Identification of Genomic Sites That Bind the Drosophila Suppressor of Hairy-wing Insulator Protein

Abstract

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 enhancerblocking 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.Genes with similar expression profiles are clustered in eukaryotic genomes (5,38,39,43,73). These observations suggest that these genomes are divided into discrete domains that facilitate the regulation of gene expression. Functional domains may be established by assembly of higher-order chromatin structures that constrain the activity of enhancers and silencers to ensure that these transcriptional elements interact with the appropriate target promoter. The demarcation of chromatin domains is associated with a conserved class of DNA elements known as insulators (6,13,17,41,61,75,79). Insulators possess two functional properties. First, they block the action of enhancers and silencers when placed between these regulatory elements and a promoter. Second, they protect gene expression from chromosomal position effects that result from ectopic placement of genes in the genome. These blocking effects are accomplished without inactivation of the intrinsic properties of any of the regulatory elements, implying that insulators disrupt signaling between enhancers, silencers, and promoters (10, 66). The mechanism by which insulators establish independent functional domains is unclear, but it might depend upon interactions between insulators and/or nuclear substructures to form loop domains that limit the action of transcriptional regulatory elements (9,23,78).One of the best-characterized insulators is the Drosophila gypsy insulator. This element resides within the 5Ј-untranslated region of the gypsy retrotransposon (33). The gypsy insulator contains twelve clustered, degenerate repeats of a consensus sequence, 5Ј-YRYTGCATAYYY-3Ј, where Y represents a pyrimidine and R represents a purine residue, that bind the Sup...

Enhancer Blocking and Transvection at the DrosophilaapterousLocus

“…Both the Su(Hw) and Modifier of mdg4 ½Mod(mdg4) proteins are necessary for su(Hw) element enhancer blocking. Su(Hw) is a DNA-binding protein containing 12 zinc-finger domains, which binds to the YRTTGCA TACCY repeats present in the su(Hw) element from the gypsy reterotransposon (Parkhurst et al 1988;Geyer and Corces 1992;Parnell et al 2006;Ramos et al 2006). Mod(mdg4) is a BTB/POZ domain-containing protein that can interact with Su(Hw), other components of the su(Hw) insulator, and itself to form insulator bodies (Parkhurst et al 1988;Geyer and Corces 1992;Gerasimova and Corces 1998;Gerasimova et al 2000;Ghosh et al 2001).…”

SUMO conjugation is required for the assembly ofDrosophilaSu(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation

“…It contains 12 degenerated repeats of the binding motif for the zinc-finger protein Suppressor of Hairy wing [Su(Hw)], which is essential for its function (Holdridge and Dorsett, 1991;Geyer and Corces, 1992). At the same time, most endogenous Su(Hw) insulators contain fewer than four Su(Hw) binding sites (Kuhn-Parnell et al, 2008;Nègre et al, 2010;Parnell et al, 2006).…”

Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

“…As far as we know, Fub and 62D are the strongest insulator elements naturally present in the Drosophila genome (Figure 8) (Golovnin et al 2003;Parnell et al 2003;Gruzdeva et al 2005;Parnell et al 2006;Ramos et al 2006;Rodin et al 2007;Schwartz et al 2012). Table S1.…”

“…As far as we know, Fub and 62D are the strongest insulator elements naturally present in the Drosophila genome (Figure 8) (Golovnin et al 2003;Parnell et al 2003;Gruzdeva et al 2005;Parnell et al 2006;Ramos et al 2006;Rodin et al 2007;Schwartz et al 2012). The strength of the gypsy insulator is explained by its unusual composition of an array of 12 binding sites for the Su(Hw) protein (Spana et al 1988 2R: 19488971-19489908; R4, X: 17613897-17614889; R5, 3R: 2737495-2738551; d2, 2R: 18984278-18985246; d3, 3R: 5925961-5926884; d4, 3R: 12934220-12935236; and d6, X: 2763148-2764144 (all coordinates are in Dm3 2006 genome release) were PCR amplified from genomic DNA and cloned into the FRT cassette of yellow-attB-mobile vector (Schwartz et al 2012) using XbaI, NotI, NheI, or Aor51HI sites.…”