INTRODUCTION
A Bombyx mori homologue of the Broad-Complex gene ( BmBR-C ) is composed of 13 exons, and is transcribed from two promoters at a distance of about 101 kbp (Fig. 1; Nishita and Takiya, 2006). The transcriptional activity of the distal promoter, Pdist, but not the proximal promoter, Pprox, is activated by ecdysone (Nishita and Takiya, 2009). Sequence analysis revealed that a candidate ecdy- sone responsive element (referred to as the cEcRE), which is highly homologous to the canonical EcRE, exists on the Pdist at -2,976 bp from the distal transcription start site (TSS). However, removal of the cEcRE did not affect the responsiveness of the Pdist to ecdysone. A sequential deletion mutant of Pdist indicated that two regions, EcRE- D  and  EcRE-P,  -4,950 bp  and  -3,480 bp  upstream  from the distal TSS, respectively, are important in the respon- siveness of the Pdist to 20-hydroxyecdysone (20E; Nishita, 2014). Ecdysone activates "'ecdysone responsive'' genes through binding to a nuclear receptor heterodimer comprising an ecdysone receptor (EcR; Koelle et al. , 1991) and an ultra- spiracle (Usp; Yao et al. , 1992, BmCF1; Swevers et al., 1996). The presence of ecdysone responsive elements bound by an EcR/Usp (EcRE) have been observed on a variety of promoters, including those on Drosophila hsp27, Fbp1, Eip28/29, Eip40, and Lsp-2 genes (Andres and Cherbas, 1992; Antoniewski et al. , 1995). Together, these data allowed the consensus EcRE binding site for EcR/ Usp to be determined as 5’-(A/G) G (G/T) T C A N T G A (C/A) C (C/T)-3’ (Antoniewski et al., 1993; Cherbas et al., 1991). No significant sequence similarities have been noted between the canonical EcRE and the EcREs on the distal promoter of BmBR-C, however, both EcRE-D and -P can sequence-specifically bind Bombyx mori protein(s) (Nishita, 2014). Interestingly, those bound proteins may not include EcR/Usp, implying that BmBR-C transcription can be indirectly activated by ecdysone through the EcRE-P. Meanwhile, the mid-region of EcRE-D, EcRE- Db, might bind a functional Bombyx ecdysone receptor that activates the expression of BmBR-C from Pdist. Ac- cordingly, it has been demonstrated that the expression of BR-C can be induced not only by juvenile hormone (JH; Zhou and Riddiford, 2002; Konopova and Jindra, 2008; Minakuchi et al., 2011), but also directly by ecdysone. Together, these results experimentally validate at a molec- ular level the fact that BmBR-C is classified as an ecdy- sone-induced gene. In this study, we demonstrated that mutations of bases that are conserved between the canonical EcRE and the EcRE-D of BmBR-C Pdist reduced the binding activity of EcR/Usp to EcRE-D, and led to a functional decline in ecdysone responsive transcription from BmBR-C Pdist.
MATERIALS AND METHODS
Preparation of nuclear extracts
Nuclear extracts of BM-N cells cultured in TC-100 (Sig- ma) supplemented with 10% fetal bovine serum ± 1 μM 20E (Sigma) were prepared as previously described (Dig- nam et al., 1983). Briefly, exponentially growing BM-N cells (1.5 × 10 ) were collected by centrifugation at 2,000 g for 1 min at 4°C. The pellet was washed with ice cold PBS, and then suspended in ice cold buffer A [10 mM HEPES-KOH  pH 7.9,  1.5 mM  MgCl²,  10 mM  KCl, 0.5 mM  dithiothreitol  (DTT),  0.4 µM  phenylmethylsulfo- nyl fluoride (PMSF)], allowed to swell on ice for 20 min, homogenized, and centrifuged. The resulting nuclear pel- let was resuspended in ice cold buffer C [20 mM HEPES- KOH  pH 7.9,  420 mM  NaCl,  1.5 mM  MgCl,,  0.2 mM EDTA pH 8.0, 25% glycerol, 0.5 mM DTT, 0.4 μM PMSF], and shaken at 4°C for 20 min. The extract was dialyzed  against  buffer  D  [20 mM  HEPES-KOH  pH 7.9, 20 mM KCl, 2 mM MgCl,, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.4 μM PMSF]. Aliquots of the nuclear ex- tract were stored at −80°C until use.
