DIRECT REPEATS BIND THE ECR USP RECEPTOR AND MEDIATE ECDYSTEROID RESPONSES IN DROSOPHILA-MELANOGASTER/

The steroid hormone 20-hydroxyecdysone plays a key role in the inducti on and modulation of morphogenetic events throughout Drosophila develo pment. Previous studies have shown that a heterodimeric nuclear recept or composed of the EcR and USP proteins mediates the action of the hor mone at the transcriptional level through binding to palindromic ecdys teroid 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 heterodime r can bind in vitro with various affinities to direct repetitions of t he motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which ar e known to be target sites for vertebrate nuclear receptors. At varian ce with these receptors, EcR/USP was also found to bind to a DRO direc t repeat with no intervening nucleotide. In cell transfection assays, direct repeats DRO to DR5 alone can render the minimum viral tk or Dro sophila Fbp1 promoter responsive to 20-hydroxyedysone, as does the pal indromic hsp27 EcRE. In a transgenic assay, however, neither the palin dromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 min imal promoter responsive to premetamorphic ecdysteroid peaks. In contr ast, DRO and DR3 elements, when substituted for the natural palindromi c EcRE in the context of the Fbp1 enhancer, can drive a strong fat bod y-specific ecdysteroid response in transgenic animals. These results d emonstrate that directly repeated EcR/USP binding sites are as effecti ve as palindromic EcREs in vivo. They also provide evidence that addit ional flanking regulatory sequences are crucially required to potentia te the hormonal response mediated by both types of elements and specif y its spatial and temporal pattern.