FEBS Letters 588 (2014) 8–14

journal homepage: www.FEBSLetters.org

Review

Chromatin insulators and long-distance interactions in Drosophila Olga Kyrchanova a, Pavel Georgiev b,⇑ a b

Group of Transcriptional Regulation, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia

a r t i c l e

i n f o

Article history: Received 19 October 2013 Revised 25 October 2013 Accepted 25 October 2013 Available online 5 November 2013 Edited by Wilhelm Just Keywords: Chromatin insulator Enhancer blocking Long-range interaction Architectural protein Drosophila

a b s t r a c t Data on long-distance enhancer-mediated activation of gene promoters and complex regulation of gene expression by multiple enhancers have prompted the hypothesis that the action of enhancers is restricted by insulators. Studies with transgenic lines have shown that insulators are responsible for establishing proper local interactions between regulatory elements, but not for defining independent transcriptional domains that restrict the activity of enhancers. It has also become apparent that enhancer blocking is only one of several functional activities of known insulator proteins, which also contribute to the organization of chromosome architecture and the integrity of regulatory elements. Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

1. Introduction The complexity of regulatory systems in higher eukaryotes, featuring many distantly located enhancers that nonetheless properly activate the target, has promoted the hypothesis that the action of enhancers is restricted by a special class of regulatory elements, named insulators (reviewed in [1,2]). Insulators were regarded as genomic regulatory elements (nucleoprotein complexes) that have two characteristic properties: they can block the action of an enhancer on a promoter when interposed between them and can protect the transgenes they flank from chromosomal position effects [3–7]. On the basis of these properties, it was initially suggested that insulators form functionally independent units of gene regulation (reviewed in [8]). However, recent advances in genome-wide studies of chromatin architecture and new insights in insulator properties suggest a revision of the initial concept on the role of these elements in gene regulation. This review is an attempt to summarize recent updates in the study of Drosophila insulators and make some general conclusions concerning the mechanisms of enhancer Abbreviations: ANT-C, Antennapedia complex, a group of five Drosophila homeotic genes controlling the development of the head and the anterior part of the thorax; BX-C, Bithorax complex, a group of three Drosophila homeotic genes controlling the development of the abdomen and the posterior part of the thorax ⇑ Corresponding author. Fax: +7 499 1354105. E-mail address: [email protected] (P. Georgiev).

blocking and the role of insulators in gene regulation and chromosome architecture. 2. Drosophila insulators 2.1. Drosophila insulators are a diverse group of elements Drosophila is a unique model organism to study the properties of insulators. Using transposon-mediated transformation or attP-phage-based integration and manipulation with recombination systems, it is possible to obtain different combinations of regulatory elements in the same genomic position and study the role of a particular regulatory element in reporter expression [9–11]. Many different insulators have been characterized in the Drosophila genome. In particular, they include the scs and scs’ insulators at the boundaries of the domain consisting of two heat shock 70 genes [5,6]; Wari [12] and 1A2 [13,14] insulators located on the 30 side of the white and yellow genes, respectively; insulators flanking the promoter regions of the Notch gene [15]; and the insulator that separates two closely spaced promoters of divergently oriented genes [16]. Insulators have been found in the regulatory regions of the homeotic gene complexes bithorax (BX-C) [17–21] and antennapedia (ANT-C) [22]. Some Drosophila retrotransposons, such as gypsy or Idefix, also contain insulators in their regulatory region [4,23].

0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.10.039

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14

2.2. Drosophila insulator proteins Insulator activity is realized through proteins bound to the insulator. For most of known insulators, there is a key protein that specifically binds to DNA. Three insulator proteins – Su(Hw), Zw5, and dCTCF, a Drosophila homolog of vertebrate insulator protein CTCF – have multiple C2H2 zinc fingers [24–26]. Some of these zinc fingers determine a specific DNA binding site for a given protein. The best studied insulator located in the regulatory region of the gypsy retrotransposon contains 12 reiterated potential binding sites for the Su(Hw) protein [4]. In contrast, many endogenous insulators contain only one or two binding sites for Su(Hw) [27–29]. Binding sites for dCTCF have been found in most boundary elements of the bithorax complex that separate regulatory iab enhancers involved in regulation of the abd-A and Abd-B genes [30,31]. Some of these boundary elements function as insulators in an enhancer-blocking assay in transgenic lines [19,21]. In addition, five out of eight randomly tested dCTCF binding regions proved to have a strong enhancer-blocking activity [32]. The Zw5 protein preferentially binds to promoter regions [29]. In particular, a Zw5 site is located in the promoter bordering the scs insulator [25,33]. The sequences consisting of four binding sites for the Su(Hw), dCTCF, and Zw5 proteins function as effective insulators in an enhancer-blocking assay [10,34,35]. Su(Hw) interacts with CP190 and the Mod(mdg4) isoform named Mod(mdg4)-67.2 [36–40]. The dCTCF protein also interacts with CP190 [30,41]. GAF has been implicated in the activity of one insulator from BX-C, named Fab-7 [20]; the SF1 insulator from ANT-C [22]; and the insulator located between two divergently expressed genes, myoglianin and eyeless [16]. Unlike Su(Hw), CTCF, and Zw5, the GAF protein has only a single C2H2 Zn finger that is required for binding to the GAG sequence [42,43]. The GAF, Mod(mdg4)-67.2, and CP190 proteins have a BTB (bric-a-brac, tramtrack and broad complex)/POZ (poxvirus and zinc finger) domain at the N-terminus. All well-studied mammalian BTB domains form obligate homodimers and, rarely, tetramers [44]. The BTB domains of the Drosophila GAF and Mod(mdg4)67.2 factors belong to the ‘‘ttk group’’ comprising several highly conserved sequences that are not found in other BTB domains and exist as higher-order multimers [42,45–47]. The BTB domains of ‘‘ttk’’ group have been shown to extensively interact with each other [42,47–50]. Su(Hw), dCTCF, and GAF are ubiquitous proteins. In contrast to most of transcription factors, Su(Hw) and dCTCF mainly have constitutive binding sites in different cell lines and Drosophila tissues [29,32,51,52], suggesting that these proteins can effectively bind to chromosomes during the cell cycle. The boundary element-associated factor, named BEAF, have been identified based on their interaction with the scs’ insulator element [53,54]. BEAF has atypical C2H2 zinc finger DNA binding domain, termed BED finger, at the N termini. The C-terminal domain is required for trimerization of the BEAF protein [54,55]. Each subunit of the BEAF complex targets one CGATA motif, while BEAF trimers bind with high affinity to clusters of CGATA motifs [54]. The results of genome-wide analysis show that BEAF preferentially binds to the clusters of such motifs in the promoter regions of active genes and is required for stimulating their transcription [56,57]. A new insulator complex recently described in Drosophila, named ELBA [58], is composed of two proteins, Elba1 and Elba2, sharing a conserved C-terminal ‘BEN domain’ that mediates DNA binding. The third protein, Elba3, is responsible for ‘‘dimerizing’’ the Elba1-2 BEN domains and is encoded by a gene that is closely linked to Elba1. Thus, in this case the dimerization domain is required for cooperative binding of two BEN domains to

9

corresponding insulator sites. ELBA activity is developmentally restricted, because two of the three Elba proteins (Elba1 and Elba3) are encoded by mid-blastula transition genes [58]. It has been shown that ELBA is required for the enhancer-blocking activity of the Fab-7 insulator. 3. Studies on Drosophila insulators in transgenic model systems 3.1. Interaction between insulators leads to partial neutralization of their activity The main property of an insulator is the ability to block the activity of an enhancer when positioned between it and a promoter. In Drosophila, scs is the strongest insulator that almost completely blocks all tested enhancers [6,59,60]. The activity of gypsy insulator depends on the enhancer–promoter pair. For example, this insulator completely blocks the yellow enhancers [4], but only slightly affects the activity of the eye enhancer that is responsible for white expression in the eyes [61]. This may be partially explained by differences in the strength of enhancer–promoter interactions. For example, the Zeste protein is specifically involved in communication between the eye enhancer and white promoter and allows the eye enhancer to bypass the gypsy insulator [62]. All other insulators studied can only partially block enhancer activity in transgenic lines [10,12,13,19–22,25,28,59,61,63–66]. As a rule, insulator activity is strongly dependent on the insertion site of a transposon. Such a position dependence of enhancer blocking may be due to the influence of the surrounding chromatin structure on the enhancers tested. Recent data obtained with transgenic lines show even weak transcription through enhancers markedly suppresses their activity [67]. Thus, it appears that the level of enhancer blocking in transgenic lines is directly correlated with enhancer strength [68]. In many experiments with different insulators in transgenic lines, it was found that two identical insulators neutralized each other’s enhancer-blocking activity when inserted between an enhancer and a promoter [59,69–71]. If the original distance between the enhancer and promoter was large or they were separated by an additional gene, the enhancer failed to effectively stimulate the promoter. When two identical insulators were inserted in close proximity to the enhancer and promoter, enhancer–promoter communication was facilitated. Thus, the pairing of insulators proved to shorten the distance between the enhancer and promoter, thereby improving their interaction. Partial neutralization of enhancer-blocking activity was also observed in case of pairing between different insulators, e.g., gypsy (12 Su(Hw) binding sites), 1A2 (two Su(Hw) binding sites), and Wari (no Su(Hw) binding sites) [10]. Similar results were obtained with Polycomb group response elements (PRE) that silence neighboring genes. In transgenic lines, PRE displayed long-range repression of two reporter genes simultaneously, which was blocked when a strong insulator, such as gypsy or scs, was inserted between PRE and reporter genes [72– 74]. However, flanking the first reporter gene by two identical gypsy insulators led to repression of the second reporter gene [74]. In this case, the interaction between the gypsy insulators was found to bring the PRE silencer in close proximity to the promoter of the second reporter gene, inducing repression. The physical interaction between two gypsy insulators in these transgenic lines was confirmed by 3C technique [75]. Testing transgenic lines with three copies of the Su(Hw) insulators inserted in different combinations between enhancers and two reporter genes (Fig. 1A) strongly suggested that all three copies interacted simultaneously [10,76]. It was found that two insulators inserted between the enhancer and promoter of the first

10

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14

Fig. 1. Interactions between Drosophila insulators: (A) interactions between three copies of the same or different insulators (gypsy, 1A2 and Wari); (B) orientationdependent interactions of two insulator copies; (C) a model of specific longdistance interaction between two copies of an insulator.

gene allowed enhancers to stimulate only the first gene. When an additional insulator was inserted between the first and second genes, the enhancers stimulated both genes equally. When two insulators flanked the first gene, the enhancers could activate only the second gene. These results suggest that interactions between insulators can positively or negatively regulate local enhancer– promoter communication. 3.2. Orientation-dependent interactions of insulators The results of the first studies on pairing between two identical copies of insulators were contradictory, because some pairs of insulators tested failed to neutralize each other’s enhancer-blocking activity [59,63]. To explain these findings, it was suggested that identical insulators interact in an orientation-dependent manner [77]. This hypothesis was confirmed by using an assay based on the inability of the yeast GAL4 activator to stimulate the white promoter across the yellow gene. Insulators tested for interaction were inserted in close proximity to the GAL4 binding sites and the white promoter (Fig. 1B). When two identical insulators were in opposite orientations, the configuration of the resulting chromatin loop was favorable for interaction between the promoter and GAL4 activator binding element located outside the loop. When the insulators were in the same orientation, GAL4 activator failed to stimulate the white promoter. All pairs of insulators included in analysis proved to interact in an orientation-dependent manner [34,35,77]. Similar results were obtained when analyzing the effect of two gypsy insulators on site-specific recombination between FRT sites catalyzed by yeast Flp recombinase [78]: the gypsy insulators inserted between the FRT sites in opposite orientations markedly facilitated their FLP-mediated recombination, while the insulators in the same orientation suppressed this process. To explain the mechanism of orientation-dependent interaction between insulators, we suggest that all tested insulators contain not only the key DNA-binding proteins (specific for each insulator) but also binding sites for one or several additional, as yet unknown insulator-like proteins, and that it is specific protein–protein

