Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila Antennapedia complex

Abstract

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. Here we provide evidence for a third mechanism: promoter-proximal tethering elements. The Scr-ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3' of the ftz gene and approximately 25 kb 5' of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1-ftz and T1-Scr interactions. A 450-bp DNA fragment located approximately 100 bp 5' of the Scr transcription start site is essential for T1-Scr interactions and can mediate long-range activation of a ftz/lacZ reporter gene when placed 5' of the ftz promoter. We suggest that the Scr450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer-promoter interactions at other complex genetic loci.

Figure 1

Summary of the Scr-ftz region of the ANT-C. (A) The diagram (Upper) shows the order of the five Hox-containing homeotic selector genes. The pair-rule gene ftz is also indicated. The second diagram presents an enlarged view of the Scr-ftz interval. The two genes are divergently transcribed, and the intron-exon organization of each gene is indicated. The AE1 enhancer is located in the intergenic region between Scr and ftz but selectively interacts with the ftz promoter to maintain the seven-stripe expression pattern in embryos undergoing germ-band elongation. Scr expression depends on the T1 enhancer, which is located 3′ of the ftz transcription unit. (B) Scr expression pattern in an 8- to 9-hr embryo undergoing germ-band retraction. Staining was visualized after hybridization with a digoxigenin-labeled Scr antisense RNA probe. Expression is detected in different tissues of parasegment 2, which includes the posterior-most regions of the head and the anterior compartment of the prothorax. (C) ftz expression pattern in a 4- to 5-hr elongating embryo. Staining is detected in seven stripes along the germ band. Staining was visualized after hybridization with a digoxigenin-labeled ftz antisense RNA probe. (D and E) Transgenic embryos were obtained from adults containing the P element transformation vector indicated in the diagram beneath the photomicrographs. Divergently transcribed CAT and lacZ reporter genes were placed under the control of the Scr and ftz promoter regions, respectively. The Scr promoter region is 1.1 kb in length and includes ≈555 bp 5′ of the transcription start site. The ftz promoter region is 200 bp and includes ≈100 bp of 5′ flanking sequence. The 430-bp AE1 enhancer and the 3.8-kb T1 enhancer were placed between the CAT and lacZ reporter genes. The leftward CAT gene is activated by the T1 enhancer in posterior head segments and the anterior compartment of the prothorax, similar to the endogenous Scr expression pattern (compare D with B). The lacZ reporter gene exhibits seven stripes of ftz expression, similar to endogenous ftz expression (compare E with C). CAT and lacZ expression patterns were visualized after hybridization with digoxigenin-labeled CAT or lacZ antisense RNA probes.

Figure 2

Identification of promoter-proximal tethering elements. The T1 enhancer was placed 5′ of a series of truncated Scr-lacZ fusion genes. The “full-length” Scr promoter region is 1.1 kb in length and includes 555 bp of 5′ flanking sequence (A). Truncations that remove a 450-bp fragment exhibit diminished levels of expression (B and C). This 450-bp fragment is sufficient to stimulate expression from the minimal Scr core promoter (Scr80 + 450; D). Inserting this fragment immediately upstream of the heterologous ftz promoter is sufficient to drive strong T1 expression (ftz80 + 450; F) on an otherwise unresponsive promoter (ftz80; E).

Figure 3

Swapping regulatory specificity in the Scr-ftz region. Transgenic embryos express the P element minigenes indicated above the photomicrographs. The AE1 enhancer selectively activates the rightward ftz/lacZ reporter gene, and T1 activates the leftward Scr/CAT gene (A and B; also see Fig. 1 D and E). Regulatory specificity is reversed on modification of the Scr and ftz promoters (C and D). The Scr450 tethering fragment was removed from the 5′ region of Scr and attached to the minimal ftz80 promoter. In addition, the minimal Scr promoter was modified to include an optimal TATA element. The AE1 enhancer now strongly activates the leftward Scr promoter, and induces seven stripes of CAT expression along the germ band. Conversely, the T1 enhancer selectively interacts with the rightward ftz/lacZ fusion gene and activates lacZ expression in the prothorax and weakly in posterior regions of the head. AE1 expression of Scr/CAT depends on the presence of T1 between AE1 and ftz/lacZ. Removal of T1 results in greatly diminished Scr/CAT staining.

Figure 4

Scr tethering elements mediate long-range enhancer–promoter interactions. Transgenic embryos express the Pelement minigenes indicated in the diagrams. In all cases, the T1 enhancer was placed 3′ of the lacZ reporter gene. Minimal Scr and ftz promoters are not activated by T1, which now maps ≈5 kb from the ftz promoter and more than 6 kb from Scr. The 3′ T1 enhancer activates the distal Scr/CAT reporter gene when 5′ tethering elements are included in the Scr promoter region (A). The proximal ftz/lacZ gene remains silent (B). Removal of the 5′ tethering elements from the Scr promoter region causes a loss in CAT expression (C). However, when the Scr450 fragment is placed 5′ of the ftz promoter, the 3′ T1 enhancer now activates the proximal lacZ reporter gene (D). E summarizes the model for enhancer tethering, whereby promoter-proximal elements selectively recruit a specific distal enhancer (Enhancer A, T1). Perhaps regulatory proteins bound to both the distal enhancer and the proximal tethering elements form homomeric complexes that stabilize enhancer-promoter loops. Enhancer B (AE1) is regulated through promoter competition, selectively interacting with the stronger promoter.