Fig. 4. Delimiting a minimal notum enhancer. (A) VISTA plot comparing the D. melanogaster IroRE^2-B sequence with those of D. pseudoobscura, D. virilis and D. mojavensis (window size: 100 bp). Pink peaks represent regions with more than 70% of identity (the most conserved one is marked by an asterisk). The light blue peak indicates the putative linc coding region. (B) Subfragments of IroRE^2-B tested for transcription enhancing activity. Only the blue regions activate transcription. Putative binding sites for ETS proteins, Ttk, GATA proteins and Mad are indicated. Filled symbols, binding sites matching the consensus sequence; open symbols, one or two mismatches compared to the consensus sequence. (C-F) lacZ expressions mediated by the indicated fragments. Note in C that IroRE^2B drives a weak expression in the prospective intervein regions, suggesting that shortening has removed some wing-pouch repressor elements. (G,G') Expression mediated by IroRE^2-B2GATAm extends into the prospective medial notum, proximal to the wg (blue) and ara/caup (green) domains. The dotted lines in E and G' outline the proximal region of the discs. (H,H') Mutation of the ETS binding site of IroRE^2-B2 that most closely matches the consensus strongly reduces enhancer activity in the wing disc. (I-I'') Up-regulation of IroRE^2-B2GATAm-lacZ expression in tkv^a12 clones, some of which are indicated by arrows.