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Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori
Insect Biochemistry and Molecular Biology 41 (2011) 592e601
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Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori Shigeharu Takiya a, b, *, Hiroshige Inoue b, Mai Kimoto b a b
Division of Biological Sciences and Center for Genome Dynamics, Faculty of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan Graduate School of Life Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 January 2011 Received in revised form 21 March 2011 Accepted 29 March 2011
Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from 1602 to þ47 was sufficient to induce MSG-specific expression. The 50 deletion mutants showed a three-step decrease in promoter activity with the key sequences located between 1362 and 1250, 201 and 116, and 115 and 37. We detected a tissueand stage-specific factor complex (MSGeintermolt-specific complex: MIC) bound to the sequence elements around the 1350, 320, 180, and 70 regions. A mutation in the 70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MICbinding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Sericin-1 gene Enhancer Promoter Gene gun Homeodomain Middle silk gland
1. Introduction The silk gland of the silkworm Bombyx mori is an interesting organ for studying the fundamental mechanisms of gene regulation and organ differentiation (Suzuki et al., 1990a) as well as for its potential applications in industry and medical technology (Hino et al., 2006; Ogawa et al., 2007; Tomita et al., 2007). The silk gland can produce vast amounts of silk proteins at the last instar, and the silk proteins fibroin and sericins are expected to be useful biomaterials beyond textiles (Terada et al., 2002; Tsubouchi et al., 2005; Kim et al., 2005; Li et al., 2006; Gobin et al., 2006; Cheema et al., 2007; MacIntosh et al., 2008). Understanding the mechanisms regulating expression of the silk protein genes is important for both basic biology and practical applications. The silk gland is divided into anterior (ASG), middle (MSG), and posterior (PSG) silk glands. The MSG is further divided into three parts: anterior, middle, and posterior regions. The fibroin-H gene (fibH) encodes the major silk fiber protein, fibroin heavy-chain, and is expressed specifically in the PSG. The sericin genes encoding glue
* Corresponding author. Division of Biological Sciences and Center for Genome Dynamics, Faculty of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan. Tel.: þ81 11 706 3590; fax: þ81 11 706 3588. E-mail address: [email protected] (S. Takiya). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.03.011
proteins are expressed specifically in the MSG with sublocal specificity. Expression of the sericin-1 gene (ser1) is restricted to the posterior region of MSG in the early instars, and expands to the middle region in the last instar (Couble et al., 1987; Matsunami et al., 1998; Takasu et al., 2010). Meanwhile, the sericin-2 and -3 genes are expressed mainly in the anterior region of MSG (Couble et al., 1987; Michaille et al., 1990; Takasu et al., 2002, 2007, 2010). Matsuno et al. (1989, 1990) revealed that the SA (around 90) and SC (around 200) sites of the ser1 promoter stimulate its transcription in vitro. Bombyx fork head (Fkh) protein and POUhomeodomain (POU-HD) protein POU-M1 bind to the SA and SC, respectively (Fukuta et al., 1993; Mach et al., 1995). However, the expression level of POU-M1 is complementary to that of ser1 (Kokubo et al., 1997; Matsunami et al., 1998), and the POU-M1binding elements of the POU-M1 gene per se actuate a repressive effect on its transcription in vitro (Xu et al., 1994). POU-M1 might negatively regulate the expression of ser1. Horard et al. (1994, 1997) and Nony et al. (1995) used a gene gun to introduce the PSG-specific fibrohexamerin gene (fhx) into silk glands, and revealed the regulatory elements necessary for its tissue specificity. Using the same method, Shimizu et al. (2007) discovered upstream enhancing elements (UEEs) of fibH necessary for the PSGspecific efficient transcription in vivo. The element around 1.6 kbp of fibH contains a sequence recognized by LIM-homeodomain (LIM-
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regions around 1350, 180 and 70 were important for the promoter activity. We detected a novel tissue- and stage-specific factor complex bound to these cis-acting elements.
HD) family proteins, and the same sequences bound by silk gland factor-2 (SGF-2) were also found in the promoter-proximal elements detected in vitro (Tsuda and Suzuki, 1983; Suzuki et al., 1986; Hui et al., 1990). In addition to the silk protein genes, Wang et al. (2009) identified upstream regulatory elements of a Bombyx wing cuticle protein gene using the transient reporter assay system with a gene gun. These results demonstrated that the in vivo assay system employing a gene gun and organ transplantation is useful for analyzing tissue-specific regulatory elements. In this study, we used biolistic particle bombardment with the gene gun and organ transplantation to analyze the mechanisms regulating ser1 gene expression. The region from 1602 to þ47 was sufficient for efficient MSG-specific expression in vivo. At least three
(Firefly / Renilla)
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2. Materials and methods 2.1. Animals Eggs of the silkworm B. mori (Kinshu Showa) were purchased from Ueda Sanshu (Ueda, Japan). Larvae were grown at 25 C on an artificial diet obtained from Nippon Nosan-Kogyo (Yokohama, Japan), and staged as described previously (Maekawa and Suzuki, 1980; Kiguchi and Agui, 1981; Suzuki et al., 1990b).
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Fig. 1. Promoter activity of the sericin-1 gene introduced into the silk gland. (A) The Photinus reporter vector PGV-ser1 (1602/þ47) or PGV-fibH (5661/þ10) was introduced into the silk glands together with the Renilla internal control vector pRL-A3 (128/þ28), and luciferase activity was measured after separating MSG and PSG. The Photinus luciferase activity was normalized to the Renilla luciferase activity. Error bars represent S.D., and the p value indicated with * is <0.05 and with ** is <0.001. (B) The series of 50 deletion mutants of PGV-ser1 shown in the left half of the panel was introduced into three silk glands, and luciferase activity in the MSG was measured individually. These experiments were repeated more than three times. The activity is indicated relative to the activity of PGV-ser1 (1602/þ47) after normalization to Renilla luciferase activity. The vertical arrows on the figure of PGV-ser1(1602/þ47) show approximate positions of the binding sites for POU-M1 (P), LIM-HD (L), MIC (M) and Fkh (F). The expression such as P,M,L or M,L indicates the overlapping of the binding sites for these factors. The open triangle indicates the TATA-box.
