Gene 546 (2014) 129–134
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3 Hanfu Xu a, Dangjun Deng a,b, Lin Yuan a, Yuancheng Wang a, Feng Wang a, Qingyou Xia a,⁎ a b
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China The Institute of Forensic Science, Chongqing Public Security Bureau, Chongqing 401147, China
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 15 May 2014 Accepted 17 May 2014 Available online 20 May 2014 Keywords: Enhancer Bmlp3 promoter 30 K protein Fat body Silkworm
a b s t r a c t 30 K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30 K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5′ upstream sequences to a luciferase reporter gene. The results indicated that the sequences from −150 to −250 bp and −597 to −675 bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between −230 and −250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. © 2014 Published by Elsevier B.V.
1. Introduction The fat body of the holometabolous insect Bombyx mori (silkworm) is a relatively large organ that is functionally similar to the vertebrate liver (Thomson, 1975). As a central storage center for nutrition and energy, the fat body synthesizes large amounts of proteins including lipoproteins, storage proteins and vitellogenins and secretes them into the hemolymph in a time-dependent manner during the life cycle of B. mori (Sakai et al., 1988; Wyatt and Pan, 1978). Among the major plasma proteins is a group of structurally related proteins with molecular weights around 30 kDa, termed B. mori low molecular lipoproteins (Bmlps or “30 K proteins”). 30 K proteins are synthesized largely in the fat body and play multiple roles in the growth and development of B. mori, including inhibiting apoptosis (Kim et al., 2001), defending against fungal infection (Ujita et al., 2005), translocating chymotrypsin inhibitor (Ueno et al., 2006), transporting lipid and/or sugar (Yang et al., 2011), improving sialylation of recombinant proteins (Wang
Abbreviations: Bmlp3, Bombyx mori low molecular lipoprotein gene 3; 30 K proteins, proteins with molecular weights around 30 kDa; JH, juvenile hormone; BmE, Bombyx mori embryonic cell line; Spli-221, Spodoptera litura embryonic cell line; FBS, fetal bovine serum; JH III, juvenile hormone III; 20E, 20-hydroxyecdysone; PCR, polymerase chain reaction; SV40, Simian virus 40; EMSA, electrophoretic mobility shift assay; HEPES, 4-(2hydroxyerhyl) piperazine-1-erhaesulfonic acid; DTT, DL-dithiothreitol; JHA, juvenile hormone analog; Luc, luciferase gene. ⁎ Corresponding author. E-mail address: [email protected] (Q. Xia).
http://dx.doi.org/10.1016/j.gene.2014.05.038 0378-1119/© 2014 Published by Elsevier B.V.
et al., 2011), and enhancing cellular uptake and stability of enzymes (Lee et al., 2014). Interestingly, changes in the level of 30 K protein mRNA in the fat body reflect changes in the hemolymph concentration of 30 K proteins, indicating that transcription is the major mode of 30 K protein gene regulation. B. mori hemolymph proteins have proven to be good models for studying the developmental regulation of gene expression (Izumi et al., 1981; Mori et al., 1991a, 1991b). The availability of the complete genome sequence of B. mori provides a unique opportunity to identify genes encoding 30 K proteins and determining how they are developmentally regulated. So far, a total of 24 genes encoding typical 30 K proteins have been identified (Bmlp1–24) (Zhang et al., 2012a, 2012b). Most of the 30 K protein genes share similar genome organizations, as well as high similarity in their amino acid sequences (Mori et al., 1991a, 1991b; Sun et al., 2007). Notable, several genes such as Bmlp3 and Bmlp7, are synthesized largely in the fat body in a developmentally regulated manner (Deng et al., 2013; Zhang et al., 2012a, 2012b). The biosynthesis of 30 K proteins appears to be repressed by juvenile hormone (JH) and appears to be the result of reduced transcription initiation (Bosquet et al., 1989; Izumi et al., 1984; Ogawa et al., 2005). These data suggest that there might be DNA elements responsive to JH in the upstream sequences of 30 K protein genes. Little is known about the promoters of 30 K protein genes and their associated enhancers. In a previous study, we cloned a 1.1-kb promoter region of Bmlp3 and demonstrated its ability to direct fat body-specific expression of DsRed in transgenic silkworms (Deng et al., 2013). To further characterize the promoter of Bmlp3, a series of promoter-containing
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Table 1 The sequence of the primers used to clone Bmlp3 promoter constructs. Promoter construct
Primer sequence (5′’–3′’)
Amplicon length
pGL3-975 pGL3-675 pGL3-597 pGL3-453 pGL3-374 pGL3-310 pGL3-250 pGL3-150 pGL3-100 pGL3-50
Forward: TCCcccgggGGAAACATCAAGCTAAGACCATT (Sma I) Forward: TCCcccgggGGAGCCGCGCGGCGCGCTACCAT (Sma I) Forward: TCCcccgggGGAGGTGTATCGCGTCCCGAACC (Sma I) Forward: TCCcccgggGGATCGGCCGGGCGTCCTTGAGT (Sma I) Forward: TCCcccgggGGAGTATAGTTACAACAGCTGCCCC (Sma I) Forward: GGggtaccCCAGGCAGGGTGGTGGTACCTA (Kpn I) Forward: GGggtaccCCAAATATACCTTTCACATGGC (Kpn I) Forward: GGggtaccCCTTGATAATTTCACTCAATCA (Kpn I) Forward: GGggtaccCCTTTCATTTGACATTTGAACT (Kpn I) Forward: GGggtaccCCAATTCAAAACAATTTCGAGT (Kpn I) Reverse: CCGctcgagCGGCTGTGTGTTACCCAAGAACG (Xho I)
999 bp 699 bp 621 bp 477 bp 398 bp 334 bp 274 bp 174 bp 124 bp 74 bp
The restriction sites are shown in lowercase and the name of the restriction enzyme is followed the primer sequence.
