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doi: 10.1242/10.1242/dev.00227

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The promoter targeting sequence facilitates and restricts a distant enhancer to a single promoter in the Drosophila embryo

Qing Lin, Di Wu and Jumin Zhou*

Molecular Genetics Division, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA



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  		Fig. 1. Cis interactions in Abd-B. (A) Abd-B has an extended 5' regulatory region that can be subdivided into four regulatory domains, termed infra-abdominal (iab)-5, iab-6, iab-7 and iab-8. Each of these controls the development of a corresponding abdominal parasegment (PS). For example, iab-5 regulates Abd-B function in PS10 (roughly the fifth abdominal segment, or A5), iab-6 controls A6, and so on. Neighboring iab genes are separated by domain boundary elements such as Fab-7 and Fab-8, which function to prevent cross-regulatory interference between neighboring iab genes. These elements are also potent insulators that can block enhancer-promoter interactions in transgenic constructs. The PTS element has an anti-insulator activity that allows an enhancer to activate a promoter despite an intervening insulator. In the Abd-B locus, PTS may mediate enhancer-promoter interactions by allowing enhancers to overcome the intervening Fab elements and activates Abd-B promoter. (B) Detailed map of the 9.5 and 5.3 kb tmr elements. B, BamHI; H, HindIII; P, PstI; and R, EcoRI.

 


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  		Fig. 2. The 5.3 kb transvection mediating region (tmr) exhibits orientation-dependent promoter targeting activity. Whole-mount RNA in situ hybridization was performed on 2- to 4-hour-old embryos using anti white or lacZ RNA probes. All embryos are oriented anterior towards the left and dorsal side upwards. Arrows in the posterior regions of the embryos indicate IAB8 activity. (A) IAB8 activates Tp-Z when located 5' proximal to the Tp (Transposase) promoter. (B) IAB8 activates Tp-lacZ from a 3' position, 4.5 kb away from the Tp promoter. (C) The 5.3 kb BamHI-HindIII tmr fragment was inserted just 3' end of the lacZ gene in a C4PLZ vector in the forwards (5'-3') orientation. It consists of 290 bp 5' of the 625 bp PTS, the entire 580 bp Fab-8 insulator, the IAB8 enhancer, a Polycomb response element (PRE) located between Fab-8 and IAB8 (Barges et al., 2000Go; Zhou et al., 1999Go), and about 500 bp of additional genomic sequence 3' to the 1.6 kb IAB8. In this orientation, IAB8 is proximally located and activates both the divergently transcribed white (w) and the Tp-lacZ fusion gene. (D) In the reverse (3'->5') orientation, IAB8 can direct strong transcriptional activation of either w or lacZ. In two out of 12 lines examined, IAB8 activates only lacZ. Many of these intensely stained embryos also exhibit anterior staining (arrowhead). This is probably caused by the lack of repressor binding sites in the transgenic regulatory regions that normally repress Abd-B enhancer activity in the anterior region of the embryo. (E) In five of these strains, IAB8 activates only w. In the remaining lines, the intervening Fab-8 insulator (data not shown) presumably blocks the IAB8 enhancer.

 


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  		Fig. 5. Quantitative analysis of the activity of PTS. (A) Representative embryos showing IAB5-lacZ promoter targeting. Red circles indicate the areas measured for relative staining intensity. (B) The graph displays staining intensity as a function of time. We detected almost no staining in the first 7 minutes after adding the substrate NBT/BCIP. This may be due to the time needed for the substrate to diffuse into the embryos. Between 10 and 60 minutes, staining intensity (2{Delta}EV-1) was linear with staining time. (C) To compare IAB8 enhancer strength in different P-elements, heterozygous embryos were fixed and stained in parallel for 45 minutes. Approximately 30 embryos were measured. Lanes 1 and 2 show the staining intensities for the IAB8 enhancer on the lacZ promoter when it is located about 100 bp (lane 1, gray), or 5.5 kb (lane 2, green) away from the lacZ promoter. Five strains for each of these transgenes were analyzed. Lane 3 (blue) and 4 (yellow) indicate IAB8-w interaction (W32-w) before and after the removal of PTS and Fab-8 in W32. Lane 5 and 6 shows IAB8-lacZ interaction (W32-Z) in the same strain before and after the removal of PTS and Fab-8. (D) Analysis of enhancer activities before (blue) and after (yellow) the removal of PTS and suHw from W81. Four different transgenic strains (W81-1, W81-2, W81-3 and W81-4) were shown.

 


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  		Fig. 3. PTS facilitates and restricts enhancer activity to a single promoter. Constructs W32 and W81 were tested. The former contains an FRT flanked PTS and Fab-8, and the IAB8 enhancer, where as the latter contains FRT flanked PTS and suHw, plus a 1.0 kb IAB5 enhancer. Large orange arrowheads indicate FRT sites. (A,B) A representative line exhibiting a selective IAB8-w interaction is shown. (C,D) The same strain after removal of the Fab-8 insulator and the PTS by Flp-mediated recombination (Golic and Lindquist, 1989Go). There is a severe reduction of IAB8-w interaction with the accompanying activation of lacZ. (E,F) Most transgenic strains carrying the W81 display an IAB5-lacZ interaction as shown here. (G,H) The same strain after Flp-FRT analysis. Note the reduction of IAB5-lacZ interaction and the concomitant appearance of IAB5-w interaction.

 


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  		Fig. 4. Anti-insulator and promoter targeting by the PTS element. (A,B) Transgenic embryos carrying construct W78. The IAB5 enhancer selectively activates the lacZ but not the w. (C,D) The same transgenic strain after the removal of the PTS by Flip-mediated recombination. Notice that the IAB5-lacZ interaction is blocked by the suHw insulator. Arrowheads indicate FRT sites.

 


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  		Fig. 6. Model for PTS function. We propose that PTS establishes a stable association between DNA sequences near the enhancer and promoter that results in a constant physical proximity between the two. This type of association could not be blocked by an insulator, and would facilitate weak, long-range enhancer-promoter interactions, while at the same time preventing the enhancer from interacting with other promoters.

 

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