Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements

Abstract

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs-scs, scs'-scs', 1A2-1A2 and Wari-Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5-Su(Hw), dCTCF-Su(Hw), or dCTCF-Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

Figure 1.

(A) Schemes of the scs, scs’, Wari and 1A2 insulators. The scs insulator contains a cryptic promoter (59) and the promoter of the CG31211 gene. The scs’ insulator contains promoters of the CG3281 and aurora genes. Arrows indicate gene promoters. The Zw5 binding site within scs is shown as a white oval. Positions of the CGATA motifs within scs’ are shown as arrowheads, with the arrow indicating the direction of the motif (5′-CGATA-3′). Clusters of three CGATA motifs form low-affinity (white arrows) and high-affinity (black arrows) binding sites for the BEAF protein (28). Su(Hw) binding sites in the 1A2 insulator are shown as white rectangles. (B) Sequences of the oligonucleotides used to produce the Zw5, Su(Hw) and dCTCF synthetic binding regions. The core binding sites are boxed.

Figure 2.

Insulator bypass depends on the relative orientation of two scs insulators inserted between the eye enhancer and the white promoter. (A and B) Experimental evidence that one copy of scs in both orientations can effectively insulate the eye enhancer. (C and D) Experimental evidence that scs insulators are capable of functional interaction. (E) Tests for the functional interaction between Zw5 binding sites. Reductive schemes of the transgenic constructs are shown (not to scale). The yellow and white genes are shown as boxes, with arrows indicating the direction of their transcription. The eye enhancer (Ee) is shown as a black circle. Downward arrows indicate target sites for Flp recombinase (frt) or Cre recombinase (lox). The same sites in construct names are denoted by parentheses. The scs insulator is shown as a white box, with a white oval indicating the Zw5 binding site. The superscript index ‘R’ indicates that the corresponding element is inserted in the reverse orientation in the construct. The ‘white’ column shows the numbers of transgenic lines with different levels of white expression. The wild-type white expression determined the bright red eye color (R); in the absence of white expression, the eyes were white (W). Intermediate levels of pigmentation, with the eye color ranging from pale yellow (pY) through yellow (Y), dark yellow (dY), orange (Or), dark orange (dOr) and brown (Br) to brownish red (BrR), reflect the increasing levels of white expression. N is the number of lines in which flies acquired a new white (w) phenotype upon deletion (Δ) of the specified DNA fragment (the eye enhancer or scs); T is the total number of lines examined for each particular construct.

Figure 3.

Testing the functional interaction between scs insulators or Zw5 binding sites in the GAL4/white model system. The GAL4 binding sites (indicated as G4) are at a distance of ∼5 kb from the white promoter. A reductive scheme of transgenic construct used to examine the functional interaction between the insulators is presented in the upper part of the figure. ‘+GAL4’ indicates that eye phenotypes in transgenic lines were examined after the induction of GAL4 expression. N is the number of lines in which flies acquired a new w phenotype upon induction of GAL4. For other designations, see Figure 2.

Figure 4.

Testing the functional interaction between Wari insulators (A and B) in the eye enhancer/white model and (C and D) in the GAL4/white model. The Wari insulator is shown as a hatched box. For other designations, see Figures 2 and 3.

Figure 5.

Testing the functional interaction between (A and B) two scs’ or (C and D) two 1A2 insulators. The scs’ insulator is shown as a gray box with the black and white arrows indicating binding sites for the BEAF protein. The 1A2 insulator is shown as a black box with white rectangles indicating Su(Hw) binding sites. For other designations, see Figures 2 and 3.

Figure 6.

Testing the functional interaction between DNA fragments containing binding sites for different insulator proteins, dCTCF (black ovals), Zw5 (white ovals), Su(Hw) (white rectangles) or composite DNA fragments containing dCTCF and Su(Hw) binding sites (black ovals and white rectangles). For designations, see Figures 2 and 3. The results with G4(C^x4)Y(C^x4)W were taken from Kyrchanova et al. (51).

Figure 7.

Two models of pairing between the insulators. Presumptive proteins responsible for insulator pairing are shown as a white cylinder and a gray cube. Solid and dotted arrows indicate high and low levels of transcription, respectively. Other designations: (P) promoter, (E) enhancer.