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.

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...

Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophilaand 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 Δ1 ) 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.Insulators in Drosophila and vertebrate genomes have been identified based on their abilities to disrupt communications between enhancers and promoters, when inserted between them, and to prevent the repression mediated by heterochromatin 1-5 . In the past few years, a special class of architectural proteins has been identified, including some well-known insulator proteins, that are responsible for global chromosomal architecture and the local regulation of enhancer-promoter interactions 6,7 . Insulator proteins are involved in the formation of topologically associated domains, which are the fundamental structural elements of the eukaryotic genome [8][9][10][11] .Today, Suppressor of Hairy-wing [Su(Hw)] is one of the best-characterized insulator proteins in Drosophila. The Su(Hw) insulator was first identified in the gypsy retrotransposon, which contains a 460-bp sequence with 12 degenerate octamer binding sites for the Su(Hw) 12,13 . The best-characterized insulator complex consists of the Su(Hw) protein and its partners, Mod(mdg4)-67.2 and CP190 14-18 . The CP190 and Mod(mdg4)-67.2 proteins are recruited to chromatin through interactions with each other and with the Su(Hw) protein 19,20 .The Su(Hw) protein is ubiquitously expressed throughout development and contains an array of 12 C 2 H 2 -type zinc finger (ZF) domains 21,22 . The 6-9 ZF domains specifically recognize a 12-bp motif 23 . In previous genome-wide studies, three classes of Su(Hw) binding regions have been identified, which are characterized by whether they bind Su(Hw) alone (SBS-O), bind both Su(Hw) and CP190 (SBS-C), or bind all three proteins (SBS-CM) [24][25][26][27] . CP190 and Mod(mdg4)-67.2 both assist the Su(Hw) complex when binding to SBS-CM sites 19,20 .The Su(Hw) C-terminal domain (aa 716-892) is responsible for both insulator function 15,21,22 and the repression of neuronal genes in oocytes 28 . Like the gypsy insulator, artificia...

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