Figure 5. E74A protein is sufficient to inhibit its own ecdysone-induced transcription.

(A) Experimental paradigm for expressing E74A protein prior to the prepupal pulse of ecdysone. Expression profile of endogenous E74A mRNA (in gray) and E74A protein (in blue) in salivary glands shown relative to hours after head eversion (AHE). Arrows mark timing of the 30 minute heat-shock treatment and the subsequent dissection of larval salivary glands. (B) Precocious expression of E74A protein (gray bars) is sufficient to repress induction of endogenous E74A mRNA in control and bel^psg9 mutant salivary glands. Control salivary glands show over 100-fold induction of E74A in response to the prepupal pulse of ecdysone (first bar from left). In contrast, ectopic expression of E74A protein (second bar), but not E75A protein (third bar), inhibits endogenous E74A transcription. Similarly, ectopic E74A protein inhibits the ecdysone-induced expression of E74A in bel^psg9 mutant salivary glands (fourth and fifth bars). (C) Although precocious expression of E74A protein is sufficient to repress the ecdysone-induced transcription of E74A, it has a minor effect on the expression of ecdysone early response genes E75A and BR-C Z1. Salivary glands dissected from heat-shock-treated control (white bars) and hs-E74A carrying (gray bars) animals. (D) E74A is not necessary for the regression of other ecdysone early response genes. Salivary glands dissected from control (white bars) and E74A^neo24/Df mutant (gray bars) animals at +1.5 AHE, when endogenous expression of early response genes E75A and BR-C have regressed to levels prior to the ecdysone pulse. E74A^neo24 allele is an RNA null, hence the barely detectable levels of endogenous E74A mRNA. y-axis plots relative expression, normalized to rp49. Expression ratios in 5B calculated relative to −4 AHE control samples to show induction in response to the prepupal pulse of ecdysone. All samples are in triplicate; asterisks indicate p-values <0.05. bel/Df: bel^psg9/Df. AHE: after head eversion.