Supplemental Material for Camino et al., 2020

Figure S1. Expression of the DsRed.T4-NLS and EGFP-NLS reporters at the single-cell level. (A) Schematic of the evaluated reporter transgenes. Here, the dimorphic element enhancer is at an equal proximity to the DsRed.T4-NLS (red oval) and EGFP-NLS (green oval) reporter due to the absence of a spacer sequence. Expression of (A’) EGFP-NLS and (A’’) DsRed.T4-NLS is prominent in the female A5 and A6 segments. (A’’’) When the DsRed.T4-NLS and EGFP-NLS expression patterns were merged, widespread co-expression is observed at the single-cell level, indicating that both reporters can be active in the same cells.

Figure S2. Promoter competition and spacer sequence regulatory activity had little to no influence on the expressions of the proximal and distal reporter genes. (A) bab locus showing the relative position of the dimorphic element (DE) core enhancer from the promoters (black arrows) for the bab1 and bab2 genes. (B-E) Schematics of the evaluated DsRed.T4-NLS (red oval) and EGFP-NLS (green oval) reporter transgenes. (B’-E’) Expression of the EGFP-NLS reporter in the abdomens of transgenic female pupae. (B’’-E’’) Expression of the DsRed.T4-NLS reporter in the abdomens of transgenic female pupae. (B) At equal spacing from the reporter genes, (B’ and B’’) the dimorphic element drove seemingly identical patterns and comparative levels of EGFP-NLS and DsRed.T4-NLS reporter expression. (C) When separated from the dimorphic element by an 8 kb spacer, (C’ and C’’) little to no expression occurred for the distal DsRed.T4-NLS reporter while expression occurred for the proximal EGFP-NLS reporter in the dimorphic element pattern. (D) In the absence of a promoter for the proximal reporter (red X), (D’ and D’’) EGFP-NLS expression was absent, and little to no expression occurred from the distal DsRed.T4-NLS reporter. (E) When the dimorphic element enhancer was absent, (E’ and E’’) the 8 kb spacer sequence drove little to no expression of the DsRed.T4-NLS and EGFP-NLS reporters.