InteractiveFly: GeneBrief

Centrosomal protein 190kD: Biological Overview | References

BIOLOGICAL OVERVIEW

Centrosomes are the main microtubule (MT)-organizing centers in animal cells (see The centrosome cycle in mammalian cells from Azimzadeh, 2007), but they also influence the actin/myosin cytoskeleton. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis (Whitfield, 1988; full text of article), and binds to MTs directly in vitro (Oegema, 1995; Kellogg, 1995). CP190 has an essential function in the nucleus as a chromatin insulator (Pai, 2004), but centrosomes and MTs appear unperturbed in Cp190 mutants (Pai, 2004; Baker, 1993). Thus, the centrosomal function of CP190, if any, is unclear. This study examined the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. It is hypothesized that coordinates CP190 myosin-driven cortical contractions with the cell-cycle state of the internal nuclei. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos (Chodagam, 2005).

CP190 and CP60 are centrosomal microtubule-associated proteins (MAPs) that form a complex and shuttle between the nucleus in interphase and the centrosome in mitosis (Oegema, 1995; Kellogg, 1995). Both proteins interact directly with MTs in vitro, but their concentration at centrosomes does not depend on MTs (Oegema, 1995; Raff, 1993). The CP190 gene is essential for viability, and homozygous mutant animals die during late stages of pupal development (Butcher, 2004). Surprisingly, these mutants have no detectable defects in mitosis, or in any aspect of centrosome or MT behavior. Moreover, a form of CP190 that cannot bind to centrosomes or MTs (CP190ΔM) can rescue the lethality associated with Cp190 mutations, demonstrating that the ability of CP190 to interact with centrosomes and MTs is not essential for fly viability. Recently, CP190 has been shown to act in the nucleus as a chromatin-insulator element that sets up boundaries between different regions of chromatin (Pai, 2004). Thus, CP190 appears to have essential functions in the nucleus, but its function at the centrosome, if any, remains unclear (Chodagam, 2005).

Several Drosophila centrosomal proteins are essential for the rapid rounds of mitosis that occur in the early embryo but are dispensable for mitosis at later stages of development. Therefore, whether CP190 might have an essential role at the centrosome during early embryogenesis was tested. This was not possible previously because CP190 mutant flies are inviable as a result of the nuclear requirements for CP190, and mutant flies rescued by CP190ΔM are generally unhealthy and are sterile (Butcher, 2004). Therefore the Cp190¹ and Cp190² mutations were recombined onto an FRT chromosome so that germline clone (GLC) embryos could be generated (hereafter referred to as CP190GLCs). These embryos develop from heterozygous females whose germline is homozygous for the Cp190 mutation. CP190GLCs from either mutant contained essentially undetectable levels of the CP190 protein, and similar results were obtained with both alleles. Although CP190 was no longer detectable at centrosomes, mitotic spindles appeared to function normally, and the centrosomal localization of γ-tubulin, CNN, D-TACC, and Msps was largely unperturbed (Chodagam, 2005).

Although centrosomes and MTs appeared to behave normally in CP190GLCs, it was noticed that these embryos had a defect in axial expansion. In syncytial Drosophila embryos, the first zygotic nucleus is usually positioned toward the anterior. During nuclear cycles 4-7, the process of axial expansion causes the nuclei to spread out along the anterior-to-posterior axis so that, by nuclear cycle 7-8, they are distributed evenly throughout the length of the embryo. In CP190GLCs, axial expansion failed, and the nuclei remained abnormally clustered at the anterior of the embryo (Chodagam, 2005).

Axial expansion is a highly coordinated contractile process that requires both actin and cytoplasmic myosin II. A live analysis of myosin behavior, labeled by virtue of GFP-tagged myosin regulatory light chain (RLC, an obligatory subunit of functional myosin II), has shown that during axial expansion myosin undergoes cycles of recruitment to and dispersion from the cortex, in coordination with the nuclear-division cycles of the internal nuclei (Royou, 2002). Recruitment occurs during mitotic interphase and promotes a cortical contraction that is thought to drive axial expansion. This cyclical cortical recruitment of myosin requires the phosphorylation of one of the activating residues of the RLC (Royou, 2002) but does not require either microtubules (Royou, 2002) or an intact actin network (it is not perturbed by cytochalasin or latrunculin injection) (Chodagam, 2005).

To test if these cycles of myosin accumulation occurred in CP190GLCs, RLC-GFP behavior was examined in CP190GLCs. In optical sections of wild-type (WT) embryos expressing one copy of RLC-GFP, cycles of myosin cortical accumulation and dispersion were observed prior to the arrival of the nuclei at the cortex, and these continued when the nuclei were at the cortex, with RLC-GFP being strongly recruited to the cortex in interphase and dispersing from the cortex during mitosis. By contrast, in CP190GLCs expressing one copy of RLC-GFP, only very weak cycles of myosin II accumulation at the cortex could be observed, and these were more uneven than those seen in WT embryos. Even after the nuclei had arrived at the cortex, the accumulation of RLC-GFP at the cortex in interphase was much weaker in CP190GLCs than in WT embryos. Surprisingly, however, the subsequent accumulation of RLC-GFP at the leading edge of the cellularization furrows was equally strong in CP190GLCs and WT embryos. Moreover, in cellularized embryos, the accumulation of RLC-GFP in contractile rings during cytokinesis also appeared to occur normally in CP190GLCs. Thus, the organization of myosin appears to be disrupted in CP190GLCs specifically during the syncytial phase of embryogenesis (Chodagam, 2005).

That myosin organization was disrupted in CP190GLCs was confirmed by immunostaining fixed embryos with an anti-myosin heavy chain (MHC) antibody. Although MHC staining was strong in the cortical regions surrounding the nuclei of WT embryos, in CP190GLCs, MHC staining was much reduced and more irregular. As was the case with RLC-GFP, the localization of MHC to the leading edge of the cellularization furrow appeared to be normal in CP190GLCs (Chodagam, 2005).

Although myosin II behavior was profoundly disrupted in CP190GLCs, actin organization appeared to be unperturbed. In CP190GLC blastoderm embryos, cortical actin caps form over each nucleus, just as in WT. In preblastoderm WT embryos, a network of actin fibers and granules lies below the actin-rich cortex, and an actin-rich 'central domain' is associated with the internal nuclei during axial expansion; actin is also concentrated around the centrosomes during these early syncytial divisions. All these features of actin organization were maintained in CP190GLCs (Chodagam, 2005).

These observations suggested that the failure in axial expansion in CP190GLCs is due to a failure to properly recruit cortical myosin. Western blotting confirmed that the levels of MHC were not altered in CP190GLCs. To test whether CP190 might act upstream of myosin activation, it was asked whether an 'activated' RLC could rescue the axial expansion defect in CP190GLCs. The phosphorylation of the myosin RLC (on Ser-19 and, secondarily, on Thr-18 in vertebrates; these correspond to Ser-21 and Thr-20 in Drosophila) is required for myosin II motor activity. Blocking RLC phosphorylation, either by using mutant forms of the RLC in which these residues have been replaced by alanines (RLC-A20,A21) or by inhibiting Rho Kinase, whose activity is required for phosphorylating these residues, renders myosin II non-functional, eliminates its cortical localization, and leads to a failure in axial expansion. In contrast, replacement of these sites by phospho-mimetic glutamates (RLC-E20,E21) restores activity, as defined genetically, and appears to render the myosin constitutively active. Thus, phosphorylation is essential for the function and localization of myosin (Chodagam, 2005).

It was found that expression of one copy of a transgene encoding the activated form of RLC (RLC-E20,E21) partially rescues both the axial-expansion defects and myosin cortical recruitment in CP190GLCs. Importantly, the expression of one copy of this transgene in WT flies had no effect on axial expansion, and the expression of one copy of a WT RLC-GFP transgene did not rescue the CP190GLC axial-expansion defect. Thus, an 'activated' form of RLC can recruit MHC to the cortex during interphase and can rescue the axial-expansion defect in CP190GLCs, strongly suggesting that CP190 normally acts upstream of myosin II activation to regulate axial expansion (Chodagam, 2005).

A form of CP190 lacking the centrosomal and MT binding domain of CP190 (CP190ΔM) can rescue the adult lethality associated with mutations in the CP190 gene (Butcher, 2004), presumably because this form of the protein can still function as a chromatin insulator in the nucleus. Therefore whether the axial-expansion defects of the CP190GLCs could also be rescued by CP190ΔM was tested. In CP190GLCs that expressed the full-length CP190 protein driven from the polyubiquitin promoter, the axial-expansion defect was strongly suppressed, and the transgenically supplied CP190 localized to centrosomes. In CP190GLCs expressing CP190ΔM driven from the polyubiquitin promoter, the axial-expansion defect was not significantly rescued and CP190ΔM did not localize to centrosomes. Thus, it appears that CP190 requires its centrosome/MT binding domain to function properly in axial expansion (Chodagam, 2005).

How might CP190 influence myosin activity? The cycles of cortical myosin II recruitment that drive axial expansion are regulated by oscillations in the activity of Cdc2-Cyclin B, with levels of cortical myosin being high in interphase and low in mitosis (Royou, 2002). This regulation is probably indirect; Cdc2-Cyclin B activity varies only locally around the nuclei during early embryo development, and cycles of myosin recruitment are initiated at the cortex long before the nuclei arrive there. Moreover, although Cdc2-Cyclin B can directly phosphorylate RLC in vitro, the removal of the potential Cdc2 phosphorylation sites in Drosophila RLC alters neither the myosin II recruitment cycles nor the ability of myosin to drive axial expansion (Royou, 2002). How local fluctuations in Cdc2-Cyclin B activity around the nuclei direct cycles of myosin recruitment at the cortex is therefore unclear, but it is speculated that CP190 plays a role in facilitating this process (Chodagam, 2005).

Cdc2-Cyclin B, for example, could regulate myosin by regulating the activity and/or localization of Drosophila rho kinase (Drok). This kinase is required for axial expansion (Royou, 2002), it regulates myosin II activity via phosphorylation of Thr-20 and Ser-21, and it is concentrated at centrosomes in at least some cell types. Perhaps CP190 facilitates the activation of Drok at centrosomes or the targeting of Drok from centrosomes to the embryo cortex (either by diffusion or along MTs). It has been shown previously that MTs are not essential for the cycling of myosin at the cortex (Royou, 2002), but these studies were performed when the nuclei had already reached the embryo cortex. Perhaps MTs are essential for the long-range signaling that must occur between the cortex and the nuclei/centrosomes during axial expansion. Because the interaction of CP190 with centrosomes and MTs is regulated during the cell cycle (Oegema, 1995; Kellogg, 1995), the involvement of CP190 in this process could ensure that the myosin-driven cortical contractions are coordinated with the cell-cycle state of the internal nuclei (Chodagam, 2005).

These data suggest that, whatever its mechanism, CP190 serves as a crucial link between the centrosome/MT and actin/myosin cytoskeletal networks during the early stages of Drosophila embryonic development. This mechanism may be specific for organisms that have a syncytial phase of development and so require that centrosomes influence actin/myosin behavior over considerable distances. Indeed, no obvious orthologs of CP190 have been identified on the basis of sequence homology in species other than insects. On the other hand, the fertilized eggs of many species are very large, and special mechanisms that allow the long-range communication between the centrosomes and the cortical myosin network may be required in these systems (Chodagam, 2005).

dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes

dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts, the distribution and localization of Drosophila E2F and dREAM proteins were examined. This study reports a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains (Korenjak, 2014).

The large number of E2F proteins has precluded all attempts to generate a comprehensive set of E2F binding sites in the human genome. This study has taken advantage of the simplicity of the Drosophila E2F family to examine the genome-wide distribution of E2F proteins. An unexpected finding from this analysis is that dE2F proteins strongly co-localize with insulator-binding proteins. The extent of overlap between dE2F2/dREAM and CP190 binding sites is comparable to the co-localization previously described for dCTCF and CP190, which are both required for insulator function at common binding sites. Furthermore, the co-localization between dREAM and Beaf-32 is even greater than that observed for Beaf-32 and CP190. This striking overlap of dREAM binding sites with proteins involved in nuclear architecture has exciting new implications for the function of dREAM complexes (Korenjak, 2014).

An interesting feature of dREAM bound genes is their strong enrichment in divergently paired genes (DPGs), genes that are transcribed in opposite direction, with their TSS separated by less than 1000 bp. Moreover, the set of genes de-regulated upon loss of dE2F2/dREAM include mostly DPGs that are differentially expressed, with one gene of the pair being stably repressed whereas its partner is actively transcribed. Inactivation of dREAM complex subunits or CP190 results in the loss of transcriptional integrity at these differentially expressed DPGs (Korenjak, 2014).

Several different models could account for the observed transcriptional up-regulation of the stably repressed and down-regulation of the actively expressed gene. First, dREAM/CP190/Beaf-32 sites might act as boundary elements at DPGs, separating an active from a repressed chromatin domain. Genome-wide binding maps for insulator-binding proteins revealed enriched binding to DPGs. In addition, these studies have shown that CP190 and Beaf-32 binding are significantly enriched at differentially expressed DPGs. The exact role of insulator-binding proteins at differentially expressed DPGs is still unclear. A recent study has shown that, upon inactivation of the SOX14 transcription regulator, DPGs that lack Beaf-32 binding show a significantly higher likelihood of concerted de-regulation of the two genes within a pair (up or down) than when Beaf-32 is present at the DPG. This suggests a role for Beaf-32 in the maintenance of independent regulation of gene expression at DPGs, consistent with a function as boundary factor. Moreover, CP190 binding sites are commonly found at the borders of large H3K27me3 domains, which are a hallmark of Polycomb-mediated silencing. These studies further show that inactivation of CP190 can, at a subset of regions, result in local spreading of H3K27me3 beyond the CP190 binding site. Although the repressive mechanisms might vary at different dREAM-regulated DPGs, several of the stably repressed genes display H3K27me3 over the length of the gene body. The possibility of dREAM and CP190 being important for the physical separation of distinct chromatin domains was tested by assessing the distribution of H3K27me3 over selected gene pairs. In agreement with the observed de-repression of the inactive gene, mutant animals displayed loss of H3K27me3 in the gene body. However, spreading of the mark into the active gene was not observed, suggesting that the down-regulation is achieved by a different mechanism or the level of reduction in gene expression is below the detection limit of our H3K27me3 ChIP (Korenjak, 2014).

Second, dREAM, together with CP190 and possibly Beaf-32, may be involved in the silencing of stably repressed genes. The repressive mechanism might include specific activities provided by CP190/Beaf-32. Both CP190 and Beaf-32 have been shown to be critical for the establishment of long-range chromatin interactions through looping mechanisms, which could be utilized to physically separate a stably repressed gene from the surrounding transcriptionally active chromatin environment. It is intriguing to speculate that dREAM, either by facilitating chromatin association of CP190 or even more directly, could be involved in the formation of these chromatin loops. Interestingly, the CP190 and Beaf-32 binding sites involved in long-range chromatin interactions are also prominent dREAM binding sites. Alternatively, Beaf-32 is known to compete for DNA binding with the transcriptional activator DNA replication-related element factor (DREF) (70). It has further been shown that, upon inactivation of Beaf-32, bound genes are specifically de-repressed when they also contain a DREF consensus site. In this scenario, loss of the repressive mechanism by inactivation of dREAM or CP190/Beaf-32 might either generate a vacant binding site for a transcription activator or result in the re-distribution of the general transcription machinery or a specific transcription activator from the actively expressed to the repressed gene (Korenjak, 2014).

Third, the possibility cannot formally be ruled out that dREAM acts on both components of differentially expressed DPGs, serving as a repressor for one and as an activator for the other gene. dREAM has been implicated in transcriptional repression as well as activation. However, dREAM complexes containing dE2F2 do not appear to be involved in gene activation and, based on genome-wide binding studies for dREAM subunits, there is no evidence for the presence of two independent dREAM peaks at differentially expressed DPGs (Korenjak, 2014).

Genome-wide association studies have identified a large number of binding sites for insulator-binding proteins, but only a few studies have addressed the potential enhancer-blocking activity of these DNA fragments. In these experiments, CP190 and dCTCF co-bound regions display strong enhancer-blocking activity compared to Su(Hw) bound sites. Moreover, it has been shown that sites associated with CP190 or CP190 and Beaf-32 exhibit strong enhancer-blocking activity, whereas sites occupied by any other combination of insulator-binding proteins show only weak or no enhancer blocking. In agreement with the importance of CP190 for the definition of elements with enhancer-blocking activity, dREAM-CP190 co-bound regions display robust enhancer-blocking in a cell-based assay system. The notion that dREAM functions as an enhancer-blocker may explain why a complex that is best known as a transcriptional repressor is almost exclusively found in euchromatic regions (Korenjak, 2014).

The dREAM subunits Mip130 and Mip120 are important for the observed enhancer-blocking activity, but the underlying mechanism is unclear. Work in Drosophila suggests that the propagation of a nucleosome-free region in insulator elements is required for enhancer blocking. It is possible that dREAM is needed for the establishment of nucleosome-free regions through recruitment of chromatin modifying activities or their maintenance through binding and stabilization of these regions, which in turn might be important for the recruitment of CP190 (Korenjak, 2014).

A detailed analysis of the enhancer-blocking function of the 1A2 insulator, which harbors Su(Hw) binding sites, shows that the region adjacent to the Su(Hw) sites is important for full enhancer-blocking activity of 1A2, even though this element by itself lacks any activity. It is conceivable that dREAM binding sites fulfill a similar 'facilitator' function for CP190 and/or Beaf-32 (Korenjak, 2014).

Although cell-based assays have been effectively used to measure the enhancer-blocking activity of characterized insulator elements, transfected plasmids are only partially chromatinized. In order to address the enhancer-blocking function of dREAM/CP190-bound regions in more detail and dissect the underlying mechanism it will, therefore, be interesting to test the identified elements in an in vivo enhancer-blocking assay (Korenjak, 2014).

