Conservation of enhancer location in divergent insects

Abstract

Dorsoventral (DV) patterning of the Drosophila embryo is controlled by a concentration gradient of Dorsal, a sequence-specific transcription factor related to mammalian NF-kappaB. The Dorsal gradient generates at least 3 distinct thresholds of gene activity and tissue specification by the differential regulation of target enhancers containing distinctive combinations of binding sites for Dorsal, Twist, Snail, and other DV determinants. To understand the evolution of DV patterning mechanisms, we identified and characterized Dorsal target enhancers from the mosquito Anopheles gambiae and the flour beetle Tribolium castaneum. Putative orthologous enhancers are located in similar positions relative to the target genes they control, even though they lack sequence conservation and sometimes produce divergent patterns of gene expression. The most dramatic example of this conservation is seen for the "shadow" enhancer regulating brinker: It is conserved within the intron of the neighboring Atg5 locus of both flies and mosquitoes. These results suggest that, like exons, an enhancer position might be subject to constraint. Thus, novel patterns of gene expression might arise from the modification of conserved enhancers rather than the invention of new ones. We propose that this enhancer constancy might be a general property of regulatory evolution, and should facilitate enhancer discovery in nonmodel organisms.

Fig. 1.

The Anopheles twist and brinker enhancers. (A and C) The locations of the Anopheles twist (A) and brinker (brk) (C) enhancers are indicated by pink rectangles. The twist enhancer is located 5′ of the gene; the brinker enhancer is ≈100 kb away in the intron of Atg5. (B and D) The Anopheles twist (twi) enhancer (B) drives lacZ reporter expression in the Drosophila mesoderm, whereas the Anopheles brinker enhancer (D) drives lacZ in the neurogenic ectoderm.

Fig. 2.

The Tribolium cactus and sim enhancers. (A) Tribolium embryo stained for cactus mRNA (red) and Twist protein to mark the mesoderm (green). (B) sim (red) is expressed in the ventral midline in beetles. DNA is stained in blue (A and B), anterior is to the left, and dorsal is up in all cases. (C and D) The locations of the Tribolium cactus (C) and sim (D) enhancers are indicated by pink rectangles. (E and F) The Tribolium cactus enhancer and the Tribolium sim enhancer drive lacZ reporter expression in transgenic Drosophila in the mesoderm (cactus) (E) and ventral midline (sim) (F).

Fig. 3.

Summary of enhancer positions. Enhancers are indicated by pink rectangles. In all cases, the Drosophila locus is shown on the left and the orthologous Tribolium or Anopheles gene on the right. Abbreviations: Dm, Drosophila melanogaster; Ag, Anopheles gambiae; Tc, Tribolium castaneum.

Fig. 4.

The Tribolium sog enhancer. (A and C) Tribolium sog is expressed first in the mesoderm and ventral neurogenic ectoderm (A) before becoming restricted to the ventral neurogenic ectoderm (C). sog is in red, Twist in green, and DNA in blue. (B and D) The Tribolium sog enhancer has the same expression pattern when driving a reporter in transgenic Drosophila. Frame E shows the location of the Tribolium sog enhancer (pink rectangle), and Pol II chIP–chip (Upper) and cDNA expression profiles (Lower) of the Tribolium sog locus. White arrow indicates the promoter.