DNA fragments of differing lengths were attached to a luciferase reporter gene and the levels of reporter-gene expression were measured. Our results indicate the presence of a 21-bp sequence located between − 230 and − 250 bp upstream of the Bmlp3 transcription start site with the ability to increase luciferase gene expression in BmE cells. 2. Materials and methods 2.1. Cell culture and chemicals The B. mori embryonic cell line BmE and the Spodoptera litura embryonic cell line Spli-221, were maintained at 27 °C in Grace's medium plus 10% fetal bovine serum (FBS, Hyclone, China), supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin. The insect hormones, juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) (Sigma, USA), were used to evaluate the effects of hormones on promoter activity.
2.2. Generation of the Bmlp3 promoter deletion constructs Bmlp3 promoter constructs containing varying amounts of 5′ upstream sequences were generated as described below. First, PCR was performed using the corresponding forward and reverse primers (Table 1) and either a plasmid containing 1.1-kb of the Bmlp3 promoter or B. mori (P50) genomic DNA as a template. Second, the PCR products were gel-purified and cloned into the vector pMD19-T simple (TaKaRa Biotechnology (Dalian), China). Third, the promoter-containing fragments were excised from pMD19-T simple vectors using restriction enzymes and inserted into compatible sites of the luciferase reporter gene-containing vector pGL3-Basic (Promega, USA), to create a series of promoter deletion constructs. 2.3. Transient transfections and Dual-luciferase assays Transient gene expression assays were carried out in BmE and Spli221 cells using previously described methods (Deng et al., 2013).
Fig. 1. Activity of 10 different promoter regions of the Bmlp3 gene. (A) Different fragments of the Bmlp3 promoter region that includes the first exon. The numbers to the left end of the construct diagrams indicate the end of each deletion. The exon (from +1 to +24) and luciferase gene (Luc) are indicated by a shared black box and open box, respectively. All the reporter constructs were transfected into BmE cells (B) and Spli-221 cells (C) and the luciferase activities were measured. Relative firefly luciferase activities were averages of three independent transfections normalized to Renilla luciferase control activities.
H. Xu et al. / Gene 546 (2014) 129–134
Briefly, a 100 μL mixture containing 1 μg luciferase reporter plasmid DNA, 0.1 μg internal control plasmid DNA expressing Renilla luciferase (pRL-SV40, Promega, USA), and 3 μL LipofectAMINE 2000 (Invitrogen, USA) were mixed with BmE and Spli-221 cells, and the transfected cells were harvested after 24 h. Luciferase activities was measured using the Dual-luciferase reporter assay system (Promega, USA) and Modulus™ single tube multimode reader (Turner BioSystems, USA) according to the manufacturer's protocol. All results were averages of at least three independent experiments and reported as mean ± standard error.
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2.4. Electrophoretic mobility shift assay (EMSA) Nuclear protein extracts of fat body (day-7 fifth instar larvae), BmE cells and Spli-221 cells were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Complementary doublestrand DNA probes were produced from chemically synthesized oligonucleotides (Sangon Biotech, China). The oligonucleotides were 5′labeled with the fluorescent cyanine dye Cy3 and purified by native polyacrylamide gel electrophoresis. The labeled oligonucleotides and unlabeled reverse complementary sequence were then annealed to
Fig. 2. Binding of nuclear proteins to the fragments of the Bmlp3 promoter. (A) Locations of probes 1, 2 and 3 in the Bmlp3 promoter and nucleotide sequences of the probes. (B) EMSA assays showed specific binding of the fat body nuclear proteins to probe 2. (C) EMSA assays confirmed the binding of nuclear proteins to probe 2. Nuclear proteins were isolated from the fat body of B. mori, Spli-221 cells and BmE cells, respectively.
