Figure 1.
Genomic structure of three nuclear receptor genes in Bombyx mori.
Exon and intron structures for (A) BmEcR-A and –B1, (B) BmE75-A and (C) BHR3-B are shown on each chromosomal location. The filled box is coding sequence (CDS); shaded box, 5′ untranslated region (UTR); striped box, 3′-UTR. The EcR/USP binding sites (EcRE or E1 for BmEcR-B1) and E2 for BmEcR-B1 identified in this study are shown by filled and blank triangles, respectively.
Figure 2.
Dose response to 20E of each promoter region for three nuclear receptor genes.
Each promoter activity for the full length plasmid of BmEcR-B1 (A, pBmEcR-B1_−3652/+248), BmE75-A (B, pBmE75-A_−5023/+85), and BHR3-B (C, pBHR3-B_−2891/+18)) under 2.0×10−4(white bar), 2.0×10−2 (grey bar) and 2.0 µg/mL (filled bar) of 20E in aff3 cells is shown at each incubation time (h). The ratio of relative luciferase activities with and without 20E (fold induction by 20E) is shown (reference 1.0 at 96 h without 20E). Error bars represent standard error (SE) (N = 4).
Figure 3.
Identification of the ecdysone responsive region in the BmEcR-B1 promoter.
(A, B) Each promoter activity for 5′-deletion series of constructs for the BmEcR-B1 promoter region (A) and A5 mutation series of constructs for BmEcR-B1 −2850 to −2800 (B) is shown, respectively. (C) BmEcRB1-E1 (15 bp, from −2835 to −2830) and BmEcRB1-E2 (20 bp, from −2825 to −2805) were identified as essential for the 20E response elements. (D, E) Each promoter activity for the 2 bp-mutated (A: C and T: G transversion) series of constructs for BmEcRB1-E1 (−2844 to −2831) (D) and for BmEcRB1-E2 (−2826 to −2817) (E) is shown. Each plasmid construct was transfected into aff3 cells and incubated 48 h with 2.0 µg/mL (4.2 µM) of 20E and measured the luciferase activity. The ratio of relative luciferase activities with and without 20E (fold induction by 20E) is shown on the right, as referred 1.0 at 48 h without 20E. Error bar represents SE (N = 4). “null” indicates the pGL4.10 vector.
Figure 4.
Responses of EcR-B1 reporters to 20E in M1 and Sf9 cells.
M1 and Sf9 cells were transfected with representative EcR-B1 reporter plasmids (see Fig. 3), treated with 1 µM of 20E during 3 days, and a dual reporter assay was conducted. −3652/+248, the full length reporter construct; mu02, an E1 region mutant; mu05, a mutant within the internal region between E1 and E2; mu06, an E2 region mutant. Bar represent SE (N = 4).
Figure 5.
Identification of EcREs for BmE75-A and BHR3-B.
(A) Each promoter activity of the A5 mutation series of constructs for BmE75-A −350 to −310 is shown. (B) The sequence of BmE75A-EcRE (20 bp). (C) Each promoter activity of the excision series of constructs for BHR3-B −485 to −285 is shown. Each excision size (white region) is 20 bp. (D) The sequence of BHR3B-EcRE (20 bp). (A, C) The fold induction by 20E of each construct is shown on the right. Error bars represent SE (N = 4).
Figure 6.
Electrophoretic mobility shift analysis for BmEcRB1-E1 and E2.
(A, B) Competition assay with cold probes for BmEcRB1-E1 (A) and for BmEcRB1-E2 (B). Mutation sites in E1 and E2 probe sequence “N” (Normal) are shown in gray region of “M” (Mutant). Two hundred (A) or 50 (B) femtomoles of 32P-probe were incubated with 5 µg (A) and 10 µg (B) of cell extracts and loaded onto the gel. 20E (6 h) represents extracts from cells cultured under 20E (2.0 µg/mL). ×1, ×10, ×50 and ×100 represent the ratio of the cold probe amount to the 32P-probe amount. Filled arrows show the shifted bands and blank arrows show the free probes. (C, D) Super shift assay with the anti-V5 or/and anti-USP antibody for E1 (C) and for E2 (D). Intact: intact cell extracts. anti-V5 AB: anti-V5 antibody. anti-USP AB: anti-USP antibody. AT, B1T, and USPT represent extracts from cells that overexpressed EcRA, EcRB1, and USP, respectively.
Figure 7.
Structural conservation of E1 and E2 structure of EcR-B1 EcRE in Lepidoptera.
(A) Schematic localization of E1 (red triangle) and E2 (green triangle) of EcR-B1 EcRE in seven Lepidoptera (upper section). +1, transcriptional start site. Shaded box, EcR-B1 gene. Sequence comparison of each element is shown in lower section. B. m, Bombyx mori; H.s, Helicoperva armigera; S. f, Spodoptera frugiperda; Bi.a, Bicyclus anynana; D. p, Danaus plexippus; M. s, Manduca sexta; P. x, Papilio xuthus. E1 (red, nucleotide sequence same to the silkworm E1 element) and E2 (green, nucleotide sequence same to the silkworm E2 element) for lepidopteran EcR-B1 and their surrounding sequences are aligned. *, consensus nucleotide among seven lepidopteran sequences. E-box in E2 sequence is underlined. (B) The alignment of the EcR/USP heterodimer binding site for ecsysone-inducible nuclear receptor genes, EcR-B1, E75-A and BHR3-B. Classical EcRE of Drosophila and 14 bp consensus motif among three nuclear receptors found in this study are show in the top and bottom, respectively. The red character shows conserved nucleotide in three elements.
Figure 8.
The genome-wide distribution of 14 bp EcRE consensus motif in the silkworm.
(A) The location of the genomic sequences identical to 64 patterns of 14 bp consensus motif (5′-KCRGGTCWWCGMWC-3′, see Fig. 7B). The arrows show the location of 138 motifs in each linkage group of the silkworm. The chromosomal locations of 12 motifs are not certified. Each grey bar shows the scaffold composed of each chromosome. The number located left side of grey bars represents the scaffold number. The white regions show the gap between scaffolds. Underlined numbers on the right of grey bars represent the location for 20E activated genes (more than 4 fold induction by 20E) identified by microarray analysis. 3 and 3-2 are located near the same 14 bp consensus motif. (B) The 20E-dependent activation of predicted-genes surrounding 14 bp consensus motifs analyzed by microarray. The relative expression patterns of 18 genes in aff3 cells treated with 20E for 0, 3, 12, 24 h are shown, based on the spot intensities for the predicted genes in the silkworm 44K cDNA microarray. Detailed information for 18 genes and 17 motifs is summarized in Table S6.