| Chromatin insulators are defined by being able to block enhancer-promoter communication and to protect transgenes from the surrounding chromatin. The ∼350 bp DNA sequence of the gypsy chromatin insulator of D. melanogaster binds a protein complex that consists of three characterized components, Su(Hw), Mod(mdg4)2.2 and CP190. The Drosophila genome contains hundreds of gypsy insulator protein binding sites, which coalesce into several nuclear foci, termed insulator bodies. These nuclear bodies are proposed to represent clusters of distant insulator loci and thus the bases of chromatin loop domains, which may aid in the establishment of gene expression autonomy. Furthermore, insulator bodies are reported to associate with the nuclear periphery, which may facilitate the role of insulators in organization of chromatin.; A novel factor dTopors, identified in a yeast two hybrid screen for interactions with Mod(mdg4)2.2, was found to associate with all three gypsy insulator proteins in vivo and to be present at a fraction of insulator chromatin binding sites. In cell nuclei, dTopors resides in the nuclear lamina, which was confirmed by a detected physical interaction between lamin and dTopors. Over-expression of dTopors in the background of a disrupted insulator, created by a null mutation in the gene coding for Mod(mdg4)2.2, rescues the enhancer blocking ability of gypsy and the nuclear coalescence of insulator bodies. We propose that dTopors is involved in tethering insulator complexes to the nuclear lamina, which promotes their nuclear organization and activity.; The conserved RING domain of dTopors led to an investigation in its possible role in post-translational modification by ubiquitin or SUMO, as this domain was found to be required for the positive effect of dTopors on the insulator. Mod(mdg4)2.2 and CP190 were found to be sumoylated, with dTopors exerting an inhibitory effect on this modification. The SUMO conjugation pathway was seen to antagonize the enhancer blocking activity of gypsy and to disrupt the formation of nuclear insulator bodies, suggesting that self-interactions of insulator proteins can be regulated by sumoylation. These results reveal the significance of self-interactions or tethering to a subnuclear scaffold in the mechanism of chromatin insulators and in the formation of autonomous chromatin domains. |
