Investigation of the properties of non-gypsy suppressor of hairy-wing-binding sites.

Insulators define interactions between transcriptional control elements in eukaryotic genomes. The gypsy insulator found in the gypsy retrovirus binds the zinc-finger Suppressor of Hairy-wing [Su(Hw)] protein that associates with hundreds of non-gypsy regions throughout the Drosophila genome. Models of insulator function predict that the gypsy insulator forms chromatin loop domains through interactions with endogenous Su(Hw) insulators (SIs) to limit the action of transcriptional control elements. Here we study SI 62D and show that interactions occur between two SI 62D elements, but not between SI 62D and the gypsy insulator, limiting the scope of genomic gypsy insulator interactions. Enhancer blocking by SI 62D requires fewer Su(Hw)-binding sites than needed for gypsy insulator function, with these target regions having distinct zinc-finger requirements for in vivo Su(Hw) association. These observations led to an investigation of the role of the Su(Hw) zinc-finger domain in insulator function. Using a combination of in vitro and in vivo studies, we find that this domain makes sequence-dependent and -independent contributions to in vivo chromosome association, but is not essential for enhancer or silencer blocking. These studies extend our understanding of the properties of Su(Hw) and the endogenous genomic regions to which this protein localizes.

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.

An endogenous suppressor of hairy-wing insulator separates regulatory domains in Drosophila.

Insulators define independent domains of gene function throughout the genome. The Drosophila gypsy insulator was isolated from the gypsy retrotransposon as a region that contains a cluster of binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. To study the effects of the gypsy insulator on gene expression within a single genomic domain, targeted gene replacement was used to exchange the endogenous yellow gene, located at cytological location 1A, with a set of gypsy-modified yellow genes. Replaced yellow genes carried a gypsy insulator positioned between the yellow promoter and either the upstream or the downstream tissue-specific enhancers. Whereas the gypsy insulator blocked the function of the upstream enhancers at the endogenous location, the downstream enhancers were not blocked. Investigation of the 1A region revealed two clustered Su(Hw)-binding sites downstream of the yellow gene, named 1A-2, that bind Su(Hw) in vivo and possess enhancer blocking function. We propose that interaction between 1A-2 and the gypsy insulator permits activation of yellow expression by enhancers in the neighboring achaete-scute complex, causing an apparent absence of the block of the downstream yellow enhancers. Based on these data, we suggest that 1A-2 is an endogenous Su(Hw) insulator that separates regulatory domains within the Drosophila genome.