Long-range enhancer-promoter interactions in the Scr-Antp interval of the Drosophila Antennapedia complex

Abstract

Long-range enhancer-promoter interactions are commonly seen in complex genetic loci such as Hox genes and globin genes. In the case of the Drosophila Antennapedia complex, the T1 enhancer bypasses the neighboring ftz gene and interacts with the distant Scr promoter to activate expression in posterior head segments. Previous studies identified a 450-bp promoter-proximal sequence, the tethering element, which is essential for T1-Scr interactions. To obtain a more comprehensive view of how individual enhancers selectively interact with appropriate target genes, we used bioinformatic methods to identify new cis-regulatory DNAs in the approximately 50-kb Scr-Antp interval. Three previously uncharacterized regulatory elements were identified: a distal T1 tethering sequence mapping >40 kb from the proximal tethering sequence, a repressor element that excludes activation of Scr by inappropriate enhancers, and a new ftz enhancer that directs expression within the limits of stripes 1 and 5. Many of the regulatory DNAs in the Scr-Antp interval are transcribed, including the proximal and distal tethering elements. We suggest that homotypic interactions between the tethering elements stabilize long-range T1-Scr interactions during development.

Fig. 1.

Summary of the Scr-Antp region of the Antennapedia complex. The Scr-Antp interval of the Antennapedia complex contains two homeotic selector genes, Scr and Antp, and the ftz segmentation gene. (A) Scr and ftz are divergently transcribed and separated by a 15-kb intergenic spacer. The AE1 enhancer, which drives localized expression of ftz in seven pairrule stripes (D), is positioned upstream of the ftz promoter within this spacer. A 30-kb intergenic spacer separates ftz from the homeotic selector gene Antp.A newly identified ftz enhancer, ftzDE, is contained within this spacer, just downstream of the ftz coding region. ftzDE directs expression specifically in ftz stripes 1 and 5. The T1 enhancer directs Scr expression in the labial head segment and anterior compartment of the first thoracic segment (C). The Scr promoter-proximal tethering element is required for the proper targeting of T1 to the Scr promoter (A). The 450-bp tethering element is positioned immediately upstream of the Scr core promoter and contains eight copies of a palindromic motif, TTCGAA. Another cluster of this motif is located within 3 kb downstream of the Antp coding region. This “distal” tethering cluster contains five copies of the palindromic motif. Colored arrows beneath the diagram indicate regions within the intergenic spacer that are transcribed. Red arrows represent regions transcribed in patterns overlapping the endogenous Scr expression domain. Blue arrows represent intergenic regions transcribed in the ftz pair-rule pattern. A 14-kb region containing the ftz gene and associated enhancers has undergone a chromosomal inversion in related Drosophila species, such as D. littoralis (B).

Fig. 3.

The distal cluster mediates tethering. (A–D) A P element expression vector was used to determine the activities of the newly identified distal cluster, which contains five copies of the TTCGAA motif in a 389-bp DNA fragment located downstream of Antp (see Fig. 1 A legend). The distal cluster was placed downstream of the 3′ T1 enhancer (top). Divergently transcribed CAT and lacZ reporter genes were placed under the control of the Scr and ftz promoters, respectively. The 450-bp proximal tethering element was positioned immediately 5′ of Scr and separated from the divergently transcribed ftz-lacZ fusion gene with a 1.6-kb spacer sequence from bacteriophage λ. The T1 enhancer activates Scr-CAT expression in the posterior head segment (A–D). However, in the absence of the distal cluster (“distal,” top), CAT exhibits variable background staining (A and C). With the distal cluster, background staining is eliminated, and CAT expression is restricted to the posterior head segment (B and D). One hundred stained embryos undergoing germband retraction were scored for CAT expression outside of the T1 expression domain. This was done for three separate lines for both transgenes. In lines containing the distal cluster, only 5% of the embryos displayed any ectopic staining. In contrast, more than half of the embryos lacking the distal cluster express lacZ outside of the T1 expression domain. (E–H) Multimers of the TTCGAA palindrome were inserted immediately upstream of a ftz-lacZ reporter to test whether it is sufficient to mediate tethering activity (center). The T1 enhancer was placed upstream of the modified reporter, and T1 activity was assayed by in situ hybridization. Copies of the palindrome were separated by 10 bp or one helical turn. None, 2, 4, and 6 copies of the palindrome were stained in parallel with a ftz-lacZ reporter modified with the full-length Scr proximal tethering (E–G). Weak activation of the lacZ reporter gene is detected on insertion of four tandem copies of the TTCGAA motif (G). An additional two copies of the palindrome do not significantly alter lacZ expression (not shown). Stronger activation of lacZ expression is obtained with either the proximal tethering element (H; “full”) or distal cluster (not shown). (I–L) The distal cluster was inserted between the 5′ T1 enhancer and ftz-lacZ fusion gene, or between AE1 and ftz-lacZ. The distal cluster mediates strong activation by T1 (J; compare with I), but diminishes activation by the AE1 enhancer (L; compare with K).