Electrophoretic mobility shift assay (EMSA)
The oligonucleotide sequences used for electrophoretic mobility shift assays are shown in Figs. 1A, 2A, and 3A. The annealed oligonucleotides used as the probes were la- beled with biotin using the DIG Gel Shift Kit, 2 nd Genera- tion (Roche). For the chemiluminescence-detected gel shifts, binding reactions were performed as described by Yao et al. (1992) with slight modifications. Nuclear ex- tracts were incubated with binding buffer (100 mM KCl, 7.5% glycerol, 20 mM HEPES pH 7.5, 2 mM DTT, and 0.1% NP-40) on ice for 20 min in the presence of 1 µg of nonspecific competitor poly [d(I-C)] or other oligonucle- otide competitors as indicated in the text. The labeled probe (15.4 fmol) was added to the reaction and incubated at room temperature for 20 min. An anti-ecdysone recep- tor antibody (Abnova, MAB9070; Immunogen: native Manduca ecdysone receptor B1; Nishita, 2014) was added 10 min into the incubation. Then, the reaction was loaded into a 5% non-denaturing polyacrylamide gel in 0.5% TBE running buffer. After electrophoresis, the gel was blotted onto a positively charged Nylon membrane (Hy- bond N+; Amersham Biosciences). Membranes were de- veloped using the DIG Gel Shift Kit, 2 nd Generation (Roche) according to the manufacturer's protocol.
Preparation of promoter-containing reporter plas-
mids
To construct luciferase reporter plasmids driven by the distal promoter of BmBR-C harboring a single base muta- tion within EcRE-D, we used PCR to amplify sequences from a PGV-BmBR-C Pdist (-5k) luciferase reporter plas- mid containing a -5,124 to +52 bp region of the BmBR-C distal promoter (Nishita and Takiya, 2009). Reporter plas- mids were amplified using a combination of one of the oligonucleotides shown in Fig. 4A as a forward primer and the reverse primer, 5’- CCCAAGGTTACAAATTC ATAGCTCCATTAGAATG-3’.
Transfections and luciferase reporter assays
BM-N cells were seeded in 24-well plates the day be- fore transfection at a density of 1.25 × 10 5 cells in TC-100 supplemented with 10% fetal bovine serum at 25°C. The cells were transiently transfected using FuGENE HD Transfection Reagent (Promega) with 0.5 μg of the test plasmid and 0.05 μg of the control plasmid, pRL-BmA3, which was used to normalize for transfection efficiency. This control plasmid (Nishita and Takiya, 2009) contained the 128 bp Bombyx mori A3 gene promoter (Mangé et al., 1997) upstream of the Renilla luciferase reporter gene in the pRL-null vector (Promega). After transfection, the cells were cultured ± 1 µM 20E (Sigma). After 48 h in culture, the cells were harvested and as- sayed for luciferase activity using the Dual-Luciferase As- say System (Promega) according to the manufacturer's instructions with a Luminescencer-PSN (AB-2200; ATTO). The firefly luciferase activity of the reporter plasmid was normalized to the Renilla luciferase activity of the control plasmid. The results were expressed as the normalized firefly luciferase activity ± 20E. Data represent the mean ± 95% confidence intervals (CI; n = 6).
results and discussion
Identification of a key EcR/Usp binding element
within EcRE-D on BmBR-C Pdist.
