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:The architectural protein Pita is critical for Drosophila embryogenesis and predominantly binds to gene promoters and insulators. In particular, Pita is involved in the organization of boundaries between regulatory domains, controlled by the expression of three hox genes in the Bithorax complex (BX-C). The best-characterized partner for Pita is the BTB/POZ-domain containing protein CP190. Here, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. The deletion of the CP190-interacting regions did not significantly affect the binding of the mutant Pita protein to most chromatin sites. The expression of the mutant protein completely complemented the null pita mutation. However, the mutant Pita protein does not support the ability of multimerized Pita sites to prevent cross-talk between the iab-6 and iab-7 regulatory domains that activate the expression of Abdominal-B (Abd-B), one of the genes in the BX-C. The recruitment of the Pita region and the interaction with CP190 and the polytene chromosomes of larvae induces the formation of a new interband, which is a consequence of the formation of open chromatin in this region. These results suggested that the interaction with CP190 is required for the primary Pita activities, but other architectural proteins may also recruit CP190 in flies expressing only the mutant Pita protein.
Project description:Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and their normal localization is correlated with proper insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
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.
Project description:Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood. Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins. Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8. Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates. We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome. ChIP-seq of exosome components. RNA-seq after control and exosome subunit knockdown in Drosophila cell lines.
Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)