interactions that determine the relative orientation of paired insulators (Fig. 1B). Indeed, the dCTCF, SU(Hw), and Zw5 proteins have homodimerization domains at the amino termini that may be important for specific long-distance interactions (A. Golovnin and A. Bonchuck, unpublished data). Strong functional interactions have been revealed between DNA fragments containing binding sites for Zw5, Su(Hw), or dCTCF alone but not between heterologous binding sites [Zw5–Su(Hw), dCTCF–Su(Hw), or dCTCF–Zw5] [35]. Thus, these insulator proteins are able to support specific long-distance interactions. Several studies suggest that BTB domains play an important role in organizing long-range interactions between regulatory elements. For example, the GAF protein was shown to support distant GAL4 stimulation of the promoter in heterologous model systems like yeast and mammalian cell line [46,79]. However, in contrast to Zw5, dCTCF, or Su(Hw), binding sites for GAF do not support distant interaction in Drosophila transgenic lines [47]. Thus, there is no conclusive experimental evidence for the ability of GAF to support long-distance interactions in Drosophila. On the other hand, oligomerization of the BTB domains is required for cooperative binding of GAF to many adjacent sites in the same regulatory region (enhancer, insulator, or promoter) [80]. A similar role may be played by the self-association domain located at the C-terminus of BEAF protein [54,55]. The relevant data are somewhat ambiguous but nevertheless indicate that the multimerized BTB domains of the Mod(mdg4)-67.2 and GAF proteins may be involved in stabilization of long-distance interactions. The BTB domain of CP190 forms an obligate homodimer [47] and is required for the interaction with Su(Hw) and dCTCF proteins (A.B. and A.G., unpublished data). Thus, the answer as to how this domain can be involved in long-distance interactions is not as obvious as is supposed in a recent study [81]. 3.3. Insulators can support functional interactions between regulatory elements across megabase distances In contrast to vertebrates, Drosophila homologous chromosomes are paired in the interphase nuclei [82]. Many tested insulators block the activity of enhancers more effectively when the corresponding construct is present on both homologous chromosomes. For example, the gypsy insulator inserted between the eye-specific enhancer and promoter of the white gene failed to effectively block enhancer–promoter communication [61]. However, in transgenic lines homozygous for the construct, the eye enhancer was completely blocked due to pairing between the gypsy insulators located on the homologous chromosomes. Likewise, pairing between the gypsy insulators blocked unequal recombination between FRT sites located on homologous chromosomes [78]. Thus, insulators may contribute to pairing of homologous chromosomes in the interphase nucleus. It is noteworthy that one isoform of the Mod(mdg4) protein is required for pairing and segregation of homologous chromosomes during meiosis in males [83]. In Drosophila, a phenomenon named transvection has been described. Its essence is that pairing between homologous chromosomes allows an enhancer located on one chromosome to stimulate promoter located on the homologous chromosome [82]. However, the level of trans-activation of a promoter by an enhancer is strongly dependent on the genomic site of transgene insertion [84,85]. The presence of gypsy insulators on both homologous chromosomes, even at a distance of 9 kb downstream from the promoter, dramatically improves the trans-activation of yellow [85]. Thus, the interaction between gypsy insulators improves the pairing between the homologous chromosomes. The gypsy insulators have also proved to stabilize trans-activation between enhancers and the promoter located even at megabase distances [85]. Likewise, Fab-7 and Mcp insulators support

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14

super-long-distance functional interactions between PREs [86–88]. These long-range functional interactions were confirmed in experiments demonstrating physical co-localization of two insulatorcontaining constructs by in vivo fluorescence imaging and chromosome conformation capture (3C) methods [86]. Such interactions were usually observed in the same arm of chromosome. These results are evidence that two identical insulators can organize and support stable interactions across megabase distances. In the model proposed to explain this property of insulators (Fig. 1C), it is assumed that different self-interacting insulator-like proteins bind to these elements, providing the possibility of multiple protein–protein interactions between two identical insulators. The set of these proteins is specific for each insulator type, which ensures specificity of long-range interactions between identical insulator complexes, despite the presence of numerous binding sites for any of individual insulator proteins in the intervening chromatin segments. 4. Mechanisms of enhancer blocking and role of insulators in transcriptional regulation 4.1. Insulators can stimulate the activity of promoters and enhancers As found in experiments with transgenic lines, insulators located on the 30 side of genes interact with promoters, and these interactions are in some cases required for the activity of promoters [61,89]. It appears that the interaction of insulators with promoters is not promoter-specific. For example, the 1A2 insulator can interact with three different promoters in transgenic constructs [89]. Insulator proteins can probably interact with general transcription factors of the TFIID complex bound to promoters. Indeed, it has been shown that TAF3, a subunit of the TFIID complex, interacts with vertebrate insulator protein CTCF [90]. In Drosophila, insulator proteins such as GAF [91] and dCTCF (A.B. unpublished) interact with TAF3, suggesting that such an interaction is conserved among higher eukaryotes. It was also found that the gypsy insulator located downstream of the white gene could stimulate its expression by the upstream eye enhancer in transgenic lines. A direct interaction between the enhancer and the gypsy insulator was confirmed by 3C [61]. Likewise, the gypsy insulator placed as far as 5 kb downstream of the yellow gene in transgenic lines could stimulate the promoter weakened by partial deletion [92]. In addition to the possible role of a gene loop in the enhancement of RNAP II recycling and mRNA export, insulators may serve to bring to the promoter the remodeling and histone modification complexes that improve the binding and stabilization of TFIID and enhancer complexes. For example, Su(Hw) insulators recruit SAGA and Brahma complexes that associate with transcriptionally active genome regions [93]. The maintenance of promoter activity can be one of the main function of insulators, since endogenous insulators have often been found in the 30 regions of genes [12–14] and insulator proteins such as Zw5, BEAF, GAF, and dCTCF preferably have binding sites in promoter regions [29,32].

