The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKrh1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loophelix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JHdependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JHmediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/ BmSRC complex activates BmKr-h1 by interacting with kJHRE. development | insecticide | steroid receptor coactivator

SUMMARYIn insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis-and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.The molting and metamorphosis of insects are intricately regulated by the actions and interactions of ecdysteroids and juvenile hormone (JH).2 In holometabolous insects, 20-hydroxyecdysone (20E, the primary active ecdysteroid) induces the larval-larval molt in the presence of JH. When the JH titer declines to a trace level in the final larval instar, 20E induces larval-pupal and pupal-adult molts (metamorphosis). Thus, JH plays a key role in preventing larvae from undergoing precocious metamorphosis (1).Our understanding of the molecular mechanism of JH-mediated repression of insect metamorphosis has significantly advanced (2): JH carried to a target cell is received by a JH receptor, methoprene tolerant (Met) (3-6); JH-liganded Met interacts with steroid receptor coactivator (7-12); the JH/Met/ steroid receptor coactivator complex activates Krüppel homolog 1 (Kr-h1), a repressor of metamorphosis, by interacting with a JH response element (kJHRE) in the Kr-h1 gene (9, 10, 12-15); Kr-h1 represses the larval-pupal metamorphosis (16 -20). To date, however, the mechanism of the repression of metamorphosis by Kr-h1, including target gene(s), binding site(s), and partner(s), has remained unknown.The Broad-Complex (BR-C) protein is a transcription factor that is composed of a Bric-a-brac/Tramtrack/Broad-Complex (BTB) domain and an alternatively spliced zinc finger domain (Z1-Z6) (21-25). BR-C expression is induced by 20E, ...

Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval-pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysoneinduced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93. Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.H olometabolous insects undergo a complete metamorphosis, which consists of egg, larval, pupal, and adult stages. Larvalpupal and pupal-adult metamorphoses are coordinated by the actions of ecdysteroids and juvenile hormone (JH) (1). In the presence of JH, 20-hydroxyecdysone (20E; the active metabolite of ecdysteroids) induces larval-larval molting, whereas, in the absence of JH, it induces larval-pupal and pupal-adult molts (1). Thus, the major function of JH is to prevent immature larvae from precociously transitioning to pupae and adults (1).The molecular action of 20E in target cells during the larvalpupal transition was initially proposed in the 1970s (2), and its molecular mechanisms were well characterized by later studies. In particular, the 20E-liganded ecdysone receptor (EcR)/ultraspiracle (USP) complex directly activates the expression of a few early ecdysone-inducible transcription factors, which then regulate a large number of late ecdysone-inducible genes involved in pupal formation (3-5). The molecular mechanism of JH signaling has been clarified more recently (6-8). The JH receptor methoprene tolerant (Met) was found to bind JH (9-12) and subsequently interact with steroid receptor coactivator (SRC; also known as FISC and Taiman) (13-15). The JH/Met/SRC complex then activates Krüppel homolog 1 (Kr-h1) (15, 16), which plays a key role in the repression of metamorphosis (17-20).The transcription factor Broa...