In contrast sellekchem to the CTCF and BEAF32 sites, signals of the three tested Su sites are significantly weaker than the signal at Fab 8 and are indistinguishable with the negative control region 1A6, suggesting that the BTB domain is critical for association of Cp190 with the Su com plexes at these loci, consistent with the results of co IP experiments and the polytene chromosome staining experiments. The CP190dBTB protein lacking the BTB domain does not associate with the Su complex. We thus tested if the BTB domain is sufficient to associate with insulators. We generated flies carrying the P which encodes the fusion pro tein containing the GFP and the BTB domain of Cp190 fused to the nuclear localization signal of the Drosophila Transformer protein.

Distribution of this GFP tagged Cp190 mutant protein in the cell nucleus is significantly different from that of the mRFP CP190. First, the GFP CP190BTB nls protein localizes to extra chromosomal spaces but the mRFP CP190 does not. Sec ond, the GFP CP190BTB nls is not present at most of the strong mRFP CP190 bands on polytene chromo somes in the cell nucleus. Third, we could not detect signals of the GFP CP190BTB nls protein, stained by the anti GFP anti body, on the polytene chromosomes spreads. These results suggest that the BTB domain alone is not sufficient to associate with the Su insu lator complexes. The BTB domain and an Aspartic acid rich region of Cp190 are sufficient for association with gypsy, CTCF and BEAF32 sites The predicted protein of CP190En15, labeled as CP190dCT, contains the BTB and CENT domains, but lacks two of the three zinc fingers and the C terminal E rich domain.

Genetic tests indicate that the CP190dCT protein cannot support insulator activity. Loss of function CP190 mutations dominantly enhance the effects of the homozygous mod T6 mutation on gypsy dependent phenotypes. The CP190En15 allele was obtained in a newly conducted genetic screen of EMS mutagenized flies for dominant enhancers of mod T6. The CP190En15 mutation dominantly enhances y2, ombP1 D11, and ct6 all three gypsy dependent phenotypes in CP190En15, mod T6 flies, indicat ing that the gypsy insulator function is reduced. Homozygous CP190En15 is pupal lethal, but we found four halfway eclosed CP190En15 CP190P11 adults that survived for some 18 hours without significant locomotion after removal from the pupal case.

The cuti cle color of these y2 w ct6, CP190En15 CP190P11 adults was darker than the y2 w ct6 flies and the wings had fully developed margins, indicating that the gypsy insula tor was non functional. Although the gypsy insulator is non functional in CP190En15 flies, the CP190dC protein is still pre sent at gypsy insulators. CP190dC binds polytene chromosomes and colocalizes with Brefeldin_A the Su protein at the y locus in y2 mutants. CP190dC also co localizes with Su and Mod 67. 2 proteins in diploid cells.