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2.2. Construction of the reporter plasmids Appropriate regions of the ser1 promoter were amplified by PCR with the primers listed in Supplementary Table 1 and inserted between the SmaI and XhoI sites of the Photinus luciferase vector PGV-Basic (Toyo Ink). The series of ser1 gene reporter plasmids was referred to as PGV-ser1. To construct PGV-ser1 (70M2) and PGV-ser1 (70M4) with mutations at the 70 region of the ser1 promoter, the promoter region between 1602 and þ42 was divided into two parts at the HindIII site at 54, and the upstream region of the HindIII site was amplified with primers incorporating each mutation. This upstream fragment was joined with the downstream fragment and inserted into the PGV vector. PGV-fibH (5661/þ10) carrying both the UEEs and promoter-proximal elements of the fibH gene from 5661 to þ10 was constructed by insertion of the PCR fragment between the SmaI and NheI sites of PGV-Basic. Construction of pRL-A3 (128/þ28) involved insertion of the PCR fragment of the Bombyx actin A3 gene promoter between the EcoRI and XhoI sites of the Renilla luciferase vector pRL-null (Promega) for use as an internal control (Nishita and Takiya, 2009).
Fig. 2. Binding of MIC to promoter-proximal regions. The oligonucleotides Sr-320, Sr180, Sr-140, and SrCP (Supplementary Table 2) were labeled and used as probes for EMSAs. Silk gland extracts were prepared from the entire MSG (MSG) or PSG (PSG) at the fourth molt (IVm) or on day 2 of the fifth instar (V), and the posterior region of MSG (MSG-P in B) on day 2 of the fifth instar. In those lanes indicated with þPOU.C, EMSA was conducted in the presence of a POU-M1-specific competitor.
Fig. 3. Binding of MIC to the 70 region of the sericin-1 gene. The Sr-70 oligonucleotide was labeled and used as a probe for EMSA (A). Competition experiments were conducted using the Sr-70S oligonucleotide and its mutants (B). The effect of two basepair changes in Sr-70S M3 was weak. Nucleotide sequences of the Sr-70 probe and competitors are shown below the panels. The putative MIC-binding element (see Fig. 9A) is indicated by a line over the nucleotide sequence of Sr-70. (C) PGV-ser1 (1602/þ42): WT, or its mutant constructs in the 70 region PGV-ser1 (70M2) and PGV-ser1 (70M4) were introduced into the silk glands and treated for the luciferase assay. The activity was normalized to activity of the internal control pRLA3(128/þ28). Error bars represent S.D. The p value indicated with * is <0.02, and with ** is <0.005.
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2.3. Luciferase reporter assay The reporter plasmids were introduced into silk glands from 1-day old larvae of the fifth instar with a gene gun (BioRad) according to Shimizu et al. (2007) with several modifications. Tangsten particles (25 mg) were mixed with PGV-ser1 or PGV-fibH DNA (250 mg) and pRL-A3 internal control DNA (25 mg) and treated according to the manufacturer’s instructions. A 70-cm Tefzel tube (BioRad) was coated with the tangsten particles coated with DNA and cut into 12-mm pieces. The particles were introduced into silk glands on day 1 of the fifth instar using the pressure of N2 gas at 100 psi. The silk glands were then transplanted into larvae at the same stage.
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Each reporter plasmid, together with the internal control, was introduced into three silk glands, and luciferase activity was measured individually at 2 days after the transplantation with a Dual luciferase assay kit (Promega). Silk glands that appeared to be unhealthy were omitted from the assay. These experiments were repeated at least three times. The Photinus luciferase activity was normalized to the Renilla luciferase activity. 2.4. Preparation of the silk gland extracts Stage- and territory-restricted silk gland extracts were prepared as previously described (Tsuda and Suzuki, 1981; Takiya et al., 1990). On day 2 of the fifth instar, ordinary MSG extracts were prepared
Fig. 4. Competition of MIC-binding with the mutant oligonucleotides of Sr-320 and Sr-180. The competitive effects of the Sr-320 short oligonucleotide Sr-320S and its mutants on MIC-binding to Sr-320 (A) and Sr-70 (B) and the effects of Sr-180S and its mutants (C) were examined. (D) Nucleotide sequences of the oligonucleotides used as competitors in (A), (B), and (C). Putative MIC-binding elements are indicated with lines and the POU-HD-binding element is indicated with a broken line over the sequences.
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To analyze the promoter activity of the ser1 gene in vivo, we first cloned the 50 upstream and core promoter regions from 1602 to þ47 into a luciferase reporter vector, introduced the reporter construct into silk glands with a gene gun, and analyzed the luciferase activity. As shown in Fig. 1A, PGV-ser1 (1602/þ47) was expressed efficiently and specifically in the MSG. The activity was comparable to that of the fibH reporter construct PGV-fibH (5661/þ10) containing the UEEs in the PSG. The 50 deletion series resulted in a three-step decrease in promoter activity in the MSG (Fig. 1B). The deletion from 1362 to 1250 decreased the activity to approximately 40% of PGV-ser1 (1602/þ47), and the deletion from 201 to 116 diminished almost all of the activity. However, the residual activity of PGV-ser1 (115/þ42) was still significant compared to PGV-ser1 (36/þ42), which included the core promoter (refer to the insert graph in Fig.1B).