The underlying mechanism(s) by which dREAM cooperates with CP190 is likely to be connected with the ability of dREAM to help establish or maintain CP190 chromatin association. Interestingly, CP190, but not Beaf-32 chromatin binding was reduced in dE2f2 mutant animals. Beaf-32 can bind to DNA in a sequence-specific manner through recognition of the CGATA motif. In contrast, CP190 does not possess known DNA-binding activity and is thought to get recruited indirectly by DNA-binding factors. This view is based on physical and functional interactions with sequence-specific insulator-binding proteins as well as the high degree of co-localization of these factors in genomic binding studies. Despite the high degree of overlap in their genomic binding profiles, the significance of Beaf-32 for CP190 chromatin association is unclear. Recent studies found contrary results regarding the significance of Beaf-32 for CP190 recruitment. CP190 chromatin association is reduced, however, in ctcf mutant animals and upon dCTCF knockdown in tissue culture cells. A recent study in Drosophila cells has shown that CP190 associates with the majority of its binding sites in distinct combinatorial patterns with other insulator-binding proteins. At a subset of binding sites, however, it does not co-localize with any known DNA-binding protein, suggesting that CP190 either has intrinsic DNA-binding activity or depends on a not yet identified factor for recruitment. Given the physical interaction between dREAM complex subunits and CP190, it is speculated that dREAM complexes may be directly involved in the recruitment of CP190 to these sites (Korenjak, 2014).

Interestingly, the strong enrichment of DPGs among dREAM-bound genes is conserved in human cells, but, to date, CTCF is the only known mammalian ortholog of fly insulator-binding proteins. Based on the extensive co-localization among insulator-binding proteins in the fly genome and the presence of CP190 as a common insulator co-factor, which suggests a shared mechanism, it has been proposed that the functions of the Drosophila proteins were integrated in CTCF. CP190 is a chromatin architectural protein, and it is conceivable that another protein with similar properties has adopted its role. Interestingly, the function of mammalian CTCF, including its role in chromatin looping, has been intimately linked to the Cohesin complex. Remarkably, inactivation of pRB in mammalian cells results in reduced chromatin association of Cohesin and the functionally related Condensin II-subunit Cap-D3. Furthermore, pRB physically interacts with Condensin II-subunits in fly and human cells and RBF1 co-localizes extensively with Cap-D3 on polytene chromosomes, raising the fascinating possibility that these specialized architectural protein complexes have taken over a CP190-related role in higher organisms (Korenjak, 2014).

Over the past years, several studies have shown that a variety of different proteins co-localize with insulator-binding proteins and/or contribute to insulator function. These factors range from different combinations of insulator proteins to factors like L(3)MBT, Topoisomerase II, the ubiquitin ligase dTopors, Ago2, the Rm62 helicase and exosome components. Further studies are clearly needed to determine how dREAM function is integrated with the array of factors acting in concert with insulator-binding proteins and to delineate a potential role of dREAM complexes in the organization of chromosome architecture (Korenjak, 2014).

Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome

Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. This study investigated the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and developed a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. BEAF32 was shown to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. A general model is proposed for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division (Vogelmann, 2014).

The physical organization of eukaryotic chromosomes is key for a large number of cellular processes, including DNA replication, repair and transcription. Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription by two independent modes of action: 'enhancer blocking' insulators (EB insulators) interfere with communications between regulatory elements and promoters, whereas 'barrier' insulators prevent the spread of silenced chromatin states into neighboring regions. Recently, insulator elements have been implicated in chromosome architecture and transcription regulation through their predicted binding to thousands of sites genome-wide. For instance, insulators were shown to regulate transcription of distinct gene ontologies, to separate distinct epigenetic chromatin states, and to recruit H3K27me3 domains to Polycomb bodies (Vogelmann, 2014).

In Drosophila, five insulator families have been identified, that differ by their DNA-binding protein (insulator binding protein, or IBP): Suppressor of Hairy-wing [Su(Hw)], boundary element-associated factor (BEAF32), Zeste-white 5 (Zw5), the GAGA factor (GAF), and dCTCF, a distant sequence homologue of mammalian CTCF. Two BEAF32 isoforms exist (BEAF32A and BEAF32B). This paper, considers BEAF32B (which will be referred to as BEAF32). BEAF32B represents more than 95% of the binding peaks detected by chip-seq in cell lines, BEAF32A binding does not play a role in the insulating function of BEAF , and BEAF32A expression is not essential for the development of embryos in adult flies. IBPs are often necessary but not sufficient to ensure insulation activity at a specific locus, and several insulator co-factors have been shown to be additionally required. Particularly, Centrosomal Protein 190 (CP190), a protein originally described for its ability to bind to the centrosome during mitosis, was shown to play a crucial role in the insulation function of various IBPs (Vogelmann, 2014).

Insulator proteins often associate in clusters of overlapping binding sites more often than would be expected by chance, suggesting that these factors often bind as a complex to the same genetic locus. For instance, BEAF32, dCTCF and CP190 binding sites most often cluster with at least another factor (~70, ~77 and >90%, respectively). In addition, insulators show a large compositional complexity, as demonstrated by the frequencies of binding of different combinations of insulator associated proteins: CP190 associates with its most common partner BEAF32 (~50%), but also to a lesser extent to dCTCF and Su(HW), while BEAF32, dCTCF, and CP190 cluster together in >15% of CP190 binding sites. This compositional complexity may be key to understanding the locus-specific functions of insulators (Vogelmann, 2014).

A critical feature of Drosophila and vertebrate insulators is their ability to form specific long-range DNA interactions (hereafter LRIs). Three-dimensional loops have been implicated in all levels of chromatin organization ranging from kb-size loops to larger intra-chromosomal loops hundreds of kb in size. To date, it is unclear what factors provide the physical interactions required for the formation and regulation of LRIs. In addition to binding the specific insulator sequences, IBPs have been proposed to be sufficient to bridge two distant DNA molecules. However, other factors such as CP190, Mod(mdg4), or cohesin have been implicated in the formation of LRIs (Vogelmann, 2014).

The observation that most CP190 binding sites co-localize with insulator binding proteins (>90%) prompted the hypothesis that CP190 is a common regulator of different insulator classes. CP190 is composed of a BTB (bric-a-brac, tramrack, and broad complex)/POZ (poxvirus and zinc-finger) domain, four predicted C2H2 zinc-finger motifs, and an E-rich, C-terminal region. Importantly, CP190 has been recently shown to preferentially mark chromatin domain barriers. These barriers are also heavily bound by other insulator proteins, such as BEAF32, dCTCF and to a lesser extent Su(HW), and have been shown to often form LRIs. Overall, these data suggest a role for CP190 in participating in the three dimensional folding of the genome by the formation of long-range interactions (Vogelmann, 2014).

Surprisingly, a second factor, called Chromator, was also shown to be overrepresented at physical domain barriers. During mitosis, Chromator forms a molecular spindle matrix with other nuclear-derived proteins (Skeletor and Megator). In contrast, during interphase Chromator localizes to inter-band regions of polytene chromosomes and plays a role in their structural regulation as well as in transcriptional regulation. Chromator can be divided into two main domains, a C-terminal domain containing a nuclear localization signal, and an N-terminal domain containing a chromo-domain (ChD) required for proper localization to chromatin during interphase (Vogelmann, 2014).

This study investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means. A unique assay was developed to determine what factors are necessary and essential for the formation of long-range DNA interactions, and BEAF32 was shown to be necessary but not sufficient to bridge long-range interactions. In contrast, addition of CP190 or Chromator is sufficient to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several IBPs. A general model is proposed for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). The multiplicity of interactions between insulator binding and associated proteins could thus explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators (Vogelmann, 2014).

Chromatin insulators promote higher-order nuclear organization through the establishment and maintenance of distinct transcriptional domains. Notably, this activity requires the formation of barriers between chromatin domains and the establishment of specific LRIs. In this paper, this paper has investigated the molecular mechanism by which insulator proteins bind DNA, interact with each other and form long-range contacts (Vogelmann, 2014).

Recently, genome-wide approaches have been used to investigate the roles of different insulator types in genome organization. Insulators enriched in both BEAF32 and CP190 are implicated in the segregation of differentially expressed genes and in delimiting the boundaries of silenced chromatin. Notably, BEAF32 and CP190 are often found to bind jointly to the same genetic locus (>50% of CP190 binding sites contain BEAF32). However, the molecular origin of this genome-wide co-localization was unknown as there was no direct proof of interaction between these proteins. This study has shown that BEAF32 is able to interact specifically with CP190 in vitro and in vivo. In particular, this interaction was shown to be mediated by the C-terminal domain of CP190, with no implication of the C2H2 zinc-finger or the BTB/POZ domains, consistent with previous studies showing that the N-terminus of CP190 was not essential for its association with BEAF32 in vivo. BEAF32 interacts specifically and cooperatively with DNA fragments containing CGATA motifs, consistent with previous observations. In contrast, the binding of CP190 to DNA showed lower affinity and no specificity and required its N-terminal domain (containing four C2H2 zinc-fingers). Overall, these data suggest that one pathway for CP190 recruitment to DNA genome-wide requires specific interactions of its C-terminal domain with BEAF32. Other factors, such as GAF, are likely also involved in the recruitment of CP190 to chromatin, explaining why RNAi depletion of BEAF32 does not lead to the dissociation of CP190 from an insulator binding class containing high quantities of BEAF32 and CP190. It is possible that post-translational modifications in CP190 may also allow it to bind DNA directly and specifically, providing a second pathway for locus-specific localization (Vogelmann, 2014).

In addition to acting as chromatin barriers, insulators have been typically characterized for their ability to block interactions between enhancers and promoters through the formation of long-range contacts. Here, this study has developed a fluorescence cross correlation-based assay that allowed investigation of the ability of BEAF32, CP190 and their complex to bridge specific DNA fragments, mimicking LRIs. Specific LRI can be stably formed between two DNA fragments containing BEAF32 binding sites, solely in the presence of both BEAF32 and CP190. Interestingly, LRI are displaced by competition in trans with the BTB/POZ domain of CP190, and LRIs are not observed in the presence of BEAF32 and CP190-C. Thus, both protein domains are required for the bridging activity of CP190. These data strongly suggest that the C-terminal domain is responsible for BEAF32-specific contacts whereas the N-terminal domain of CP190 is involved in the formation of LRI through CP190/CP190 contacts. The role of the N-terminal domain of CP190 in protein-protein interactions is consistent with previous studies showing that N-terminal fragments of CP190 containing the BTB/POZ domains co-localize with full-length CP190 in polytene chromosomes (Vogelmann, 2014).

BTB/POZ are a family of protein-protein interaction motifs conserved from Drosophila to mammals, and present in a variety of transcriptional regulators. BTB/POZ are found primarily at the N-terminus of proteins containing C2H2 zinc-finger motifs, and can be monomeric, dimeric, or multimeric. In fact, a recent study proposed that isolated CP190-BTB/POZ domains can exist as dimers or tetramers in solution. The oligomerization behavior of CP190-BTB/POZ could have important implications for the role and mechanism by which CP190 bridges LRIs. This study showed that the BTB/POZ domains of CP190 forms homo-dimers with a large, conserved interaction surface, consistent with these domains being responsible for the formation of the direct protein-protein interactions required for the establishment of long-range contacts. Interestingly, the oligomerization of CP190-BTB/POZ into homo-dimers implies a binary interaction between two distant DNA sequences, imposing important constraints for the mechanisms of DNA bridging by CP190 (Vogelmann, 2014).

In addition to interacting with BEAF32, CP190 is able to directly interact with other insulator binding proteins, such as dCTCF, Su(HW), and Mod(Mdg4), or with the RNA interference machinery. These interactions are usually mediated by the C-terminal domain of CP190, but a role for the C2H2 zinc-finger or the BTB/POZ domains in providing specific protein-protein contacts cannot be discarded. In fact, an interesting feature of several homo-dimeric BTB/POZ domains is their ability to recruit a multitude of protein partners using a single protein-protein binding interface. For instance, several transcriptional co-repressors (BCOR, SMRT and NCor) are able to bind with micromolar affinity (2:2 stoichiometry) to the BTB/POZ domain of BCL6, despite their low sequence homology. In this case, the mechanism of binding involves the formation of a third strand by the N-terminus of co-repressors folding onto the two strands exchanged by the BCL6-BTB/POZ monomers on their interface, with the rest of the minimal domain of interaction (10 residues) winding up along the lateral groove of the BCL6-BTB/POZ dimer. In the case of CP190, the sequence and structural features of the conserved peptide binding groove within insect CP190-BTB/POZ domains suggest that the dimer interface of CP190 may act as a protein-protein interaction platform. Thus, the ability of BTB/POZ domains to form dimers and the promiscuous binding of CP190 to different insulator binding proteins (Su(HW), dCTCF, and BEAF32) suggest not only that insulators share protein components, but also that CP190 may bridge long-range contacts involving distinct factors at each end of the DNA loop. This model is consistent with previous proposals, and with the requirement of both C- and N-terminal domains of CP190 for fly viability. Importantly, it provides a rationale for CP190 being a common factor between insulator binding proteins (Vogelmann, 2014).

CP190 frequently binds with additional insulator binding proteins (~85%), with BEAF32 and dCTCF being the most common partners (~50% and ~25%, respectively), and Su(Hw) amongst the least frequent partner (~20%). Importantly, BEAF32 does not show clustering with either dCTCF or Su(HW) in the absence of CP190 (<0.5% or ~0.1%, respectively), suggesting that the clustering of two insulator binding proteins requires CP190. The ability of CP190 to mediate LRIs between sites harboring different insulator binding proteins raises important questions: Are these LRIs specific? How is this specificity regulated? Are other factors or post-translational modifications involved in this selectivity? Future research will be needed to address these important questions (Vogelmann, 2014).

Chromator localizes to inter-band regions of polytene chromosomes and binds to the barriers of physical domains genome-wide, however the mechanism leading to these localization patterns has been lacking. Previous studies showed that BEAF32 and Chromator co-localize at some genomic sites, and suggested that these proteins may participate in the formation of a single comple. This study showed that BEAF32 directly and specifically interacts with Chromator in vivo and in vitro. This interaction is mediated by the C-terminal domain of Chromator, thus the ChD domain does not seem to be directly involved in interactions with BEAF32. The results show that Chromator possesses a reduced affinity for DNA and binds with no sequence specificity to loci displaying strong Chromator binding peaks at the site tested (Tudor-SN locus). Thus, it is suggested that specific interactions between BEAF32 and Chromator may be responsible for its recruitment to polytene inter-band regions and domain barriers. Significantly, most BEAF32 binding sites genome-wide (>90%) contain Chromator, suggesting an almost ubiquitous interaction between the two factors (Vogelmann, 2014).

Interestingly, Chromator also co-localizes with the JIL-1 kinase at polytene inter-band regions and the two proteins directly interact by their C-terminal domains. JIL-1 is an ubiquitous tandem kinase essential for Drosophila development and key in defining de-condensed domains of larval polytene chromosomes. Importantly, JIL-1 participates in a complex histone modification network that characterizes active, de-condensed chromatin, and is thought to reinforce the status of active chromatin through the phosphorylation of histone H3 at serine 10 (H3S10). Thus, BEAF32 could be responsible for the recruitment of the Chromator/JIL-1 complex to active chromatin domains to prevent heterochromatin spreading. This mechanism would be consistent with the observation that BEAF32 localizes primarily to de-condensed chromatin regions in polytene chromosomes, is implicated in the regulation of active genes and delimits the boundaries of chromatin silencing (Vogelmann, 2014).

CP190 is a common partner of BEAF32, dCTCF, and Su(HW), and has been thus proposed to play a role in the formation of long-range interactions at these insulators. On the other hand, both CP190 and Chromator have been recently shown to be massively overrepresented at barriers between transcriptional domains. This pape shows that only when CP190 or Chromator are present can long-range interactions between BEAF32-bound DNA molecules be generated. Strong evidence is provided that the formation of in vitro LRI requires three ingredients: (1) binding of BEAF32 to its specific DNA binding sites; (2) specific interactions between the C-terminal domains of CP190/Chromator and BEAF32; and (3) homo- interactions between CP190/Chromator molecules mediated by their N-terminal ends (Vogelmann, 2014).

To further investigate the roles of CP190 and Chromator in the formation of LRIs, statistically relevant contacts containing specific combinations of insulator factors were aggregated together from Hi-C data from embryos. This analysis shows a relatively high correlation between the presence of BEAF32 and both CP190 and Chromator in sites displaying a high proportion of interacting bins between distant BEAF32 sites, as compared with neighboring sites (16.9% of interacting bins for Chromator and CP190 sites). Thus, CP190 and Chromator may play a role at a subset of genetic loci by mediating and/or stabilizing interactions between BEAF32 and a distant locus bound by BEAF32 or a different insulator binding protein. Interestingly, the binding of BEAF32 to CGATA sites as multimers, and the existence of CP190-Chromator interactions suggest that long-range interactions at a single locus could involve hybrid/mixed complexes comprising at least these three factors (Vogelmann, 2014).

These observations suggest a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). Direct or indirect interactions of first layer insulator proteins with additional factors (e.g. JIL-1, NELF, mediator) are very likely involved in directing alternative activities (e.g. histone modifications, regulation of RNAPII pausing) to specific chromatin loci. This model provides a rationale for the compositional complexity of insulator sequences and for the multiplicity of functions often attributed to insulators (e.g. enhancer blocker, chromatin barrier, transcriptional regulator). Ultimately, a characterization of the locus-specific composition of insulator complexes and their locus-specific function may be required to obtain a general picture of insulator function. In mammals, CTCF is the only insulator protein identified so far, but other factors, such as cohesin have been identified as necessary and essential for the formation of CTCF-mediated long-range interactions. Mammalian CTCF contains eleven zinc-fingers, and it has been shown that different combinations of zinc-fingers could be used to bind different DNA sequences. Thus, in mammals CTCF may play the role of first layer insulator protein, whereas other factors such as cohesin or mediator may play the role of second layer insulator proteins (Vogelmann, 2014).

