Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene

Abstract

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, ...

Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval–adult metamorphosis

“…The pupal specifier BR-C is differentially regulated by Kr-h1 in holometabolous insects; it is induced in response to JHmediated Kr-h1 expression at the prepupal and pupal stages, whereas Kr-h1 represses BR-C during the larval stage (17,18,24,25,28,36). The transcription factor CSL [CBF1/Su(H)/Lag-1] recruits a corepressor complex in the absence of Notch signaling to repress its target genes (51); however, Notch signaling converts the CSL repressor complex to an activator complex, resulting in transcriptional activation of the target genes (51).…”

“…In Lepidoptera, JH was shown to repress 20E-dependent induction of BR-C in the larval epidermis (26,27). We have found that Kr-h1 directly binds to a site within the BR-C promoter and represses 20E-dependent BR-C activation (28). Through these molecular actions, JH prevents immature larvae from undergoing precocious larval-pupal transition.…”

“…A previous study demonstrated that BmKrh1 directly binds to a Kr-h1 binding site (KBS) in the BmBR-C promoter and represses 20E-dependent BmBR-C transcription (28). Thus, we hypothesized that BmKr-h1 is also involved in the JH-mediated repression of BmE93A.…”

Molecular Mechanisms of Larval Color Pattern Switch in the Swallowtail Butterfly

“…In a simplified model, a lepidopteran progresses through a larvato-larva molt when JH is present and a larva-to-pupa metamorphosis when JH is absent at the final molting stage. It has been hypothesized that JH modulates the action of ecdysteroid-molting hormones, but the detailed mechanisms of the modulation are still unclear (Jindra et al 2013;Urena et al 2014;Kayukawa et al 2016). There is some evidence that JH has an effect on larval pigmentation.…”

Something “hairy” in juvenile hormone signaling for mosquito reproduction

“…One obvious candidate is the zinc-finger protein Kr-h1, which plays a critical role as a JHinduced repressor of insect metamorphosis (11,14). Interestingly, a recently published paper shows that Kr-h1 indeed acts via direct DNA binding to repress transcription of the Broad-Complex gene (15), which is absolutely necessary to initiate metamorphosis (larva-to-pupa transition) in holometabolous insects (11). Whether the transcriptional repressor Kr-h1 also regulates genes important for the mosquito PE development remains to be investigated.…”