GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo

Abstract

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl-GAGA increases the stability of enhancer-promoter interactions by creating an open chromatin configuration at the core promoter.

Figure 1

The eve promoter possesses an enhancer blocking activity. Transgenic embryos are undergoing cellularization and are oriented with anterior to the left and dorsal up. Embryos were hybridized with digoxigenin-labeled antisense white, CAT, and lacZ RNA probes and stained with anti-digoxigenin antibodies. (A) Embryo contains the eve/CAT–eve/lacZ P-transformation vector indicated in the diagram. It was hybridized with a CAT probe to monitor the expression of the distal eve/CAT reporter gene. Replacing the proximal eve promoter with the white promoter sequence results in the full induction of eve/CAT expression (see Fig. 2A). (B) Same as A except that the embryo was hybridized with a lacZ probe to monitor the expression of the proximal eve/lacZ reporter gene. The weak staining in head regions is due to the P-transformation vector used in these experiments (Small et al. 1992). (C) Embryo contains the white/white–white/CAT P-transformation vector indicated in the diagram. It was hybridized with a white probe to monitor expression of the distal white/white reporter gene. (D) Same as C except that a CAT probe was used to monitor the expression of the proximal white/CAT reporter gene.

Figure 2

A chimeric eve–white promoter functions in a dominant-negative fashion to block linked genes. Transgenic embryos, oriented with anterior to the left and dorsal up, are at various stages of cellularization and express the transgenes shown in the diagrams beneath the photomicrographs. All embryos shown were stained in parallel, thereby permitting a direct comparison of expression levels. (A,B) eve/CAT–white/lacZ transgenic embryos with the eve promoter upstream of the distal CAT reporter gene and the white promoter attached to the proximal lacZ gene. The distal CAT gene is fully activated by the 3′ IAB5 enhancer (A) and exhibits intense expression in the presumptive abdomen. In contrast, hybridization with a lacZ probe indicates that the proximal white/lacZ gene is inactive. (C,D) Embryos carry a P-transformation vector that is similar to the one shown in A and B except that the proximal white promoter was replaced with the chimeric promoter, white^eve. This promoter contains the 5′ TATA region from eve and the 3′ Inr region from white, as indicated in the diagram. (D) Hybridization with the lacZ probe reveals that the white^eve promoter is virtually inactive. (C) Hybridization with the CAT probe demonstrates that expression of the distal eve/CAT gene, which contains a completely wild-type eve promoter, is severely attenuated. (E,F) Same as C and D except that the white^eve promoter was mutagenized to disrupt the GAGA element (TATA is intact; see diagram). The modified promoter essentially fails to direct expression of the lacZ reporter (F). However, the distal eve/CAT reporter gene is nearly fully active (E; cf A).

Figure 3

GAGA–Trl interactions mediate the enhancer blocking activity of the eve promoter. Transgenic embryos carry the indicated P-transformation vectors and were hybridized and oriented as described. (A,B) Embryos express CAT and lacZ transgenes that are both driven by the eve promoter, except that the proximal eve/lacZ reporter gene was mutagenized to disrupt the GAGA element (see diagram). The distal eve/CAT gene exhibits moderate levels of expression (A, cf. C); the proximal eve/lacZ gene is expressed at normal levels (B, cf. D). It is conceivable that the residual enhancer blocking activity is due to a ‘cryptic’ GAGA element located in the transcribed region. (C,D) Same as A and B except that both the proximal lacZ and distal CAT genes are attached to normal eve promoter sequences (same as Fig. 1A,B). The proximal, wild-type eve promoter (D) has full enhancer blocking activity so that the distal eve/CAT gene is silent (C). (E,F) Same as C and D except that the transgene was crossed into an embryo derived from an R85/+ heterozygous female. R85 is a null mutation in the trl gene, so that these embryos contain half the normal dose of trl⁺ gene activity. This reduction in Trl attenuates the enhancer blocking activity of the proximal, wild-type eve promoter (F), so that the distal eve/CAT gene is active (E). The levels of eve/CAT expression are similar to those obtained when the proximal GAGA element was mutagenized (A). These results suggest that Trl–GAGA interactions are critical for the enhancer blocking activity of the eve promoter.

Figure 4

Uncoupling competition and enhancer trapping. The IAB5 enhancer was placed 5′ of the divergently transcribed CAT and lacZ reporter genes. Transgenic embryos were oriented as described and stained with CAT (left) or lacZ (right) probes. (A,B) The transgene contains the white and eve promoters. As shown previously (Ohtsuki et al. 1998), IAB5 preferentially interacts with the TATA-containing eve promoter (B) and only weakly activates the TATA-less white promoter (A). (C,D) Same as A and B except that the rightward eve promoter was mutagenized to eliminate the GAGA element. The resulting promoter, eve^ΔGAGA, contains TATA and continues to be the preferred target of the IAB5 enhancer (D). (E,F) The CAT and lacZ reporter genes are regulated by the wild-type eve promoter. Both reporter genes are strongly activated by IAB5 and exhibit intense staining in the presumptive abdomen. (G,H) Same as E and F except that the rightward eve promoter was mutagenized to eliminate the GAGA element. The resulting eve^ΔGAGA promoter continues to direct intense expression of lacZ (H) and is essentially as active as the distal eve promoter (G).

Figure 5

Changing the location of GAGA. Transgenic embryos carry the indicated P-transformation vectors and were hybridized and oriented as described. Embryos were stained in parallel, thereby alowing direct comparison of relative expression levels. (A,B) The distal CAT gene is under the control of the normal eve promoter; the proximal lacZ gene was attached to a modified white promoter containing an optimal TATA element (see Ohtsuki et al. 1998). The 3′ IAB5 enhancer interacts with both promoters to direct nearly equal levels of CAT (A) and lacZ (B) expression. The normal white promoter, lacking TATA, is not activated by IAB5 in similar transgenes (e.g., Fig. 2B). (C,D) Same as A and B except that the proximal white^TATA promoter was further modified to include a GAGA element between TATA and the Inr. This promoter exhibits the same type of dominant-negative activity as white^eve (see Fig. 2D). (E,F) Same as C and D except that the GAGA element was placed 28 bp 5′ of TATA. The resulting promoter, white^GAGA+TATA, traps the IAB5 enhancer so that CAT expression (E) is weak (cf. A). However, unlike the white^TATA+GAGA promoter (D), this promoter is fully active (F).