Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation.

BACKGROUND: Chromatin 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. RESULTS: Here 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. CONCLUSIONS: These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.

A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.

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.