Pairing between gypsy Insulators Facilitates the Enhancer Action in trans throughout the Drosophila Genome

Abstract

The Suppressor of the Hairy wing [Su(Hw)] binding region within the gypsy retrotransposon is the best known chromatin insulator in Drosophila melanogaster. According to previous data, two copies of the gypsy insulator inserted between an enhancer and a promoter neutralize each other's actions, which is indicative of an interaction between the protein complexes bound to the insulators. We have investigated the role of pairing between the gypsy insulators located on homologous chromosomes in trans interaction between yellow enhancers and a promoter. It has been shown that trans activation of the yellow promoter strongly depends on the site of the transposon insertion, which is evidence for a role of surrounding chromatin in homologous pairing. The presence of the gypsy insulators in both homologous chromosomes even at a distance of 9 kb downstream from the promoter dramatically improves the trans activation of yellow. Moreover, the gypsy insulators have proved to stabilize trans activation between distantly located enhancers and a promoter. These data suggest that gypsy insulator pairing is involved in communication between loci in the Drosophila genome.The enhancer-mediated activation is the basic mechanism of gene regulation in eukaryotes. Enhancers can act over large distances to activate transcription independently of their orientation and position relative to the promoter without affecting adjacent genes (6, 11). Insulators represent a class of DNA sequences that restrain regulatory interactions within eukaryotic genomes (21,35,36,53,63,65). These elements restrict the enhancer and silencer functions, contributing to the establishment of independent gene regulation within heterochromatic and euchromatic domains.The best known insulator was identified in Drosophila melanogaster within the 5Ј untranslated region of the gypsy retrotransposon (42). It consists of 12 binding sites for the Su(Hw) protein (44,62). Recently, another protein, CP190, was shown to bind to the gypsy insulator (54). These DNA-binding proteins are important for the gypsy insulator function, as mutations in the su(Hw) and CP190 genes reverse the mutagenic effects of the gypsy retrotransposon (46, 54). Mutations in another gene, mod(mdg4), alter the phenotypes of the gypsyinduced mutations (17,18,22). Previous studies indicate that the Mod(mdg4)-67.2 protein isoform interacts with the Su(Hw) and CP190 proteins (14,25,54). The prevalent model suggests that boundary elements, or insulators, subdivide eukaryotic chromosomes into functionally and structurally autonomous domains (65). The insulators determine the limits of higher-order "looped" chromatin domains by interacting either with each other or with some nuclear structures (8,36,65). This interaction might be responsible, at least partially, for the establishment of independent chromatin domains. In this context, it is noteworthy that the gypsy insulators have been found to coalesce into a few "insulator bodies" located at the periphery of the nucleus (19), which are partially or complete...

The Capacity to Act in Trans Varies Among Drosophila Enhancers

“…For example, pairing the same enhancer fragment with transgenes carrying different genomic fragments upstream of a trans-promoter can result in varying levels of transvection, suggesting that "compatibility" of paired sequences is important for robust enhancer action in trans (Mellert and Truman 2012). Furthermore, insulator sequences have previously been shown to have a positive influence on transvection and other long-distance enhancer-promoter interactions (Kravchenko et al 2005;Schoborg et al 2013;Fujioka et al 2016). In addition, some examples of transvection at the abdominal-B locus require a 10-kb sequence in addition to the enhancer (Hopmann et al 1995), and transvection of the Men locus varies greatly across genetic backgrounds (Lum and Merritt 2011).…”

“…Evidence for enhancer action in trans has been uncovered at a handful of genes in the Drosophila genome, often providing an explanation for unexpected intragenic complementation of loss-of-function alleles (Lewis 1954;Gelbart 1982;Geyer et al 1990;Leiserson et al 1994;Hendrickson and Sakonju 1995;Casares et al 1997;Morris et al 1998;Southworth and Kennison 2002;Marin et al 2004;Coulthard et al 2005;Gohl et al 2008;Juni and Yamamoto 2009) paired mutant and wild-type alleles (Gibson et al 2000;Lum and Merritt 2011;Bing et al 2014). More recently, transgenic approaches based on site-specific recombination have been developed for Drosophila to query specific enhancer fragments and genomic positions for support of transvection (Chen et al 2002;Kravchenko et al 2005;Bateman et al 2012a;Mellert and Truman 2012; also see Kassis et al 1991). Thus far, transvection has been observed at all genomic insertion sites tested, suggesting that the Drosophila genome is generally permissive to enhancer action in trans.…”

“…More recently, transgenic approaches based on site-specific recombination have been developed for Drosophila to query specific enhancer fragments and genomic positions for support of transvection (Chen et al 2002;Kravchenko et al 2005;Bateman et al 2012a;Mellert and Truman 2012; also see Kassis et al 1991). Thus far, transvection has been observed at all genomic insertion sites tested, suggesting that the Drosophila genome is generally permissive to enhancer action in trans.…”

Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

“…Another genetic element implicated in long-range interactions between homologous chromosomal regions is an insulator carried by the gypsy retrotransposon (reviewed by Kuhn and Geyer 2003;Capelson and Corces 2004;Brasset and Vaury 2005;Gaszner and Felsenfeld 2006;Valenzuela and Kamakaka 2006; also see Kravchenko et al 2005). Of note, two factors that work in conjunction with the gypsy insulator, Suppressor of Hairy Wing ½Su(Hw) and Modifier of mdg4 ½Mod(mdg4) (Kravchenko et al 2005), colocalize at hundreds of chromosomal sites that do not correspond to gypsy elements and, along with another protein, Centrosomal Protein 190 (CP190), mediate the clustering of these sites into ''insulator bodies'' (reviewed by Kuhn and Geyer 2003;Capelson and Corces 2004;Brasset and Vaury 2005;Gaszner and Felsenfeld 2006;Valenzuela and Kamakaka 2006).…”

“…Of note, two factors that work in conjunction with the gypsy insulator, Suppressor of Hairy Wing ½Su(Hw) and Modifier of mdg4 ½Mod(mdg4) (Kravchenko et al 2005), colocalize at hundreds of chromosomal sites that do not correspond to gypsy elements and, along with another protein, Centrosomal Protein 190 (CP190), mediate the clustering of these sites into ''insulator bodies'' (reviewed by Kuhn and Geyer 2003;Capelson and Corces 2004;Brasset and Vaury 2005;Gaszner and Felsenfeld 2006;Valenzuela and Kamakaka 2006). Consistent with this finding, loss of Su(Hw) function has been observed to compromise homolog pairing (Fritsch et al 2006).…”