between 115 and 37 (Mach et al., 1995; Takiya et al., 2003), a candidate element for POU-M1 binding was found near the 1350 region (Fig. 1B). However, we reinvestigated the binding of factors to the upstream region of ser1 because the region where the POU-M1 gene is expressed does not correspond to the region where ser1 is expressed (Kokubo et al., 1997; Matsunami et al., 1998). The ser1 gene is expressed in the middle and posterior parts of the MSG at the fifth instar, while the POU-M1 gene is strongly expressed in the ASG and anterior part of the MSG. We synthesized oligonucleotides including or excluding the binding elements for POU-M1, Fkh and LIM-homeodomain (LIMHD) proteins of the ser1 promoter (Supplementary Table 2), and used them as probes and competitors for EMSA. Several probes gave specific patterns with MSG extracts from the fifth instar, but not with MSG extracts from the fourth molt larvae or PSG extracts (Fig. 2A). Using ordinary extracts prepared from the entire MSG, a few vague bands were observed, but we could not examine the binding specificity of the factors responsible for such vague bands. We prepared a new extract exclusively of the region where ser1 is expressed. The posterior section of the MSG on day 2 of the fifth instar was collected and treated for the preparation of tissue extracts, as previously described (Takiya et al., 1990). We designated this extract “MSG-P extract”. EMSAs of MSG-P extracts generated specific patterns with the Sr-320 and Sr-180 probes (Fig. 2B). Competition experiments using oligonucleotides containing POU-M1-binding elements showed that these complexes were different from POU-M1 (see þPOU.C lanes in Fig. 2B). These complexes were named MSGeintermolt-specific complex-A (MICA) for Sr-180 and -B (MIC-B) for Sr-320.
3.2. Binding of MSG-specific factors to the promoter-proximal elements
3.3. The region around 70 of the sericin-1 gene was indispensable for promoter activity in vivo
In addition to two POU-M1-binding elements between 201 and 116 (Matsuno et al., 1990), and one Fkh-binding element
The Sr-180 oligonucleotide contained the typical POU-M1binding element ATGAATAA, and the Sr-320 oligonucleotide
from the entire middle silk glands, and MSG-P extracts were prepared only from the posterior region of the middle silk gland. 2.5. Electrophoretic mobility shift assay The electrophoretic mobility shift assay (EMSA) was carried out as previously described by Matsuno et al. (1989) and Takiya et al. (1997, 2005) using the oligonucleotides shown in Supplementary Table 2 as the probes and competitors. The proteineprobe complexes were separated on 7% polyacrylamide gels. For the competition experiments, a 100-fold molar excess of unlabeled oligonucleotides was added to the reaction. 3. Results 3.1. Deletion assay of the sericin-1 gene promoter in vivo
Fig. 5. EMSAs using the probes Sr-1350, Sr-340, and Sr-310. (A) The binding factors in each extract shown above the panel were analyzed using the Sr-1350, Sr-340, and Sr-310 oligonucleotides as probes. MIC-A detected with Sr-310 probe and factor B detected with Sr-340 probe (see Fig. 9B) are marked. (B) Competition of MIC-A detected with the Sr310 probe with the Sr-70S and its mutant oligonucleotides.
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contained the typical LIM-HD-binding element CAATTA. We performed additional EMSAs using probes including a core sequence (TAAT) recognized by homeodomain (HD) proteins or probes including AT-rich cis-elements. It was found that MIC-A bound strongly to the Sr-70 probe (Fig. 3A and Supplementary Fig. 1), although this probe contained neither typical POU-M1-binding elements nor LIM-HD-binding elements. MIC-A on the Sr-70 probe competed effectively with the oligonucleotides Sr-320, Sr180, and Sr-70 itself, and partly with Sr-1350 (Supplementary Fig. 2). Competition experiments with mutant oligonucleotides of Sr-70 clearly demonstrated the sequence specificity of MIC-A binding (Fig. 3B). The Sr-70S M1, M2, M3, and M5 mutants did not compete with the MIC-A binding to the Sr-70 probe, whereas the Sr-70S M4 competed as efficiently as the wild type oligonucleotides. We constructed reporter plasmids, PGV-ser1 (70M2) and PGVser1 (70M4), with the same two base-pair mutations as Sr-70S M2 and M4 oligonucleotides, respectively. These plasmids were introduced into silk glands and analyzed for promoter activity. As shown in Fig. 3C, PGV-ser1 (70M2) lost almost all promoter activity, while PGV-ser1 (70M4) indicated the same activity as the wild type construct. Thus, the region around 70 of ser1 was indispensable for promoter activity in vivo.
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3.4. MIC-B contained MIC-A and had the same DNA-binding specificity The oligonucleotide, Sr-320, effectively competed with MIC-A binding to the Sr-70 probe (Supplementary Fig. 2). We further analyzed the relationship between MIC-A and MIC-B by examining their binding specificity with various mutant oligonucleotides as competitors (Fig. 4). MIC-A with the Sr-70 probe and MIC-B with Sr320 were similarly competed with the Sr-320S mutant oligonucleotides (compare Fig. 4A with Fig. 4B). Sr-320S M3 and M4 lost the ability to compete with both MIC-A and MIC-B, and the effects of other mutants were partial. The wild type and mutant Sr-180 oligonucleotides also competed with both MIC-A and MIC-B with similar efficiency (Fig. 4C), to the extent that DNA-binding specificity of MIC-A and MIC-B was essentially identical. During the course of this study, we found that the Sr-320 oligonucleotide contains an atypical LIM-HD-binding element CATAAT around 340 (Supplementary Fig. 3). Two LIM-HD-binding elements around 340 and 310 within the Sr-320 probe were separated into the oligonucleotides, Sr-340 and Sr-310, and these oligonucleotides were used for EMSAs as probes. As shown in Fig. 5, these probes detected different complexes; the first one was denoted as MIC-A for the Sr-310 probe and the other one was
Fig. 6. Competition of MIC-binding to Sr-70 or Sr-320 probes with Sr-1350 region. Sr-1350S and its mutants were used as competitors for the binding of MIC to Sr-70 (A) or Sr-320 (B). Effects of Sr-340 and Sr-750 oligonucleotides on MIC-binding to Sr-70 were also examined. (C) Nucleotide sequences of the competitors. The putative MIC-binding element and LIM-HD-binding elements are shown with lines.
