Identification of kJHRE in BmKr-h1. Reporter assays with progressive deletion and mutation constructs were used to identify the JHRE. NIAS-Bm-aff3 cells were cotransfected with pGL4.14 reporter plasmids carrying the indicated promoter regions conjugated to firefly luciferase and a reference reporter plasmid carrying Renilla luciferase. The cells were treated with 10 μM methoprene (JHA) for 24 h, and reporter activities were measured by using the Dual-Luciferase reporter assay system. The activities of firefly luciferase were normalized against those of Renilla luciferase in the same samples. Data represent means ± SD (n = 3). Means with the same letter are not significantly different (Tukey–Kramer test, P < 0.05). Some data were analyzed using Student’s t test (***P < 0.001; NS, P > 0.05). (A) Reporter plasmids containing the 5′-flanking and first intron regions of BmKr-h1α were assayed. The structure of BmKr-h1 is shown at the top. Numbers indicate the distance from the transcription start site (+1), and white and shaded boxes represent the untranslated and coding regions of exons, respectively. Reporter activities of progressive deletion constructs are shown below. (B) The insert in the plasmid used in A, the −2165 to +116 region, was reduced progressively from −49 toward −2079, and the effects were measured by reporter assays. (C) Schematic representation of the location of kJHRE (−2165 to −2025). The nucleotide sequence is shown below the gene structure. Boxed letters are indispensable sequences (kJHREc). (D) The functionality of kJHREc was assayed with mutations causing a triplet transversion (Left) or transition (Right) in the −2082 to −2065 region of the kJHRE reporter (−2165 to −2025 and −49 to +116, pGL4.14). (E) The JH response of a reporter carrying three tandem copies of kJHREc was examined.

Functional analysis of BmMet2 and kJHRE in mammalian HEK293 cells. HEK293 cells were treated as described below, and reporter activity was examined by using the Dual-Luciferase reporter assay system. Data represent means ± SD (n = 3). Means with the same letter are not significantly different (Tukey–Kramer test, P < 0.05). Some data were analyzed using Student’s t test (***P < 0.001; NS, P > 0.05). (A) Cells were cotransfected with a UAS reporter plasmid carrying firefly luciferase, a reference reporter plasmid (pRL-TK) carrying Renilla luciferase, and an expression plasmid carrying GAL4DBD fused with BmMet1, BmMet2, or VP16AD and were treated with 10 μM methoprene (JHA) for 24 h. (B) Cells were cotransfected with the UAS reporter plasmid and the expression plasmid carrying GAL4DBD fused with BmMet2 and were treated with the indicated concentrations of JH, JHA, MF, or FA for 24 h. (C) Cells were cotransfected with a kJHRE-reporter plasmid and an expression plasmid carrying native BmMet2 or BmSRC, and were treated with 0.1 μM JH I for 24 h. (D) Cells were cotransfected with a kJHRE-reporter plasmid, BmMet2 expression plasmid, and an expression plasmid carrying BmMet1, BmUSP, BmARNT, BmHIF-1α, or BmTimeless. (E) Cells were cotransfected with the UAS-reporter plasmid and an expression plasmid carrying GAL4DBD or VP16AD fused with BmMet2 or BmSRC and were treated with 0.1 μM JH I for 24 h. (F) A model for JH-mediated transcriptional induction of BmKr-h1. The physical interaction between kJHRE and BmMet2/BmSRC complex remains to be determined.

Regulation of BmKr-h1 expression by JH in B. mori larvae and NIAS-Bm-aff3 cells. BmKr-h1 expression levels were determined by qPCR. Data represent means ± SD (n = 3 except in A, where n = 2). Means with the same letter are not significantly different (Tukey–Kramer test, P < 0.05). (A) Developmental expression profiles of BmKr-h1 in the epidermis. (Inset) Expression levels of BmKr-h1α and BmKr-h1β in fourth-instar larvae at day 0. Roman and Arabic numerals under the horizontal axis indicate the instar and days in the instar, respectively. HCS, head capsule slippage; SP, spinning. (B) Effects of allatectomy (CAX) and methoprene (JHA) treatment on BmKr-h1 expression. Fourth-instar larvae at day 0 were treated with either JHA (1 μg) or acetone (JHA−) 3 h after allatectomy. Twelve hours later the epidermis was dissected, and BmKr-h1 expression was measured. (C) NIAS-Bm-aff3 cells were treated with 10 μM JHA, and temporal changes in BmKr-h1 expression were monitored. (Inset) Expression levels of BmKr-h1α and BmKr-h1β in cells treated with JHA for 0 and 12 h. (D) NIAS-Bm-aff3 cells were treated with different concentrations of JH (JH I, JH II, and JH III), JHA, MF, or FA, and the relative expression levels of BmKr-h1 were determined after 2 h. (E) Untreated cells (Ctrl) or cells precultured in a medium with 50 μM CHX or solvent only (DMSO, 3% vol/vol) for 1 h were treated with 1 μM JHA or solvent for 2 h, and the relative expression levels of BmKr-h1 were determined.

Predicted kJHREc of Kr-h1 in other insect species. (A) Genomic structures and putative kJHREc positions are shown: Am, A. mellifera; Ap, A. pisum; Bm, B. mori; Dm, D. melanogaster; Nv, N. vitripennis; Tc, T. castaneum. White boxes, black boxes, and arrows represent the UTRs, ORFs, and transcription start sites, respectively. Ellipses indicate putative kJHREc, and the numbers below represent distances from the transcription start site (BmKr-h1, TcKr-h1, AmKr-h1, and DmKr-h1) or translation start site (NvKr-h1 and ApKr-h1). The position marked with an asterisk in AmKr-h1 represents a gap in the available genomic information. (B) Alignment of the putative kJHREc sequences in the 5′-flanking region of Kr-h1.