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produce double-strand probes. The unlabeled probes used in competition experiments were synthesized using the same procedure. The nonspecific probe was a 54-bp random oligonucleotide. EMSA analysis was performed using previously described methods (Zhang et al., 2004). Briefly, 6 μg nuclear protein extract was incubated with 150 pmol DNA probes in a buffer containing 10 mM HEPES (pH 8.0), 40 mM KCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol. To avoid non-specific protein binding, 5 mg/mL heparin was added to the mixtures and incubated for 30 min at room temperature. For competition experiments, unlabeled probes were added to the reaction mixtures and incubated for 10 min at room temperature before the addition of Cy3-labeled probes. Finally, the reaction mixtures were fractionated by electrophoresis in a 5% native polyacrylamide for 2 h at 200 V, 4 °C and visualized with a TYPHON scanner (Amersham, USA).
were treated with 0, 0.1, 1.0 or 10.0 μM exogenous JH III and 20E, however, no apparent differences were observed in luciferase expression (Fig. S1), suggesting that hormones had no significant effects on transcriptional activity of the 1.1-kb Bmlp3 promoter in BmE cells. It has been reported that cultured silkworm fat body cells can respond to juvenile hormone analogs (JHA) by turning off the expression of 30 K protein genes (Ogawa et al., 2005). We speculated that this might be due to the lack of specific factors in BmE cells that are necessary for hormone regulation of 30 K protein genes or alternatively that the binding sites for JH III and 20E are not present in the 1.1-kb Bmlp3 promoter. Thus, the mechanism by which Bmlp3 is hormonally regulated remains to be investigated.
3. Results and discussion
To determine which regions of the Bmlp3 promoter were essential for activity, we constructed a series of luciferase reporter plasmids containing different Bmlp3 promoter truncations along with the 24-bp first exon of Bmlp3. The constructs designated as pGL3-975–pGL3-50 (Fig. 1A), were transfected into BmE and Spli-221 cells, transiently expressed and promoter activity was assessed by measuring luciferase activity. As shown in Fig. 1B and C, all constructs, when introduced into both cell types, were capable of expressing luciferase compared with the control plasmid pGL3-Basic. The luciferase activity from pGL3-675 was about 2.8- and 3.5-fold higher than that from pGL3-975 in BmE and Spli-221 cells respectively, suggesting that the sequences between −675 and −975 might contain negative regulatory elements. Progressive deletion of the Bmlp3 promoter revealed that removing the
3.1. Effects of insect hormones on the Bmlp3 promoter activity In silkworms, hormones and especially 20E and JH mainly function as regulators of ecdysis and metamorphosis (Soin et al., 2008; Tang et al., 2003). Previous studies indicated that there were putative ecdysone- and JH-response elements within 1.1-kb of the Bmlp3 promoter, and that the biosynthesis of 30 K proteins could be repressed directly or indirectly by JH (Deng et al., 2013; Izumi et al., 1984; Ogawa et al., 2005). Therefore, we investigated further whether insect hormones were involved in the regulation of Bmlp3 promoter activity. After BmE cells were transfected with pGL3-374 plasmid DNA they
3.2. Characterization of the Bmlp3 promoter
Fig. 3. Binding of nuclear proteins to core sequences of the Bmlp3 promoter. (A) Locations of probes 2-1, 2-2 and 2-3 in the Bmlp3 promoter and nucleotide sequences of the probes. (B) EMSA assays showed obvious binding of fat body nuclear proteins to probe 2-1. Nuclear proteins were isolated from the fat body of B. mori and BmE cells, respectively.
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sequences between −597 to −675 and −150 to −250 had significant effects on luciferase expression, indicating that cis-regulatory elements positively regulating the expression of Bmlp3 were present in these regions. Together, the results indicate that the promoter of Bmlp3 spanned from −1 to −675 from the start of transcription and that sequences between − 597 to − 675 and −150 to −250 contain cis-regulatory elements that played important roles in maintaining transcription. We next focused on the identification of the cis-regulatory elements in these two regions.
unique 4- or 5-basepair polymorphisms, M1, M2 and M3 (Fig. 4A), to form DNA–protein complexes in EMSA assays. The results show that mutations in the 21-bp core sequence could reduce the binding of fat body nuclear proteins (Fig. 4B). Taken together, these data show that the 21-bp core sequence at position − 230 to − 250, was capable of binding fat body nuclear proteins and may be involved in mediating transcriptional activity of Bmlp3.
3.3. Identification of cis-regulatory elements in the Bmlp3 promoter
To further test the function of the 21-bp core sequence in Bmlp3 expression, we attached the 21-bp sequence and its variants M1, M2, and M3 to Bmlp3 promoter sequences −50 to +24 in the luciferase reporter plasmid pGL3-Basic (designated as pGL3-C21-50, pGL3-C21M1-50, pGL3-C21M2-50, and pGL3-C21M3-50, respectively) (Fig. 5A). The plasmids were transfected into BmE cells and as shown by luciferase assays (Fig. 5B), resulted in 2.8-fold more luciferase activity compared to identical experiments using plasmid pGL3-50. Mutations in the 21-bp core sequence could abolish or partially reduce its activity, which was consistent with the EMSA results shown in Fig. 4. These data indicate that the 21-bp sequence led to a significant enhancement of promoter activity. In other words, the 21-bp core sequence appears to function as a cisregulatory element necessary for transcriptional activity of the Bmlp3 promoter.