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denoted as factor B for the Sr-340 probe. MIC-A detected with the Sr-310 probe showed identical DNA-binding specificity as those detected with the Sr-70 and Sr-180 probes (Fig. 5B and Supplementary Fig. 4). 3.5. MIC-A bound to the region around 1350 of the sericin-1 gene When the Sr-1350 oligonucleotide was used as a probe in EMSA, a vague but specific band that was detected in MSG-P extracts appeared around the MIC position (Fig. 5A and Supplementary Fig. 1). The affinity of MIC-binding to the Sr-1350 probe was not strong enough to directly examine the binding specificity. Competition experiments were conducted with its mutant oligonucleotides as competitors. Competition for MIC-A binding to Sr-70 with the Sr-1350 and Sr-1350S oligonucleotides was partial, but sequence specificity for the competition was clearly observed (Fig. 6A and C). Sr-1350S M3 and M4 could not compete with MIC-A binding. The effects of these mutant oligonucleotides on MIC-B were the same as on MIC-A (Fig. 6B). The results indicate that the 1350 region of ser1 is a MIC-A binding site. 3.6. The region around 1350 of the sericin-1 gene enhanced the promoter activity in MSG The region around 1350 of ser1 contains overlapping sequence elements recognized by many HD proteins, such as LIM-HD, POU-HD, Antp, Ubx, Dfd, and Ftz (Fig. 7). We analyzed function of the region around 1350 using a deletion mutant PGVser1(1340/þ42). The deletion from 1362 to 1340, lacking all the HD-protein-binding sequences, decreased the promoter activity to the same level as PGV-ser1 (1249/þ42) (Fig. 7). Thus, the 1350 region maintained a tissue (MSG)-specific enhancerlike activity.
Fig. 7. Enhancement of the expression by the 1350 region of the sericin-1 gene. PGVser1 (1602/þ47), (1362/þ47), (1340/þ47), (1249/þ47), and (201/þ47) were introduced into the silk glands, and the luciferase activity was measured after separating the MSG and PSG. The activity is indicated relative to the activity of PGVser1(1602/þ47) in the MSG. The p value indicated with * is <0.05 and with ** is <0.02. The nucleotide sequence around the 1350 region is shown above. HD-binding elements overlapping around 1350 are shown with thick bars below the sequence. The deletion positions of PGV-ser1 (1362/þ47) and PGV-ser1 (1340/þ47) are indicated with arrows over the sequence.
3.7. Coexistence of oligonucleotides, including LIM-HD-binding elements, affected MIC-A binding to the ser1 promoter The Sr-70 oligonucleotide did not contain either typical (CAATTA) or atypical (CATAAT) LIM-HD elements; however, the oligonucleotides, Sr-1350, Sr-320 and Sr-180 contained the typical LIM-HD element. We investigated the effects of oligonucleotides containing the LIM-HD-binding elements on MIC-A binding to the Sr-70 probe. Addition of the Sr-340 oligonucleotide to the reaction weakened the MIC-A band on Sr-70. The intensity of the MIC-A band was recovered with a mutation of the LIM-HD element in Sr-340 (Fig. 6A). On the other hand, Sr-750, including both typical and atypical LIM-HD elements (Fig. 6C), stimulated MIC-A binding to the Sr-70 probe, and its mutant lost such ability. Similar phenomena were clearly observed with the Sr-320 probe (Fig. 8A). In the presence of the Sr-340 oligonucleotide, a weak but sharp MIC-A band was detected instead of a MIC-B band. In the presence of the Sr-750 oligonucleotide, a highly strengthened MIC-A band was seen, and mutations of the LIM-HD-binding elements in Sr-750 diminished the effect. Because a clear and strong MIC-A band was observed with the Sr-320 probe in the presence of the Sr-750 oligonucleotide, the effects of the Sr-1350 and its mutant oligonucleotides on MIC-A on the Sr-320 probe were examined in the presence of Sr-750. The binding specificity of MIC-A to the Sr-1350 region was reconfirmed. The mutants, M3 and M4 did not compete MIC-A binding, and M5 and M6 competed partially (Fig. 8B).
3.8. Consensus sequence recognized by MIC-A MIC-A seemed to bind elements recognized by HD proteins. We selected several sequence elements from the intron of ser1 (Supplementary Table 2) and examined the ability of the oligonucleotides to compete with MIC-binding. Of these oligonucleotides, the Srþ1380 and Srþ2440 competed with MIC-binding almost completely, while Srþ1890, containing a typical LIM-HD element,
Fig. 8. Effects of LIM-HD-binding elements on MIC-A-binding to the Sr-320 probe. (A) Competition effects of the LIM-HD-binding elements on the binding of MIC to Sr-320 were examined in EMSAs with the oligonucleotides Sr-340, Sr-340S, Sr-750 and Sr750M as competitors. (B) Effects of Sr-1350S and its mutant oligonucleotides on MIC-binding to the Sr-320 probe in the presence of Sr-750. In addition to the competitors, all EMSA reactions contained a 100-fold excess amount of Sr-750.