Previous work suggests that the mid-region of EcRE-D, termed EcRE-Db, might be bound by a functional Bom- byx ecdysone receptor and contributes to the induction of BmBR-C expression from Pdist (Nishita, 2014). To eluci- date the key functional elements of EcRE-Db (Fig. 1), four overlapping oligonucleotides (EcRE-Dbs1-4), shown in Figure 2A were synthesized, DIG-labeled, and used as probes in electrophoretic mobility shift assays (EMSAs). Each probe was incubated with nuclear extracts obtained from cultures of the Bombyx mori cell line, BM-N. As shown  in  Figure 2B,  an  appreciable  band  representing  a DNA-protein complex was observed only when EcRE- Dbs3 was used as a probe (Lane 11). Since the addition of a 50- or 100-fold excess of unlabeled probe successful- ly inhibited probe-protein binding (Lanes 12 and 13), the bound factor(s) appeared to interact in a sequence-specific manner with the EcRE-Dbs3 probe. Furthermore, the bind- ing of the EcRE-Dbs3 probe to the factor(s) was com- Isoform specific pletely inhibited by the addition of the Drosophila hsp27 EcRE oligonucleotide (Riddihough and Pelham, 1987). These results demonstrated that EcR/Usp may interact with the EcRE-Dbs3 sequence within EcRE-D of the BmBR-C Pdist. To obtain additional evidence that the protein(s) that bound to the EcRE-Dbs3 represented EcR/ Usp, we tested the ability of an anti-EcR antibody (Abno- va, MAB9070) to interfere with EcRE-Dbs3 probe-protein complex formation. We noted that the signal intensity re- sulting from the EcRE-Dbs3 probe-protein(s) complex (arrowhead) decreased in the presence of increasing con- centrations of the anti-EcR antibody (Fig. 2C). These observations suggested that an EcR formed part of the protein-probe complex, and that 5’- TGGAGCGGT CATTCACACCGTG-3’, a sequence within EcRE-Dbs3 containing an element exhibiting weak similarity to the canonical EcRE (underlined), was the target of EcR/Usp binding. Shirai et al. (2012) advocated that a 14 bp con- sensus motif is conserved among EcREs for three Bombyx mori nuclear receptor genes ( Bm-EcRB1, BmE75A, BHR3B ). However, the EcRE-Db3 EcR/Usp binding sequence bears a higher sequence similarity to the canonical EcRE than does the 14 bp consensus motif (Fig. 2A).
Importance of the residue at position -2 of cE-
cRE for EcR/Usp binding.
Visual inspection indicated improved matching of ho- mologous sequences at 2,976 bp upstream from the distal transcription start site (TSS) within BmBR-C Pdist, re- ferred to as the candidate EcRE (cEcRE; Fig. 1), which differed from the canonical EcRE by only a single base pair (Nishita and Takiya, 2009). A comparison of the ca- nonical EcRE sequence with the cEcRE showed that the cEcRE has one mutation at the -2 position within the Usp-binding site (Fig. 3A; Grad et al., 2001). It has been suggested that as an EcR/Usp heterodimer binding to EcRE, Usp may rigidly recognize a specific DNA se- quence, while EcR alone may be more promiscuous in its binding (Devarakonda et al., 2003). To test the impor- tance of the nucleotide at the -2 position within the Usp- binding site, an oligonucleotide containing an A to C base substitution at t the -2 position of the cEcRE was synthe- sized and used as a probe for EMSA (Fig. 3B; Lanes 6–10). Binding of the cEcRE -2 (A→C) probe to the un- defined factor(s) was observed (Lane 6); this binding was completely inhibited by the addition of either a 50- or 100-fold excess of the Drosophila hsp27 EcRE oligonu- cleotide (Lanes 9–10) or unlabeled probe (Lanes 7–8). To confirm that the protein(s) bound to the cEcRE -2 (A→C) probe represented EcR/Usp, we tested the ability of an anti-EcR antibody to interfere with the probe-protein com- plex formation (Nishita, 2014). We noted that the signal intensity resulting from the cEcRE -2 (A→C) probe-pro- tein complex (arrowhead) decreased in response to in- creasing concentrations of the anti-EcR antibody (Fig. 3C). Although the results strongly suggested that EcR/Usp could bind to EcRE-Dbs3, the latter's sequence similarity to the canonical EcRE (Fig. 2A) was less than that ob- served for the cEcRE that did not bind to EcR/Usp (Figs. 3A and B; Lanes 1–5). Compared to the canonical EcRE sequence, the cEcRE has one mutation in the Usp- binding site, but EcRE-Dbs3 is not mutated at this site (compare Figs. 2A  and  3A).  These  results  demonstrated that the C residue located at -2 position within an Usp- binding site may be one of the most important residues for the binding of EcR/Usp to the EcRE-D of BmBR-C Pdist.
