Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster

Abstract

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.