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indispensable for promoter activity of ser1 in the MSG. The 70 region contains the core sequence (TAAT) recognized by many HD proteins, whereas the 1350 and 180 regions contain sequences recognized by LIM-HD proteins. To investigate whether there is a factor that specifically binds to these elements, a new MSG extract (MSG-P extract) was prepared from the posterior region of MSG. With this MSG-P extract, tissue- and stage-specific binding factors (MIC-A and -B) were clearly detected. The factors were related to each other in their DNA-binding specificity and were bound to the 1350, 320, 180, and 70 regions with slightly different EMSA patterns. MIC-B was first detected with the Sr-320 probe was thought to contain at least two DNA-binding factors or factor complexes (Fig. 9B): one (factor B) bound to the sequence element CATAAT around 340, and the other (MIC-A) bound to the 10-bp AT-rich element around 310, overlapping with the LIM-HDbinding element CAATTA. MIC-A bound to the 10-bp region which might be divided into two overlapping sequence elements recognized independently by factors A and A0 , representing different surfaces of the protein. Mutations at the overlapping nucleotides inhibited the binding of MIC-A completely, while mutations at the nucleotides recognized by either A or A0 inhibited it partly. Therefore, MIC-A was competed partially even with the mutant oligonucleotides at the 10-bp MICA-binding sequence. The element recognized by factor A0 around 1350 was likely destroyed in part by the T nucleotide, shown in Fig. 9, and the intensity of the MIC band with the Sr-1350 probe was low. The broad band (MIC-B) observed with the Sr-320 probe seemed to be due to interactions of multiple factors. The band (MIC-B)
did not (Supplementary Fig. 5). Comparison of the nucleotide sequences of the MIC-binding elements and other LIM-HD-binding elements indicated that the 10-bp sequence occasionally overlapped with the LIM-HD-binding sequence CAATTA, and the region extended 5-bp from the side of the CAATTA element (Fig. 9A). 4. Discussion Genes introduced transiently into cultured cells were reported to be modified and to form a chromatin structure similar to the endogenous genes within several hours (Agelopoulos and Thanos, 2006). The in vivo expression system for analyzing promoter activity using a gene gun and organ transplantation in the silkworm B. mori has led to the identification of enhancer and promoter elements necessary for the PSG-specific expression of fhx and fibH (Horard et al., 1994, 1997; Nony et al., 1995; Shimizu et al., 2007). Moreover, the promoter elements of a Bombyx wing cuticle protein gene were recently identified using a transient assay system with the gene gun (Wang et al., 2009). We applied this method to the ser1 gene expressed specifically in the MSG. The region from 1602 to þ47 of ser1 was sufficient for MSG-specific transcription, and the promoter elements that had not been found in vitro were detected. Although the efficiency was as high as that of the fibH gene carrying UEEs under the conditions used in this study, we do not exclude the possibility that additional enhancer elements exist in the further upstream region and intron of the ser1 gene. A series of deletions in the ser1 promoter showed that at least three regions around 1350, 180, and 70 were important for specificity and efficiency in vivo. The 70 region was particularly
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Fig. 9. Consensus sequence found in MIC-binding elements. (A) Nucleotide sequences of the 6 MIC-binding sites and 4 MIC-unbound but putative binding sites for LIM-HD are shown. The elements corresponding to the consensus sequence for MIC-binding are boxed, and putative binding sites for POU-M1 and LIM-HD are indicated with lines. The dotted T nucleotide at 1341 is probably not part of the consensus sequence and weakens the binding of MIC to the 1350 region. Because the mutant oligonucleotide Sr-320 M4 competed almost completely with MIC-binding to the Sr-320 probe (Supplementary Fig. 3), a change of the A nucleotide at position 2 to the G nucleotide is probably occurring, but we did not find an endogenous MIC-binding element of which the nucleotide at position 2 is G. (B) A model for MIC-binding to the ser1 promoter. MIC-A has two DNA-binding surfaces or is composed of at least two factors A and A0 and binds to the elements around 1350, 320, 180, and 70, containing the consensus sequence. Factors A and A0 independently recognize overlapping half sites of the consensus sequence, and MIC-A can bind weakly to the half sites. Factor B, recognizing the element CATAAT, forms MIC-B with MIC-A and likely stabilizes MIC-A-binding; thus, MIC-B was easily detected with the Sr-320 probe. Factor C, recognizing CAATTA, disturbs the binding of MIC-A to the 1350, 320, and 180 regions. Competition of factor C with Sr-750 enhanced the binding of MIC-A to these sites.
