20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

Abstract

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing.Animals undergo developmental transitions from the embryo to juvenile to adulthood, and these processes are determined by steroid hormones and their corresponding nuclear receptors (NRs).2 In insects, 20-hydroxyecdysone (20E; ecdysone is the immediate precursor of 20E; 20E and ecdysone are the main ecdysteroids) is the actual steroid hormone. The ecdysone receptor (EcR) and its partner molecule, Ultraspiracle (USP), form the functional NR complex of 20E. In conjunction with EcR-USP, 20E activates a small set of early response genes encoding several transcription factors that further activate a large set of downstream late response genes. Pulses of 20E signals initiate major developmental transitions in insects, including egg hatching, larval-larval molting, and larval-pupal-adult metamorphosis (1, 2). NRs form a large and conserved superfamily of ligand-activated transcription factors that are essential for growth, development, reproduction, homeostasis, and metabolism. NRs are defined by the presence of a highly conserved DNA binding domain (DBD) and a less conserved ligand binding domain (3, 4). There are 18 -19 NRs in insects, including the fruit fly, Drosophila melanogaster, and the silkworm, Bombyx mori (3,5,6). Apart from the EcR, ligand was only identified for another insect NR, ecdysone-induced protein 75B (E75). E75 is a crucial 20E response gene that affects ecdysteroid titer. E75 binds to heme, which responds to gases . The E75 orthologs in mammals are Rev-erb ␣ (NR1D1) and Rev-erb ␤ (NR1D2), and NR1D2 binds to heme, responds to NO, and regulates circadian rhythm (13-15).In Drosophila, the E75 locus encodes four E75 mRNA isoforms, E75A, E75B, E75C, and E75D, which are generated by differential promoter usage and alternative splicing of 5Ј exons. The DBD of E75A/C possesses two C4 zinc fingers; E75B is incomplete and contains only one zinc finger, whereas E75D lacks a DBD. 20E-...

20‐Hydroxyecdysone‐upregulated proteases involved in Bombyx larval fat body destruction

“…20E triggers the cascades of its downstream signaling pathway by the heterodimer receptors EcR–USP, and the interference of the 20E receptor BmEcR or Bmusp leads to the disruption of the 20E signalling and downregulation of 20E primary response gene expression in Bombyx , thus further arresting larval–pupal metamorphosis (Tian et al ., ; Li et al ., ). After Bmusp RNAi for 24 h, the protein levels of both basic and phosphorylated BmUSP were dramatically reduced (Fig.…”

Juvenile hormone signaling – a mini review

“…Thus, Kr-h1 is regarded as an anti-metamorphic factor that mimics JH action (Jindra et al, 2013;Jindra et al, 2015a). Kr-h1 directly mediates transcriptional suppression via binding to the Kr-h1 binding site (KBS) on the promoters of several 20E primary-response genes, such as Br, E93 and E75 (Li et al, 2015;Liu et al, 2015b;Li et al, 2016;Liu et al, 2018), partially explaining the sophisticated JH-20E cross-talk (Fig. 1).…”

Transcriptomic analysis at the first instar larval stage of nonmolting Bombyx mori mutant (a42)

“…Overexpression or deletion of BmJHE ( juvenile hormone esterase ), the primary enzyme responsible for JH‐specific degradation, resulted in shortened or extended larval stages, respectively (Tan, Tanaka, Tamura, & Shiotsuki, 2005; Z. Zhang et al, 2017). Ecdysone‐induced protein 75B ( E75 ) encodes a nuclear receptor for ecdysone and is an important 20E response gene (Li et al, 2015, 2016). RNA interference (RNAi) knockdown of E75 corresponds with decreases in the synthesis of 20E, and CRISPR/Cas9 knockout larvae exhibit a significant reduction in the wandering stage and have high ecdysteroid titers.…”

“…RNA interference (RNAi) knockdown of E75 corresponds with decreases in the synthesis of 20E, and CRISPR/Cas9 knockout larvae exhibit a significant reduction in the wandering stage and have high ecdysteroid titers. Correspondingly, overexpression of E75A/C (two messenger RNA [mRNA] isoforms of E75 ) promotes 20E signaling and accelerates metamorphosis (Li et al, 2016).…”

“…Zhang et al, 2017). Ecdysone-induced protein 75B (E75) encodes a nuclear receptor for ecdysone and is an important 20E response gene (Li et al, , 2016. RNA interference (RNAi) knockdown of E75 corresponds with decreases in the synthesis of 20E, and CRISPR/Cas9 knockout larvae exhibit a significant reduction in the wandering stage and have high ecdysteroid titers.…”