Boundary elements in Drosophila and their role in nuclear organization

Chromatin insulators are defined functionally as DNA sequences that interfere with enhancer promoter interaction when placed between them and protect genes from surrounding chromosomal environments. A 350 by sequence located in the 5′ transcribed, untranslated region of the gypsy retrovirus behaves as a classical insulator element. Genetic studies have led to the identification of three protein components of the gypsy insulator, Su(Hw), Mod(mdg4) and CP190. Su(Hw) and Mod(mdg4) colocalize in ∼200 discrete bands on polytene chromosomes that presumably function as endogenous insulators, but are present only in 20–25 distinct spots in the diploid interphase nucleus. These observations have led to the hypothesis that several independent insulator sites might cluster together at a single nuclear location to form rosette-like structures.; Using the yeast two-hybrid assay, I have shown that all three proteins components of the gypsy insulator interact directly with one another, In addition Mod(mdg4) interacts with itself to form homodimers. The BTB domain located at N-terminus of Mod(mdg4) mediates self-interaction as well as interaction with CP190, whereas the carboxy terminal acidic domain is involved in interactions with leucine zipper and adjacent regions of Su(Hw). Interactions through BTB domain could lead to aggregation of multiple protein molecules. These results provide a molecular mechanism of how multiple insulator sites could be brought together through protein-protein interaction and support the role of gypsy insulator in nuclear organization.; To elucidate the role of insulators in chromatin organization it is essential to identify and characterize insulator sequences in their native context. I have isolated genomic Su(Hw) binding sites. Analyses of some of these binding sites show the presence of a conserved sequence that is also present in gypsy insulator sequence. Previous experiments have shown that this conserved sequence is the Su(Hw) binding site. DNA in situ hybridization with putative endogenous insulators followed by immunolocalization shows that they are in vivo localization sites for Su(Hw). These endogenous insulators are located mostly in intergenic regions. This finding is consistent with the idea that the normal role of insulators in the genome might be to mark boundaries of transcriptionally related genes.