11

not the main mechanism of enhancer blocking. In most cases, even flanking of an enhancer or a gene by interacting insulators resulted in only a slight increase in blocking activity [10,21,60,61,77]. The only exception was the chromatin loop formed by the gypsy insulators, which proved to be clearly involved in blocking the eye enhancer–white promoter interactions in transgenic lines [61]. The enhancers placed in the loop flanked by these insulators were blocked [61,76], which could be explained, in particular, by constraints imposed by this tight loop on the ability of proteins bound to the enhancer and promoter to interact properly so as to stimulate transcription (Fig. 2A). An increase in the distance between the insulators to 7–9 kb restored communication between the enhancers and promoter [76,97], indicating that the interaction between the insulators in this case did not interfere with the proper interaction between proteins bound to the enhancer and promoter (Fig. 2B). A specific situation for enhancer blocking is when the enhancers and promoters are located in close proximity to the paired insulators [61,77]. For example, the eye enhancer was inserted in a 5-kb loop flanked by the gypsy insulators, close to the upstream insulator. In this configuration, the gypsy insulators located in the same orientation proved to bring together the eye enhancer located within the loop and the white promoter located outside the loop (Fig. 2C). When the gypsy insulators were in opposite orientations, they blocked enhancer–promoter communication (Fig. 2D). Therefore, the position of an enhancer relative to gypsy insulators within the loop appears to be critical for the functional outcome of loop formation. Taken together, the results obtained with Drosophila transgenic lines strongly suggest that chromatin loops formed by insulators play only an auxiliary role in enhancer blocking. A decade ago, Pamela Geyer proposed a ‘‘decoy model’’ postulating that the insulator complex binds to an enhancer or promoter complex to neutralize it or traps its vital components [98]. A growing body of experimental evidence that insulators interact with enhancers and promoters strongly supports the model that insulator-bound proteins directly interfere with proper enhancer–promoter interactions. The enhancer-blocking activity of several insulators depends on general transcription factors that inhibit RNAP II elongation [91]. The recruitment of Mod(mdg4)-67.2 to

4.2. Mechanisms of insulator actions and role in gene regulation Different variants of structural models have been proposed to explain the action mechanism of insulators (reviewed in [8]). According to these models, insulators interact with each other or with some nuclear structures (e.g., the lamina or nuclear matrix), which leads to formation of chromatin loops and, consequently, constrains the interaction between an enhancer and a promoter located in different loops. To date, the chromatin loop models have been supported only by a few pieces of indirect experimental evidence [33,66,94–96]. On the other hand, many studies with transgenic lines have shown that the formation of chromatin loops is

Fig. 2. Mechanisms of insulator action: (A) enhancer blocking is produced by a tight insulator loop; (B) enhancer–promoter interaction is restored when the enhancer is located in a large chromatin loop formed by interacting insulators; (C and D) orientation-dependent enhancer blocking with the enhancer inserted in a loop between insulators in close proximity to the upstream one; (E) enhancer–promoter interaction and gene expression are improved by an insulator located outside the enhancer–promoter loop; (F) an insulator interferes with proper organization of enhancer–promoter communication.

12

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14

the Su(Hw) insulators directly interferes with Zeste protein binding, which is essential for the enhancer–promoter communication in the white locus [61]. In general, when an insulator is located outside an enhancer–promoter pair, interactions of insulator proteins with promoter and enhancer proteins stabilize the formation of the transcriptional complex and thereby increase gene expression (Fig. 2E). Conversely, when the insulator is placed between the enhancer and the promoter, interactions of insulator proteins with the general transcription machinery or enhancer bound proteins interfere with organization of a functional complex between the latter and the TFIID complex on the promoter (Fig. 2F). As a result, enhancer-dependent gene stimulation is decreased. This model better explains why enhancers are only partially blocked by most of known insulators and why paired insulators neutralize each other. The ability of insulators to efficiently interact in an orientationdependent manner and interfere with proper enhancer–promoter interactions strongly suggests that insulators are involved in local organization of enhancer–promoter interactions. An illustrative example is their role in organizing interactions between the iab enhancers and Abd-B promoter in the bithorax complex (reviewed in [99]). 5. Conclusions and perspectives In recent years, considerable progress has been made in understanding chromosome organization (reviewed in [100,101]). Highresolution chromosome conformation capture approaches have shown that chromosomes in the human, mouse, and Drosophila genomes are partitioned into topologically associating domains (TADs) [102]. Insulator proteins are frequently found at the boundaries of these domains. Experiments with Drosophila insulators show that they can organize and support super-long-range functional interactions between regulatory elements at distances of up to several megabases [85,86]. However, the results obtained with transgenic lines argue against a general role of chromatin loops formed by insulators in blocking enhancer–promoter interactions, suggesting that TADs are likely to shape the architecture of chromosomes in the nucleus but not to form independent transcriptional domains. On the other hand, insulator proteins are frequently bound to promoters, being probably involved in their activity. Since enhancer blocking is only one of several functional activities of the insulator proteins, it appears better to give them a more general name of ‘‘architectural proteins.’’ It may well be that more proteins of this type exists in the Drosophila genome. For example, we have recently identified two new proteins, Pita and CG7928, that support long-distance interactions and block enhancer or silencer activity (manuscript in preparation). It would be interesting to know whether the same diversity of insulator/architectural proteins exist in vertebrate genome. Acknowledgements We are grateful to N.A. Gorgolyuk for his help in preparing the manuscript. This study was supported by 12-04-92423-EMBL-a, SS-2591.2012.4 (to P.G.), and the Ministry of Science and Education of the Russian Federation (project no. 8052). The literature regarding Drosophila insulator proteins is overwhelming, and we apologize to the authors whose studies we have failed to cite. References [1] Ghirlando, R., Giles, K., Gowher, H., Xiao, T., Xu, Z., Yao, H. and Felsenfeld, G. (2012) Chromatin domains, insulators, and the regulation of gene expression. Biochim. Biophys. Acta 1819, 644–651. [2] Herold, M., Bartkuhn, M. and Renkawitz, R. (2012) CTCF: insights into insulator function during development. Development 139, 1045–1057.