To precisely identify the potential cis-regulatory elements within the regions −597 to −675 and −150 to −250 of the Bmlp3 promoter, mobility shift assays were performed using three probes (Fig. 2A), probe 1 (− 598/− 675), 2 (− 196/− 250) and 3 (− 151/− 205). As shown in Fig. 2B, both probes 1 and 3 did not interact with nuclear proteins isolated from the fat body of B. mori, whereas probe 2 formed complexes with the nuclear proteins isolated from the fat body of B. mori and Spli-221 cells. Mobility shift assays performed in the presence of an unlabeled competitor probe confirmed the specific binding of nuclear proteins to probe 2 (Fig. 2C). To further define the core sequences specific for binding nuclear proteins in the −196/−250 fragment, three shorter probes (Fig. 3A), probe 2-1 (− 230/− 250), 2-2 (− 213/− 234) and 2-3 (−196/−217), were synthesized and used in mobility shift assays. As revealed in Fig. 3B, DNA–protein complexes were clearly detected after incubating probe 2-1 (− 230/−250) with nuclear extracts from the fat body of B. mori, and the formation of the complexes was effectively inhibited by adding unlabeled probes to the reaction mixture. Furthermore, the binding specificity of the − 230/− 250 sequence was investigated by testing the ability of three variants, each containing a
Fig. 4. Mutational analysis of the 21-bp core sequence of the Bmlp3 promoter. (A) The 21-bp core sequences of the wild type and mutated oligonucleotides M1, M2 and M3. The mutated nucleotides are underlined. (B) EMSA assays showed that mutations partly abolished the binding of fat body nuclear proteins to the 21-bp core sequence. Nuclear proteins were isolated from the fat body of B. mori.
3.4. Functional analysis of the 21-bp core sequence of the Bmlp3 promoter
Fig. 5. Functional analysis of the 21-bp core sequence of the Bmlp3 promoter. (A) The diagram of the pGL3-50, pGL3-C21-50, pGL3-C21M1-50, pGL3-C21M2-50, and pGL3-C21M3-50 constructs. The 21-bp core sequence and its variants M1, M2 and M3 were inserted into the upstream region of pGL3-50 vector. The first exon of Bmlp3 (from + 1 to +24) and luciferase gene (Luc) are indicated by a shared black box and open box, respectively. (B) The constructs were transfected into BmE cells and luciferase activities were measured. Relative firefly luciferase activities were averages of three independent transfections normalized to Renilla luciferase control activities (*p b 0.05, **p b 0.01, ***p b 0.001).
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4. Conclusions In this study, the promoter of a fat body-specific gene encoding a member of the 30 K protein family, Bmlp3, was characterized by creating various luciferase reporter gene constructs with differing amounts of 5′ promoter-proximal sequences. A 21-bp core sequence located between − 230 and − 250 upstream of transcription start site of Bmlp3 was identified using the EMSA method. We demonstrated that the 21-bp sequence can bind specifically with the nuclear proteins from the fat body of B. mori and increase luciferase gene expression resulting in 2.8-fold more luciferase activity in BmE cells. These data indicate that the 21-bp sequence is an enhancer-like element responsible for maintaining the transcriptional activity of the Bmlp3 promoter. This study provides new insights into the function and developmental regulation of Bmlp3. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.038. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Professor Qili Feng of South China Normal University, China, for providing the Spodoptera litura embryonic cell line Spli-221. We are grateful to Professor David O'Brochta of the University of Maryland, USA, and Huawei He of Southwest University, China, for improving this manuscript. This work was supported by the National Basic Research Program of China (2012CB114600), and Grant (XDJK2014B014) from the Fundamental Research Funds for the Central Universities. References Bosquet, G., et al., 1989. The regulation of major haemolymph protein synthesis: changes in mRNA content during the development of Bombyx mori larvae. Insect Biochem. 19, 29–39. Deng, D.J., et al., 2013. The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, Bombyx mori. Transgenic Res. 22 (5), 1055–1063.