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obtained with the Sr-320 probe became a sharp band (MIC-A) upon the addition of the oligonucleotide Sr-340 or Sr-750 as a competitor. These oligonucleotides include the sequence element, CATAAT, in Sr-340 or CATAAT and CAATTA in Sr-750 (Fig. 6C), recognized by the LIM-HD protein family but not the consensus sequence recognized by MIC-A. Furthermore, competition with the oligonucleotides, Sr-140, SC and POU.C, including the POU-M1-binding element, weakened the intensity of the MIC-B bands (Supplementary Fig. 6). Together with factors B and C, and POU-M1, might be responsible for the formation of the broad band (MIC-B). The addition of Sr-750, but not Sr-340, to the EMSA reactions with the Sr-320 probe enhanced the MIC-A band remarkably. Therefore, factor C seemed to compete for MIC-A-binding sites with factor A, and might have an inhibitory effect on ser1 gene expression. LIM-HD proteins are candidates for the identification of factor C. Effects of the Sr-340 and Sr-750 oligonucleotides were also observed on MIC-A bound to the Sr-70 probe. Factors B and C may induce their effect via the core sequence (TAAT), recognized by HD proteins, or through direct interactions with MIC-A. The Sr-180 oligonucleotide overlaps partly with the SC site of the promoter-proximal enhancing element, detected in vitro, and binds POU-M1 (Matsuno et al., 1989, 1990). In the 1350 region, many HD-binding elements, including the POU-HD-binding element, overlapped, but competition with SC oligonucleotide and POU-specific competitor POU.C did not disturb the formation of MIC-A on the Sr-70 and Sr-180 probes. Although the role of POUM1 in ser1 gene expression was not investigated in this study, we support the assumption that POU-M1 plays an inhibitory role in ser1 gene expression. The expression level of POU-M1 is complementary to that of ser1 (Kokubo et al., 1997; Matsunami et al., 1998), and POU-M1-binding sites of the POU-M1 gene promoter tend to inhibit its transcription in vitro (Xu et al., 1994). Moreover, cointroduction of a POU-M1 expression vector together with the ser1-reporter construct into silk glands suppressed ser1 gene expression (Kimoto et al., unpublished results). In contrast, MIC-A was detected in the posterior part of MSG, where ser1 is expressed efficiently, and bound to the promoter and enhancer elements indispensable for the expression of ser1 in MSG. The results suggested that MIC-A is intrinsic for tissue- and stagespecific expression of ser1 in vivo. Acknowledgments We thank Dr. Y. Nishita for the discussions and advice regarding the luciferase assay. We also deeply appreciate the anonymous reviewers for their helpful advice. Appendix. Supplementary material Supplementary material related to this article can be found at doi:10.1016/j.ibmb.2011.03.011. References Agelopoulos, M., Thanos, D., 2006. Epigenetic determination of a cell-specific gene expression program by ATF-2 and the histone variant macroH2A. EMBO J. 25, 4843e4853. Cheema, S.K., Gobin, A.S., Ohea, R., Lopez-Berestein, G., Newman, R.A., Mathur, A.B., 2007. Silk fibroin mediated delivery of liposomal emodin to breast cancer cells. J. Pharmaceutics 341, 221e229. Couble, P., Michaille, J.-J., Garel, A., Couble, M.L., Prudhomme, J.-C., 1987. Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silk glands. Dev. Biol. 124, 431e440. Fukuta, M., Matsuno, K., Hui, C.-c., Nagata, T., Takiya, S., Xu, P.-X., Ueno, K., Suzuki, Y., 1993. Molecular cloning of a POU-domain-containing factor involved in the regulation of the Bombyx sericin-1 gene. J. Biol. Chem. 268, 19471e19475. Gobin, A.S., Rhea, R., Newman, R.A., Mathur, A.B., 2006. Silk-fibroin-coated liposomes for long-term and targeted drug delivery. J. Nanomedicine 1, 81e87.
Hino, R., Tomita, M., Yoshizato, K., 2006. The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor. Biomaterials 27, 5715e5724. Horard, B., Mange, A., Pelisser, B., Couble, P., 1994. Bombyx gene promoter analysis in transplanted silk gland transformed by particle delivery system. Insect Mol. Biol. 3, 261e265. Horard, B., Julien, E., Nony, P., Garel, A., Couble, P., 1997. Differential binding of the Bombyx silk gland-specific factor SGFB to its target DNA sequence drives posterior-cell-restricted expression. Mol. Cell. Biol. 17, 1572e1579. Hui, C.-c., Matsuno, K., Suzuki, Y., 1990. Fibroin gene promoter contains a cluster of homeodomain binding sites that interact with three silk gland factors. J. Mol. Biol. 213, 651e670. Kiguchi, K., Agui, N., 1981. Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombyx mori. Insect Physiol. 27, 805e812. Kim, K.-H., Lim, J., Park, H.-N., Shin, S.-Y., Park, W.-H., Lee, S.-C., Kim, T.-I., Park, Y.-J., Seol, Y.-J., Lee, Y.-M., Ku, Y., Rhyu, Y.-J., Han, S.-B., Chung, C.-P., 2005. Biological efficacy of silk fibroin nanofiber membranes for guide bone regeneration. J. Biotech. 120, 327e339. Kokubo, H., Xu, P.-X., Xu, X., Matsunami, K., Suzuki, Y., 1997. Spatial and temporal expression pattern of POU-M1/SGF-3 in Bombyx mori embryogenesis. Dev. Genes Evol. 206, 494e502. Li, C., Vepari, C., Jin, H.