Interaction of Su(Hw) with gypsy-like sequences. All lanes contain ³²P-labeled gypsy DNA plus the proteins or extracts indicated in A–C. The location of the unbound gypsy DNA is indicated by an arrow and the location of the protein–DNA complexes is indicated by an asterisk. (A) Su(Hw) purified from Drosophila S2 cells binds specifically to the 351-bp gypsy insulator in EMSAs. The different lanes contain gypsy probe alone (lane 1), gypsy probe with the addition of S2-Su(Hw) (lane 2), gypsy probe with rabbit reticulocyte extract (lane 3), and gypsy probe with luciferase protein (lane 4). (B) Specificity of the myc–Su(Hw)–gypsy interaction. Gypsy probe (lane 1); gypsy DNA with in vitro-translated myc-tagged Su(Hw) (lane 2); and gypsy DNA with myc-tagged Su(Hw) and myc antibody, which causes a supershift in the mobility of the gypsy/myc–Su(Hw) complex (lane 3). (C) myc–Su(Hw) binds specifically to gypsy insulator DNA. Free gypsy probe (lane 1), gypsy probe plus a 100-fold excess unlabeled Bluescript (pBS) DNA (lane 2), and gypsy probe plus a 100-fold excess unlabeled gypsy DNA (lane 3). (D) myc–Su(Hw) can bind genomic DNA containing putative Su(Hw)-binding sites. DNA sequences 3.08, 3.09, and 3.10 were radiolabeled (lane 1 in each gel) and incubated with rabbit reticulocyte (lane 2), myc–Su(Hw) extract (lane 3), and myc–Su(Hw) with anti-myc antibody (lane 4).

MARs plots for DNA fragments containing genomic Su(Hw)-binding sites. The x-axis denotes position within the DNA sequences and the y-axis indicates the probability of MARs activity. Peaks >0.75 show areas of high MARs potential. Red arrows indicate the location of Su(Hw)-binding sites in DNA fragments containing one (3.08, 3.09, 3.28), two (y-454), or three (X-103, 2L-203) Su(Hw)-binding sites.

Genomic Su(Hw)-binding sites have insulator activity that is dependent on Su(Hw) protein. DNA fragments containing one (3.08 and 3.28), two (y-454), or three (X-103 and 2L-203) binding sites for Su(Hw) were tested for their ability to inhibit promoter–enhancer interactions in the yellow gene. A representative example is given for each series of the transgenic insulating lines. Male flies are shown except in D where female flies are displayed. In all cases, the insulating activity is visible in both sexes. In each panel, the fly on the left contains the transgene in the background of a wild-type su(Hw) gene, whereas the fly on the right is mutant for su(Hw); only the phenotypes for su(Hw)²/su(Hw)^V mutants are shown but other allele combinations, su(Hw)²/su(Hw)^f and su(Hw)^f/su(Hw)^V, gave similar results. (A) Effect of a single Su(Hw)-binding site present in fragment 3.08 on the yellow enhancer-blocking assay (left). The wings and body cuticle appear yellow. When placed in a su(Hw) mutant background, the coloration of the wing and body reverts back to a wild-type phenotype (right). (B) A similar analysis carried out with fragment 3.28 (one binding site) also shows insulating activity (left) that is dependent on Su(Hw) (right). (C–E) Transgenic animals carrying DNA fragments y-454 (two binding sites), X-103 (three binding sites), and 2L-203 (three binding sites) in the absence (leftmost in each panel) or presence of mutations in su(Hw) (rightmost in each panel). (F) Adult flies from different transgenic lines carrying DNA fragment 2L-203 showing a series of insulator-induced phenotypes ranging from weak (i) to strong (iv).

Sequence and binding strength of genomic Su(Hw)-binding sites. (A) The weighted consensus recognition sequence for gypsy-like genomic Su(Hw)-binding sites. (B) EMSA competition analysis. All lanes contain a truncated radiolabeled gypsy probe containing the first three Su(Hw)-binding sites found in gypsy. (Lane 1) Truncated-gypsy probe alone. (Lane 2) Truncated-gypsy probe with Su(Hw) protein. (Lane 3) Unlabeled truncated-gypsy DNA containing Su(Hw)-binding sites. (Lanes 4 and 5) Unlabeled DNA containing either one or two genomic binding sites for Su(Hw).

Predicted Su(Hw)-binding sites colocalize with Su(Hw) protein on polytene chromosomes. Fluorescently labeled genomic DNA fragments containing Su(Hw)-binding sites were used for DNA in situ hybridization on polytene chromosomes. Chromosomes were simultaneously labeled with antibodies against Su(Hw). The DNA is visualized with DAPI and shown in blue. The DNA FISH signals corresponding to probes 3.08 (A), X-103 (B), and 2L-203 (C) are shown in red. The location of Su(Hw) protein is shown in green. The overlapping signals for FISH and Su(Hw) protein appear yellow.

Organization of Su(Hw)-binding sites in the Drosophila genome. Representative examples of the location of Su(Hw)-binding sites with respect to the location of neighboring genes. The red arrow indicates the location of the Su(Hw)-binding sequences. (A) An example of an intergenic Su(Hw)-binding site cluster separating gene-rich regions. (B) An example of an intergenic Su(Hw)-binding site cluster separating genes >20 kb apart. (C) An example of an intragenic insulator sequence located within the intron of the large rdgA gene. (D) An example of a genomic insulator located, at least partially, within the coding region of an annotated gene. A, B, C, and D each display a 100-kb genomic region.