Izumi, S., et al., 1981. Molecular properties and biosynthesis of major plasma proteins in Bombyx mori. Biochim. Biophys. Acta 670, 222–229. Izumi, S., Kiguchi, K., Tomino, S., 1984. Hormonal regulation of biosynthesis of major plasma proteins in Bombyx mori. Zool. Sci. 1, 223–228. Kim, E.J., Rhee, W.J., Park, T.H., 2001. Isolation and characterization of an apoptosisinhibiting component from the hemolymph of Bombyx mori. Biochem. Biophys. Res. Commun. 285 (2), 224–228. Lee, H.J., et al., 2014. Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles. Biomaterials 35 (5), 1696–1704. Mori, S., Izumi, S., Tomino, S., 1991a. Complete nucleotide sequences of major plasma protein genes of Bombyx mori. Biochim. Biophys. Acta 1090, 129–132. Mori, S., Izumi, S., Tomino, S., 1991b. Structures and organization of major plasma protein genes of the silkworm Bombyx mori. J. Mol. Biol. 218 (1), 7–12. Ogawa, N., et al., 2005. The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 35 (3), 217–229. Sakai, N., et al., 1988. Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori. Biochim. Biophys. Acta 949 (2), 224–232. Soin, T., et al., 2008. Juvenile hormone analogs do not affect directly the activity of the ecdysteroid receptor complex in insect culture cell lines. J. Insect Physiol. 54 (2), 429–438. Sun, Q., et al., 2007. Analysis of the structure and expression of the 30 K protein genes in silkworm, Bombyx mori. Insect Sci. 14, 5–14. Tang, S.M., et al., 2003. Functional analysis of the larval serum protein gene promoter from silkworm, Bombyx mori. Chin. Sci. Bull. 48 (23), 2611–2615. Thomson, J.A., 1975. Major patterns of gene activity during development in holometabolous insects. Adv. Insect Physiol. 11, 321–398. Ueno, Y., et al., 2006. Silkworm midgut proteins interacting with a hemolymph protease inhibitor, CI-8. Biosci. Biotechnol. Biochem. 70 (7), 1557–1563. Ujita, M., et al., 2005. Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection. Biosci. Biotechnol. Biochem. 69 (6), 1178–1185. Wang, Z.S., et al., 2011. Enhancement of recombinant human EPO production and sialylation in Chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression. Biotechnol. Bioeng. 108 (7), 1634–1642. Wyatt, G.R., Pan, M.L., 1978. Insect plasma proteins. Annu. Rev. Biochem. 47, 779–817. Yang, J.P., et al., 2011. Crystal structure of the 30 K protein from the silkworm Bombyx mori reveals a new member of the β-trefoil superfamily. J. Struct. Biol. 175 (1), 97–103. Zhang, T.Y., et al., 2004. Identification of a POU factor involved in regulating the neuronspecific expression of the gene encoding diapause hormone and pheromone biosynthesis-activating neuropeptide in Bombyx mori. Biochem. J. 380 (Pt 1), 255–263. Zhang, Y., et al., 2012a. Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family. Funct. Integr. Genomics 12 (4), 705–715. Zhang, Y., et al., 2012b. The synthesis, transportation and degradation of BmLP3 and BmLP7, two highly homologous Bombyx mori 30 K proteins. Insect Biochem. Mol. Biol. 42 (11), 827–834.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3 Hanfu Xu a, Dangjun Deng a,b, Lin Yuan a, Yuancheng Wang a, Feng Wang a, Qingyou Xia a,⁎ a b
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China The Institute of Forensic Science, Chongqing Public Security Bureau, Chongqing 401147, China
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 15 May 2014 Accepted 17 May 2014 Available online 20 May 2014 Keywords: Enhancer Bmlp3 promoter 30 K protein Fat body Silkworm
a b s t r a c t 30 K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30 K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5′ upstream sequences to a luciferase reporter gene. The results indicated that the sequences from −150 to −250 bp and −597 to −675 bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between −230 and −250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. © 2014 Published by Elsevier B.V.
1. Introduction The fat body of the holometabolous insect Bombyx mori (silkworm) is a relatively large organ that is functionally similar to the vertebrate liver (Thomson, 1975). As a central storage center for nutrition and energy, the fat body synthesizes large amounts of proteins including lipoproteins, storage proteins and vitellogenins and secretes them into the hemolymph in a time-dependent manner during the life cycle of B. mori (Sakai et al., 1988; Wyatt and Pan, 1978). Among the major plasma proteins is a group of structurally related proteins with molecular weights around 30 kDa, termed B. mori low molecular lipoproteins (Bmlps or “30 K proteins”). 30 K proteins are synthesized largely in the fat body and play multiple roles in the growth and development of B. mori, including inhibiting apoptosis (Kim et al., 2001), defending against fungal infection (Ujita et al., 2005), translocating chymotrypsin inhibitor (Ueno et al., 2006), transporting lipid and/or sugar (Yang et al., 2011), improving sialylation of recombinant proteins (Wang
Abbreviations: Bmlp3, Bombyx mori low molecular lipoprotein gene 3; 30 K proteins, proteins with molecular weights around 30 kDa; JH, juvenile hormone; BmE, Bombyx mori embryonic cell line; Spli-221, Spodoptera litura embryonic cell line; FBS, fetal bovine serum; JH III, juvenile hormone III; 20E, 20-hydroxyecdysone; PCR, polymerase chain reaction; SV40, Simian virus 40; EMSA, electrophoretic mobility shift assay; HEPES, 4-(2hydroxyerhyl) piperazine-1-erhaesulfonic acid; DTT, DL-dithiothreitol; JHA, juvenile hormone analog; Luc, luciferase gene. ⁎ Corresponding author. E-mail address: [email protected] (Q. Xia).
http://dx.doi.org/10.1016/j.gene.2014.05.038 0378-1119/© 2014 Published by Elsevier B.V.
