Identification of genomic sites that bind the Drosophila suppressor of hairy-wing insulator protein

Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, we identified 37 putative Su(Hw) insulators (pSIs) that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. Our data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators.