Project description:80% of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces the number of Gcn5 binding sites and binding strength to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190
Project description:80% of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces the number of Gcn5 binding sites and binding strength to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190
Project description:CCCTC-binding factor (CTCF) is a conserved protein able to block communication between regulatory elements and gene promoters in flies and mammals. Here, we studied CTCF function in the Drosophila central nervous system (CNS) in which we find CTCF plays a vital role. Chromatin immunoprecipitation (ChIP)-sequencing of CTCF and its partner protein Centrosomal protein 190 kDa (Cp190) in CNSs of wildtype (WT), CTCF[0] mutants completely lacking CTCF and Cp190[KO] mutants with a CRISPR/Cas9-mediated deletion of the Cp190 open reading frame identified CTCF and Cp190 peaks defined as enriched in WT relative to each respective mutant control. These experiments also revealed that CTCF recruits Cp190 to CTCF's binding sites, but Cp190 is dispensable for CTCF binding to most of CTCF's sites.
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:ChIP-chip experiments with NimbleGen whole-genome tiling arrays to compare Su(Hw), dCTCF, BEAF, and CP190 localization on DNA in Kc and Mbn2 cells revealed 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.
Project description:ChIP-chip experiments with NimbleGen whole-genome tiling arrays to compare Su(Hw), dCTCF, BEAF, and CP190 localization on DNA in Kc and Mbn2 cells revealed 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. Three replicates of Su(Hw), dCTCF, and CP190 ChIP-chip experiments in both Kc and Mbn2 cells as well as two replicates of BEAF ChIP-chip experiments in both Kc and Mbn2 cells were compared.
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.
