InteractiveFly: GeneBrief

Centrosomal protein 190kD: Biological Overview | References

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).

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

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

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. Medline abstract: 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. Medline abstract: 16209949

Capelson, M. and Corces V. G. (2006). SUMO conjugation attenuates the activity of the gypsy chromatin insulator. EMBO J. 25(9): 1906-14. Medline abstract: 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. Medline abstract: 16051175

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. Medline abstract: 8590797

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

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

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. Medline abstract: 8522588

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. Medline abstract: 15574329

Raff, J. W., Kellogg, D. R. and Alberts, B. M. (1993). Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs. J. Cell Biol. 121: 823-835. Medline abstract: 8491775

Royou, A., Sullivan, W. and Karess, R. (2002). Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: Its role in nuclear axial expansion and its regulation by Cdc2 activity. J. Cell Biol. 158: 127-137. Medline abstract: 12105185

Whitfield, W. G. F., Millar, S. E., Saumweber, H., Frasch, M. and Glover, D. M. (1988). Cloning of a gene encoding an antigen associated with centrosome in Drosophila. J. Cell Sci. 89: 467-480. Medline abstract: 3143740

Zhang, R., et al. (2004). Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody. Exp. Cell Res. 294(1): 86-93. PubMed citation: 14980504

date revised: 8 January 2008

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