Multiple interacting elements delineate an ecdysone-dependent regulatory region with secondary responsive character

Within the 2.2 kb region between hsp23 and gene 1 of the small heat shock gene locus 67B1 of Drosophila melanogaster, an approximately 1 kb perturbation of the chromatin architecture has previously been observed to occur in response to the steroid hormone ecdysone. Transient expression assays in hormonally-responsive Drosophila tissue culture cells utilizing hsp70-lacZ chimeric reporter constructs revealed the presence of ecdysone-dependent regulatory sequences in this hsp23-gene 1 intergenic region. The analysis delimited five functional segments: three core regions which were completely encompassed within the region of chromatin perturbation, and two gene-proximal regions which appear to be functionally equivalent under some circumstances. None of the delineated regions was capable of stimulating expression independently, while sub-maximal expression was obtained when combinations of two or three regions were monitored. This requirement for multiple DNA segments to drive maximal transcription suggested that cooperative interactions between the regions were essential for full hormonal responsiveness. Unexpectedly, no binding of the ecdysone receptor was detectable within any of the delineated regions, implying the involvement of multiple non-receptor factors in the observed hormonal responsiveness. The ecdysone-dependent activation of reporter constructs driven by these sequences showed a significant time lag and was coupled with a marked sensitivity to low concentrations of cycloheximide. The data obtained strongly suggest that the cis-acting elements delimited within the hsp23-gene 1 intergenic region respond to ecdysone in a secondary manner, presumably by requiring interaction with the product(s) of primary ecdysone-responsive genes.