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).

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).

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

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

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: 1505-1509. PubMed ID: 19515973

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

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

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

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

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

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. PubMed ID: 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. PubMed ID: 12105185

Schwartz, Y. B., et al. (2012). Nature and function of insulator protein binding sites in the Drosophila genome. Genome Res 22: 2188-2198. PubMed ID: 22767387

Smith, S. T., et al. (2009). Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization. Dev. Biol. 328: 518-528. PubMed ID: 19210964

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. PubMed ID: 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 ID: 14980504

date revised: 10 February 2013

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