This model proposing different functional roles for insulator factors could also explain the mechanism by which insulators are able to help establish and reinforce the transcriptional state of chromatin domains throughout cell division. First layer proteins remain bound to chromatin at all stages of the cell cycle. In contrast, both CP190 and Chromator are chromatin-bound during interphase but display a drastic redistribution during mitosis: CP190 strongly binds to centrosomes while Chromator co-localizes to the spindle matrix. Thus, the dissociation and cellular redistribution of second layer insulator proteins during cell division would be responsible for the massive remodeling of chromosome architecture occurring during mitosis, and for the re-establishment of higher-order contacts at the onset of interphase. In contrast, first layer insulator proteins would act as anchor points for the re-establishment of higher-order interactions after mitosis, and for the maintenance of the transcriptional identity of physical domains. Thus, this model suggests distinct roles for insulator binding proteins and co-factors in actively shaping the organization of chromatin into physical domains during the cell cycle. This model is consistent with recent genome-wide data suggesting that, overall, first layer insulator proteins remain bound to their binding sites during mitosis, whereas second layer insulator proteins tend to show a large change in binding patterns. Further genome-wide and microscopy experiments will be needed to quantitatively test this model (Vogelmann, 2014).

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

Insulators are multi-protein - DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. This study has identified two DNA-binding zinc-finger proteins, Pita and a new factor named ZIPIC (CG7928), that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins (Maksimenko, 2014).

EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. This study has shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization (Golovnin, 2015).

Insulators belong to the class of regulatory elements that organize the architecture of chromatin compartments. Insulators, or chromatin boundaries, are characterized by two properties: they interfere with enhancer-promoter interactions when located between them and buffer transgenes from chromosomal positions effects. To date, chromatin insulators have been characterized in a variety of species, indicative of their involvement in the global regulation of gene expression (Golovnin, 2015).

The well-studied Drosophila insulator proteins, dCTCF (homolog of vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. The Su(Hw) protein, encoded by the suppressor of Hairy wing [su(Hw)] gene, was one of the first insulator proteins identified in Drosophila. The best-studied Drosophila insulator found within the 5'-untranslated region of the gypsy retrovirus consists of 12 directly repeated copies of Su(Hw) binding sites. Genetic and molecular approaches have led to the identification and characterization of three proteins recruited by Su(Hw) to chromatin-Mod(mdg4)-67.2, CP190, and E(y)2/Sus1-that are required for the activity of the Su(Hw)-dependent insulators. The mod(mdg4) gene, also known as E(var)3-93D, encodes a large set of BTB/POZ protein isoforms. One of these isoforms, Mod(mdg4)-67.2, by its specific C-terminal domain interacts with the enhancer-blocking domain of the Su(Hw) protein. The BTB domain is located at the N-terminus of Mod(mdg4)-67.2 and mediates homo-multimerization (Golovnin, 2015).

Su(Hw), dCTCF, and most of other identified insulator proteins interact with Centrosomal Protein 190 kD (CP190). This protein (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc finger motifs, and a C-terminal E-rich domain. The BTB domain of CP190 forms stable homodimers that may be involved in protein-protein interactions. In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis. It has been shown that CP190 is recruited to chromatin via its interaction with the DNA insulator proteins in interphase nucleus (Golovnin, 2015).

The Su(Hw), dCTCF, Mod(mdg4)-67.2, and CP190 proteins colocalize in discrete foci, named insulator bodies, in the Drosophila interphase cell nucleus. Contradictory reports have been published in which the insulator bodies are described either as protein-based bodies in the interchromatin compartment or as chromatin domains. As shown recently, insulator proteins rapidly coalesce from diffusely distributed speckles into large punctate insulator bodies in response to osmotic stress (Golovnin, 2015).

Cell exposure to hypertonic treatment, which enhances molecular crowding, makes it possible to discriminate between nucleoplasmic bodies formed mainly of RNA and proteins (such as PML bodies) and chromatin compartments such as Polycomb bodies formed due to the interaction of distantly located chromatin regions bound by Polycomb proteins. Nucleoplasmic bodies disappear under less crowded conditions and reassemble under normally crowded conditions, which can be interpreted as a consequence of increased intermolecular interactions between components of nucleoplasmic bodies. Similar to PML bodies, insulator bodies are preserved under hypertonic treatment, in contrast to chromatin-based structures that disappear as proteins dissociate from chromatin. The CP190 protein is suggested to be critical for the activity of insulators and to regulate the entry of other insulator proteins into the speckles. At the same time, CP190 associates with centrosomes throughout the nuclear division cycle in syncytial Drosophila embryos. Nuclear localization of CP190 is also sensitive to various kinds of stress, suggesting that this process is highly regulated. However, the mechanisms and proteins responsible for localization of CP190 in different nucleus compartments are unknown. This study has shown that the nuclear distribution of CP190 depends on the level of EAST, which is located mainly in the interchromatin compartment of the nucleus. EAST is a nuclear protein of 2362 amino acids which, except for 9 potential nuclear localization sequences and 12 potential PEST sites, contains no previously characterized motifs or functional domains. Together with Skeletor, Chromator, and Megator proteins, EAST forms the spindle matrix during mitosis. In the interphase nuclei, EAST localizes to the extrachromosomal compartment of the nucleus and is essential for the spatial organization of chromosomes (Golovnin, 2015).

Despite that the bulk of interphase EAST resides in the interchromosomal domain, the current model assumes that EAST can transiently interact with chromosomes. EAST physically interacts with Megator, a 260-kDa protein with a large N-terminal coiled-coil domain capable of self-assembly. It has been speculated that Megator can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The results show that EAST interacts with CP190 and Mod(mdg4)-67.2 proteins and modulates their aggregation into the nuclear speckles. In case of EAST overexpression, CP190 binding to chromatin is reduced; consequently, the binding of Mod(mdg4)-67.2 and Su(Hw) is reduced as well, since CP190 is essential for it. On the basis of these results, it is hypothesized that EAST regulates localization of CP190 and insulator protein complexes in the interchromatin compartment, with these complexes subsequently determining organization of chromatin insulators (Golovnin, 2015).

The results suggest that insulator bodies are sensitive to the concentration of EAST in interphase cells. The properties of insulator bodies described previously and in this study suggest that they are formed by multiple interactions between proteins and resemble nuclear bodies composed of aggregated proteins and RNAs. As shown previously, the CP190 and Mod(mdg4) proteins interact with Su(Hw) and dCTCF and help the latter to enter the insulator bodies (Golovnin, 2015).

Taking into account the high level of dCTCF and Mod(mdg4) co-binding to chromosomes, it appears that dCTCF interacts with an as yet unidentified Mod(mdg4) isoform. Mod(mdg4)-67.2 and CP190 conjugate to the small ubiquitin-like modifier protein (SUMO). Specific interactions mediated by SUMO, the ability of Mod(mdg4) BTB to form oligomers, and the interaction between the BTB domain of Mod(mdg4)-67.2 and CP190 contribute to specific aggregation of the Su(Hw)/Mod(mdg4)-67.2/CP190 and dCTCF/CP190 complexes into the insulator bodies (Golovnin, 2015).

According to current views, the Megator protein can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The overexpression of EAST leads to an extension of the EAST-Megator compartment, with consequent reduction in the effective volume available for the insulator proteins in the cell. As a result, the concentration of the insulator proteins increases, contributing to stabilization of the compact protein conformations visualized as insulator bodies. By interacting with Mod(mdg4)-67.2 and CP190, EAST may also be directly involved in nucleation of insulator bodies. It is possible that the truncated version of EAST (from 933 to 2362 aa) can more easily interact with the insulator proteins, which leads to noticeable enlargement of insulator bodies in S2 cell expressing EAST^933-2362. The overexpression of EAST leads to segregation of the CP190 protein in independent speckles. The results suggest that EAST interacts with the CP190 region that includes BTB, D, and M domains. These domains are also required for CP190 interactions with other insulator proteins (Golovnin et al., in preparation). Thus, an increase in the EAST concentration may lead to displacement of the insulator proteins from the complex with CP190 (Golovnin, 2015).

The results do not exclude the possibility that EAST overexpression directly leads to dissociation of CP190 from chromatin. During mitosis, CP190 colocalizes with EAST in the spindle matrix, and the increase in the amount of EAST may well be responsible for dissociation of CP190 prior to chromosome condensation (Golovnin, 2015).

According to the current model, the insulator bodies help to form protein complexes that subsequently bind to regulatory elements such as insulators and promoters. In view of this hypothesis, it is likely that disturbances in the insulator bodies caused by EAST overexpression are responsible for the decrease in CP190 binding to the regulatory regions such as dCTCF- and Su(Hw)-dependent insulators and promoters. As shown recently, CP190 is required for recruiting Su(Hw) and Mod(mdg4)-67.2, but not dCTCF, to chromatin. Accordingly, it was observed that EAST overexpression affects the chromosomal binding of Su(Hw), but not of dCTCF. CP190 specifically interacts with the Mod(mdg4)-67.2 isoform, and Mod(mdg4)-67.2 at all Su(Hw) binding sites is colocalized with CP190. Thus, CP190 may be essential for recruiting the specific Mod(mdg4)-67.2 isoform to the Su(Hw) binding sites, with subsequent decrease in the amount of CP190 at the Su(Hw) binding sites, which leads to the substitution of Mod(mdg4)-67.2 by other Mod(mdg4) isoforms, as has been observed in this study (Golovnin, 2015).

Strong inactivation of EAST in S2 cells reduces the entry of the Mod(mdg4)-67.2/ Su(Hw) complex, but not of CP190, into the nucleus. It appears that EAST is involved in the regulation of nuclear localization of Mod(mdg4)-67.2, whose BTB domain can form multimeric complexes. Further study is required to elucidate this issue (Golovnin, 2015).

The Dm-Myb oncoprotein contributes to insulator function and stabilizes repressive H3K27me3 PcG domains

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. This study 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. This study now reveals 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 (Santana, 2020).

This study identified four novel roles for the Drosophila Myb oncoprotein. First, it was shown that Myb acts as a potentiator of target gene transcription, not merely as an epigenetic factor that maintains activated expression. Potentiation is associated with increased H3K4me3 as well as recruitment and pause release/elongation of RNA Pol II. Next, it was shown that in addition to binding promoter potentiator sites, Myb binds to insulator sites genome wide, is necessary to promote the binding of BEAF-32 and CP190 at Myb-shared sites, and is required for the function of at least a subset of insulators. Third, it was shown that Myb plays a critical role in formation and/or stabilization of H3K27me3 domains, preventing silent genes within these domains from becoming derepressed, in stark contrast to Myb's well-known role as an activator of gene expression. Finally, this study showed that Myb is enriched at TAD boundaries and chromatin loop anchors (Santana, 2020).

Similar to what has previously been reported, an enrichment was identified of cell cycle genes directly regulated by Myb, with the majority being G2/M but also several S phase genes (consistent with studies on B-Myb, supporting its role in S phase). Notably, a recent study reported that Cyclin A (the cyclin that regulates aspects of both S phase and G2/M) directly binds to Myb and is required for expression of many of its targets (Rotelli, 2019). Further supporting a role for Myb in transcriptional regulation, this study found that Myb is required for optimal levels of H3K4 promoter methylation, with Myb playing a key role in RNA Pol II dynamics. Analysis of the promoter regions of Myb-potentiated targets in Myb mutants revealed two distinct classes of targets: one that requires Myb for efficient recruitment of RNA Pol II and both that use Myb for efficient RNA Pol II pause release/elongation. Myb may use NURF to establish a nucleosomal-free region that allows recruitment of RNA Pol II, similar to what has been observed with GAF at several targets. Further suggesting a critical role for Myb in transcriptional regulation, the failure to potentiate key targets in Myb mutants (e.g., okr and Rad9 for S phase; Cap-D2 for chromosome condensation; mad2, Mps1, Pen, dgt6, and msd5 for M phase) likely explains the varied phenotypes observed in Myb mutants (including increased mitotic index, partially condensed chromosomes, aneuploidy, and S phase defects) (Santana, 2020).

Recent studies have shown that several components of dREAM can bind insulator sites, and RNAi depletion of Mip40, Mip130, and E2f2 resulted in impairment of enhancer-blocking function at specific sites. Furthermore, components of dREAM (Myb, Mip120, Mip130, Rbf1, E2f2, and DP) were shown to directly interact with dCTCF and/or CP190. Interestingly, double knockdown of dCTCF and CP190 resulted in loss of dREAM (Mip40, Mip120, Mip130, and E2f2) at some shared sites. The results presented in this study further extend these studies and demonstrate that Myb directly contributes to boundary insulator function (including sites located at divergently paired genes). Intriguingly, in many cases where one gene of a divergently paired genes (DPGs) is transcribed and the other is silent, it was found that Myb has no role in expression of the activated gene; rather, Myb prevents inappropriate activation of the silent gene. This cannot be explained by loss of the primary repressive arm of the Myb complex (E2f2/Rbfs) because knockdown of E2f2 or Rbf1/2 in Kc167 cells led to derepression of only 17 out of the 96 genes upregulated when Myb is knocked down. Furthermore, out of the 46 genes upregulated in SL2 cells upon knockdown of E2f2 or Rbf1/2, only 3 are upregulated in Myb mutant wing discs. Finally, the absence of E2f2 in 3rd instar larvae led to upregulation of genes from four DPGs; yet, these genes are not upregulated in Myb mutant wing discs. Of the insulator sites shown in this study to require Myb, the vast majority (89%) contain Myb but not E2f2 consensus sites, similar to what was observed in Kc167 cells for genes requiring Myb for repression (Santana, 2020).

Consistent with Myb playing a role in insulators that separate different chromatin neighborhoods, this study found a striking correlation between Myb occupancy and active, null, PcG H3K27me3 (all 99%), and APBS TAD boundaries (>93%). TAD boundaries have been associated with chromatin looping, and studies on CTCF and CP190 in vertebrates and invertebrates, respectively, have proposed or confirmed a role for these insulator factors in tethering the chromatin loops. Further work has shown that chromatin loop anchors can also exist within TADs. It is particularly noteworthy that Myb occupies the majority of chromatin loop anchor points identified in another study Eagen (2017), in addition to significant overlaps with several other chromatin factors (including dCTCF, Rad21, and Pc) at anchor points. Further work is needed to determine whether Myb plays a role in tethering chromatin loops, although it is intriguing to speculate that the loss of the H3K27me3 mark in Myb or dCTCF mutants could result from disrupting H3K27me3 loops, which, in turn, might diminish that region's ability to associate with an H3K27me3 nuclear subcompartment. The observation that dCTCF has been shown to directly interact with Myb further suggests that these two chromatin factors can collaborate, and a remarkable 73% of the anchors bound by dCTCF are also bound by Myb (similar to the Rad21-bound anchors, for which 75% are also occupied by Myb). It is postulated that B-Myb (the most closely related vertebrate Myb family member to Dm-Myb) and CTCF might be playing similar collaborative roles in humans (Santana, 2020).

Although a role for Myb in transcriptional activation has been recognized for some time, its role in insulator function is particularly intriguing, as this activity appears to be separate from its transcriptional role. Indeed, the chromatin changes observed at many DPGs and other insulator elements strongly support the direct role of Myb in promoting a barrier function between chromatin states (in part by promoting binding of BEAF-32/ CP190). However, unlike previously described examples of barrier insulator loss, which results in heterochromatin spreading into euchromatin, the opposite scenario were observed in Myb mutants, namely the spreading of H3K4me3, which may be enabled by the significant loss of the H3K27me3 mark. This is the very definition of barrier insulator function, namely, maintaining a discrete separation of chromatin environments. Whether Myb is playing a role in chromatin loop formation is unclear at this point, but its overlap with the majority of loop anchor points identified in at least one study in flies is intriguing. Further work will be needed to address Myb's role at the loop anchors and whether this role relates to stabilization of H3K27me3 domains and/or barrier insulator function. For example, directed mutagenesis of Myb-dependent insulator sites by using tools, such as CRISPR-Cas9, to specifically target Myb, dCTCF, or BEAF-32 sites can provide additional insight into the respective roles of these insulator factors. In addition, assay for transposase-accessible chromatin using sequencing (ATAC-seq) or chromosome conformation capture (3C)-type techniques performed in both controls and Myb mutants can be used to determine whether important chromatin/ 3D structure changes might underlie the altered transcriptional (Santana, 2020).

Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes

After centrosome duplication, centrioles elongate before the M phase. To identify genes required for this process and understand the regulatory mechanism, this study investigated the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles. An essential microtubule polymerisation factor, Orbit/CLASP, was identified that accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically for determining centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. Loss of centriole integrity was observed, including fragmentation of centrioles and earlier separation of the centriole pairs in Cp110 null mutant cells either overexpressing Orbit or harbouring Klp10A depletion. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they are unpaired. These findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells (Shoda, 2021).

The centrosome plays an indispensable role as the major microtubule-organising centre (MTOC) in a cell. During mitosis, centrosomes duplicated in the S phase move apart from each other and reach the opposite poles of the cell. Each centrosome is involved in the assembly of spindle poles, which enables construction of the bipolar spindle microtubule structure. A centrosome consists of two components: a pair of centrioles and the surrounding pericentriolar matrix (PCM). After replication of the centrioles, the longer centriole (mother centriole) engages with the shorter one (daughter procentriole) in a V-shape. When a cell enlarges in the G2 phase, the short daughter procentriole undergoes elongation to a certain length before the subsequent M phase. A single centriole consists of a microtubule doublet or triplet, which is equivalent to the cytoplasmic microtubule. Several factors localised on centrioles have been shown to be involved in the centriole elongation process. The most critical step in centrosome duplication is the duplication of centrioles, which requires stringent regulation. However, the entire mechanism underlying regulation of centriole elongation and the regulatory factors required for the process are not known. Among the centriole-associated proteins, those belonging to the kinesin-13 family are known to act as microtubule-severing kinesins. Klp10A, a Drosophila member of the family, has been shown to play an indispensable role in the regulation of centriole elongation. Based on these observations, it is speculated that some of the factors regulating microtubule length might overlap with those required for centriole elongation. It is possible that the production of centrioles of specific length requires a balance between polymerisation and depolymerisation of the triplet microtubules. However, the main factor(s) counteracting Klp10A and promoting centriole elongation remain to be identified (Shoda, 2021).