[3] Cai, H. and Levine, M. (1995) Modulation of enhancer–promoter interactions by insulators in the Drosophila embryo. Nature 376, 533–536. [4] Geyer, P.K. and Corces, V.G. (1992) DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6, 1865–1873. [5] Kellum, R. and Schedl, P. (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64, 941–950. [6] Kellum, R. and Schedl, P. (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol. Cell. Biol. 12, 2424–2431. [7] Scott, K.S. and Geyer, P.K. (1995) Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes. EMBO J. 14, 6258–6267. [8] West, A.G., Gaszner, M. and Felsenfeld, G. (2002) Insulators: many functions, many mechanisms. Genes Dev. 16, 271–288. [9] Bischof, J., Maeda, R.K., Hediger, M., Karch, F. and Basler, K. (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317. [10] Maksimenko, O., Golovnin, A. and Georgiev, P. (2008) Enhancer–promoter communication is regulated by insulator pairing in a Drosophila model bigenic locus. Mol. Cell. Biol. 28, 5469–5477. [11] Venken, K.J. and Bellen, H.J. (2012) Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and UC31 integrase. Methods Mol. Biol. 859, 203–228. [12] Chetverina, D., Savitskaya, E., Maksimenko, O., Melnikova, L., Zaytseva, O., Parshikov, A., Galkin, A.V. and Georgiev, P. (2008) Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs. Nucleic Acids Res. 36, 929–937. [13] Golovnin, A., Biryukova, I., Romanova, O., Silicheva, M., Parshikov, A., Savitskaya, E., Pirrotta, V. and Georgiev, P. (2003) An endogenous Su(Hw) insulator separates the yellow gene from the achaete-scute gene complex in Drosophila. Development 130, 3249–3258. [14] Parnell, T.J., Viering, M.M., Skjesol, A., Helou, C., Kuhn, E.J. and Geyer, P.K. (2003) An endogenous suppressor of hairy-wing insulator separates regulatory domains in Drosophila. Proc. Natl. Acad. Sci. USA 100, 13436– 13441. [15] Vazquez, J. and Schedl, P. (2000) Deletion of an insulator element by the mutation facet-strawberry in Drosophila melanogaster. Genetics 155, 1297– 1311. [16] Sultana, H., Verma, S. and Mishra, R.K. (2011) A BEAF dependent chromatin domain boundary separates myoglianin and eyeless genes of Drosophila melanogaster. Nucleic Acids Res. 39, 3543–3557. [17] Hagstrom, K., Muller, M. and Schedl, P. (1996) Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev. 10, 3202–3215. [18] Zhou, J., Barolo, S., Szymanski, P. and Levine, M. (1996) The Fab-7 element of the bithorax complex attenuates enhancer–promoter interactions in the Drosophila embryo. Genes Dev. 10, 3195–3201. [19] Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, M., Shanower, G., Schedl, P., Gyurkovics, H. and Karch, F. (2000) The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain. Development 127, 779–790. [20] Schweinsberg, S., Hagstrom, K., Gohl, D., Schedl, P., Kumar, R.P., Mishra, R. and Karch, F. (2004) The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites. Genetics 168, 1371–1384. [21] Gruzdeva, N., Kyrchanova, O., Parshikov, A., Kullyev, A. and Georgiev, P. (2005) The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer–promoter communication. Mol. Cell. Biol. 25, 3682–3689. [22] Belozerov, V.E., Majumder, P., Shen, P. and Cai, H.N. (2003) A novel boundary element may facilitate independent gene regulation in the Antennapedia complex of Drosophila. EMBO J. 22, 3113–3121. [23] Brasset, E., Hermant, C., Jensen, S. and Vaury, C. (2010) The Idefix enhancerblocking insulator also harbors barrier activity. Gene 450, 25–31. [24] Kim, J., Shen, B., Rosen, C. and Dorsett, D. (1996) The DNA-binding and enhancer-blocking domains of the Drosophila suppressor of Hairy-wing protein. Mol. Cell. Biol. 16, 3381–3392. [25] Gaszner, M., Vazquez, J. and Schedl, P. (1999) The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer–promoter interaction. Genes Dev. 13, 2098–2107. [26] Moon, H., Filippova, G., Loukinov, D., Pugacheva, E., Chen, Q., Smith, S.T., Munhall, A., Grewe, B., Bartkuhn, M., Arnold, R., Burke, L.J., Renkawitz-Pohl, R., Ohlsson, R., Zhou, J., Renkawitz, R. and Lobanenkov, V. (2005) CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep. 6, 165–170. [27] Parnell, T.J., Kuhn, E.J., Gilmore, B.L., Helou, C., Wold, M.S. and Geyer, P.K. (2006) Identification of genomic sites that bind the Drosophila suppressor of Hairy-wing insulator protein. Mol. Cell. Biol. 26, 5983–5993. [28] Kuhn-Parnell, E.J., Helou, C., Marion, D.J., Gilmore, B.L., Parnell, T.J., Wold, M.S. and Geyer, P.K. (2008) Investigation of the properties of non-gypsy suppressor of hairy-wing-binding sites. Genetics 179, 1263–1273. [29] Schwartz, Y.B., Linder-Basso, D., Kharchenko, P.V., Tolstorukov, M.Y., Kim, M., Li, H.B., Gorchakov, A.A., Minoda, A., Shanower, G., Alekseyenko, A.A., Riddle, N.C., Jung, Y.L., Gu, T., Plachetka, A., Elgin, S.C., Kuroda, M.I., Park, P.J., Savitsky, M., Karpen, G.H. and Pirrotta, V. (2012) Nature and function of insulator protein binding sites in the Drosophila genome. Genome Res. 22, 2188–2198.

