Bowl binds directly to LAE sequences in vitro to regulate LAE activity in vivo.

(A) LOGOs of consensus Odd-family ZF binding sites [21] are shown in the upper part. The dotted line box indicates this consensus in the binding sites under investigation. The double-stranded sequences encompassing a bona fide Odd ZF-binding site [22] as well as the two consensus Bowl ZF-binding sites within the LAE are shown beneath. Corresponding probed double-stranded DNA sequences tested in EMSA are framed. The shared CAG sub-motifs in wild-type (+) sequences, substituted by TTT in each mutant (m) probe, are underlined. For the small (1S) and large (1L) CR2 probes, near palindromic sub-sequences are indicated by converging arrows. (B) EMSA experiments examining Bowl-DNA complexes. Each tested DNA probe, shown in (A), was incubated with either purified non-fused GST (as a negative control) or purified GST-Bowl^ZF. Specific complexes are indicated by black arrowheads. Note that Bowl ZF binding to LAE sequences was fully abolished for each CAG mutant probe. (C) Wild-type as well as mutant LAE sequences encompassing the biochemically-defined Bowl ZF binding sites are shown, with mutated nucleotides in red. (D-E) Wild-type late L3 leg discs co-expressing LAEwt-RFP (red) and either LAEm2-GFP (D) or LAEm3-GFP (E) (green). Bowl ZF cognate binding sites are critical for restraining LAE reporter activity in distal ts5 and in proximal ts1. (F-G) Mosaic late L3 leg discs expressing either LAEwt-GFP (F) or LAEm3-GFP (G) and harboring FO clones (RFP+) expressing lines dsRNA (i.e., ectopically-stabilizing nuclear Bowl). Merged LAE-driven GFP (green) and FO RFP (red) fluorescence are shown, as well as GFP expression in isolation, in (F’) and (G’), respectively. Representative Clones deficient for lines activity are circled with white dashed lines. While ectopically-stabilized nuclear Bowl induced LAEwt-GFP repression (F’), it was unable to down-regulate the mutant LAE reporter (G’) (as revealed by the merged yellow instead of red color obtained in F).