Another characteristic complex containing Cp110 is localised at the distal tip of the centriole, where it regulates the accessibility of the distal end to the shrinking and hypothetical lengthening factors, thereby regulating centriole elongation at this end. In the absence of Klp10A, the longer centrioles harbour incomplete ninefold symmetry at their ends in Drosophila cultured cells and tend to undergo fragmentation. Importantly, Cp110 depletion differentially affects centriole elongation in a species- and/or cell type-specific manner. In Drosophila S2 cultured cells, Cp110 depletion results in centriole length diminution. This effect is rescued by simultaneous depletion of Klp10A. In contrast, CP110 (also known as CCP110) depletion results in centriole elongation in mammalian cells. The centriolar microtubules were dramatically elongated in somatic cells, such as wing discs and larval brain cells, in the Drosophila Cp110-null mutant, whereas subtle elongation of the structure was observed in the premeiotic spermatocytes of the mutant (Shoda, 2021).

The premeiotic spermatocyte in Drosophila is a good model for investigating centrosomes and centrioles. Drosophila spermatogenesis involves four mitotic and two meiotic cycles for the formation of haploid spermatids. In the same spermatocyte cyst, each of the 16 cells undergoes synchronous cell growth, which can be divided into the S1 stage, corresponding to S phase, and five subsequent stages, S2 to S6, before initiation of meiosis I. The centrioles, in particular, can be studied more easily in this cell type, since these organelles dramatically elongate until the onset of meiosis and the centriole cylinder is composed of microtubule triplets. In early spermatocytes that possess a pair of centrioles initially, centrioles duplicate at S1 stage. As primary spermatocytes enter in the growth phase, centrioles migrate toward the surface where they assemble the primary cilium at the distal end of basal body. At the beginning of meiotic division I, centrioles move close to the nucleus with their associated 'membrane pocket' on the distal end of the cilium-like region (CLR). Between the CLR and the basal body there is the transition zone (TZ), which plays an important role in elongating the primary cilium of the spermatocyte. Centrioles are no longer duplicated between the two meiotic divisions. Primary spermatocytes hold two pairs of centrioles composed of nine triplet microtubules and engaged by a cartwheel structure at the proximal ends. The centriole pair is disengaged during prophase II, and, consequently, singlet centrioles organise the centrosomes of secondary spermatocytes (Shoda, 2021).

Previous studies have shown that Orbit (the Drosophila CLASP orthologue, encoded by chb) is essential for microtubule polymerisation, as it adds tubulin dimers to the plus end of the microtubules; however, its role in centriole elongation has not been examined. Hence, this study aimed to investigate whether Orbit was involved in centriole elongation in the mature premeiotic spermatocytes before male meiosis. As Orbit antagonises Klp10A, a severing factor determining the length of spindle microtubules in cultured Drosophila cells, whether Orbit was also involved in centriole length regulation was assessed (Shoda, 2021).

In addition, this study highlighted the importance of these regulators of centriole dynamics and the distal end capping proteins in the centriole elongation process using Drosophila spermatocytes. The importance of regulating the elongation of duplicated centrioles to a certain length for proper chromosome inheritance during male meiotic divisions is also discussed (Shoda, 2021).

Centrioles in Drosophila spermatocytes consist of ninefold triplets of microtubules. This study showed that the centrioles elongated to a certain length as the cells grew before male meiosis. Klp10A, which is a Drosophila kinesin-13 orthologue essential for shortening the microtubules, plays an indispensable role in regulation of centriole length. Microtubule length can be determined by the balance between polymerisation and depolymerisation of tubulin heterodimers and protofilaments. Thus, dynamic factors that promote microtubule elongation might also play critical roles in the determination of centriole length. This study has presented evidence suggesting that overexpression of Orbit results in excessively long centrioles in premeiotic spermatocytes. Conversely, shorter centrioles were observed in hypomorphic orbit mutants. These results are consistent with the idea that Orbit is essential for promotion of centriole elongation in spermatocytes. Orbit was initially identified as a microtubule-associated protein that adds tubulin heterodimers at the plus end of microtubules. Hence, the probability that Orbit might also elongate the triplet microtubules of centrioles by adding tubulin dimers, similar to its function in microtubule elongation, is high. In contrast, the kinesin-13 orthologue Klp10A acts as a microtubule depolymerising factor at the plus ends of microtubules in interphase and is an important regulator of centriole elongation. In Drosophila S2 cells, Klp10A antagonises Orbit in bipolar spindle formation and its maintenance. This study has shown that simultaneous overexpression of Orbit and depletion of Klp10A further enhanced centriole elongation. Based on these findings, it is proposed that these two factors act antagonistically to produce centrioles of specific length. Very long GFP-Orbit signals were observed that extended from basal bodies in spermatocytes overexpressing the protein. Accordingly, it is hypothesised that these are overly long axoneme microtubules produced as a consequence of excessively stimulated polymerisation of tubulins by the overexpression. Orbit, like other CLASP proteins, has a microtubule-binding activity to stimulate tubulin polymerisation at the plus end. Alternatively, a possibility cannot be excluded that Orbit polymerises itself to construct microtubule-like structures on the distal tip of the basal body. It would be interesting to investigate whether basal body and axonemal microtubules overly elongate in cells overexpressing Orbit without a fluorescent tag (Shoda, 2021).

In addition to these two factors regulating centriole microtubules, it is hypothesised that Cp110 plays a role as a cap to restrict these factors acting on the distal ends of the microtubules at an earlier stage. After Cp110 releases from the ends at mid-stage, Orbit can access the distal ends more easily and stimulate the centriole microtubules to extend to a certain length. Although the loss of the cap protein resulted in only a subtle extension of centrioles, Orbit overexpression in the absence of Cp110 can change the centriole microtubule dynamics significantly. Consequently, remarkably long centriole microtubules would be produced in the spermatocytes. Consistent with this hypothesis, Cp110Δ1 mutation also significantly enhanced the overly long centriole phenotype in Klp10A-depleted spermatocytes. By contrast, the Cp110-null mutation did not enhance the shorter centriole phenotype caused by Klp10A overexpression. Further experiments need to be performed to verify the model. The fact that Cp110 influences centriole length depending on the cell type and cellular context has been demonstrated in previous studies. It is likely that differential regulation of the conserved core components underlies ciliary basal diversity in different cell types. As argued previously, cellular-specific and tissue-specific regulation in centriole duplication may be indispensable to regenerate diverse centriole structures (Shoda, 2021).

Previous reports have not investigated whether Orbit and Klp10A are localised on centrioles or around the PCM in Drosophila mitotic cells; however, a considerable amount of Orbit accumulates in centrosomes in early embryos, Drosophila cultured cells and germline stem cells during Drosophila oogenesis and spermatogenesis. Whether the protein is localised in the PCM or in the centrioles is not clear. Similarly, studies on the cellular localisation of Orbit/CLASP orthologues in other species have also shown centrosome localisation of these proteins. Anti-human CLASP1 immunostaining in Hela cells has demonstrated that the protein is localised on centrosomes during M phase (Maiato et al., 2003). Furthermore, a CLASP orthologue in Caenorhabditis elegans has been observed in the centrosomes of its embryonic cells. These reports did not mention whether the orthologues were associated with the centrioles. Recently, it has been reported that Klp10A is dominantly localised in the TZ of the ciliary structures in spermatocytes, spermatids and sensory neurons (Persico, 2019). Centrosomal localisation of the protein has also been reported in mitotic cells and germline stem cells in both sexes. Whether Klp10A localises on the cylindrical microtubules of the centrioles or in the PCM of mitotic cells has not been demonstrated. Therefore, whether these antagonistic regulators are localised on centrioles in mitotic cells should be determined at a higher resolution. Furthermore, whether these two regulators are required for centriole length determination in somatic cells warrants further investigation (Shoda, 2021).

In addition to excessively elongated centrioles, this study observed several abnormalities in centriole organisation and structure in spermatocytes overexpressing Orbit and/or harbouring Klp10A depletion. In the absence of Cp110, the loss-of-centriole-integrity phenotypes were also enhanced. Small pieces of centrioles observed may be broken pieces of over-elongated centrioles, as observed in cancer cells. Alternatively, they may have been unpaired centrioles separated precociously from centriole pairs containing daughter procentrioles, which are smaller than normal centrioles. By contrast, the loss-of-centriole-integrity phenotypes were not observed in cells overexpressing a shortening factor, Klp10A. Hence, it is considered that improvised construction of the basal body microtubules may be associated with the loss-of-integrity phenotype; thereby, centriole engagement would be lost. Centriole fragments were found with reduced diameter in the centriole microtubules. The presence of disintegrated centrioles supports this idea, but further investigations are necessary to test the current hypothesis (Shoda, 2021).

Spermatocytes homozygous for loss-of-function mutation of Sas6 and of Ana2 commonly demonstrate premature centriole separation before meiosis. Hence, Sas6 and Ana2 are considered to be required for centriole engagement and/or maintenance of the pairs. Orbit overexpression and/or Klp10A depletion may affect centriole engagement through interfering with Sas6 (or Ana2) function. It is also possible that the premature disengagement can occur independently of Sas6 or Ana2. In addition, it has been reported that APC/C activation and activation of separate, thereby unexpected, cleavage of Scc1 (also known as RAD21) cohesin can take place in mammalian cultured cells. The possibility cannot be excluded that alteration of microtubule dynamics in centrioles by altered expression of Orbit and/or Klp10A led to unexpected APC/C activation. This hypothesis will be tested by several experiments in future work (Shoda, 2021).

Previous studies have also mentioned that Cp110-null mutant spermatocytes or syncytial-stage embryos do not show detectable defects in centrosome behaviour, spindle formation or chromosome segregation. By contrast, this study has show that disintegrated centrioles and multipolar spindle microtubules emanating from the centriolar fragments existed in Cp110-null mutant spermatocytes and cells overexpressing Orbit, as well as in Klp10A-depleted cells less frequently. Cells homozygous for the loss-of-function Klp10A mutation also display multipolar spindle structures. However, it cannot be excluded that the spindle phenotype would result from abnormal microtubule organisation caused by Klp10A mutation, rather than centriole disintegration. Surprisingly, in Cp110 mutants overexpressing Orbit and undergoing meiosis I, it was observed that unpaired single centrioles, even a part of them, could act as the MTOC. Centrosomes must be 'licensed' to function as an MTOC that nucleates microtubules, although the mechanism whereby the 'license' is granted remains unclear. Nevertheless, a recent study has reported that excessive elongation of centrioles in cancer cells is related to the generation of overduplicated, fragmented or hyperactive centrosomes that nucleate considerably more microtubules during cell division. Chromosome segregation is disturbed in cells harbouring these abnormal centrioles. Interestingly, generation of cells harbouring extra centrosomes has been suggested to be able to drive spontaneous tumorigenesis in mice. Additional studies have reported that excessively elongated centrioles in Drosophila spermatocytes affect spermatogenesis via the production of defective flagella. Consistently, immotile sperm production and significant decrease in male fertility were observed in the Cp110-mutant males with spermatocyte-specific Klp10A depletion. These observations suggested that production of excessively elongated centrioles can affect cell division and subsequent spermatogenesis. Once an abnormal spindle microtubule structure is constructed, the extra MTOC results in chromosome mis-segregation and eventually chromosome instability, as observed in cancer cells (Shoda, 2021).

Certain abnormalities such as mis-segregation of chromosomes and the resulting aneuploidy might appear in the subsequent division of cells containing abnormal centrioles. Conversely, excessively short centrioles may be inadequate as templates for the duplication of centrioles in S phase. Hence, regulation of centrosome length via the antagonistic activities of elongation and shrinking factors, such as Orbit and Klp10A, is important. However, the loss of centriole integrity and the resulting aneuploidy may occur in meiotic cells, but not in mitotic cells, which have stricter microtubule assembly checkpoints. Further investigations are required to understand how centrosome length is regulated in mitotic cells (Shoda, 2021).

Current findings suggest the presence of mutually antagonistic regulation to determine centriole length and the significance of the production of centrioles with a certain length for centriole integrity, and for assurance of proper chromosome segregation. It is believed that these findings may enable the identification of a mechanism whereby the loss of centriole integrity causes chromosome mis-segregation in cancer cells (Shoda, 2021).

M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. This study identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. These results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization (Bag, 2021).

In eukaryotic cells, the three-dimensional organization of the genome plays a critical role in achieving proper spatial and temporal patterns of gene expression during development. Chromatin insulators are DNA-protein complexes involved in the establishment, maintenance, and regulation of nuclear organization to modulate gene expression. Insulators regulate interactions between cis-regulatory elements such as enhancers and promoters and demarcate silent and active chromatin regions to ensure their proper regulation. They can inhibit the interaction between an enhancer and a promoter when positioned between the two elements and can act as a barrier to stop repressive chromatin from spreading over active genes. Furthermore, chromatin insulators can promote intra- and inter-chromosomal looping to control topology of the genome. Certain insulator proteins are highly enriched at the self-interacting boundaries of topologically associating domains (TADs) throughout the genome. In mammals, only a single insulator protein, CCCTC-binding Factor (CTCF), has thus far been identified, and CTCF indeed is enriched at TAD borders and is required for TAD formation. In contrast, Drosophila melanogaster CTCF is not particularly enriched at TAD borders, and a recent study indicates that CTCF plays a limited role in TAD formation in flies. In fact, Drosophila harbors a variety of insulator protein complexes, all of which contain the protein Centrosomal protein 190 (CP190). CP190 is highly enriched at TAD borders, suggesting a possible role in TAD formation. Another notable feature of genome organization that has been explored in detail in Drosophila is the key role of transcription and the presence of constitutively active genes at TAD borders. General inhibition of transcription using chemical treatments or heat shock results in disruption of TADs and compartments, but the mechanistic details of how transcription contributes to genome organization are yet to be elucidated (Bag, 2021).

The Drosophila gypsy insulator, also known as the Suppressor of Hairy wing [Su(Hw)] insulator, was the first characterized CP190-containing insulator complex. The zinc-finger DNA-binding protein Su(Hw) provides binding specificity of the complex, and both CP190 and the Modifier of mdg4 [Mod(mdg4)] 67.2 kDa isoform [Mod(mdg4)67.2] contain an N-terminal Broad-Complex, Tramtrack, and Bric a brac (BTB) domain that can homodimerize or heterodimerize to facilitate insulator-insulator interactions and promote formation of long range insulator-mediated loops. Initially, the gypsy insulator complex was characterized as binding the 5'-untranslated region of the gypsy retroelement. However, the core complex also binds thousands of endogenous sites throughout the genome and can function similarly at least at a subset of those sites. Moreover, the three gypsy insulator core components do not colocalize absolutely at all binding sites throughout the genome, and each protein can interact with other insulator proteins. In diploid interphase nuclei, gypsy insulator proteins coalesce into large foci termed insulator bodies. These structures can be induced by stress, and insulator bodies have also been proposed to serve as storage depots for insulator proteins. Nevertheless, there is a high correlation between proper insulator function and insulator body localization. In summary, the gypsy insulator complex contributes to higher order nuclear organization on several levels (Bag, 2021).

CP190 also associates with a variety of additional DNA-binding proteins that likely impart specificity of the respective complex. The BED finger-containing proteins BEAF-32, Ibf1, and Ibf2 interact with CP190 and promote insulator function (Cuartero, 2014). Three additional zinc-finger proteins Pita, ZIPIC, and CTCF also interact with CP190 and contribute to insulator activity. Recently, the zinc-finger protein CLAMP was demonstrated to positively affect gypsy insulator activity and to colocalize particularly with CP190 at promoters throughout the genome. In addition, previous work showed that CP190 preferentially binds Motif 1-containing promoters, but the functional significance of this observation is currently unknown. The precise functions of CP190, its associated factors, as well as their relationship with transcription regulation have not yet been elucidated (Bag, 2021).

Motif 1 binding protein (M1BP) is a ubiquitously expressed transcriptional activator that is required for the expression of predominantly constitutive genes. A zinc-finger DNA-binding protein, M1BP, specifically binds to the core promoter element Motif 1 consensus sequence that is distinct from the canonical TATA box and mainly controls the expression of constitutively active genes that are transiently paused (Li, 2013). For example, M1BP interacts with the TATA-binding protein-related factor 2 (TRF2) to activate transcription of ribosomal protein genes in a Motif 1-dependent manner (Baumann, 2017a). Finally, recent studies found that Motif 1 and M1BP are highly enriched at TAD boundaries along with CP190 and BEAF-32 (Cubenas-Potts, 2017; Ramirez, 2018; Sexton, 2012; Hou, 2012). Depletion of M1BP led to increased inter-chromosomal Hi-C contacts; however, concomitant cell cycle disruption precluded interpretation of these results (Ramirez, 2018). The possible role of M1BP-dependent transcriptional regulation in genome organization has not yet been interrogated in detail (Bag, 2021).

This study identified M1BP as a physical interactor and positive regulator of the gypsy insulator complex. Depletion of M1BP decreases gypsy-dependent enhancer blocking and barrier activities and reduces the association of the core insulator complex with the gypsy insulator sequence. ChIP-seq analysis reveals extensive genome-wide overlap of M1BP particularly with promoter-bound CP190, and depletion of M1BP results in extensive loss of CP190 chromatin association genome-wide. Depletion of CP190 also disrupts M1BP binding at many of its binding sites. Nascent euRNA-seq (neuRNA-seq) analysis of M1BP- or CP190-depleted cells indicates that both factors co-regulate a similar set of genes genome-wide. In particular, loss of gene activation correlates with disrupted M1BP and CP190 binding, and these events are frequently observed at TAD borders. Depletion of M1BP disrupts gypsy insulator body localization within the nucleus and alters both inter- and intra-TAD local genome compaction. Finally, knockdown of M1BP decreases chromatin accessibility at its binding sites, including genes that it activates and regions in proximity of TAD borders. Taken together, these findings identify a mechanistic relationship between M1BP and CP190 to activate Motif 1-dependent transcription as well as to promote chromatin insulator activity and nuclear organization (Bag, 2021).