-J., Kim, H.J., Kaplan, D.L., 2006. Electron silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 27, 3115e3124. Mach, V., Takiya, S., Ohno, K., Handa, H., Imai, T., Suzuki, Y., 1995. Silk gland factor-1 involved in the regulation of Bombyx sericin-1 gene contains fork head motif. J. Biol. Chem. 270, 9340e9346. MacIntosh, A.C., Kearns, V.R., Crawford, A., Hatton, P.V., 2008. Skeletal tissue engineering using silk biomaterials. J. Tissue Eng. Regen. Med. 2, 71e80. Maekawa, H., Suzuki, Y., 1980. Repeated turn-off and turn-on of fibroin gene transcription during silk gland development of Bombyx mori. Dev. Biol. 78, 394e406. Matsunami, K., Kokubo, H., Ohno, K., Suzuki, Y., 1998. Expression pattern analysis of SGF-3/POU-M1 in relation to sericin-1 gene expression in the silk gland. Develop. Growth Differ. 40, 591e597. Matsuno, K., Hui, C.-c., Takiya, S., Suzuki, T., Ueno, K., Suzuki, Y., 1989. Transcription signals and protein binding sites for sericin gene transcription in vitro. J. Biol. Chem. 31, 18707e18713. Matsuno, K., Takiya, S., Hui, C.-c., Suzuki, T., Fukuta, M., Ueno, K., Suzuki, Y., 1990. Transcriptional stimulation via SC site of Bombyx sericin-1 gene through an interaction with a DNA binding protein SGF-3. Nucleic Acids Res. 18, 1853e1858. Michaille, J.-J., Garel, A., Prudhomme, J.-C., 1990. Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori. Gene 86, 177e184. Nishita, Y., Takiya, S., 2009. Differential responsiveness of BmBR-C promoters to ecdysone signals. J. Insect Biotech. Sericol. 78, 127e138. Nony, P., Prudhomme, J.-C., Couble, P., 1995. Regulation of the P25 gene transcription in the silk gland of Bombyx. Biol. Cell 84, 43e52. Ogawa, S., Tomita, M., Shimizu, K., Yoshizato, K., 2007. Generation of a transgenic silkworm that secretes recombinant proteins in sericin layer of cocoon: production of recombinant human serum albumin. J. Biotech. 128, 531e544. Shimizu, K., Ogawa, S., Hino, R., Adachi, T., Masahiro, T., Yoshizato, K., 2007. Structure and function of 50 -flanking regions of Bombyx mori fibroin heavy chain gene: identification of a novel transcription enhancing element witha homeodomain protein-binding motif. Insect Biochem. Mol. Biol. 37, 713e725. Suzuki, Y., Tsuda, M., Takiya, S., Hirose, S., Suzuki, E., Kameda, M., Ninaki, O., 1986. Tissue-specific taranscription enhancement of the fibroin gene characterized by cell-free system. Proc. Natl. Acad. Sci. USA 83, 9522e9526. Suzuki, Y., Takiya, S., Suzuki, T., Hui, C.-c., Matsuno, K., Fukuta, M., Nagata, T., Ueno, K., 1990a. Developmental regulation of silk gene expression in Bombyx mori. In: Hagedorn, H.H., et al. (Eds.), Molecular Insect Science. Plenum Press, New York, pp. 83e89. Suzuki, Y., Obara, T., Takiya, S., Hui, C.-c., Matsuno, K., Suzuki, T., Suzuki, E., Ohkubo, M., Tamura, T., 1990b. Differential transcription of the fibroin and sericin-1 genes in cell-free extracts. Develop. Growth Differ. 32, 179e187. Takasu, Y., Yamada, H., Tsubouchi, K., 2002. Isolation of three main sericin components from cocoon of silkworm, Bombyx mori. Biosci. Biotech. Biochem. 66, 2715e2718. Takasu, Y., Yamada, H., Tamura, T., Sezutsu, H., Mita, K., Tsubouchi, K., 2007. Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 37, 1234e1240. Takasu, Y., Hata, T., Uchino, K., Zhang, Q., 2010. Identification of ser2 p.oteins as major sericin components in the non-cocoon silk of Bombyx mori. Insect Biochem. Mol. Biol. 40, 339e344. Takiya, S., Hui, C.-c., Suzuki, Y., 1990. A contribution of the core-promoter and its surrounding regions to the preferential transcription of the fibroin gene in posterior silk gland extracts. EMBO J. 9, 489e496. Takiya, S., Kokubo, H., Suzuki, Y., 1997. Transcriptional regulatory elements in the upstream and intron of the fibroin gene bind three specific factors POU-M1, BmFkh and FMBP-1. Biochem. J. 321, 645e653. Takiya, S., Gazi, M., Mach, V., 2003. The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases. Insect Biochem. Mol. Biol. 33, 1145e1154.
S. Takiya et al. / Insect Biochemistry and Molecular Biology 41 (2011) 592e601 Takiya, S., Ishikawa, T., Ohtsuka, K., Nishita, Y., Suzuki, Y., 2005. Fibroin-modulatorbinding protein-1 (FMBP-1) contains a novel DNA-binding domain, repeats of the score and three amino acid peptide (STP), conserved from Caenorhabditis elegans to humans. Nucleic Acids Res. 33, 786e795. Terada, S., Nishimura, T., Sasaki, M., Yamada, H., Miki, M., 2002. Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma. Cytotech 40, 3e12. Tomita, M., Hino, R., Ogawa, S., Izuka, M., Adachi, T., Shimizu, K., Satoshiro, H., Yoshizato, K., 2007. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Transgenic Res. 16, 449e465. Tsubouchi, K., Igarashi, Y., Takasu, Y., Yamada, H., 2005. Sericin enhances attachment of cultured human skin fibroblasts. Biosci. Biotech. Biochem. 69, 403e405.
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Tsuda, M., Suzuki, Y., 1981. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 50 flanking sequence far upstream. Cell 27, 175e182. Tsuda, M., Suzuki, Y., 1983. Transcription modulation in vitro of the fibroin gene exerted by a 200-base-pair region upstream from the “TATA” box. Proc. Natl. Acad. Sci. USA 80, 7442e7446. Wang, H.-B., Nita, M., Iwanaga, M., Kawasaki, H., 2009. bFTZ-F1 and Braod e complex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori. Insect Biochem. Mol. Biol. 39, 624e633. Xu, P.-X., Fukuta, M., Takiya, S., Matsuno, K., Xu, X., Suzuki, Y., 1994. Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes. J. Biol. Chem. 269, 2733e2742.
