Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein

The E74 early ecdysone-inducible gene plays a key role in the regulatory hierarchy activated by ecdysone at the onset of Drosophila metamorphosis. We show here that E74A protein binds to three adjacent sites in the middle of the E74 gene. The consensus sequence for E74A protein binding, determined by random-sequence oligonucleotide selection, contains an invariant purine-rich core sequence, C/AGGAA. This sequence is also present in the binding sites of two mammalian proteins that, like E74A, are related to the ets oncoprotein. Antibody staining of larval salivary gland polytene chromosomes revealed that E74A protein binds to both early and late ecdysone-inducible puffs. This study supports Ashburner's proposal that the early puffs encode site-specific DNA binding proteins that directly interact with the early and late ecdysone-inducible puffs.     

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

The 82F late puff contains the L82 gene, an essential member of a novel gene family.

Metamorphosis in Drosophila results from a hierarchy of ecdysone-induced gene expression initiated at the end of the third larval instar. A now classical model of this hierarchy was proposed based on observations of the activity of polytene chromosome "puffs" which distinguished "early" puffs as those directly induced by ecdysone and "late" puffs as those which become active as a secondary response to the hormone. We report here the isolation and characterization of the L82 gene corresponding to the extensively characterized late puff at 82F. L82 is a complex gene that spans at least 50 kb of genomic DNA, produces at least seven different nested mRNAs, and has homology to a novel gene family. In contrast to most previously characterized puff genes, the broad developmental expression pattern of L82 suggests that it is controlled by both ecdysone-dependent and ecdysone-independent regulatory mechanisms. L82 mutations were identified by transgene rescue of developmental delay and eclosion lethal phenotypes.     

Ecdysone-regulated puff genes 2000.

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.