Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.

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.

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Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the three-dimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and three-dimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction.

BackgroundChromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes.ResultsHere we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190.ConclusionsThese results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0140-6) contains supplementary material, which is available to authorized users.