Ibf1 and Ibf2 are DNA-binding proteins required for insulator function in Drosophila [ChIP-seq]
ABSTRACT: Here we report on the identification of two previously uncharacterized proteins as CP190 interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNAbinding proteins that form obligated hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancerblocking assays. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity. Overall design: ChIP-Seq peak calling of CP190, Ibf1 and Ibf2 against Input sample in Drosophila melanogaster S2 cells
Project description:Here we report on the identification of two previously uncharacterized proteins as CP190 interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNAbinding proteins that form obligated hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancerblocking assays. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity. ChIP-Seq peak calling of CP190, Ibf1 and Ibf2 against Input sample in Drosophila melanogaster S2 cells
Project description:Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.
Project description:Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.
Project description:Insulators are multiprotein-DNA complexes thought to affect gene expression by mediating inter- and intrachromosomal interactions. Drosophila insulators contain specific DNA-binding proteins plus common components, such as CP190, that facilitate these interactions. Here, we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA-binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to DNA to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.
Project description:Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.
Project description:Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.
Project description: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, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes 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. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
Project description:Chromatin insulators are remarkable regulatory elements that can bring distant genomic sites together and block unscheduled enhancer-promoter communications. Insulators act via associated insulator proteins of two classes: sequence-specific DNA binding factors and "bridging" proteins. The latter are required to mediate interactions between distant insulator elements. Chromatin insulators are critical for correct expression of complex loci; however, their mode of action is poorly understood. Here, we use the Drosophila bithorax complex as a model to investigate the roles of the bridging proteins Cp190 and Mod(mdg4). The bithorax complex consists of three evolutionarily conserved homeotic genes Ubx, abd-A, and Abd-B, which specify anterior-posterior identity of the last thoracic and all abdominal segments of the fly. Looking at effects of CTCF, mod(mdg4), and Cp190 mutations on expression of the bithorax complex genes, we provide the first functional evidence that Mod(mdg4) acts in concert with the DNA binding insulator protein CTCF. We find that Mod(mdg4) and Cp190 are not redundant and may have distinct functional properties. We, for the first time, demonstrate that Cp190 is critical for correct regulation of the bithorax complex and show that Cp190 is required at an exceptionally strong Fub insulator to partition the bithorax complex into two topological domains.
Project description:Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720-892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (Hipp?1) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.
Project description:dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts we examined the distribution and localization of Drosophila E2F and dREAM proteins. Here we report a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains.