It is assumed that heterochromatic proteins recruit transcription factors to gene promoters. Two proteins, Ouib and Nom, were previously shown to bind to the promoters of the heterochromatic genes nvd and spok. Interestingly, Ouib and Nom are paralogs of the M1BP protein, which binds to the promoters of euchromatic genes. We have shown that, like M1BP, the Quib and Nom proteins bind to CP190, which is involved in the recruitment of transcription complexes to promoters. Unlike heterochromatic proteins, Ouib and Nom do not interact with the major heterochromatic protein HP1a and bind to euchromatic promoters on polytene chromosomes from the larval salivary glands. The results suggest a new mechanism for the recruitment of transcription factors into the heterochromatic compartment of the nucleus. [ABSTRACT FROM AUTHOR]

CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes. [ABSTRACT FROM AUTHOR]

CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators. [ABSTRACT FROM AUTHOR]

The Su(Hw) protein was first identified as a DNA-binding component of an insulator complex in Drosophila. Insulators are regulatory elements that can block the enhancer-promoter communication and exhibit boundary activity. Some insulator complexes contribute to the higher-order organization of chromatin in topologically associated domains that are fundamental elements of the eukaryotic genomic structure. The Su(Hw)-dependent protein complex is a unique model for studying the insulator, since its basic structural components affecting its activity are already known. However, the mechanisms involving this complex in various regulatory processes and the precise interaction between the components of the Su(Hw) insulators remain poorly understood. Our recent studies reveal the fine mechanism of formation and function of the Su(Hw) insulator. Our results provide, for the first time, an example of a high complexity of interactions between the insulator proteins that are required to form the (Su(Hw)/Mod(mdg4)-67.2/CP190) complex. All interactions between the proteins are to a greater or lesser extent redundant, which increases the reliability of the complex formation. We conclude that both association with CP190 and Mod(mdg4)-67.2 partners and the proper organization of the DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators. In this review, we demonstrate the role of multiple interactions between the major components of the Su(Hw) insulator complex (Su(Hw)/Mod(mdg4)-67.2/CP190) in its activity. It was shown that Su(Hw) may regulate the enhancer–promoter communication via the newly described insulator neutralization mechanism. Moreover, Su(Hw) participates in direct regulation of activity of vicinity promoters. Finally, we demonstrate the mechanism of organization of “insulator bodies” and suggest a model describing their role in proper binding of the Su(Hw) complex to chromatin.

Abstract Background Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Results Here we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. Conclusions These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.

Chromatin insulators are DNA-protein complexes that establish independent higher order DNA domains to influence transcription. Insulators are functionally defined by two different properties: they can block communication between an enhancer and a promoter and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is depleted, gypsy-dependent enhancer blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, but depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity. [ABSTRACT FROM AUTHOR]

Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the three-dimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and three-dimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

Background: Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Results: Here we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. Conclusions: These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation. [ABSTRACT FROM AUTHOR]

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators. [ABSTRACT FROM AUTHOR]

CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.

Insulators are regulatory DNA elements that modulate the effect of enhancers and silencers on gene transcription. However, the role of the insulator 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 depends on the Su(Hw) factor. At the same time, the CP190 protein interacts with the promoter and terminator regions in the absence of Su(Hw). In addition, the Su(Hw) protein does not affect the level and efficiency of termination of model genes transcription. 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. [ABSTRACT FROM AUTHOR]

Drosophila polytene interphase chromosomes provide an ideal test system to study the rules that define the structure of chromatin domains. We established a transgenic condensed chromatin domain cassette for the insertion of large pieces of DNA by site-specific recombination. Insertion of this cassette into open chromatin generated a condensed domain, visible as an extra band on polytene chromosomes. Site-specific recombination of DNA sequence variants into this ectopic band allowed us to compare their capacity for open chromatin formation by cytogenetic methods. We demonstrate that the 61C7-8 interband DNA maintains its open chromatin conformation and epigenetic state at an ectopic position. By deletion analysis, we mapped the sequences essential for open chromatin formation to a 490-bp fragment in the proximal part of the 17-kb interband sequence. This fragment overlaps binding sites for the chromatin protein Chriz (also known as Chro), the histone kinase Jil-1 and the boundary element protein CP190. It also overlaps a promoter region that locates between the Rev1 and Med30 transcription units. [ABSTRACT FROM AUTHOR]

Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, d CTCF, Su( Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity. [ABSTRACT FROM AUTHOR]

Alteration of regulatory DNA elements or their binding proteins may have drastic consequences for morphological evolution. Chromatin insulators are one example of such proteins and play a fundamental role in organizing gene expression. While a single insulator protein, CTCF (CCCTC-binding factor), is known in vertebrates, Drosophila melanogaster utilizes six additional factors. We studied the evolution of these proteins and show here that-in contrast to the bilaterian-wide distribution of CTCF-all other D. melanogaster insulators are restricted to arthropods. The full set is present exclusively in the genus Drosophila whereas only two insulators, Su(Hw) and CTCF, existed at the base of the arthropod clade and all additional factors have been acquired successively at later stages. Secondary loss of factors in some lineages further led to the presence of different insulator subsets in arthropods. Thus, the evolution of insulator proteins within arthropods is an ongoing and dynamic process that reshapes and supplements the ancient CTCF-based system common to bilaterians. Expansion of insulator systems may therefore be a general strategy to increase an organism's gene regulatory repertoire and its potential for morphological plasticity. [ABSTRACT FROM AUTHOR]

Insulators help in organizing the eukaryotic genomes into physically and functionally autonomous regions through the formation of chromatin loops. recent findings in Drosophila and vertebrates suggest that insulators anchor multiple loci through long-distance interactions which may be mechanistically linked to insulator function. important to such processes in Drosophila is CP190, a common co-factor of insulator complexes. CP190 is also known to associate with the nuclear matrix, components of the RNAI machinery, active promoters and borders of the repressive chromatin domains. Although CP190 plays a pivotal role in insulator function in Drosophila, vertebrates lack a probable functional equivalent of CP190 and employ CTCF as the major factor to carry out insulator function/chromatin looping. In this review, we discuss the emerging role of CP190 in tethering genome, specifically in the perspective of insulator function in Drosophila. Future studies aiming genome-wide role of CP190 in chromatin looping is likely to give important insights into the mechanism of genome organization. [ABSTRACT FROM AUTHOR]

Chromatin insulators are special regulatory elements involved in modulation of enhancer-promoter interactions. The best studied insulators in Drosophila require Suppressor of Hairy Wing [Su(Hw)], Modifier of mdg4 [Mod(mdg4)] and centrosomal 190 kDa (CP190) proteins to be functional. These insulator proteins are colocalized in nuclear speckles named insulator bodies. Here, we demonstrate that post-translational modification of insulator proteins by small ubiquitin-like modifier (SUMO) and intact CP190 protein is crucial for insulator body formation. Inactivation of SUMO binding sites in Mod(mdg4)-67.2 leads to the inability of the mutant protein and Su(Hw) to be assembled into insulator bodies. In vivo functional tests show that a smaller amount of intact Mod(mdg4)-67.2, compared with the mutant protein, is required to restore the normal activity of the Su(Hw) insulator. However, high expression of mutant Mod(mdg4)-67.2 completely rescues the insulator activity, indicating that sumoylation is not necessary for enhancer blocking. These results suggest that insulator bodies function as a depot of sumoylated proteins that are involved in insulation and can facilitate insulator complex formation, but are nonessential for insulator action. [ABSTRACT FROM AUTHOR]