Fig. 2.

Identification of an ftz enhancer. The ftz regulatory region was scanned for clusters of cis-regulatory elements that are recognized by transcription factors encoded by maternal (bicoid and caudal), gap (hb, Kr, kni), and pair-rule (ftz) genes. Three clusters were found in the 10-kb region flanking the ftz coding region. The first two clusters correspond to previously identified cis-regulatory elements: AE1 and the zebra element, respectively (A). The third cluster is located between the ftz coding region and the T1 enhancer (B). A 1.25-kb genomic DNA fragment that encompasses this cluster was inserted into a P element expression vector that contains divergently transcribed CAT and lacZ reporter genes (B). CAT is under the control of the Scr promoter region, whereas lacZ contains the ftz promoter region. Cluster 3 selectively activates the lacZ reporter gene but fails to induce CAT expression (C). ftz-lacZ expression is detected in two stripes in cellularizing embryos (D). These stripes are expanded into a broad band in mutant embryos homozygous for a null mutation in the Kr gene (E). Double-staining experiments using a probe that visualizes the endogenous ftz stripes indicate the newly identified enhancer directs expression in stripes 1 and 5 (F).

Fig. 4.

A negative element upstream of the Scr core promoter interferes with AE1–Scr interactions. (A and B) P element expression vectors contain the AE1 enhancer positioned 5′ of the ftz-lacZ fusion gene. The minimal 450-bp proximal tethering element does not block activation of lacZ expression by the AE1 enhancer (A). However, a larger 520-bp tethering element, containing 70 bp of flanking sequence in the Scr promoter-proximal region, attenuates AE1–ftz interactions (B). (C and D) A regulatory swap in the activities of the AE1 and T1 enhancers was obtained by inserting a TATA element in the minimal Scr promoter and placing the 450-bp proximal tethering element 5′ of the ftz promoter (6). AE1 now activates the Scr-CAT reporter gene in seven stripes (C), whereas T1 activates ftz-lacZ in the posterior head segment (D). The ftz-lacZ fusion gene is weakly activated by AE1, but these interactions are attenuated by the 3.8-kb T1 (not shown). A more complete regulatory swap was obtained in the following experiment. (E and F) The AE1 enhancer was positioned between the divergently transcribed CAT and lacZ reporter genes, whereas the T1 enhancer was downstream of lacZ. The “full-length” 520-bp proximal tethering element was positioned 5′ of the ftz-lacZ fusion gene. It contains the negative elements that impede AE1–Scr interactions. This tethering sequence completely blocks AE1–ftz interactions, but AE1 is able to activate the TATA-containing Scr promoter and direct seven stripes of CAT expression (E). Conversely, the 3′ T1 enhancer strongly activates the ftz-lacZ fusion gene in posterior head segments and the anterior compartment of the first thoracic segment.

Fig. 5.

Transcription of intergenic DNA in the Scr-Antp interval. A series of 2-kb DNA fragments covering the Antp-Scr interval were used to prepare 20 digoxigenin-labeled RNA probes for in situ hybridization (diagram). Colored arrows indicate the orientation of the transcripts detected by each probe. Minus signs denote regions where no transcription is detected. Extensive transcription is seen throughout the ftz-Antp intergenic spacer (probes 10–20). Staining is detected in parasegments 2 and 3 (G–J). The anterior component of this expression pattern overlaps the normal Scr profile in parasegment 2 (A and B). Staining does not overlap the endogenous Antp pattern, which encompasses the presumptive middle thorax and is centered on parasegment 4 (C and D). Staining persists during germband elongation for probe 19 (H), which encompasses the distal TTCGAA cluster, but expression does not persist for the other detectable transcripts in the ftz-Antp spacer (e.g., J). One region, 15, exhibits transcription from both DNA strands. Double-stranded transcription is also detected throughout the AE1-ftz interval (6–8). The staining pattern observed is similar to the endogenous ftz pattern and consists of seven pair-rule stripes (data not shown). This same pattern of transcription is seen for probe 9. There is little or no transcription in the interval between AE1 and the proximal tethering element (1–5), although the tethering element (probe 1) is itself transcribed (E and F). Like transcription from the distal cluster (probe 19), staining persists during germband elongation (F).