This study has shown that M1BP is required for proper gypsy insulator function and insulator body formation and that M1BP and CP190 together activate transcription at TAD borders. M1BP was shown to physically associates with CP190 as well as core gypsy components and promotes enhancer blocking and barrier activities. Genome-wide M1BP colocalizes mainly with CP190 at Motif 1-containing promoters, which are enriched at TAD borders. M1BP is required for CP190 binding at many sites throughout the genome and vice versa, and loss of either factor reduces gene expression at TAD borders. M1BP is required for proper nuclear localization of insulator bodies, and loss of M1BP increases local genome compaction across TAD borders as well as within large TADs. Finally, M1BP promotes local chromatin accessibility at its binding sites, including transcriptionally activated genes and regions near TAD borders. Taken together, these findings suggest that M1BP may play a role in 3D genome organization through a CP190- and transcription-dependent mechanism (Bag, 2021).

As M1BP is ubiquitously expressed throughout development, effects on insulator activity and complex localization after M1BP depletion were observed in all tissues and stages of development tested. M1BP associates physically with chromatin at the Su(Hw)-binding sites of the gypsy insulator in conjunction with core insulator proteins, and M1BP is required for the binding of all three factors. These findings suggest that M1BP directly affects insulator activity by aiding the recruitment of gypsy core components to gypsy insulator sites, all three of which are required for proper insulator activity. Interestingly, Motif 1 is not present at this sequence, and M1BP binding at this site is also dependent on the presence of CP190. One scenario is that binding of the two factors could be cooperative, and M1BP recruitment may additionally heo stabilize the multimerization and/or higher order organization of insulator complexes. Consistent with this hypothesis, depletion of M1BP results in increased numbers of smaller insulator bodies, similar to the effect of complete loss of the BTB-containing core insulator protein Mod(mdg4)67.2. Another possibility is that depletion of M1BP results in cellular stress that induces insulator body formation. Since CP190 is a universal insulator protein in Drosophila, mislocalization of CP190 may result in, or at least serve as an indicator of, disrupted genome organization when M1BP is depleted (Bag, 2021).

Although M1BP physically interacts with each of the core gypsy insulator components, it was observed that M1BP colocalizes mainly with just CP190 throughout the genome, particularly at Motif 1-containing promoters. Distinct binding of the transcriptional activator M1BP compared to Su(Hw) and Mod(mdg4)67.2 genome-wide is not entirely surprising considering sub-stoichiometric levels of co-immunoprecipitation that could also reflect interaction off of chromatin. Furthermore, it has been observed that Su(Hw) binding can correlate with transcriptional repression rather than insulator activity, which may depend on the presence, or absence, of particular interacting proteins. Importantly, recruitment of CP190 is dependent on M1BP and vice versa at many co-occupied sites genome-wide. Why M1BP is only partially dependent on CP190 for binding is unclear, but these results are consistent with the known ability of M1BP to bind DNA directly (Li, 2013), whereas CP190 is believed to require interaction with a specific DNA-binding protein in order to associate with chromatin. Aside from the insulator protein BEAF-32, which binds an AT-rich dual core sequence, a large extent of overlap between M1BP and other DNA-binding insulator proteins that have been shown to be involved in recruiting CP190 to DNA was not generally observed. Future studies may reveal a possible functional relationship between M1BP and BEAF-32 in insulator activity or regulation of gene expression (Bag, 2021).

The results suggest that M1BP promotes gypsy insulator function through interaction with CP190 in a manner distinct from CP190 interaction with the zinc-finger DNA-binding protein CLAMP. A large extent of genome-wide overlap between M1BP and CLAMP, a recently identified positive regulator of gypsy insulator activity, was not observed. The sequence binding specificity of CLAMP is similar to that of GAF45, while M1BP and GAF bind to and regulate distinct sets of promoters (Li, 2013). Although either CLAMP or M1BP depletion reduces gypsy enhancer blocking and barrier activities as well as alter insulator body localization, unlike M1BP, CLAMP depletion does not affect CP190 chromatin association throughout the genome. In fact, CP190 depletion had a substantial effect on CLAMP chromatin association, again suggesting that CP190 may affect the ability of certain DNA-binding proteins, including M1BP, to associate with chromatin through cooperative or higher order physical interactions (Bag, 2021).

The results suggest that CP190 may play a more direct role in transcriptional regulation than previously appreciated, in part through interaction with M1BP. Genome-wide profiling studies have shown that CP190 preferentially associates with promoters genome-wide. CP190 was found to be enriched particularly at active promoters and was shown to affect steady-state gene expression when depleted; however, direct and indirect effects as well as transcriptional and posttranscriptional effects could not be separated. In order to avoid the complication of interpreting steady-state gene expression profiles, neuRNA-seq was performed after either CP190 or M1BP depletion in order to measure newly synthesized transcripts. Intriguingly, nascent RNA expression profiles of CP190 or M1BP-depleted cells showed a remarkably high level of correlation. Since both M1BP and CP190 are particularly associated with promoters of genes that require either factor for adequate expression, it is likely that both factors mainly function in transcriptional activation rather than repression. M1BP was previously shown to activate transcriptionally paused genes, and the ATAC-seq analysis of M1BP-depleted cells supports this conclusion and further demonstrates that M1BP promotes chromatin accessibility surrounding the TSS. Interestingly, depletion of CP190 has no effect on promoter accessibility. Furthermore, this study found that CP190 is specifically required for Motif 1-dependent expression of two previously characterized ribosomal protein genes. Whether TRF2 recruitment to Motif 1-containing promoters is affected by CP190 as well as the precise mechanism by which CP190 contributes to M1BP-dependent transcription will be important topics of further study (Bag, 2021).

This study found that genes that require M1BP and CP190 for adequate expression are frequently located at TAD borders, as are both proteins. It has previously been proposed that constitutively active transcription, particularly at/near TAD borders (also referred to as 'compartmental domains'), may be a defining or at least key feature of overall genome organization in cells and throughout development. CP190 was previously shown to be associated particularly with Motif 1-containing promoters (Rach, 2011), and CP190 was observed to be specifically enriched at TAD borders. Recently, Motif 1 was also found to be an enriched sequence at TAD borders present in Kc cells (Ramirez, 2018) and is apparent when TAD borders first appear in embryonic development, findings consistent with these previous studies. A previous study of M1BP involvement in genome organization provided limited evidence using Hi-C to suggest that chromosome intermingling may be increased after M1BP depletion (Ramirez, 2018). However, the extent and duration of M1BP depletion in their study caused a major disruption of cell cycle and cellular growth, thus obscuring interpretation of those results. No changes were observed in CT intermingling in Oligopaint FISH experiments in M1BP-depleted G1 cells, nor did was any difference observed in distance between two distant regions on the same chromosome. Therefore, large-scale changes in genome organization were not observed after M1BP depletion (Bag, 2021).

Increased local genome compaction was observed after M1BP depletion. This finding led to a test of whether TAD borders may be specifically disrupted, perhaps leading to fusion of neighboring TADs. Because Oligopaint FISH probes are limited to a minimum of 30 kb, this study was restricted to intra-TAD analysis of larger TADs, which are typically lower in transcriptional activity (PcG, inactive, null). Increased compaction was found to occur both across TAD borders and within large TADs after M1BP depletion. These effects occur in the vicinity of altered local transcription and reduced chromatin accessibility particularly near TAD borders, suggesting that M1BP-dependent transcriptional changes might alter local chromatin structure that culminates in changes in genome compaction in surrounding regions not restricted to TAD borders. However, the extent of reduced chromatin accessibility observed in M1BP-depleted cells by ATAC-seq is modest relative to the genomic sequence space interrogated by FISH, suggesting that loss of accessibility likely does not directly explain increased local genome compaction. In contrast, CP190 depletion affected transcription to a similar degree, although not identically, yet did not result in changes in local compaction or extensive loss of chromatin accessibility genome-wide. These differences perhaps reflect the multifunctional nature of CP190 as a universal insulator protein contributing to opposing forces, or alternatively, effects on transcription in M1BP-depleted cells may be functionally unrelated to increased genome compaction. This work shows the requirement of M1BP for accurate CP190 binding throughout the genome as well as gypsy-dependent chromatin insulator activity and nuclear localization through interaction with CP190 and other core insulator proteins. Overall, the results provide evidence that M1BP and CP190 at TAD borders and perhaps their ability to activate transcription of constitutively expressed genes located at TAD borders, combined with the capacity of M1BP to promote chromatin accessibility, may play a role in genome organization (Bag, 2021).

M1BP has been shown to activate transcription of genes at which RNA Pol II is transiently paused in the promoter proximal region, and these promoter regions may themselves possess insulator activity. Intriguingly, a previous study showed that stalled Hox promoters, including Motif 1-containing Abd-B, possess intrinsic enhancer blocking insulator activity47. However, the second paused promoter identified in this study, Ubx, does not harbor Motif 1; thus, M1BP may not necessarily be involved in the enhancer blocking activity of all stalled promoters. Recently, it was shown that M1BP promoter binding can prime the recruitment of the Hox protein Abd-A to the promoter in order to release paused Pol II and activate transcription (Chopra, 2009). This study found that M1BP is similarly required for CP190 recruitment at a large number of sites throughout the genome and thus propose that M1BP and CP190 are together required to maintain active gene expression near TAD borders. Active transcription and increased accessibility at these sites may be needed for higher order chromatin organization such as TAD insulation, formation of active compartmental domains, and/or proper local genome structure. Future studies will elucidate the precise mechanisms by which M1BP, CP190, and transcription contribute to higher order chromatin organization (Bag, 2021).

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). Chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments were performed with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines; evidence was found that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression (Bushey, 2009).

Su(Hw), dCTCF, and BEAF have all been implicated in chromatin loop formation, and the interaction of these different DNA-binding proteins with CP190 could have functional implications for the arrangement of the chromatin fiber within the nucleus. The work presented in this study provides critical insight into the genome-wide distribution of these four insulator proteins and is a first, crucial step toward understanding the role that they play in chromatin organization and the regulation of gene expression (Bushey, 2009).

Although insulator elements containing Su(Hw), dCTCF, and BEAF could, in principle, play similar roles, it was found that they have very different distribution patterns with respect to gene location. Only 20% of Su(Hw) sites are located within 1 kb of the 5' or 3' ends of genes. In contrast, 47% of dCTCF sites and 84% of BEAF sites are found within 1 kb of gene ends, and their distributions are highly skewed toward the 5' end of highly expressed genes. dCTCF and BEAF appear to display further functional compartmentalization in their roles, since BEAF tends to be present at the 5' end of genes involved in metabolic processes and dCTCF is enriched near genes involved in developmental processes. This could indicate that BEAF plays a specific role in the regulation of gene units consisting of metabolic genes, whereas Su(Hw) may play a more general role by setting the foundation for chromatin organization. dCTCF, which shows an intermediate distribution compared with Su(Hw) and BEAF, may sometimes function in large-scale organization and sometimes work at the level of individual developmental gene units. Together, the three CP190 insulator subclasses could create a chromatin web that is part of the framework organizing DNA in the nucleus (Bushey, 2009).

Insulators have been typically characterized as sequences capable of regulating interactions between transcriptional regulatory sequences and/or chromatin states. This function can easily be envisioned in the case of Su(Hw) and dCTCF sites located far from genes, where these sites could flank a group of transcription units that would then represent a domain of coregulated genes. If this is the case, what is the function of the remaining dCTCF and BEAF sites located close to the 5' and 3' ends of genes? This distribution is surprising in the context of what is normally think of as insulator function; however, when CTCF-binding sites were mapped in the human genome, a similar distribution pattern was observed. This is suggestive of a wider role for insulator proteins than just the establishment of chromatin domains, and, in fact, alternative insulator protein functions have been suggested. For example, CTCF in humans is present in the Igh locus in many of the VH as well as DH and JH exons, suggesting a role in V(D)J recombination. Additionally, this study provides evidence that insulator proteins near genes play a role in the regulation of expression of specific genes and suggests that the mechanism behind this regulation differs from classic transcription factors, since the same insulator complexes were seen to both activate and repress transcription. These functions could be a consequence of the ability of these proteins to both interact with each other and mediate intra- and interchromosomal loops. Bringing together various insulator protein-binding sites could facilitate localization to either transcriptionally active or transcriptionally repressed regions of the nucleus depending on the genomic context of the sites (Bushey, 2009).

Comparison of the genome-wide distribution of the three insulator subclasses in two different cell lines has led to insights into possible mechanisms employed during cell differentiation to establish different patterns of gene expression. Overall, the analysis suggests that cells may control insulator function at multiple levels and that these forms of regulation occur throughout the genome. Regulation of insulator function seems to begin at the level of DNA binding, since differential binding was observed at 5%-37% of sites for Su(Hw), dCTCF, and BEAF between two different cell lines even with the most conservative statistical analysis. Similar percentages of cell type-specific binding sites were observed for vertebrate CTCF between different cell lines. Previous analysis of Su(Hw) binding in various tissues has not revealed any significant tissue-specific binding sites, perhaps because only a small number of sites was analyzed in these studies. Alternatively, the discrepancy could be due to the use of whole tissues in previous studies that contain multiple cell types, making it difficult to detect cell type-specific sites (Bushey, 2009).

After Su(Hw), dCTCF, and BEAF bind DNA, they are thought to recruit other proteins such as CP190. Regulation at this level was observed throughout the genome, where a subset of the Su(Hw), dCTCF, and BEAF sites recruit CP190 in a cell type-specific manner. The additional Su(Hw), dCTCF, and BEAF sites that do not recruit CP190 in either Kc or Mbn2 cells may do so in other cell types or other growth conditions not tested in this study. This idea is supported by the two dCTCF sites in the bithorax region that were found to contain CP190 in third instar larvae brains but not in the data sets collected in Kc cells or Mbn2 cells. Although further study is needed to determine which sites of insulator protein localization participate in chromatin organization, it is expected that sites lacking CP190 do not, since mutations in CP190 are known to disrupt insulator body formation and only those sites that recruit CP190 seem to affect gene expression. Therefore, these sites may represent insulators that are poised for incorporation into chromatin loops upon recruitment of CP190. On the other hand, these sites could function through the recruitment of an alternative cofactor and in this way represent a functionally distinct subset of Su(Hw)-, dCTCF-, and BEAF-binding sites (Bushey, 2009).

An additional layer of regulation may then occur at the level of protein-protein interactions mediated by CP190. This type of regulation cannot be gleaned from ChIP-chip data, but other experiments have shown that sumoylation of insulator proteins is able to inhibit protein-protein interactions affecting Su(Hw) insulator body formation but not association of insulator proteins with DNA. Similarly, vertebrate CTCF insulator function has been linked to poly(ADP-ribosyl)ation (PARlation), and it has been suggested that PARlation facilitates CTCF self-interaction. Furthermore, the presence of RNA and RNA-binding proteins may also contribute to the formation or maintenance of insulator bodies required to create chromatin loops. Finally, insulator bypass that results in the inactivation of insulator activity through pairing of nearby insulator elements, and specialized sequences such as the promoter targeting sequences (PTS), can allow an enhancer to bypass an insulator. These forms of regulation may alter the ability of insulator proteins to interact with one another to regulate insulator loop formation (Bushey, 2009).

It is expected that these various forms of regulation including DNA binding, CP190 recruitment, and loop formation result in changes in gene expression between different cell lines. However, transcription analysis with insulator proteins is difficult since insulator elements are thought to control regulatory elements such as enhancers and silencers that can be found far away from their target promoters. Therefore, determining which genes are controlled by an insulator site is not a trivial process. In the transcription analysis, genes were considered with a cell type-specific insulator site only within the gene or the 1 kb surrounding region. Despite this limitation, a significant enrichment was still seen for genes that change expression between cell types when they have a cell type-specific insulator site nearby, supporting the idea that insulator proteins are involved in the regulation of gene expression. Genes that did not change expression despite being located near a cell type-specific insulator protein-binding site may not be the actual target genes of the insulator sites; therefore, this analysis probably greatly underestimates the effect of insulator proteins on gene expression. Additionally, it was found that insulator protein-binding sites that localize to genes are enriched at genes with certain expression signals, high expression for dCTCF and BEAF, and low expression for Su(Hw). However, comparison between the two cell lines revealed that expression can be either positively or negatively regulated by sites with each DNA-binding protein. Therefore, although an insulator protein associates with a highly expressed gene, it may lead to either an increase or decrease in transcription of this gene. The observed level of expression may be an additive effect of many different regulatory elements, including multiple insulator sites. Different mechanisms may be used to regulate a highly transcribed gene versus a gene with low levels of transcription, and therefore the different insulator subclasses may target these different mechanisms (Bushey, 2009).

The transcription analysis in this study suggests that insulator proteins play a role in the regulation of gene expression, but has just begun to explore the depth of their effect. Numerous steps at which insulator activation can be subject to regulation allow for a vast amount of variation between different cell types and could play a major role in establishing the diverse patterns of chromatin organization necessary for cell type-specific gene expression. The different CP190 insulator subclasses might have distinct roles in this cell type-specific nuclear organization. In vertebrates, CTCF is the only insulator known thus far, and an important question to address in the future is the apparent disparity between genome complexity and insulator diversification between flies and vertebrates. It is possible that vertebrates have insulator subclasses represented by DNA-binding proteins other than CTCF that have not yet been identified. Alternatively, it is possible that vertebrate CTCF has acquired all the functions of the various Drosophila insulator subclasses. The distribution pattern of dCTCF suggests that it can play a role in both global organization and in the regulation of individual genes, making it the most likely candidate of the three Drosophila subclasses to play this overarching organizational role in vertebrates. Therefore, vertebrates may use methods other than variant DNA-binding proteins to distinguish insulator subclasses, such as recruitment of different CTCF interaction partners at different insulator sites. This would make it difficult to distinguish between the various layers of insulator control in the vertebrate genome. If this is the case, Drosophila could provide a powerful model system to dissect the various functions and levels of regulation of chromatin insulators (Bushey, 2009).

