The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.

suppressor of Hairy-wing [su(Hw)] is a DNA-binding architectural protein that participates in the organization of insulators and repression of promoters in Drosophila. this protein contains acidic regions at both ends and a central cluster of 12 zinc finger domains, some of which are involved in the specific recognition of the binding site. One of the well-described in vivo function of su(Hw) is the repression of transcription of neuronal genes in oocytes. Here, we have found that the same su(Hw) C-terminal region (aa 720-892) is required for insulation as well as for promoter repression. The best characterized partners of Su(Hw), CP190 and Mod(mdg4)-67.2, are not involved in the repression of neuronal genes. taken together, these results suggest that an unknown protein or protein complex binds to the C-terminal region of su(Hw) and is responsible for the direct repression activity of su(Hw).High-resolution chromosome conformation capture techniques have provided evidence that regulatory elements form loops that are essential for gene regulation in higher eukaryotes 1-6 . In particular enhancers can activate target promoters at large distances (up to hundreds of kb in some cases), which raises the question of the mechanisms regulating such long-distance enhancer-promoter interactions. More than 25 years ago, a special class of regulatory elements, named insulators, was suggested to delimit the activity of enhancers 7-12 . Insulators are defined as regulatory elements that disrupt the communication between an enhancer and a promoter when inserted between them. Some insulator complexes contribute to higher-order organization of chromatin in topologically associated domains that are fundamental elements of the eukaryotic genomic structure 13,14 .One of the first insulators was identified in the gypsy retrotransposon, whose integration into genes often resulted in inactivation of enhancers that were separated from promoters by the gypsy insertion [15][16][17][18][19] . The phenotypes of the gypsy-induced mutations were suppressed by inactivation of the gene encoding the Suppressor of hairy wing protein [Su(Hw)] 20 . The gypsy insulator consists of 12 reiterated binding sites for Su(Hw) 21,22 . Today, Su(Hw) is one of the best characterized insulator proteins. It has been shown that artificial reiterated binding sites for Su(Hw) or gypsy insulator can block various enhancers at all stages of Drosophila development 21,23-27 .The Su(Hw) protein contains the N-terminal region involved in the interaction with CP190, an array of 12 C 2 H 2 -type zinc finger domains, and the C-terminal region (aa 716-892) responsible for enhancer blocking activity [28][29][30][31][32] . Several Su(Hw) partners were identified, including Mod(mdg4), CP190, ENY2, Shep, Rump, and HIPP1 28,33-38 . Mod(mdg4)-67.2 is one of the isoforms encoded by the mod(mdg4) locus 39,40 . The Mod(mdg4)-67.2 protein contains the N-terminal BTB/POZ domain and glutamine-rich (Q-rich) region which is common to all isoforms and the unique C-terminal region that is r...

The looping model of enhancer-promoter interactions predicts that these specific long-range interactions are supported by a certain class of proteins. In particular, the Drosophila transcription factor Zeste was hypothesized to facilitate long-distance associations between enhancers and promoters. We have re-examined the role of Zeste in supporting long-range interactions between an enhancer and a promoter using the white gene as a model system. The results show that Zeste binds to the upstream white promoter region and the enhancer that is responsible for white activation in the eyes. We have confirmed the previous finding that Zeste is not required for the activity of the eye enhancer and the promoter when they are located in close proximity to each other. However, inactivation of Zeste markedly affects the enhancer-promoter communication in transgenes when the eye enhancer and the white promoter are separated by a 3-kb spacer or the yellow gene. Zeste is also required for insulator bypass by the eye enhancer. Taken together, these results show that Zeste can support specific long-range interactions between enhancers and promoters.