Sequence specificity of EcR/Usp binding to the
EcRE-D
Although the sequence similarity was weak, a known binding site for EcR/Usp was discovered within EcRE- A psition No. canonical EcRE BmBR-C Pdist EcRE-Db
14-bp consensus B Dbs3 (Fig. 2B). To further dissect the exact sequence re- sponsible for EcR/Usp binding, we synthesized a series of EcRE-Dbs3 oligonucleotides differing by a single nucleo- tide mutation from the canonical EcRE within the putative EcR/Usp binding element (Fig. 4A). Then, we tested the ability of these oligonucleotides to compete EcR/Usp- probe complex formation as detected by EMSA. As shown in Figure 4B, the addition of excess amounts of the cold EcR/Usp-probe or Drosophila hsp27 EcRE abolished the shifted band representing the DNA-protein complex (Lanes 2–5). Oligonucleotides containing a mutation at either po- sition -3 or -1 within the Usp-binding half site were also effective competitors of complex formation (Lanes 10, 11, 14, and 15). By contrast, oligonucleotides containing a A
B C mutation at either position -5, -4, or -2 (with the most striking effects observed at -4 or -2) did not abrogate DNA-protein complex formation (Lanes 6, 7, 8, 9, 12, and 13). In addition, an oligonucleotide bearing a muta- tion at +3 within the EcR-binding half site effectively ab- rogated probe-protein complex formation (Fig. 4C; Lanes 8, and 9). However, oligonucleotides containing mutations at positions +1, +4, or +6 (with the weakest effect ob- served at +4) did not affect complex formation (Lanes 6, 7, 10, 11, 12, and 13). Together, these results suggested that the conserved res- idues between EcRE-Dbs3 and the canonical EcRE are essential for EcR/Usp binding. In particular, the G and C residues at -4, -2 and +4 were critically essential for EcR/ Usp binding to EcRE-Dbs3. The sequence corresponding to the canonical EcRE within EcRE-D is essential for BmBR-C Pdist re- sponsiveness to 20E To confirm the effect of the mutations in the key se- quence within EcRE-Dbs3 on the responsiveness of BmBR-C Pdist to ecdysone, single base mutations corre- sponding to the nucleotides shown in Figure 4A were in- troduced into the luciferase reporter plasmid PGV-BmBR-C Pdist -5k (Nishita and Takiya, 2009), in which luciferase expression is driven by a 5 kb region of the distal promot- er of BmBR-C. Changes in 20E responsiveness were mea- sured by a luciferase assay (Fig. 5). Reporter plasmids were transiently transfected into BM-N cells. After 48 h in culture ± 1 µ 20E, luciferase activity was measured. In accord with the results shown in Fig. 4B, point mutations at -5, -4, or -2 within the Usp-binding half site or at +1, +4, or +6 within the EcR-binding half site of EcRE-D re- duced 20E responsiveness to almost the same level as a reporter plasmid bearing a 75 bp deletion of the EcRE-D sequence (ΔEcRE-D). In general, a good correlation was observed between DNA binding affinity and transactivation levels. Together, the results identified a sequence, 5'- TGGAGCGGTCAT TCACACCGTG-3', found in the mid-region of EcRE-D (termed EcRE-Dbs3) on the BmBR-C distal promoter that bears a weak sequence similarity to the canonical EcRE (underlined). Furthermore, this identified sequence sup- ported the binding of EcR/Usp. Conserved residues be- tween the EcREs, at positions -5, -4, -2, +1, +4, and +6, were important for EcR/Usp binding. In particular, the G and C residues positioned at -4, -2 and +4 were critically essential for both EcR/Usp binding to EcRE-D and the subsequent ecdysone-dependent transcriptional activation of BmBR-C from Pdist. BR-C has long been known as a gene induced by ecdysone (Kiss et al. , 1988), although the molecular mechanism of this induction, and especially the location of the EcRE, has been unclear. It has been suggested that as an EcR/Usp heterodimer binding to EcRE, Usp may rigidly recognize a specific DNA se- quence [5’-TC (A/T)-3’; Devarakonda et al., 2003, Grad et al., 2001], while EcR may be more promiscuous in its binding. DNA sequence diversity in the EcR-binding half site of EcRE-D (-Dbs3) may make it difficult to identify the EcRE bound by EcR/Usp on the BmBR-C promoter by homology to the canonical EcRE sequence.