A comprehensive map of insulator elements for the Drosophila Genome

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. The genome-wide binding sites of 6 insulator associated proteins (dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF) was studied to obtain the first comprehensive map of insulator elements in Drosophila embryos. Over 14,000 putative insulators, including all classically defined insulators, were identified. Two major classes of insulators were defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila (Négre, 2010).

This study the embryonic binding profile of six factors previously known to be associated with insulator function in Drosophila. The analysis of insulator binding site distributions and protein composition suggest there exist 2 principal categories of insulator elements (Class I and Class II). In particular, it was shown that Class I insulators, identified by the binding of CTCF, CP190 or BEAF-32, segregate differentially expressed genes and delimit the boundaries of chromatin silencing, while they are depleted between known CRMs and their target genes. No evidence was found supporting a significant distinction between CP190/BEAF and CP190/CTCF or CTCF/BEAF. In contrast, the analyses suggest that BEAF-32, CP190, and CTCF are distributed and function quite similarly, while Su(Hw) appears distinct. The Class II insulators, bound by Su(Hw), are often exceptional in this analyses. It is noted that the analysis of genome-wide mapping data, expression data, and genome annotation provides an endogenous boundary assay that demonstrates that, while Su(Hw) has been described as an insulator before, it is not systematically associated with the boundaries of the gene units (Négre, 2010).

By helping to delimit the regulatory boundaries of genes, the Class I insulator map presented in this study will aid in the identification of transcription factor target genes and the construction of transcriptional regulatory networks. As an example of this concept, the distribution of known regulatory elements and insulators across the Antennapedia Complex (ANT-C) of homeotic genes is presented. This region quite strikingly demonstrates the potential utility of insulator binding data for cis-regulatory annotation. Across approximately 500 kb, cis-regulatory elements and their target promoters are found between insulator pairs. For example, a single insulator separates the lab and Edg84A genes, with their respective cis-regulatory elements narrowly partitioned on either side. The adjacent regulatory elements and promoters of zen and bcd are similarly insulator segregated (Négre, 2010).

Consistent with their observed regulatory boundary functions, Class I insulators are embedded within local regions of active chromatin and are frequently associated with syntenic breakpoints between species. Previous work has demonstrated that active promoters in yeast and Drosophila are associated with reduced nucleosome occupancy and low-salt soluble and high-salt insoluble chromatin. Therefore, surprisingly, dynamic chromatin is a shared feature between promoters and most classes of insulators. It is notable however that some studies have revealed functional similarities between insulators and promoters in transgenic assays. These results have been described as paradoxical, as insulators can negatively affect promoters by blocking communication between enhancers and promoters. One proposed model for insulator function is that they act as promoter 'decoys' by recruiting away factors necessary for transcriptional initiation. Alternatively, insulators and promoters might require common chromatin features to function by mechanisms that are still unknown. One potential interpretation is that the dynamic chromatin at insulators forms a flexible chromatin joint that would affect the probability of productive contact between separated regulatory elements. In this way, the similarity between promoters and insulators would be a consequence of their common requirement for dynamic chromatin, although with very different consequences. This model may explain why promoters are so frequently scored as insulators in the classical insulator assay, when an element is placed between an enhancer and a promoter (Négre, 2010).

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq was performed in two different embryonic cell lines; AGO2 was found to localize to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome (Moshkovich, 2011).

This study provides the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is dependent on CTCF and CP190 binding and may be partially achieved via looping interactions among cis-regulatory regions and promoters. It is proposed that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Moshkovich, 2011).

These results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 on polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in nonrepetitive euchromatic but not repeat-rich regions, as determined by genome-wide ChIP-seq. It is suggested that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the post-transcriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function (Moshkovich, 2011).

Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2. Second, AGO2, but not Dcr-2, is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity (Moshkovich, 2011).

An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. CP190 mutants were found to remain competent for silencing, suggesting that AGO2 chromatin association is not required for RNAi. Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated (Moshkovich, 2011).

A unique positive role for AGO2 but not other RNA silencing factors was identified in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wild-type levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis (Moshkovich, 2011).

This study shows that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, it was found that the Abd-B RB promoter interacts frequently with Fab-7, Fab-8, and the iab-8 enhancer and, moreover, that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high-frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus (Moshkovich, 2011).

AGO2 is recruited to chromatin insulator sites as well as noninsulator sites in a CTCF/CP190-dependent manner. It is speculated that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions. Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190-dependent looping interactions (Moshkovich, 2011).

AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities. However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis-regulatory elements that can maintain looping interactions of this locus, suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body (Moshkovich, 2011).

AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2. This study found that the AGO2^51B-null mutation has no effect on Fab-X PRE pairing-dependent silencing on sd as assayed in that study, and genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements that long-range interactions are dependent on insulator sequences and not PREs. Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures (Moshkovich, 2011).

It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In Caenorhabditis elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA. Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation. Recently, it has been shown that Schizosaccharomyces pombe Ago1 participates in surveillance mechanisms to prevent readthrough transcription of mRNA. However, the majority of Ago1 associates with heterochromatic regions, and it is not clear thus far whether Ago1 directly associates with euchromatin or acts post-transcriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms (Moshkovich, 2011).

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, it was 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 (Erokhin, 2011).

Insulators regulate gene activity in a variety of organisms. The defining feature of insulators as a class of regulatory elements is their ability to block enhancer-promoter interactions only when positioned between them (Erokhin, 2011).

Two mutually non-exclusive but rather complementary mechanisms can account for the ability of insulators to block enhancers and support long-distance interactions. Experiments with transgenic lines suggest that the interaction between insulators can result in the formation of chromatin loops that either block or facilitate long-distance enhancer-promoter communication depending on the nature of the interacting insulators as well as on the distances between all the elements involved (enhancers, insulators and promoters) and their relative 'strength'. Alternatively, insulator action can be explained by the ability of insulators to form direct contacts with either an enhancer (the decoy model) or a promoter, thereby inactivating them. For example, the insulator protein CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and blocks enhancer-promoter communication by directly interacting with Igf2 promoters. Insulators of the Drosophila Abd-B gene can establish contact with a region upstream of the promoter that is required for proper enhancer-promoter communication. Several Drosophila insulators [scs, scs', IdefixU3 and Fa^swb] have been shown to contain promoters, which, according to the decoy model, may tether enhancers in nonproductive interactions. The stalled promoters of the bithorax complex display insulator activity in embryos. Many insulator proteins, such as CTCF, CP190, Mod(mdg4)-67.2 [Mod(mdg4) - FlyBase] and BEAF (BEAF-32), are frequently found bound to the promoters (Erokhin, 2011 and references therein).

Previously, two well-studied tissue-specific Drosophila genes, yellow and white, were shown to contain insulators immediately downstream of their coding regions. The yellow gene is responsible for dark pigmentation of the larval and adult cuticle and its derivatives, whereas the white locus determines eye pigmentation. The 1A2 insulator located on the 3' side of the yellow gene contains two binding sites for the Su(Hw) protein. Additional proteins, Mod(mdg4)-67.2, CP190 and E(y)2, interact with Su(Hw) and are required for the activity of Su(Hw)-dependent insulators. None of the known DNA-binding insulator proteins binds to the Wari insulator located on the 3' side of the white gene. However, stage-specific binding of CP190 and E(y)2 to the Wari insulator has been observed (Erokhin, 2010), which was indicative of its relationship to Su(Hw) insulators (Erokhin, 2011).

This study presents evidence that the 1A2 and Wari insulators interact with their target promoters and that this facilitates the formation of a gene loop between the promoter and terminator regions (Erokhin, 2011).

These insulators can support a gene loop that brings together a promoter and a terminator. The results obtained by ChIP assay suggest that insulator-promoter interactions are transcription dependent. To date, transcription-dependent gene looping has been demonstrated in yeast and HIV provirus. In yeast, loop formation was reported to be organized by TFIIB and the Ssu72 and Pta1 components of the 3'-end processing machinery. It is possible that this mechanism is conserved between eukaryotes and that the interaction between an insulator and a promoter is required to facilitate the formation of a gene loop and/or its stabilization (Erokhin, 2011).

It has been suggested that gene loop formation might be a common feature of gene activation that serves to promote efficient transcriptional elongation and transcription reinitiation by facilitating RNAP II recycling from the terminator to the promoter, reinforcing the coupling of transcription with mRNA export and enhancing terminator function. This study has found that the interaction of insulators with promoters is required for the basal activity of the white and yellow promoters in the eye. In addition to the possible role of a gene loop in the enhancement of RNAP II recycling and mRNA export, insulators might serve to bring to the promoter the remodeling and histone modification complexes that improve the binding and stabilization of the TFIID complex (Erokhin, 2011).

Recently, Chopra (2009) have found that the enhancer-blocking activity of several promoters and insulators depends on general transcription factors that inhibit RNAP II elongation. That study suggests that insulators interact with components of the RNAP II complex at stalled promoters and that the resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with the neighboring locus. This study found that the upstream promoter regions required for interactions with enhancers are not necessary for insulator-promoter interactions, which provides evidence that insulator proteins can interact with general transcription factors or proteins involved in the organization of promoter architecture. Certain types of insulators [the Su(Hw)-dependent 1A2, the Zw5-dependent scs, and Wari] can effectively interact with the yellow promoter, whereas others appear not to (the GAF-dependent Fab-7 and CTCF-dependent Mcp). GAF and CTCF are frequently found bound to promoter regions (Smith, 2009; Bartkuhn, 2009; Bushey, 2009; Nègre, 2010), which indicates that insulators that utilize these proteins are also involved in long-distance interactions with some promoters. For example, it is speculated that the Fab-7 insulator can interact with stalled promoters, such as the Abd-B promoter (Erokhin, 2011).

This study has shown that the GAL4 activator is unable to stimulate the promoter when GAL4 binding sites are placed downstream of the insulator. It appears likely that the loop is also formed between the insulator and promoter in this case, but that GAL4 is rendered outside the loop and blocked by the insulator. Thus, a chromatin loop formed by the promoter and insulator can prevent undesirable interactions with downstream regulatory elements. This provides evidence that the promoter-binding capacity of at least some insulators might contribute to their enhancer-blocking activity (Erokhin, 2011).

The genome-wide analysis of binding sites for insulator proteins has shown that they are present at the 3' and 5' UTRs of many Drosophila genes (Nègre, 2010). The 1A2 and Wari insulators at the 3' end of the yellow and white genes were identified only as a result of the extensive use of these genes in insulator assays. Thus, it appears that insulators are likely to be located at the 3' UTRs of many genes. Further experiments are required to resolve this issue and to elucidate the mechanisms and functional role of insulator-promoter interactions in transcriptional regulation (Erokhin, 2011).

Nature and function of insulator protein binding sites in the Drosophila genome

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. This study tested this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. Distinct combinatorial binding was found of insulator proteins to different classes of sites, and a novel type of insulator element was uncovered that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. Insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes (Schwartz, 2012).

The binding sites of insulator proteins are often taken to represent elements that partition the genome into independent regulatory domains and demarcate chromosomes into regions of 'active' and 'repressed' chromatin. The results presented in this study give little support to this view as a general principle of genome organization although it may be true in certain regions. Instead it is argued that: 1) insulator proteins bind to genomic sites in specific combinatorial patterns, 2) the properties of sites bound by key insulator proteins SU(HW) and CTCF are markedly different depending on whether the two co-bind with CP190, 3) many of the known insulator proteins sites do not function as robust enhancer blockers, and 4) at least in cultured cells the depletion of insulator proteins has a limited impact on genome-wide gene expression (Schwartz, 2012).

Classifications of combinatorial binding of insulator proteins have been described previously. These classifications relied on the overlapping of bound regions defined according to arbitrary statistical thresholds and the position of these regions relative to TSSs. Because they did not take into account the relative strengths of binding, such classifications grouped together binding sites with very different biochemical and functional properties (Schwartz, 2012).

In contrast, this study defines the persistent co-binding patterns based on the strength of binding of the associated proteins, treating regions strongly bound by a combination of proteins differently from regions at which the same proteins are detected according to a statistical threshold but where the extent of their binding is disproportional. It is argued that this approach retains the information on biochemical interrelations between the co-bound proteins and separates the sites with different functional properties. The strongest support for this argument comes from RNAi knock-down experiments which demonstrate that the effect of the loss of one insulator protein on the binding of another insulator protein is constrained to a specific class of co-bound regions. For example, the knock-down of SU(HW) results in the loss of CP190 from class 3 (gypsy-like) sites but not from class 9 (CTCF+CP190) or class 5 (BEAF-32+CP190) sites (Schwartz, 2012).

The approach to select the sites representative of each co-binding class is conservative and inevitably excluded a fraction of binding sites from downstream analyses. For example, strong SU(HW) binding sites assigned to class 14 by initial overlap comparison were not analyzed further due the uncertainty of their co-binding by CP190. It is therefore cautioned that selection of representative binding sites is not a complete genomic catalogue and readers are advised to use the ChIP-chip binding profiles, deposited to GEO and modMINE, to gauge whether their locus of interest has a strong insulator protein binding site (Schwartz, 2012).

The prevailing model in the field suggests that CP190 is recruited to different insulator elements by DNA binding proteins where it serves as a universal adapter that mediates interactions between different insulator elements. The current results present a more complex picture. First, RNAi knock-down experiments demonstrate that the binding of SU(HW) protein to class 3 (gypsy-like) sites is dependent on CP190, indicating that CP190 is not passively tethered to common sites by SU(HW) and instead plays an active role in recruitment and/or stabilization of the bound complex. Second, the sequence analysis of class 9 (CTCF+CP190) sites suggests that the binding of both proteins to these sites is likely due to the coincidence of cognate recognition sequences. Third, RNAi knock-down experiments indicate that BEAF-32 is dispensable for CP190 binding at shared sites. Clearly CP190 plays an active role in the selection of sites shared with SU(HW), CTCF or BEAF-32. It is still possible that once it co-binds, or binds sufficiently close to another insulator protein, it may mediate the trans-interactions of the bound sites. However, such interactions would have to be rather transient, at least in cultured cells, as they are not easily detected in the ChIP-chip data (Schwartz, 2012).

SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation

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. This study demonstrates 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 (Golovnin, 2012).

Posttranslational modification by SUMO has been shown to regulate subcellular localization of many targets, including RanGAP, PML, SATB2 and others. This study presents data that SUMO is necessary for co-localizing the Su(Hw), Mod(mdg4)-67.2, and CP190 proteins in nuclear speckles, named insulator bodies. Previously an opposite model has been proposed according to which sumoylation of Mod(mdg4)-67.2 and CP190 leads to disruption of insulator bodies. This model was mainly based on the observation that, in diploid cells from the larval brain, mutations in the gene encoding Ubc9 restored aggregation of the CP190 protein in the mod(mdg4)^u1 background. This study found that inactivation of Mod(mdg4)-67.2 did not affect the ability of CP190 to form insulator bodies in S2 cells (Golovnin, 2012).

mod(mdg4)^u1 mutation also did not affect CP190 incorporation into the insulator bodies in diploid cells of wing and eye imaginal discs. Thus, the significance of Mod(mdg4)- 67.2 for CP190 recruitment to the insulator bodies is confined to diploid cells of the larval brain. To test the role of sumoylation in the formation of insulator bodies, the lwr5 mutation, generated by a single amino acid substitution in the Ubc9 region (R104H) located on the loop between strand 7 and helix B, has been used. This region of Ubc9 is required for the interaction of its active site with the substrate. Although untested, it appears that R104H makes the surface of the mutant enzyme (Ubc95) more hydrophobic, thereby strengthening binding interactions for certain enzyme-substrate pairs. Thus, lwr5 is not a null-mutation in the gene, and Ubc95 can either increase or decrease sumoylation, depending on the protein substrate. Therefore, additional studies are required to demonstrate role of Ubc95 in the formation of insulator bodies in the imaginal disks of larvae (Golovnin, 2012).

The data provide evidence for a critical role of CP190 and a passive role of Su(Hw), a DNA-binding protein, in the formation of insulator bodies. In addition to Su(Hw), CP190 forms complexes with dCTCF that is also co-localized in the insulator bodies. Thus, it is likely that Mod(mdg4)-67.2 and CP190 proteins recruit DNA-binding dCTCF and Su(Hw) proteins to the insulator bodies (Golovnin, 2012).

As shown previously, SUMO is necessary for the formation of PML nuclear bodies (PML-NBs). These bodies are formed due primarily to the self-assembly ability of the PML N-terminal domain. Moreover, SUMO-1 modification of PML was shown to target the protein from the nucleoplasm to the NBs. The occurrence of both sumoylation sites and SUMO-interacting motifs (SIMs) in the PML protein provides a basis for the network of interactions that constitute the nucleation event for subsequent recruitment of sumoylated proteins and SIM-containing proteins (Golovnin, 2012).

Cells that lack PML are unable to form NBs, with other NB components remaining diffusely distributed in the nucleus. While analysis of the CP190 sequence suggests the presence of two SIMs, no direct interaction was observed between CP190 and SUMO in vitro. At the same time, CP190 and Mod(mdg4)-67.2 contain several protein-protein interaction domains, including BTB/POZ that might be involved in direct interaction with many DNA-binding transcription factors, such as Su(Hw) and dCTCF, to facilitate their assembly into the insulator bodies. It is noteworthy that heat shock has proved to induce redistribution of CP190 to the nuclear periphery, in complex with SUMO. This is evidence that the formation of insulator bodies requires interactions with additional proteins, which are disrupted as a result of heat shock treatment (Golovnin, 2012).

Sumoylation is essential for the functional activity of proteins in transcriptional repression, activation, and recruitment of modifying complexes. This study has demonstrated that inactivation of sumoylation sites in the Mod(mdg4)-67.2 protein does not affect its functional activity in the insulator complex. This finding is in accordance with the previous observation that only 10% of Su(Hw) binding sites coincide with SUMO on polytene chromosomes (Golovnin, 2012).

