A putative direct repeat element plays a dual role in the induction and repression of insect vitellogenin-1 gene expression

Abstract

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.

Keywords: 20-Hydroxyecdysone; Cockroaches; Direct repeat; Hormone response elements; Luciferase reporter assay; Promoter region; Vitellogenin.