Abstract: The BTB (bric-a-brac, tramtrack and broad complex)/POZ (poxvirus and zinc finger) domain is a conserved protein–protein interaction motif contained in a variety of transcription factors involved in development, chromatin remodeling, insulator activity, and carcinogenesis. All well-studied mammalian BTB domains form obligate homodimers and, rarely, tetramers. Only the BTB domain of the Drosophila GAGA factor (GAF) has been shown to exist as higher-order multimers. The BTB domain of GAF belongs to the “ttk group” that contains several highly conserved sequences not found in other BTB domains. Here, we have shown by size-exclusion chromatography, chemical cross-linking, and nondenaturing PAGE that four additional BTB domains of the ttk group—Batman, Mod(mdg4), Pipsqueak, and Tramtrack—can form multimers, like GAF. Interestingly, the BTB domains of GAF and Batman have formed a wide range of complexes and interacted in the yeast two-hybrid assay with other BTB domains tested. In contrast, the BTB domains of Mod(mdg4), Pipsqueak, and Tramtrack have formed stable high-order multimer complexes and failed to interact with each other. The BTB domain of Drosophila CP190 protein does not belong to the ttk group. This BTB domain has formed stable dimers and has not interacted with domains of the ttk group. Previously, it was suggested that GAF oligomerization into higher-order complexes facilitates long-range activation by providing a protein bridge between an enhancer and a promoter. Unexpectedly, experiments in the Drosophila model system have not supported the role of GAF in organization of long-distance interaction between the yeast GAL4 activator and the white promoter. [Copyright &y& Elsevier]

Abstract: The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions. [Copyright &y& Elsevier]

For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 ‘islands’. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190. [ABSTRACT FROM AUTHOR]

Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF-32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. Here we find that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlapped with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal-B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, we demonstrate the importance of dCTCF in fly development and in the regulation of abdominal segmentation. [ABSTRACT FROM AUTHOR]

The Drosophila CP190 and CP60 proteins interact with each other and shuttle between the nucleus in interphase and the centrosome in mitosis. Both proteins can bind directly to microtubules in vitro, and have been shown to associate with a specific pattern of loci on salivary gland polytene chromosomes, but their functions are unknown. Here we show that reducing the level of CP190 or CP60 by >90% in tissue culture cells does not significantly interfere with centrosome or microtubule organisation, with cell division, or with cell viability. However, CP190 is an essential protein, as flies homozygons for mutations in the Cp190 gene die at late pupal stages of development. In larval brains of Cp190 mutants, mitosis is not radically perturbed, and a mutated form of CP190 (CP190ΔM), that cannot bind to microtubules or associate with centrosomes, can rescue the lethality associated with mutations in the Cp190 gene. Thus, CP190 plays an essential role in flies that is independent of its association with centrosomes or microtubules. [ABSTRACT FROM AUTHOR]

Drosophila Myb (Dm- Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation. • Myb transcriptional potentiation optimizes H3K4 methylation and RNA Pol II function • Myb contributes to insulator function and promotes binding of BEAF-32 and CP190 • Myb is required to stabilize H3K27me3 domains genome wide • Myb is enriched at TAD boundaries and chromatin loop anchors Myb has been considered a transcriptional activator of primarily M phase genes. Here, Santana et al. show that Myb also contributes to insulator function, in part by promoting binding of insulator factors, and is required to stabilize repressive domains in the genome. [ABSTRACT FROM AUTHOR]

We explored functional roles of two H3K9-specific histone methyltransferases of Drosophila melanogaster , SetDB1 (also known as Eggless) and Su(var)3-9. Using the DamID approach, we generated the binding profile for SetDB1 in Drosophila salivary gland chromosomes, and matched it to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartments, and is largely restricted to transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of ubiquitously expressed genes. Significant SetDB1 association is also observed at binding sites for the insulator protein CP190. SetDB1 and H3K9 di- and tri-methylated (me2 and me3)-enriched sites tend to display poor overlap. At the same time, SetDB1 has a clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, we speculate that, in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes., (© 2017 The Authors.)

Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the three-dimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and three-dimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)