This study confirms the role of Mod(mdg4)-67.2 in recruiting the Su(Hw) protein to the insulator bodies and insulators. When the mutant Mod(mdg4)-67.2 protein was expressed at a low level, Su(Hw) binding was reduced, whereas low expression of the wild-type Mod(mdg4)-67.2 protein was sufficient for completely restoring Su(Hw) binding to insulators. Therefore, the assembly of the Su(Hw) and Mod(mdg4)-67.2 proteins in insulator bodies is essential for subsequent recruitment of insulator complexes to DNA. A higher level of the mutant Mod(mdg4)-67.2 protein increases the probability of formation of the Su(Hw)/Mod(mdg4)-67.2 complex out of insulator bodies, thereby providing for more effective binding of the Su(Hw) and mutant Mod(mdg4)-67.2 proteins to the insulators (Golovnin, 2012).

Taken together, these results support the model of insulator bodies as a depot of proteins involved in transcription regulation and insulation. According to these results, the insulator proteins can interact and form complexes without SUMO. However, partial sumoylation of the Mod(mdg4)-67.2 and CP190 proteins lead to further aggregation of the protein complexes in insulator bodies. The sumoylated Mod(mdg4)-67.2 and CP190 proteins interact with Su(Hw) and recruit it to the insulator bodies. The insulator bodies possibly protect the insulator complex from degradation and facilitate the formation of complexes between Su(Hw)/Mod(mdg4)-67.2/CP190 and other transcription factors. 'Mature' insulator complexes may then transiently interact with the chromatin fibril and detach from the insulator bodies by means of desumoylation. As was suggested for PML bodies, proteins deposited in the insulator bodies may be used during cell stress. For example, it was found that heat shock treatment induced relocation of CP190 from the insulator bodies to the nuclear periphery but did not affect the insulator complexes bound to DNA. Such an unusual relocation of the CP190 protein resulted in a diffuse distribution of the Su(Hw) and Mod(mdg4)-67.2 proteins. Thus, it appears that insulator proteins may have an as yet unknown yet role in cell response to heat shock stress. During DNA replication, a large amount of insulator proteins is required for newly synthesized chromosomes. It is possible that desumoylation of insulator bodies during DNA replication results in the release of protein complexes that form functional insulators on the newly synthesized DNA.Further studies are required to verify this model (Golovnin, 2012).

Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila

Insulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. This study demonstrates that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADPribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)-ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation (Ong, 2013).

Insulator proteins play an important role in chromatin organization, but the mechanisms by which insulator activity can be regulated to orchestrate the establishment of distinct patterns of intra- and interchromosomal interactions during cell differentiation are poorly understood. This study presents evidence suggesting that Drosophila insulator proteins CP190, dCTCF, Mod(mdg4)2.2, and Su(Hw) are PARylated and that mutations in the Parp gene impair the activity of the gypsy and Fab-8 insulators in vivo. Consistent with reports indicating that binding of vertebrate CTCF to DNA is independent of PARylation, this study found that inhibition of PARylation causes only a moderate change in the genome-wide occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). Instead, interaction of CP190 with insulator DNA-binding proteins is decreased in the absence of PARylation. Because CP190 is involved in mediating interactions among insulator sites, it is likely that PARylation regulates the ability of insulators to mediate contacts between distant sites in the genome. This conclusion is strongly supported by the fact that PARylation of CP190 protein at lysine 566 is required for its in vivo function (Ong, 2013).

Eukaryotic genomes are organized into physical domains that are remarkably stable between cell types and even species. Although borders between topologically associating domains (TADs) are enriched in aligned insulators in Drosophila and contain CTCF, SINE elements, and tRNA genes in mice and humans, the majority of insulator binding sites lie within TADs (Dixon, 2012; Hou, 2012; Sexton, 2012). This differential distribution points to the possible existence of two functional classes of insulator sites in the genome. One class is composed of sites that are relatively constant during cell differentiation and are present at TAD borders. The second one may be composed of independent insulator sites within TADs that may have a role in regulating intradomain interactions to affect specific transcriptional outcomes. Consistent with this hypothesis, CTCF is primarily involved in mediating intradomain interactions in pre-pro B cells (Lin, 2012). Moreover, recent studies indicating that the large, invariant TADs can be hierarchically organized by CTCF, cohesin, and/or Mediator complexes into constitutive and cell-type-specific subtopologies support the idea that interactions within TADs can be regulated during cell differentiation (Phillips-Cremins, 2013). The current results suggest that PARylation of insulator proteins may represent a mechanism used by cells to regulate intrachromosomal contacts during their response to stimuli and cell lineage commitment. Significant disruption in the formation of insulator bodies in Parp mutants suggests that nuclear clustering of distant insulator sites may require PARylation. This clustering is mediated by CP190 and Mod(mdg4), which in turn interact with the insulator DNA-binding proteins dCTCF and Su(Hw). By modulating the interactions between these two sets of proteins, PARylation may influence insulator-mediated chromatin looping both within topological domains and between TAD borders to elicit either a local transcriptional response or global architectural reorganization of the genome. One observation from the current studies is that PARylation-sensitive CP190 binding sites are enriched within H3K27me3-marked chromatin domains. Although Polycomb group proteins are recruited by PARP-1 to DNA lesions during the UV damage response, it remains to be seen whether PARylation of CP190 can be targeted by the Polycomb complex at specific genomic sites. A recent report found that Tip60-mediated H2AK5 acetylation at the 5' end of the Hsp70 genes is critical for the activation and spread of Parp prior to nucleosome eviction (Petesch, 2012), suggesting that additional mechanism may be present to target PARylation to specific insulator binding sites (Ong, 2013).

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. This study identifies chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. The results reveal an extensive list of known and novel HP1a-interacting proteins, of which three were selected for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, this study found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. It is proposed that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms (Alekseyenko, 2014).

The centrosomal protein CP190 is a component of the gypsy chromatin insulator

Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190<) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2 (Pai, 2004).

A genetic screen for dominant enhancers of mod(mdg4) has resulted in the identification of CP190 as a third component of the gypsy insulator. CP190 is present at gypsy retrotransposon insulator sites and overlaps extensively with Su(Hw) and Mod(mdg4)2.2 at presumed endogenous insulators. CP190 displays a specific distribution pattern on polytene chromosomes, showing significant overlap with Su(Hw) and Mod(mdg4)2.2 at the junctions between transcriptionally inert bands and transcriptionally active interbands. Similar localization patterns have been reported for other insulators. For example, the fa^swb insulator at the notch locus and the BEAF-32 protein of the scs' insulator are also present at the boundaries between bands and interbands. Results suggest that CP190 can bind DNA on its own or can be tethered to the chromosome through interactions with Su(Hw). Mutations in the CP190 gene impair the function of the insulator present in the gypsy retrotransposon without affecting the presence of Su(Hw) and Mod(mdg4)2.2, suggesting an essential task for CP190 in the activity of this insulator. In addition, the lethality of CP190 mutants suggests a critical role for the CP190 protein in the function of gypsy endogenous insulators. This essential role may be a consequence of the requirement of CP190 for the formation of insulator bodies in the nuclei of diploid cells (Pai, 2004).

The insulator present in the gypsy retrotransposon contains only Su(Hw) binding sites, and CP190 is present in this insulator through direct interactions with Su(Hw). The gypsy insulator contains 12 Su(Hw) binding sites, and at least four are needed for insulator activity. However, clusters of three or more Su(Hw) binding sites are rare in the genome. Therefore, a critical question is whether the sites of Su(Hw) and Mod(mdg4)2.2 localization present throughout the genome truly function as insulators. The presence of CP190 at these sites and its ability to bind DNA might explain this apparent paradox. For example, the endogenous insulator present in the yellow-achaete region has only two binding sites for Su(Hw). Nevertheless, the y454 fragment containing this insulator is able to bind CP190, suggesting that this protein might act in concert with Su(Hw) to confer insulator activity. It is therefore possible that endogenous gypsy insulators are composed of binding sites for Su(Hw) and/or for CP190 and, together with Mod(mdg4)2.2, form a complex. Endogenous gypsy insulators may have few or no Su(Hw) binding sites, and they may rely on CP190 to bind DNA and tether other insulator components such as Mod(mdg4)2.2 via protein-protein interactions (Pai, 2004).

Previous studies have suggested that gypsy insulators separated at a distance in the genome may come together and form large insulator bodies in the nucleus during interphase. These aggregates represent higher order structures of chromatin and are implicated in the regulation of gene expression by compartmentalizing the genome into transcriptionally independent domains. The formation of these aggregates appears to require Mod(mdg4) function because the large aggregates are missing in mod(mdg4) mutants. The formation of gypsy insulator bodies is severely impaired also in CP190 mutants, suggesting that CP190 plays an essential role in the formation of these bodies and in the establishment of the chromatin domain organization mediated by gypsy endogenous insulators. It is possible that the BTB/POZ protein-protein interaction domains of both CP190 and Mod(mdg4)2.2 are required for and contribute to the stability of the interactions among insulator sites. In vitro-expressed CP190 lacking the BTB/POZ domain is soluble, whereas the wt protein is not, further suggesting that CP190 might exist as a complex with itself or other proteins in vivo, and the formation of this complex is likely mediated by the BTB/POZ domain. However, because CP190 is present at the gypsy insulator in the absence of Mod(mdg4)2.2 protein, the interaction between these two proteins may not be crucial for CP190 recruitment to the insulator (Pai, 2004).

Previous studies have identified CP190 as a centrosome-specific protein during mitosis that also associates with chromatin during interphase. Although many of these studies have focused on the possible role of CP190 during cell division, the current results suggest that centrosomal function and cell division are not affected in CP190 mutants. This conclusion is supported by independent studies of CP190 function during the cell cycle. The main function of CP190 might then be to regulate chromosome-related processes during interphase. Several lines of evidence suggest that this role is related to the function of the gypsy insulator: mutations in CP190 alter gypsy-induced phenotypes; CP190 colocalizes with Su(Hw) and Mod(mdg4)2.2 on polytene chromosomes and in diploid cell nuclei, and CP190 associates physically with gypsy insulator components in vitro and in vivo. However, the centrosomal localization of CP190 might also be important for its role in the gypsy insulator despite being unnecessary for cell cycle progression. The centrosome could either be a temporary storage site for CP190 during mitosis, or a site for a mitosis-specific modification that could be important for CP190 reassociation with chromosomes later in the cell cycle. The presence of CP190 in the centrosome could also be related to the regulation of the level of this protein in the cell. In fact, it has been shown that some chromatin-binding proteins are targeted to the centrosome for degradation. Alternatively, the presence of CP190 at the centrosome might be related to a possible role in the ubiquitin modification pathway. Recent findings have linked BTB/POZ domain proteins to ubiquitin E3 ligase function, some of which are known to be present at the centrosome. CP190 may be involved in similar types of interactions as an adaptor for ubiquitin E3 ligases and might target associated insulator proteins to the centrosome during mitosis for ubiquitination and/or degradation, which in turn may be required for properly reestablishing chromosome domain boundaries after mitosis (Pai, 2004).

The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator

Chromatin insulators are gene regulatory elements implicated in the establishment of independent chromatin domains. The gypsy insulator of D. melanogaster confers its activity through a protein complex that consists of three known components, Su(Hw), Mod(mdg4)2.2 (a spliced variant encoded by the modifier of mdg4), and CP190. Drosophila Topoisomerase I-interacting RS protein (dTopors) interacts with the insulator protein complex and is required for gypsy insulator function. In the absence of Mod(mdg4)2.2, nuclear clustering of insulator complexes is disrupted and insulator activity is compromised. Overexpression of dTopors in the mod(mdg4)2.2 null mutant rescues insulator activity and restores the formation of nuclear insulator bodies. dTopors associates with the nuclear lamina, and mutations in lamin disrupt dTopors localization as well as nuclear organization and activity of the gypsy insulator. Thus, dTopors appears to be involved in the establishment of chromatin organization through its ability to mediate the association of insulator complexes with a fixed nuclear substrate (Capelson, 2005).

A yeast two-hybrid screen for proteins that interact with Mod(mdg4)2.2 resulted in identification of dTopors as a factor involved in the activity of the gypsy insulator. dTopors was found to interact with the three known insulator components, Su(Hw), Mod(mdg4)2.2, and CP190, and to associate with the gypsy insulator complex on chromosomes and in diploid nuclei. Additionally, dTopors appears to physically associate with the nuclear lamina. Genetically, dTopors was shown to behave as a positive factor involved in gypsy insulator activity. Consistently, reduction in levels of dTopors, observed in the background of a dTopors-spanning deletion or of an inducible dTopors RNAi construct, results in the disruption of insulator activity. The effects of elevated levels of dTopors are particularly dramatic as they restore the activity of a compromised gypsy insulator on multiple levels. The enhancer blocking function of the insulator, the binding of Su(Hw) to chromatin, and the formation of insulator bodies in cell nuclei -- all compromised in mod(mdg4)^u1 mutants -- are rescued by overexpression of dTopors (Capelson, 2005).

These effects can be explained by a model in which dTopors acts as a nuclear lamina-associated factor that serves to tether the gypsy insulator complexes to a fixed substrate. In the wild-type situation, Mod(mdg4)2.2 mediates the coalescence of distant insulator sites and the subsequent establishment of chromatin compartments, whereas dTopors may be involved in further organization of insulator bodies at specific nuclear attachment points through its direct interaction with both Mod(mdg4)2.2 and Su(Hw). The absence of Mod(mdg4)2.2 leads to the breakdown of nuclear organization and the destabilization of Su(Hw)-chromatin association. Through tethering distant insulator sites to a nuclear substrate, dTopors, when present at elevated levels, may be able to compensate for the loss of a component such as Mod(mdg4)2.2. By stabilizing the nuclear organization of insulator complexes, dTopors may also promote the binding of Su(Hw) to chromatin. This explanation is further reinforced by the observed disruptive effects of a lamin mutation on the nuclear organization and the enhancer blocking activity of the gypsy insulator (Capelson, 2005).

The connection between gypsy insulator activity and nuclear insulator bodies has relied predominantly on the effects of the mutations in Mod(mdg4)2.2 and CP190 on both enhancer blocking function and insulator body integrity. The activity of dTopors provides further evidence for a functional relationship between insulators and their nuclear localization, since rescue of insulator phenotypes by dTopors is accompanied by the recovery of insulator bodies. Establishment of independent chromatin domains, which has been proposed as the main function of insulators, is thought to rely on structural partitioning of chromatin through physical interactions between distant loci or through interactions with a fixed nuclear substrate. It has been previously intimated that gypsy insulators may employ both types of structural organization to ensure the establishment of domain autonomy. This work suggests that the gypsy insulator may undergo physical clustering through the BTB domains of Mod(mdg4)2.2 and of CP190 and may utilize the attachment to the nuclear lamina via dTopors. The interaction of the insulator with a nuclear substrate is further supported by a recent report that gypsy insulator proteins associate with the nuclear matrix, of which lamin is a principal component. Tethering to a subnuclear surface has also been implicated in the activity of the chicken β-globin insulator, where β-globin insulator loci were observed to interact with the nucleolar surface, perhaps via a direct association between the insulator protein CTCF and the nucleolar component nucleophosmin (Capelson, 2005).

The E3 ubiquitin ligase activity of dTopors was not found to act directly on the known insulator proteins, yet the RING domain of dTopors appears to be essential for its positive effect on the gypsy insulator. It thus remains possible that an unknown factor involved in insulator activity may be a substrate for dTopors-mediated ubiquitination. A connection between the gypsy insulator complex and the ubiquitin conjugation pathway is also suggested by the presence of BTB domains in Mod(mdg4)2.2 and CP190, since BTB domain proteins have been proposed to act as substrate adaptors for the ubiquitin RING E3 ligases. It is feasible that BTB-containing insulator proteins and RING-containing dTopors are involved in ubiquitin conjugation with functional consequences for the insulator (Capelson, 2005).

The association of dTopors with a subset of insulator binding sites on polytene chromosomes implies that its presence is not required by all insulator complexes. This may be a consequence of the proposed function of dTopors as a tethering factor, such that the interaction between distant insulator loci may alleviate the need for dTopors at every binding site of the insulator complex. Alternatively, it may suggest that endogenous insulator complexes are not all functionally equivalent, and that the enzymatic properties of dTopors may be important for specific insulator complexes. The ubiquitin ligase activity of dTopors may be involved in regulation of insulator complexes, such that modification of a yet uncharacterized component by ubiquitin can lead to variation in function of endogenous insulators (Capelson, 2005).

SUMO conjugation attenuates the activity of the gypsy chromatin insulator

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. Evidence is presented suggesting that two protein components of the gypsy chromatin insulator of Drosophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation (Capelson, 2006).

Two protein components of the gypsy chromatin insulator, Mod(mdg4)2.2 and CP190, were found to be modified by SUMO in vitro and in vivo. dTopors was observed to interfere with their sumoylation by possibly disrupting the contacts between the SUMO E2 enzyme Ubc9 and substrate insulator proteins. The inhibitory effect of dTopors, although relatively subtle, is consistent across the various assays utilized such that any time dTopors was introduced at higher levels, either by direct addition in vitro or by increasing expression in vivo, it was found to result in reduced sumoylation of Mod(mdg4)2.2 and CP190. Disruption of SUMO conjugation by mutations in genes coding for Ubc9 and SUMO exerts a positive effect on gypsy insulator activity, suggesting that the normal role of SUMO modification is to antagonize insulator function. A fraction of chromatin-bound insulator proteins appears to be associated with SUMO, yet mutations in the SUMO pathway are not seen to affect the chromatin-binding properties of CP190 or Mod(mdg4)2.2. Instead, sumoylation interferes with the formation of nuclear insulator bodies, such that overexpression of Ubc9 leads to breakdown of nuclear insulator structures, whereas lower levels of Ubc9 and sumoylation result in a partial recovery of coalescence lost in the absence of Mod(mdg4)2.2 (Capelson, 2006).