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14 [30] Mohan, M., Bartkuhn, M., Herold, M., Philippen, A., Heinl, N., Bardenhagen, I., Leers, J., White, R.A., Renkawitz-Pohl, R., Saumweber, H. and Renkawitz, R. (2007) The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J. 26, 4203–4214. [31] Holohan, E.E., Kwong, C., Adryan, B., Bartkuhn, M., Herold, M., Renkawitz, R., Russell, S. and White, R. (2007) CTCF genomic binding sites in Drosophila and the organisation of the bithorax complex. PLoS Genet. 3, e112. [32] Negre, N., Brown, C.D., Ma, L., Bristow, C.A., Miller, S.W., Wagner, U., Kheradpour, P., Eaton, M.L., Loriaux, P., Sealfon, R., Li, Z., Ishii, H., Spokony, R.F., Chen, J., Hwang, L., Cheng, C., Auburn, R.P., Davis, M.B., Domanus, M., Shah, P.K., Morrison, C.A., Zieba, J., Suchy, S., Senderowicz, L., Victorsen, A., Bild, N.A., Grundstad, A.J., Hanley, D., MacAlpine, D.M., Mannervik, M., Venken, K., Bellen, H., White, R., Gerstein, M., Russell, S., Grossman, R.L., Ren, B., Posakony, J.W., Kellis, M. and White, K.P. (2011) A cis regulatory map of the Drosophila genome. Nature 471, 527–531. [33] Blanton, J., Gaszner, M. and Schedl, P. (2003) Protein:protein interactions and the pairing of boundary elements in vivo. Genes Dev. 17, 664–675. [34] Kyrchanova, O., Toshchakov, S., Podstreshnaya, Y., Parshikov, A. and Georgiev, P. (2008) Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene. Mol. Cell. Biol. 28, 4188–4195. [35] Kyrchanova, O., Chetverina, D., Maksimenko, O., Kullyev, A. and Georgiev, P. (2008) Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements. Nucleic Acids Res. 36, 7019– 7028. [36] Büchner, K., Roth, P., Schotta, G., Krauss, V., Saumweber, H., Reuter, G. and Dorn, R. (2000) Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila. Genetics 155, 141–157. [37] Gause, M., Morcillo, P. and Dorsett, D. (2001) Insulation of enhancer– promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between suppressor of hairy-wing and modifier of mdg4 proteins. Mol. Cell. Biol. 21, 4807–4817. [38] Ghosh, D., Gerasimova, T.I. and Corces, V.G. (2001) Interactions between the Su(Hw) and Mod(mdg4) proteins required for gypsy insulator function. EMBO J. 20, 2518–2527. [39] Pai, C.Y., Lei, E.P., Ghosh, D. and Corces, V.G. (2004) The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol. Cell 16, 737– 748. [40] Golovnin, A., Mazur, A., Kopantseva, M., Kurshakova, M., Gulak, P.V., Gilmore, B., Whitfield, W.G., Geyer, P., Pirrotta, V. and Georgiev, P. (2007) Integrity of the Mod(mdg4)-67.2 BTB domain is critical to insulator function in Drosophila melanogaster. Mol. Cell. Biol. 27, 963–974. [41] Gerasimova, T.I., Lei, E.P., Bushey, A.M. and Corces, V.G. (2007) Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila. Mol. Cell 28, 761–772. [42] Espinas, M.L., Jimenez-Garcia, E., Vaquero, A., Canudas, S., Bernues, J. and Azorin, F. (1999) The N terminal POZ domain of GAGA mediates the formation of oligomers that bind DNA with high affinity and specificity. J. Biol. Chem. 274, 16461–16469. [43] Lu, Q., Wallrath, L.L., Granok, H. and Elgin, S.C. (1993) (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene. Mol. Cell. Biol. 13, 2802–2814. [44] Stogios, P.J., Downs, G.S., Jauhal, J.J., Nandra, S.K. and Prive, G.G. (2005) Sequence and structural analysis of BTB domain proteins. Genome Biol. 6, R82. [45] Zollman, S., Godt, D., Prive, G.G., Couderc, J.L. and Laski, F.A. (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl Acad. Sci. USA 91, 10717–10721. [46] Mahmoudi, T., Katsani, K.R. and Verrijzer, C.P. (2002) GAGA can mediate enhancer function in trans by linking two separate DNA molecules. EMBO J. 21, 1775–1781. [47] Bonchuk, A., Denisov, S., Georgiev, P. and Maksimenko, O. (2011) Drosophila BTB/POZ domains of ‘‘ttk group’’ can form multimers and selectively interact with each other. J. Mol. Biol. 412, 423–436. [48] Pagans, S., Ortiz-Lombardía, M., Espinás, M.L., Bernués, J. and Azorín, F. (2002) The Drosophila transcription factor tramtrack (TTK) interacts with Trithoraxlike (GAGA) and represses GAGA-mediated activation. Nucleic Acids Res. 30, 4406–4413. [49] Melnikova, L., Juge, F., Gruzdeva, N., Mazur, A., Cavalli, G. and Georgiev, P. (2004) Interaction between the GAGA factor and Mod(mdg4) proteins promotes insulator bypass in Drosophila. Proc. Natl. Acad. Sci. USA 101, 14806–14811. [50] Bartoletti, M., Rubin, T., Chalvet, F., Netter, S., Dos Santos, N., Poisot, E., PacesFessy, M., Cumenal, D., Peronnet, F., Pret, A.M. and Théodore, L. (2012) Genetic basis for developmental homeostasis of germline stem cell niche number: a network of Tramtrack-Group nuclear BTB factors. PLoS One 7, e49958. [51] Soshnev, A.A., He, B., Baxley, R.M., Jiang, N., Hart, C.M., Tan, K. and Geyer, P.K. (2012) Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary. Nucleic Acids Res. 40, 5415–5431. [52] Soshnev, A.A., Baxley, R.M., Manak, J.R., Tan, K. and Geyer, P.K. (2013) The insulator protein suppressor of Hairy-wing is an essential transcriptional repressor in the Drosophila ovary. Development 140, 3613–3623.