et al., 2011), and enhancing cellular uptake and stability of enzymes (Lee et al., 2014). Interestingly, changes in the level of 30 K protein mRNA in the fat body reflect changes in the hemolymph concentration of 30 K proteins, indicating that transcription is the major mode of 30 K protein gene regulation. B. mori hemolymph proteins have proven to be good models for studying the developmental regulation of gene expression (Izumi et al., 1981; Mori et al., 1991a, 1991b). The availability of the complete genome sequence of B. mori provides a unique opportunity to identify genes encoding 30 K proteins and determining how they are developmentally regulated. So far, a total of 24 genes encoding typical 30 K proteins have been identified (Bmlp1–24) (Zhang et al., 2012a, 2012b). Most of the 30 K protein genes share similar genome organizations, as well as high similarity in their amino acid sequences (Mori et al., 1991a, 1991b; Sun et al., 2007). Notable, several genes such as Bmlp3 and Bmlp7, are synthesized largely in the fat body in a developmentally regulated manner (Deng et al., 2013; Zhang et al., 2012a, 2012b). The biosynthesis of 30 K proteins appears to be repressed by juvenile hormone (JH) and appears to be the result of reduced transcription initiation (Bosquet et al., 1989; Izumi et al., 1984; Ogawa et al., 2005). These data suggest that there might be DNA elements responsive to JH in the upstream sequences of 30 K protein genes. Little is known about the promoters of 30 K protein genes and their associated enhancers. In a previous study, we cloned a 1.1-kb promoter region of Bmlp3 and demonstrated its ability to direct fat body-specific expression of DsRed in transgenic silkworms (Deng et al., 2013). To further characterize the promoter of Bmlp3, a series of promoter-containing
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Table 1 The sequence of the primers used to clone Bmlp3 promoter constructs. Promoter construct
Primer sequence (5′’–3′’)
Amplicon length
pGL3-975 pGL3-675 pGL3-597 pGL3-453 pGL3-374 pGL3-310 pGL3-250 pGL3-150 pGL3-100 pGL3-50
Forward: TCCcccgggGGAAACATCAAGCTAAGACCATT (Sma I) Forward: TCCcccgggGGAGCCGCGCGGCGCGCTACCAT (Sma I) Forward: TCCcccgggGGAGGTGTATCGCGTCCCGAACC (Sma I) Forward: TCCcccgggGGATCGGCCGGGCGTCCTTGAGT (Sma I) Forward: TCCcccgggGGAGTATAGTTACAACAGCTGCCCC (Sma I) Forward: GGggtaccCCAGGCAGGGTGGTGGTACCTA (Kpn I) Forward: GGggtaccCCAAATATACCTTTCACATGGC (Kpn I) Forward: GGggtaccCCTTGATAATTTCACTCAATCA (Kpn I) Forward: GGggtaccCCTTTCATTTGACATTTGAACT (Kpn I) Forward: GGggtaccCCAATTCAAAACAATTTCGAGT (Kpn I) Reverse: CCGctcgagCGGCTGTGTGTTACCCAAGAACG (Xho I)
999 bp 699 bp 621 bp 477 bp 398 bp 334 bp 274 bp 174 bp 124 bp 74 bp
The restriction sites are shown in lowercase and the name of the restriction enzyme is followed the primer sequence.
DNA fragments of differing lengths were attached to a luciferase reporter gene and the levels of reporter-gene expression were measured. Our results indicate the presence of a 21-bp sequence located between − 230 and − 250 bp upstream of the Bmlp3 transcription start site with the ability to increase luciferase gene expression in BmE cells. 2. Materials and methods 2.1. Cell culture and chemicals The B. mori embryonic cell line BmE and the Spodoptera litura embryonic cell line Spli-221, were maintained at 27 °C in Grace's medium plus 10% fetal bovine serum (FBS, Hyclone, China), supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin. The insect hormones, juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) (Sigma, USA), were used to evaluate the effects of hormones on promoter activity.
2.2. Generation of the Bmlp3 promoter deletion constructs Bmlp3 promoter constructs containing varying amounts of 5′ upstream sequences were generated as described below. First, PCR was performed using the corresponding forward and reverse primers (Table 1) and either a plasmid containing 1.1-kb of the Bmlp3 promoter or B. mori (P50) genomic DNA as a template. Second, the PCR products were gel-purified and cloned into the vector pMD19-T simple (TaKaRa Biotechnology (Dalian), China). Third, the promoter-containing fragments were excised from pMD19-T simple vectors using restriction enzymes and inserted into compatible sites of the luciferase reporter gene-containing vector pGL3-Basic (Promega, USA), to create a series of promoter deletion constructs. 2.3. Transient transfections and Dual-luciferase assays Transient gene expression assays were carried out in BmE and Spli221 cells using previously described methods (Deng et al., 2013).
Fig. 1. Activity of 10 different promoter regions of the Bmlp3 gene. (A) Different fragments of the Bmlp3 promoter region that includes the first exon. The numbers to the left end of the construct diagrams indicate the end of each deletion. The exon (from +1 to +24) and luciferase gene (Luc) are indicated by a shared black box and open box, respectively. All the reporter constructs were transfected into BmE cells (B) and Spli-221 cells (C) and the luciferase activities were measured. Relative firefly luciferase activities were averages of three independent transfections normalized to Renilla luciferase control activities.
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Briefly, a 100 μL mixture containing 1 μg luciferase reporter plasmid DNA, 0.1 μg internal control plasmid DNA expressing Renilla luciferase (pRL-SV40, Promega, USA), and 3 μL LipofectAMINE 2000 (Invitrogen, USA) were mixed with BmE and Spli-221 cells, and the transfected cells were harvested after 24 h. Luciferase activities was measured using the Dual-luciferase reporter assay system (Promega, USA) and Modulus™ single tube multimode reader (Turner BioSystems, USA) according to the manufacturer's protocol. All results were averages of at least three independent experiments and reported as mean ± standard error.