These findings suggest that modification of CP190 and Mod(mdg4)2.2 by SUMO may prevent self-association and thus interfere with long-range interactions between distant insulator complexes required to form insulator bodies. Thereby, sumoylation may preclude formation of closed chromatin loops and the consequent establishment of autonomous gene expression domains (Capelson, 2006).

Multiple lines of evidence point to a role for SUMO modification in transcriptional repression. Sumoylation of histones has been characterized as a mark of repressed chromatin, whereas SUMO conjugation to certain transcriptional regulators leads to their association with histone deacetylases, which remove the active acetylation marks from histones. SUMO modification of the Polycomb group (PcG) protein SOP-2 is required for its function in stable repression of Hox genes, and another PcG repressor, Pc2, acts as a SUMO E3 ligase. Modification of gypsy insulator proteins by SUMO does not seem to associate them exclusively with transcriptional repression, as reduction of sumoylation in lwr/smt3 mutants results in the upregulation of expression from the omb^P1-D1 locus, but in the downregulation of transcription at y² and ct⁶. In these cases, transcriptional output appears to correlate only with the enhancer-blocking activity of the insulator. Nevertheless, it is possible that one of the roles of sumoylation involves association of selected insulator sites in the genome with transcriptional repression. Sumoylated insulator complexes may not participate in the formation of expression domains, but instead, could target silencing factors to the surrounding chromatin (Capelson, 2006).

In mammalian nuclei, the homolog of dTopors localizes to PML bodies, which are enriched in the SUMO conjugation machinery. If inhibition of sumoylation is also a property of mammalian Topors, it may play a role in preventing further sumoylation of factors that are targeted to these nuclear compartments. In this manner, ICP0 also localizes to the PML bodies, where it causes desumoylation of two primary components, PML and SP100. It has been reported that Topors may function as a SUMO E3 ligase for the tumor suppressor p53 protein. This apparent contradiction with the current results may be due to several reasons. Topors and dTopors may have diverged their functions regarding the SUMO pathway, such that Topors functions as a SUMO E3 while dTopors interferes with SUMO addition due to its conserved interaction with Ubc9. Alternatively, the involvement of dTopors in the SUMO pathway may be substrate-specific, since it may bind to Ubc9 in ways that allow for interaction with a given target protein or prevent it. In the context of the gypsy insulator, the interference of dTopors with sumoylation is consistent with previous observations that dTopors promotes insulator activity, whereas sumoylation appears to disrupt it (Capelson, 2006).

It has been suggested that SUMO conjugation may affect the function of the modified protein even after the SUMO tag itself has been removed, creating a cellular memory for protein regulation. This idea has arisen partly to explain the commonly observed contradiction between the small percentage of a given protein that is modified by SUMO and the dramatic consequences of the modification on the protein's cellular function. Sumoylation may be needed for proteins to enter stable complexes or functional states, but the persistence of the SUMO modification may not be required after the initial establishment. Thus, the actual effect of sumoylation may far exceed that of the detectable sumoylated population since the function of a much larger proportion of molecules has been altered by SUMO conjugation and subsequent deconjugation. Similarly to other reported cases, the sumoylated forms of Mod(mdg4)2.2 and of CP190 represent a small fraction of the total pool of the insulator proteins, yet the phenotypic effects of the loss of these forms are quite striking. It is possible that SUMO attachment regulates the initial organization of chromatin domains, perhaps in earlier development or following mitosis, yet once established, the domains may be stably maintained without SUMO. Additionally, the rapid conjugation and deconjugation cycle of the SUMO tag implies that sumoylation may be used by processes that require reassembly upon signal. In that sense, SUMO modification seems particularly suitable for the regulation of gene expression domains as it can result in 'remembered' yet flexible states (Capelson, 2006).

The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning

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. This study found that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlaps 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, this study demonstrates the importance of dCTCF in fly development and in the regulation of abdominal segmentation (Mohan, 2007).

The CP190 protein contains three classical C2H2 zinc-finger motifs and an N-terminal BTB/POZ domain. Both domains could potentially be involved in chromatin binding. In contrast, chromatin binding might be achieved by interaction with other factors, such as dCTCF. A possible interaction of dCTCF with CP190 was tested using co-immunoprecipitation. Precipitation of CP190 from Schneider cell extracts resulted in the detection of dCTCF. To confirm the interaction a FLAG-dCTCF fusion protein was expressed in Schneider cells and precipitated with either an antibody against CP190 or an antibody against FLAG. The CP190 precipitate contained endogenous dCTCF as well as FLAG-dCTCF in the same ratio as the input, suggesting that both dCTCF proteins are similarly associated with CP190. Furthermore, the reverse experiment using FLAG precipitation demonstrated that dCTCF and CP190 interact in vivo (Mohan, 2007).

Because CP190 and dCTCF colocalize on polytene chromosomes and interact in vivo, it was asked whether the overall amount of dCTCF protein might be changed in CP190-deficient third instar larvae. A Western blot analysis of both Cp190¹ homozygotes (deficient in CP190) and wild-type larval extracts showed that the amount of dCTCF is reduced in Cp190¹ homozygotes (Mohan, 2007).

Next it was of interest to know whether the reduced amount of dCTCF caused by the loss of CP190 affects dCTCF binding on the polytene chromosomes. It was found that the total number of dCTCF labeled sites is reduced in the Cp190¹ mutant, whereas the number of CP190 sites was not affected by dCTCF mutants. The analysis of dCTCF binding in the two hypomorphic mutants CTCF^EY15833/CTCF^EY15833 and GE24185/GE24185 revealed that that the number of bound sites is reduced to about 50% and 25%, respectively. By close inspection of the chromosomes it was found that the set of dCTCF sites missing in the CP190 or in the dCTCF mutants overlap but are not identical. Thus, different sites vary in their requirement for CP190/dCTCF cooperation (Mohan, 2007).

Insulator elements with enhancer blocking activity establish independent regulatory domains. An analysis of binding sites (CTS) for the enhancer blocking factor dCTCF on salivary gland polytene chromosomes resulted in the identification of several hundred sites bound by dCTCF. All of these sites are found in interbands, and when inspected more precisely are often at the borders of interbands next to bands. Interbands harbor active housekeeping genes or regulatory regions of inactive genes, whereas bands contain the bodies of inactive genes. Interbands and bands differ in chromatin composition and modification. Thus, there is a clear border between interbands and bands. Any factors generating functional chromatin boundaries would be expected to be localized to the interband/band transition. This is not only the case for dCTCF, as a similar location has been found for Su(Hw). Also, BEAF-32 and Zw5 are located in interbands at hundreds of binding sites throughout the genome (Mohan, 2007).

The obvious question was whether dCTCF has a redundant function and therefore similar targets as the other Drosophila enhancer blocking factors. No significant colocalization of dCTCF with either BEAF-32 or with Su(Hw) on polytene chromosomes was detected. This may provide an explanation of how an organism with a small genome, such as Drosophila, can prevent promiscuous enhancer interaction with any nearby gene. Apparently, an elaborate system of different enhancer blockers and barrier factors fulfills the insulation of regulatory units (Mohan, 2007).

The biochemical composition and function of insulator complexes involving Su(Hw) have been studied in detail. The best studied binding site is the gypsy transposon with a 350-bp sequence containing 12 binding sites for Su(Hw). A functional complex of Su(Hw), Mod(mdg4)67.2, CP190, and possibly other factors has been documented (Capelson, 2005; Lei, 2006). Although there is no colocalization of Su(Hw) with dCTCF on polytene chromosomes, and only partial colocalization with Mod(mdg4), it was of interest to examine whether CP190 plays a role in dCTCF function. Vertebrate CTCF is a centrosomal factor during mitosis and a nuclear protein during interphase (Zhang, 2004), and that CP190 (centrosome binding protein) is associated with centrosomes as well. CP190 is essential for viability, but is not required for cell division (Butcher, 2004). CP190 knockdown in Schneider cells has no effect, whereas a null mutation in flies leads to pharate lethality. A similar phenotype is seen after dCTCF depletion in Schneider cells and in the pharate lethality in flies. The centrosomal function of CP190 is not required for the insulator activity in the context of Su(Hw) bound to gypsy (Pai, 2004). The localization of CP190 on polytene chromosomes overlaps with sites bound by Su(Hw) or by Mod(mdg4)67.2. In addition, CP190 is found at loci devoid of Su(Hw) or Mod(mdg4)67.2, suggesting that other factors might recruit CP190 to these sites (Pai, 2004). There is a significant overlap in dCTCF with CP190 binding sites. A functional dependence is seen, because at many sites binding of dCTCF depends on CP190. Although there is an overall reduction in the dCTCF amount observed in the CP190 mutant, differences in dCTCF occupancy in dCTCF and CP190 mutants indicate a discrimination between CP190-independent and -dependent sites. Furthermore, the previously characterized insulator Fab-8 is impaired in the absence of dCTCF (Moon, 2005) and by the reduction of CP190 (Mohan, 2007).

Another perspective on the requirement of insulators comes from the fact that many genes are controlled by several regulatory elements that are required for tissue and cell-specific expression. A prominent example is the Drosophila BX-C. This is one of two Hox gene clusters, which contain regulator genes controlling development. The BX-C is responsible for the correct specification of the posterior thorax segment (T3) and all of the abdominal segments. Within BX-C, only three protein coding genes, Ubx, abd-A and Abd-B, are responsible for the segment-specific development of organs and tissues. On the other hand, nine separate groups of many mutations are affecting segment-specific functions. The borders of some of these domains are genetically defined by elements Fab-6, Fab-7, Fab-8 and by Mcp. Proteins involved in such a functional separation are the GAGA factor in case of the Fab-7 element, and dCTCF for the Fab-8 sequence. Recently, it has been demonstrated that six of the BX-C domain junctions are bound by dCTCF. Consequently, if these sites contribute to boundary function, gene activity within this locus should be changed. Indeed, a homeotic phenotype and a reduced expression of Abd-B was found in larval nerve cord. If dCTCF plays a central role in separating the different regulator domains in the BX-C and elsewhere in the genome, it is difficult to predict the dCTCF phenotype. The situation could be complicated as the three BX-C genes are controlling realizator genes as well as other regulators. Furthermore, individual BX-C genes repress others, for example Abd-B as well as the miRNA iab-4 and bxd expression repress Ubx. In addition, other factors, such as CP190 and perhaps additional unknown factors may contribute to the enhancer blocking function of dCTCF. For all of the CTS in the BX-C, dCTCF and CP190 binding was found. Although both factors clearly interact as seen by co-immunoprecipitation, CP190 may contact other DNA-bound factors as well, or may be directly targeted to chromatin (Mohan, 2007).

Thus, dCTCF shares several biochemical and functional features with Su(Hw), but is clearly targeted to dCTCF-specific sites. Overall, this study has shown that dCTCF is important for fly development, and has important functions in the regulation of abdominal segmentation (Mohan, 2007).

Search PubMed for articles about Drosophila cp190

Alekseyenko, A. A., Gorchakov, A. A., Zee, B. M., Fuchs, S. M., Kharchenko, P. V. and Kuroda, M. I. (2014). Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs. Genes Dev 28: 1445-1460. PubMed ID: 24990964

Azimzadeh, J. and Bornens, M. (2007). Structure and duplication of the centrosome J. Cell. Sci. 120: 2139-2142. Full text of article: http://jcs.biologists.org/cgi/content/full/120/13/2139

Bag, I., Chen, S., Rosin, L. F., Chen, Y., Liu, C. Y., Yu, G. Y. and Lei, E. P. (2021). M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity. Nat Commun 12(1): 4170. PubMed ID: 34234130

Bartkuhn, M., et al. (2009). Active promoters and insulators are marked by the centrosomal protein 190. EMBO J. 28: 877-888. PubMed ID: 19229299

Baumann, D. G. and Gilmour, D. S. (2017). A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes. Nucleic Acids Res 45(18): 10481-10491. PubMed ID: 28977400

Bushey, A. M., Ramos, E., and Corces, V. G. (2009). Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev. 23: 1338-1350. PubMed ID: 19443682

Butcher, R.D.J., et al. (2004). The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division. J. Cell Sci. 117: 1191-1199. PubMed ID: 14996941

Capelson, M. and Corces, V. G. (2005). The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol Cell 20: 105-116. PubMed ID: 16209949

Capelson, M. and Corces V. G. (2006). SUMO conjugation attenuates the activity of the gypsy chromatin insulator. EMBO J. 25(9): 1906-14. PubMed ID: 16628226

Chodagam, S., Royou, A., Whitfield, W., Karess, R. and Raff, J. W. (2005). The centrosomal protein CP190 regulates myosin function during early Drosophila. development. Curr. Biol. 15(14): 1308-13. PubMed ID: 16051175

Chopra, V. S., Cande, J., Hong, J. W. and Levine, M. (2009). Stalled Hox promoters as chromosomal boundaries. Genes Dev 23(13): 1505-1509. PubMed ID: 19515973

Cuartero, S., Fresan, U., Reina, O., Planet, E. and Espinas, M. L. (2014). Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function. EMBO J 33(6): 637-647. PubMed ID: 24502977

Cubenas-Potts, C., Rowley, M. J., Lyu, X., Li, G., Lei, E. P. and Corces, V. G. (2017). Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucleic Acids Res 45(4): 1714-1730. PubMed ID: 27899590

Dixon, J. R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J. S. and Ren, B. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485: 376-380. PubMed ID: 22495300

Eagen, K. P., Aiden, E. L. and Kornberg, R. D. (2017). Polycomb-mediated chromatin loops revealed by a subkilobase-resolution chromatin interaction map. Proc Natl Acad Sci U S A 114(33): 8764-8769. PubMed ID: 28765367

Erokhin M., Parshikov A., Georgiev P. and Chetverina D. (2010). E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster. Chromosoma 119: 243-253. PubMed ID: 20082086

Erokhin, M., et al. (2011). Insulators form gene loops by interacting with promoters in Drosophila. Development 138(18): 4097-106. PubMed ID: 21862564

Golovnin, A., Volkov, I. and Georgiev, P. (2012). SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation. J. Cell Sci. 125(Pt 8): 2064-74. PubMed ID: 22375064

Golovnin, A., Melnikova, L., Shapovalov, I., Kostyuchenko, M. and Georgiev, P. (2015). EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin. PLoS One 10: e0140991. PubMed ID: 26489095.

Hou, C., Li, L., Qin, Z. S. and Corces, V. G. (2012). Gene density, transcription, and insulators contribute to the partition of the Drosophila genome into physical domains. Mol Cell 48: 471-484. PubMed ID: 23041285

Kellogg, D. R., Oegema, K., Raff, J., Schneider, K. and Alberts, B. M. (1995). Cp60 a microtubule associated protein that is localized to the centrosome in a cell cycle specific manner. Mol. Biol. Cell 6: 1673-1684. PubMed ID: 8590797

Korenjak, M., Kwon, E., Morris, R. T., Anderssen, E., Amzallag, A., Ramaswamy, S., Dyson, N. J. (2014). dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes. Nucleic Acids Res 42(14): 8939-53. PubMed ID: 25053843

Lei, E. P. and Corces, V. G (2006). RNA interference machinery influences the nuclear organization of a chromatin insulator. Nat. Genet. 38: 936-941. PubMed ID: 16862159

Lin, Y. C., Benner, C., Mansson, R., Heinz, S., Miyazaki, K., Miyazaki, M., Chandra, V., Bossen, C., Glass, C. K. and Murre, C. (2012). Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat Immunol 13: 1196-1204. PubMed ID: 23064439

Maksimenko, O., Bartkuhn, M., Stakhov, V., Herold, M., Zolotarev, N., Jox, T., Buxa, M. K., Kirsch, R., Bonchuk, A., Fedotova, A., Kyrchanova, O., Renkawitz, R. and Georgiev, P. (2014). Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin. Genome Res 25(1):89-99. PubMed ID: 25342723

Mohan, M., et al. (2007). The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J. 26(19): 4203-14. PubMed ID: 17805343

Moshkovich, N., et al. (2011). RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function. Genes Dev. 25(16): 1686-701. PubMed ID: 21852534

Négre, N., et al. (2010). A comprehensive map of insulator elements for the Drosophila genome. PLoS Genet. 6(1): e1000814. PubMed ID: 20084099

Oegema, K., Whitfield, W. G. F. and Alberts, B. (1995). The cell cycle dependent localization of the Cp190 centrosomal protein is determined by the coordinate action of 2 separable domains, J. Cell Biol. 131: 1261-1273. PubMed ID: 8522588

Ong, C. T., Van Bortle, K., Ramos, E., Corces, V. G. (2013). Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila. Cell 155(1): 148-59. PubMed ID: 24055367

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. PubMed ID: 15574329

Persico, V., Callaini, G. and Riparbelli, M. G. (2019). The male stem cell niche of Drosophila melanogaster: Interactions between the germline stem cells and the hub. Exp Cell Res 383(1): 111489. PubMed ID: 31278899

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Rach, E. A., Winter, D. R., Benjamin, A. M., Corcoran, D. L., Ni, T., Zhu, J. and Ohler, U. (2011). Transcription initiation patterns indicate divergent strategies for gene regulation at the chromatin level. PLoS Genet 7(1): e1001274. PubMed ID: 21249180

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Ramirez, F., Bhardwaj, V., Arrigoni, L., Lam, K. C., Gruning, B. A., Villaveces, J., Habermann, B., Akhtar, A. and Manke, T. (2018). High-resolution TADs reveal DNA sequences underlying genome organization in flies. Nat Commun 9(1): 189. PubMed ID: 29335486

Rotelli, M. D., Policastro, R. A., Bolling, A. M., Killion, A. W., Weinberg, A. J., Dixon, M. J., Zentner, G. E., Walczak, C. E., Lilly, M. A. and Calvi, B. R. (2019). A Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles. PLoS Genet 15(7): e1008253. PubMed ID: 31291240

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date revised: 23 July 2023

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