Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between suppressor of hairy-wing and modifier of mdg4 proteins.

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein.

The su(Hw) insulator can disrupt enhancer-promoter interactions when located more than 20 kilobases away from the Drosophila achaete-scute complex.

Here we report that the su(Hw) insulator may not necessarily separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. For this purpose we used the strains of Drosophila melanogaster which carry inversion of the region containing the yellow gene and the achaete-scute complex (AS-C). Despite the reverse orientation of the region, the AS-C enhancers continue to activate achaete and scute gene expression. The su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers.

hobo Induced rearrangements in the yellow locus influence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster.

The su(Hw) protein is responsible for the insulation mediated by the su(Hw)-binding region present in the gypsy retrotransposon. In the y2 mutant, su(Hw) protein partially inhibits yellow transcription by repressing the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. y2 mutation derivatives have been induced by the insertion of two hobo copies on the both sides of gypsy: into the yellow intron and into the 5' regulatory region upstream of the wing and body enhancers. The hobo elements have the same structure and orientation, opposite to the direction of yellow transcription. In the sequence context, where two copies of hobo are separated by the su(Hw)-binding region, hobo-dependent rearrangements are frequently associated with duplications of the region between the hobo elements. Duplication of the su(Hw)-binding region strongly inhibits the insulation of the yellow promoter separated from the body and wing enhancers by gypsy. These results provide a better insight into mechanisms by which the su(Hw)-binding region affects the enhancer function.

Interactions between su(Hw)-binding regions in neighboring y2 and scD1 alleles hinder trans-activation of the y2 promoter by yellow enhancers located on a homologous chromosome.

The phenomenon of transvection has been well characterized for the yellow locus in Drosophila. Enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other when its own enhancers are blocked by the su(Hw) insulator introduced by the gypsy retrotransposon. Insertion of another gypsy into the neighboring scute locus hinders transvection presumably owing to disruption of chromosomal synapsis between the yellow alleles. We determined the sequences of gypsy required for inhibition of transvection. Two partial revertants of the scD1 mutation were obtained in which transvection between the yellow alleles was restored. Both sc revertants were generated by deletion of nine of the twelve su(Hw)-binding sites of gypsy inserted into the scute locus. This result suggests that the su(Hw) region is required for an interaction between two gypsy elements that disrupts trans activation of the yellow promoter by enhancers located on the homologous chromosome.

Phenotypic reversion of the gypsy-induced mutation scD1 of Drosophila melanogaster by replicative transposition of a sc enhancer to the yellow gene and by mutations in the enhancer of yellow and zeste loci.

Both mutations of Drosophila melanogaster in the y2scD1 strain are caused by the insertion of gypsy, but the scD1 mutation contains, in addition, an insertion of the jockey mobile element at a distance of 1.8 kb from gypsy. The su(Hw)-binding region in the copy of gypsy in scD1 inhibits scute expression in several specific areas of the epithelium, and in particular, blocks the formation of anterior notopleural (ANP) bristles. The y2 reversions y+2MC and y2#+9 restore the formation of ANP bristles. Molecular analysis revealed that both revertants were induced by transposition into the gypsy element at the yellow locus of jockey and an adjacent 1.8-kb region of the scute gene which contains an enhancer responsible for the development of ANP bristles. The duplicated enhancer is not insulated by the su(Hw)-binding region and can interact with the scute promoter in spite of the presence of two other promoters between them. Hypomorphic mutation in the e(y)2 gene and zOp6 allele partially inhibit ANP bristle formation in flies with y+2MC and y2+9 mutations. In addition, we found that mutations in the e(y)1, e(y)3 and zeste genes may interfere with transcriptional insulation by the su(Hw)-binding region in the scD1 allele.