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2.4. Electrophoretic mobility shift assay (EMSA) Nuclear protein extracts of fat body (day-7 fifth instar larvae), BmE cells and Spli-221 cells were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Complementary doublestrand DNA probes were produced from chemically synthesized oligonucleotides (Sangon Biotech, China). The oligonucleotides were 5′labeled with the fluorescent cyanine dye Cy3 and purified by native polyacrylamide gel electrophoresis. The labeled oligonucleotides and unlabeled reverse complementary sequence were then annealed to
Fig. 2. Binding of nuclear proteins to the fragments of the Bmlp3 promoter. (A) Locations of probes 1, 2 and 3 in the Bmlp3 promoter and nucleotide sequences of the probes. (B) EMSA assays showed specific binding of the fat body nuclear proteins to probe 2. (C) EMSA assays confirmed the binding of nuclear proteins to probe 2. Nuclear proteins were isolated from the fat body of B. mori, Spli-221 cells and BmE cells, respectively.
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produce double-strand probes. The unlabeled probes used in competition experiments were synthesized using the same procedure. The nonspecific probe was a 54-bp random oligonucleotide. EMSA analysis was performed using previously described methods (Zhang et al., 2004). Briefly, 6 μg nuclear protein extract was incubated with 150 pmol DNA probes in a buffer containing 10 mM HEPES (pH 8.0), 40 mM KCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol. To avoid non-specific protein binding, 5 mg/mL heparin was added to the mixtures and incubated for 30 min at room temperature. For competition experiments, unlabeled probes were added to the reaction mixtures and incubated for 10 min at room temperature before the addition of Cy3-labeled probes. Finally, the reaction mixtures were fractionated by electrophoresis in a 5% native polyacrylamide for 2 h at 200 V, 4 °C and visualized with a TYPHON scanner (Amersham, USA).
were treated with 0, 0.1, 1.0 or 10.0 μM exogenous JH III and 20E, however, no apparent differences were observed in luciferase expression (Fig. S1), suggesting that hormones had no significant effects on transcriptional activity of the 1.1-kb Bmlp3 promoter in BmE cells. It has been reported that cultured silkworm fat body cells can respond to juvenile hormone analogs (JHA) by turning off the expression of 30 K protein genes (Ogawa et al., 2005). We speculated that this might be due to the lack of specific factors in BmE cells that are necessary for hormone regulation of 30 K protein genes or alternatively that the binding sites for JH III and 20E are not present in the 1.1-kb Bmlp3 promoter. Thus, the mechanism by which Bmlp3 is hormonally regulated remains to be investigated.
3. Results and discussion
To determine which regions of the Bmlp3 promoter were essential for activity, we constructed a series of luciferase reporter plasmids containing different Bmlp3 promoter truncations along with the 24-bp first exon of Bmlp3. The constructs designated as pGL3-975–pGL3-50 (Fig. 1A), were transfected into BmE and Spli-221 cells, transiently expressed and promoter activity was assessed by measuring luciferase activity. As shown in Fig. 1B and C, all constructs, when introduced into both cell types, were capable of expressing luciferase compared with the control plasmid pGL3-Basic. The luciferase activity from pGL3-675 was about 2.8- and 3.5-fold higher than that from pGL3-975 in BmE and Spli-221 cells respectively, suggesting that the sequences between −675 and −975 might contain negative regulatory elements. Progressive deletion of the Bmlp3 promoter revealed that removing the
3.1. Effects of insect hormones on the Bmlp3 promoter activity In silkworms, hormones and especially 20E and JH mainly function as regulators of ecdysis and metamorphosis (Soin et al., 2008; Tang et al., 2003). Previous studies indicated that there were putative ecdysone- and JH-response elements within 1.1-kb of the Bmlp3 promoter, and that the biosynthesis of 30 K proteins could be repressed directly or indirectly by JH (Deng et al., 2013; Izumi et al., 1984; Ogawa et al., 2005). Therefore, we investigated further whether insect hormones were involved in the regulation of Bmlp3 promoter activity. After BmE cells were transfected with pGL3-374 plasmid DNA they
3.2. Characterization of the Bmlp3 promoter
Fig. 3. Binding of nuclear proteins to core sequences of the Bmlp3 promoter. (A) Locations of probes 2-1, 2-2 and 2-3 in the Bmlp3 promoter and nucleotide sequences of the probes. (B) EMSA assays showed obvious binding of fat body nuclear proteins to probe 2-1. Nuclear proteins were isolated from the fat body of B. mori and BmE cells, respectively.
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sequences between −597 to −675 and −150 to −250 had significant effects on luciferase expression, indicating that cis-regulatory elements positively regulating the expression of Bmlp3 were present in these regions. Together, the results indicate that the promoter of Bmlp3 spanned from −1 to −675 from the start of transcription and that sequences between − 597 to − 675 and −150 to −250 contain cis-regulatory elements that played important roles in maintaining transcription. We next focused on the identification of the cis-regulatory elements in these two regions.
unique 4- or 5-basepair polymorphisms, M1, M2 and M3 (Fig. 4A), to form DNA–protein complexes in EMSA assays. The results show that mutations in the 21-bp core sequence could reduce the binding of fat body nuclear proteins (Fig. 4B). Taken together, these data show that the 21-bp core sequence at position − 230 to − 250, was capable of binding fat body nuclear proteins and may be involved in mediating transcriptional activity of Bmlp3.