13

[53] Zhao, K., Hart, C.M. and Laemmli, U.K. (1995) Visualization of chromosomal domains with boundary element-associated factor BEAF-32. Cell 81, 879– 889. [54] Hart, C.M., Zhao, K. and Laemmli, U.K. (1997) The scs’ boundary element: characterization of boundary element associated factors. Mol. Cell. Biol. 17, 999–1009. [55] Gilbert, M.K., Tan, Y.Y. and Hart, C.M. (2006) The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure. Genetics 173, 1365–1375. [56] Emberly, E., Blattes, R., Schuettengruber, B., Hennion, M., Jiang, N., Hart, C.M., Käs, E. and Cuvier, O. (2008) BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dualcore binding sites. PLoS Biol. 6, 2896–2910. [57] Jiang, N., Emberly, E., Cuvier, O. and Hart, C.M. (2009) Genome-wide mapping of boundary element-associated factor (BEAF) binding sites in Drosophila melanogaster links BEAF to transcription. Mol. Cell. Biol. 29, 3556–3568. [58] Aoki, T., Sarkeshik, A., Yates, J. and Schedl, P. (2012) Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex. Elife 1, e00171. [59] Kuhn, E.J., Viering, M.M., Rhodes, K.M. and Geyer, P.K. (2003) A test of insulator interactions in Drosophila. EMBO J. 22, 2463–2471. [60] Kyrchanova, O., Leman, D., Parshikov, A., Fedotova, A., Studitsky, V., Maksimenko, O. and Georgiev, P. (2013) New properties of Drosophila scs and scs’ insulators. PLoS One 8, e62690. [61] Kyrchanova, O., Maksimenko, O., Stakhov, V., Ivlieva, T., Parshikov, A., Studitsky, V.M. and Georgiev, P. (2013) Effective blocking of the white enhancer requires cooperation between two main mechanisms suggested for the insulator function. PLoS Genet. 9, e1003606. [62] Kostyuchenko, M., Savitskaya, E., Koryagina, E., Melnikova, L., Karakozova, M. and Georgiev, P. (2009) Zeste can facilitate long-range enhancer–promoter communication and insulator bypass in Drosophila melanogaster. Chromosoma 118, 665–674. [63] Majumder, P. and Cai, H.N. (2003) The functional analysis of insulator interactions in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 100, 5223– 5228. [64] Rodin, S., Kyrchanova, O., Pomerantseva, E., Parshikov, A. and Georgiev, P. (2007) New properties of Drosophila Fab-7 insulator. Genetics 177, 113–121. [65] Chopra, V.S., Cande, J., Hong, J.W. and Levine, M. (2009) Stalled Hox promoters as chromosomal boundaries. Genes Dev. 23, 1505–1509. [66] Gohl, D., Aoki, T., Blanton, J., Shanower, G., Kappes, G. and Schedl, P. (2011) Mechanism of chromosomal boundary action: roadblock, sink, or loop? Genetics 187, 731–748. [67] Erokhin, M., Davydova, A., Parshikov, A., Studitsky, V.M., Georgiev, P. and Chetverina, D. (2013) Transcription through enhancers suppresses their activity in Drosophila. Epigenetics Chromatin 6, 31. [68] Scott, K.C., Taubman, A.D. and Geyer, P.K. (1999) Enhancer blocking by the Drosophila gypsy insulator depends upon insulator anatomy and enhancer strength. Genetics 153, 787–798. [69] Cai, H.N. and Shen, P. (2001) Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291, 493–495. [70] Muravyova, E., Golovnin, A., Gracheva, E., Parshikov, A., Belenkaya, T., Pirrotta, V. and Georgiev, P. (2001) Loss of insulator activity by paired Su(Hw) chromatin insulators. Science 291, 495–498. [71] Brasset, E., Bantignies, F., Court, F., Cheresiz, S., Conte, C. and Vaury, C. (2007) Idefix insulator activity can be modulated by nearby regulatory elements. Nucleic Acids Res. 35, 2661–2670. [72] Sigrist, C.J.A. and Pirrotta, V. (1997) Chromatin insulator elements block the silencing of a target gene by the Drosophila Polycomb response element (PRE) but allow trans interactions between PREs on different chromosomes. Genetics 147, 209–221. [73] Mallin, D.R., Myung, J.S., Patton, J.S. and Geyer, P.K. (1998) Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator. Genetics 148, 331–339. [74] Comet, I., Savitskaya, E., Schuettengruber, B., Nègre, N., Lavrov, S., Parshikov, A., Juge, F., Gracheva, E., Georgiev, P. and Cavalli, G. (2006) PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter. Dev. Cell 11, 117–124. [75] Comet, I., Schuettengruber, B., Sexton, T. and Cavalli, G. (2011) A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber. Proc. Natl. Acad. Sci. USA 108, 2294–2299. [76] Savitskaya, E., Melnikova, L., Kostuchenko, M., Kravchenko, E., Pomerantseva, E., Boikova, T., Chetverina, D., Parshikov, A., Zobacheva, P., Gracheva, E., Galkin, A. and Georgiev, P. (2006) Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer– promoter communication in Drosophila melanogaster. Mol. Cell. Biol. 26, 754–761. [77] Kyrchanova, O., Toshchakov, S., Parshikov, A. and Georgiev, P. (2007) Study of the functional interaction between Mcp insulators from the Drosophila bithorax complex: effects of insulator pairing on enhancer–promoter communication. Mol. Cell. Biol. 27, 3035–3043. [78] Krivega, M., Savitskaya, E., Krivega, I., Karakozova, M., Parshikov, A., Golovnin, A. and Georgiev, P. (2010) Interaction between a pair of gypsy insulators or between heterologous gypsy and Wari insulators modulates Flp site-specific recombination in Drosophila melanogaster. Chromosoma 119, 425–434.

