Dynamics and function of intron sequences of thewinglessgene during the evolution of theDrosophilagenus

Abstract

To understand the function and evolution of genes with complex patterns of expression, such as the Drosophila wingless gene, it is essential to know how their transcription is regulated. However, extracting the relevant regulatory information from a genome is still a complex task. We used a combination of comparative genomics and functional approaches to identify putative regulatory sequences in two introns (1 and 3) of the wingless gene and to infer their evolution. Comparison of the sequences obtained from several Drosophila species revealed colinear and well-conserved sequence blocks in both introns. Drosophila willistoni showed a rate of evolution, in both introns, faster than expected from its phylogenetic position. Intron 3 appeared to be composed of two separate modules, one of them lost in the willistoni group. We tested whether sequence conservation in noncoding regions is a reliable indicator of regulatory function and, if this function is conserved, by analyzing D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. melanogaster (Sophophora subgenus) and the species from the Drosophila subgenus presenting the most divergent sequence, D. americana. The analysis indicated that intron 3 contains pupal enhancers conserved during the evolution of the genus, despite the fact that only 30% of the D. melanogaster intron 3 sequences lie in conserved blocks. Additional analysis of D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. willistoni revealed the absence of an abdomen-specific expression pattern, probably due to the above-mentioned loss of a regulatory module in this species.

A 3′ cis‐regulatory region controls wingless expression in the Drosophila eye and leg primordia

“…We next focused on the regulation of the dorsal domain of wg expression in the eye disc, as it is through this expression that wg controls the DV organization of the disc and has a major role in repressing dorsal eye fate (Ma and Moses, 1995; Treisman and Rubin, 1995; Von Ohlen and Hooper, 1997; Maurel‐Zaffran and Treisman, 2000). Considering the previously described wg enhancers (Neumann and Cohen, 1996a, b; Von Ohlen and Hooper, 1997; Lessing and Nusse, 1998; Costas et al, 2004), and the noncoding wg gene regions surveyed in this study (FIG. 1), the 502‐bp wg 2.10 sequence is unique in its ability to direct expression in the eye disc.…”

“…As a first step to molecularly dissect wg regulation during eye disc development, we set to identify the cis ‐regulatory elements (CREs) responsible for driving wg expression in this disc. We generated a series of transgenic strains harboring genomic fragments of the wg gene upstream of a heat‐shock minimal promoter driving the lacZ reporter gene from previously unexplored regions (see Costas et al, 2004, and references therein). A ∼ 6.0‐kb DNA fragment ( wg 2), derived from the wg 3′ genomic region (Fig.…”

“…Nevertheless, much less is known about these wg imaginal‐specific enhancers. Neumann and Cohen (1996a) and Costas and coworkers (2004) have characterized a wing disc margin/hinge enhancer in the wg 5′ intergenic region and a pupal enhancer within its third intron, respectively (Costas et al, 2004; Neumann and Cohen, 1996a). Therefore, other disc‐specific cis ‐regulatory elements must exist to regulate wg expression.…”

Evolution of cis-regulatory sequence and function in Diptera

“…Sequences comprising transcription factor binding sites may thus be more constrained than sequences not used as binding sites. Surprisingly, the pattern of nucleotide substitutions is similar within characterized binding sites and in surrounding regions of DNA, suggesting this may not be the case (Emberly et al, 2003;Costas et al, 2004;Phinchongsakuldit et al, 2004;Balhoff and Wray, 2005). It remains to be seen how often 'surrounding' sequences contain unidentified binding sites, and how sequence divergence affects gene expression.…”

“…Sequence comparisons of orthologous cis-regulatory elements generally show blocks of conserved sequence surrounded by more divergent sequences (Kassis et al, 1985;Wilde and Akam, 1987;Kassis et al, 1989;Langeland and Carroll, 1993;Lukowitz et al, 1994;Pan et al, 1994;Sackerson, 1995;Ludwig et al, 1998;Wolff et al, 1999;Kim, 2001;Dellino et al, 2002;Emberly et al, 2003;Berman et al, 2004;Costas et al, 2004). Sequence similarity to D. melanogaster cis-regulatory regions has been used to identify enhancers in other Dipterans, including Anopheles gambiae (Papatsenko and Levine, 2005), Scaptodrosophila lebanonensis (Papaceit et al, 2004), and Calliphora vicina (Gibert and Simpson, 2003).…”