3.3. Identification of cis-regulatory elements in the Bmlp3 promoter
To further test the function of the 21-bp core sequence in Bmlp3 expression, we attached the 21-bp sequence and its variants M1, M2, and M3 to Bmlp3 promoter sequences −50 to +24 in the luciferase reporter plasmid pGL3-Basic (designated as pGL3-C21-50, pGL3-C21M1-50, pGL3-C21M2-50, and pGL3-C21M3-50, respectively) (Fig. 5A). The plasmids were transfected into BmE cells and as shown by luciferase assays (Fig. 5B), resulted in 2.8-fold more luciferase activity compared to identical experiments using plasmid pGL3-50. Mutations in the 21-bp core sequence could abolish or partially reduce its activity, which was consistent with the EMSA results shown in Fig. 4. These data indicate that the 21-bp sequence led to a significant enhancement of promoter activity. In other words, the 21-bp core sequence appears to function as a cisregulatory element necessary for transcriptional activity of the Bmlp3 promoter.
To precisely identify the potential cis-regulatory elements within the regions −597 to −675 and −150 to −250 of the Bmlp3 promoter, mobility shift assays were performed using three probes (Fig. 2A), probe 1 (− 598/− 675), 2 (− 196/− 250) and 3 (− 151/− 205). As shown in Fig. 2B, both probes 1 and 3 did not interact with nuclear proteins isolated from the fat body of B. mori, whereas probe 2 formed complexes with the nuclear proteins isolated from the fat body of B. mori and Spli-221 cells. Mobility shift assays performed in the presence of an unlabeled competitor probe confirmed the specific binding of nuclear proteins to probe 2 (Fig. 2C). To further define the core sequences specific for binding nuclear proteins in the −196/−250 fragment, three shorter probes (Fig. 3A), probe 2-1 (− 230/− 250), 2-2 (− 213/− 234) and 2-3 (−196/−217), were synthesized and used in mobility shift assays. As revealed in Fig. 3B, DNA–protein complexes were clearly detected after incubating probe 2-1 (− 230/−250) with nuclear extracts from the fat body of B. mori, and the formation of the complexes was effectively inhibited by adding unlabeled probes to the reaction mixture. Furthermore, the binding specificity of the − 230/− 250 sequence was investigated by testing the ability of three variants, each containing a
Fig. 4. Mutational analysis of the 21-bp core sequence of the Bmlp3 promoter. (A) The 21-bp core sequences of the wild type and mutated oligonucleotides M1, M2 and M3. The mutated nucleotides are underlined. (B) EMSA assays showed that mutations partly abolished the binding of fat body nuclear proteins to the 21-bp core sequence. Nuclear proteins were isolated from the fat body of B. mori.
3.4. Functional analysis of the 21-bp core sequence of the Bmlp3 promoter
Fig. 5. Functional analysis of the 21-bp core sequence of the Bmlp3 promoter. (A) The diagram of the pGL3-50, pGL3-C21-50, pGL3-C21M1-50, pGL3-C21M2-50, and pGL3-C21M3-50 constructs. The 21-bp core sequence and its variants M1, M2 and M3 were inserted into the upstream region of pGL3-50 vector. The first exon of Bmlp3 (from + 1 to +24) and luciferase gene (Luc) are indicated by a shared black box and open box, respectively. (B) The constructs were transfected into BmE cells and luciferase activities were measured. Relative firefly luciferase activities were averages of three independent transfections normalized to Renilla luciferase control activities (*p b 0.05, **p b 0.01, ***p b 0.001).
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4. Conclusions In this study, the promoter of a fat body-specific gene encoding a member of the 30 K protein family, Bmlp3, was characterized by creating various luciferase reporter gene constructs with differing amounts of 5′ promoter-proximal sequences. A 21-bp core sequence located between − 230 and − 250 upstream of transcription start site of Bmlp3 was identified using the EMSA method. We demonstrated that the 21-bp sequence can bind specifically with the nuclear proteins from the fat body of B. mori and increase luciferase gene expression resulting in 2.8-fold more luciferase activity in BmE cells. These data indicate that the 21-bp sequence is an enhancer-like element responsible for maintaining the transcriptional activity of the Bmlp3 promoter. This study provides new insights into the function and developmental regulation of Bmlp3. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.038. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Professor Qili Feng of South China Normal University, China, for providing the Spodoptera litura embryonic cell line Spli-221. We are grateful to Professor David O'Brochta of the University of Maryland, USA, and Huawei He of Southwest University, China, for improving this manuscript. This work was supported by the National Basic Research Program of China (2012CB114600), and Grant (XDJK2014B014) from the Fundamental Research Funds for the Central Universities. References Bosquet, G., et al., 1989. The regulation of major haemolymph protein synthesis: changes in mRNA content during the development of Bombyx mori larvae. Insect Biochem. 19, 29–39. Deng, D.J., et al., 2013. The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, Bombyx mori. Transgenic Res. 22 (5), 1055–1063.
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