14

O. Kyrchanova, P. Georgiev / FEBS Letters 588 (2014) 8–14

[79] Petrascheck, M., Escher, D., Mahmoudi, T., Verrijzer, C.P., Schaffner, W. and Barberis, A. (2005) DNA looping induced by a transcriptional enhancer in vivo. Nucleic Acids Res. 33, 3743–3750. [80] Katsani, K.R., Hajibagheri, M.A. and Verrijzer, C.P. (1999) Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology. EMBO J. 18, 698–708. [81] Wood, A.M., Van Bortle, K., Ramos, E., Takenaka, N., Rohrbaugh, M., Jones, B.C., Jones, K.C. and Corces, V.G. (2011) Regulation of chromatin organization and inducible gene expression by a Drosophila insulator. Mol. Cell 44, 29–38. [82] Wu, C.T. and Morris, J.R. (1999) Transvection and other homology effects. Curr. Opin. Genet. Dev. 9, 237–246. [83] Soltani-Bejnood, M., Thomas, S.E., Villeneuve, L., Schwartz, K., Hong, C.S. and McKee, B.D. (2007) Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis. Genetics 176, 161–180. [84] Chen, J.L., Huisinga, K.L., Viering, M.M., Ou, S.A., Wu, C.T. and Geyer, P.K. (2002) Enhancer action in trans is permitted throughout the Drosophila genome. Proc. Natl. Acad. Sci. USA 99, 3723–3728. [85] Kravchenko, E., Savitskaya, E., Kravchuk, O., Parshikov, A., Georgiev, P. and Savitsky, M. (2005) Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome. Mol. Cell. Biol. 25, 9283– 9291. [86] Li, H.B., Müller, M., Bahechar, I.A., Kyrchanova, O., Ohno, K., Georgiev, P. and Pirrotta, V. (2011) Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster. Mol. Cell. Biol. 31, 616–625. [87] Muller, M., Hagstrom, K., Gyurkovics, H., Pirrotta, V. and Schedl, P. (1999) The Mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions. Genetics 153, 1333–1356. [88] Vazquez, J., Müller, M., Pirrotta, V. and Sedat, J.W. (2006) The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila. Mol. Biol. Cell. 17, 2158–2165. [89] Erokhin, M., Davydova, A., Kyrchanova, O., Parshikov, A., Georgiev, P. and Chetverina, D. (2011) Insulators form gene loops by interacting with promoters in Drosophila. Development 138, 4097–4106. [90] Liu, Z., Scannell, D.R., Eisen, M.B. and Tjian, R. (2011) Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146, 720– 731.

[91] Chopra, V.S., Srinivasan, A., Kumar, R.P., Mishra, K., Basquin, D., Docquier, M., Seum, C., Pauli, D. and Mishra, R.K. (2008) Transcriptional activation by GAGA factor is through its direct interaction with dmTAF3. Dev. Biol. 317, 660–670. [92] Golovnin, A., Melnick, E., Mazu, R.A. and Georgiev, P. (2005) Drosophila Su(Hw) insulator can stimulate transcription of a weakened yellow promoter over a distance. Genetics 170, 1133–1142. [93] Vorobyeva, N.E., Mazina, M.U., Golovnin, A.K., Kopytova, D.V., Gurskiy, D.Y., Nabirochkina, E.N., Georgieva, S.G., Georgiev, P.G. and Krasnov, A.N. (2013) Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome. Nucleic Acids Res. 41, 5717–5730. [94] Bondarenko, V.A., Jiang, Y.I. and Studitsky, V.M. (2003) Rationally designed insulator like elements can block enhancer action in vitro. EMBO J. 22, 4728– 4737. [95] Hou, C., Zhao, H., Tanimoto, K. and Dean, A. (2008) CTCF-dependent enhancer blocking by alternative chromatin loop formation. Proc. Natl. Acad. Sci. USA 105, 20398–20403. [96] Mukhopadhyay, S., Schedl, P., Studitsky, V.M. and Sengupta, A.M. (2011) Theoretical analysis of the role of chromatin interactions in long-range action of enhancers and insulators. Proc. Natl. Acad. Sci. USA 108, 19919–19924. [97] Gause, M., Hovhannisyan, H., Kan, T., Kuhfittig, S., Mogila, V. and Georgiev, P. (1998) Hobo Induced rearrangements in the yellow locus influence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster. Genetics 149, 1393–1405. [98] Geyer, P.K. (1997) The role of insulator elements in defining domains of gene expression. Curr. Opin. Genet. Dev. 7, 242–248. [99] Maeda, R.K. and Karch, F. (2009) The bithorax complex of Drosophila an exceptional Hox cluster. Curr. Top. Dev. Biol. 88, 1–33. [100] Gibcus, J.H. and Dekker, J. (2013) The hierarchy of the 3D genome. Mol. Cell 49, 773–782. [101] Tanay, A. and Cavalli, G. (2013) Chromosomal domains: epigenetic contexts and functional implications of genomic compartmentalization. Curr. Opin. Genet. Dev. 23, 197–203. [102] Sexton, T., Yaffe, E., Kenigsberg, E., Bantignies, F., Leblanc, B., Hoichman, M., Parrinello, H., Tanay, A. and Cavalli, G. (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148, 458– 472.