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Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb
Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori Shigeharu Takiya a, b, *, Hiroshige Inoue b, Mai Kimoto b a b
Division of Biological Sciences and Center for Genome Dynamics, Faculty of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan Graduate School of Life Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 January 2011 Received in revised form 21 March 2011 Accepted 29 March 2011
Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from 1602 to þ47 was sufficient to induce MSG-specific expression. The 50 deletion mutants showed a three-step decrease in promoter activity with the key sequences located between 1362 and 1250, 201 and 116, and 115 and 37. We detected a tissueand stage-specific factor complex (MSGeintermolt-specific complex: MIC) bound to the sequence elements around the 1350, 320, 180, and 70 regions. A mutation in the 70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MICbinding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Sericin-1 gene Enhancer Promoter Gene gun Homeodomain Middle silk gland
1. Introduction The silk gland of the silkworm Bombyx mori is an interesting organ for studying the fundamental mechanisms of gene regulation and organ differentiation (Suzuki et al., 1990a) as well as for its potential applications in industry and medical technology (Hino et al., 2006; Ogawa et al., 2007; Tomita et al., 2007). The silk gland can produce vast amounts of silk proteins at the last instar, and the silk proteins fibroin and sericins are expected to be useful biomaterials beyond textiles (Terada et al., 2002; Tsubouchi et al., 2005; Kim et al., 2005; Li et al., 2006; Gobin et al., 2006; Cheema et al., 2007; MacIntosh et al., 2008). Understanding the mechanisms regulating expression of the silk protein genes is important for both basic biology and practical applications. The silk gland is divided into anterior (ASG), middle (MSG), and posterior (PSG) silk glands. The MSG is further divided into three parts: anterior, middle, and posterior regions. The fibroin-H gene (fibH) encodes the major silk fiber protein, fibroin heavy-chain, and is expressed specifically in the PSG. The sericin genes encoding glue
* Corresponding author. Division of Biological Sciences and Center for Genome Dynamics, Faculty of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan. Tel.: þ81 11 706 3590; fax: þ81 11 706 3588. E-mail address: [email protected] (S. Takiya). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.03.011
proteins are expressed specifically in the MSG with sublocal specificity. Expression of the sericin-1 gene (ser1) is restricted to the posterior region of MSG in the early instars, and expands to the middle region in the last instar (Couble et al., 1987; Matsunami et al., 1998; Takasu et al., 2010). Meanwhile, the sericin-2 and -3 genes are expressed mainly in the anterior region of MSG (Couble et al., 1987; Michaille et al., 1990; Takasu et al., 2002, 2007, 2010). Matsuno et al. (1989, 1990) revealed that the SA (around 90) and SC (around 200) sites of the ser1 promoter stimulate its transcription in vitro. Bombyx fork head (Fkh) protein and POUhomeodomain (POU-HD) protein POU-M1 bind to the SA and SC, respectively (Fukuta et al., 1993; Mach et al., 1995). However, the expression level of POU-M1 is complementary to that of ser1 (Kokubo et al., 1997; Matsunami et al., 1998), and the POU-M1binding elements of the POU-M1 gene per se actuate a repressive effect on its transcription in vitro (Xu et al., 1994). POU-M1 might negatively regulate the expression of ser1. Horard et al. (1994, 1997) and Nony et al. (1995) used a gene gun to introduce the PSG-specific fibrohexamerin gene (fhx) into silk glands, and revealed the regulatory elements necessary for its tissue specificity. Using the same method, Shimizu et al. (2007) discovered upstream enhancing elements (UEEs) of fibH necessary for the PSGspecific efficient transcription in vivo. The element around 1.6 kbp of fibH contains a sequence recognized by LIM-homeodomain (LIM-
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regions around 1350, 180 and 70 were important for the promoter activity. We detected a novel tissue- and stage-specific factor complex bound to these cis-acting elements.
HD) family proteins, and the same sequences bound by silk gland factor-2 (SGF-2) were also found in the promoter-proximal elements detected in vitro (Tsuda and Suzuki, 1983; Suzuki et al., 1986; Hui et al., 1990). In addition to the silk protein genes, Wang et al. (2009) identified upstream regulatory elements of a Bombyx wing cuticle protein gene using the transient reporter assay system with a gene gun. These results demonstrated that the in vivo assay system employing a gene gun and organ transplantation is useful for analyzing tissue-specific regulatory elements. In this study, we used biolistic particle bombardment with the gene gun and organ transplantation to analyze the mechanisms regulating ser1 gene expression. The region from 1602 to þ47 was sufficient for efficient MSG-specific expression in vivo. At least three
(Firefly / Renilla)
Luciferase Activity
A
2. Materials and methods 2.1. Animals Eggs of the silkworm B. mori (Kinshu Showa) were purchased from Ueda Sanshu (Ueda, Japan). Larvae were grown at 25 C on an artificial diet obtained from Nippon Nosan-Kogyo (Yokohama, Japan), and staged as described previously (Maekawa and Suzuki, 1980; Kiguchi and Agui, 1981; Suzuki et al., 1990b).
300
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n=9
n=3
n=9
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Sr-1602
B
P,L,M
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L,M L,M F L P P M
L L
-1602
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-1462 -1362 -1249 -1018 -964 -698 -484 -345 -201 -115
-115
-36
-36 0
vector 0
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Fig. 1. Promoter activity of the sericin-1 gene introduced into the silk gland. (A) The Photinus reporter vector PGV-ser1 (1602/þ47) or PGV-fibH (5661/þ10) was introduced into the silk glands together with the Renilla internal control vector pRL-A3 (128/þ28), and luciferase activity was measured after separating MSG and PSG. The Photinus luciferase activity was normalized to the Renilla luciferase activity. Error bars represent S.D., and the p value indicated with * is <0.05 and with ** is <0.001. (B) The series of 50 deletion mutants of PGV-ser1 shown in the left half of the panel was introduced into three silk glands, and luciferase activity in the MSG was measured individually. These experiments were repeated more than three times. The activity is indicated relative to the activity of PGV-ser1 (1602/þ47) after normalization to Renilla luciferase activity. The vertical arrows on the figure of PGV-ser1(1602/þ47) show approximate positions of the binding sites for POU-M1 (P), LIM-HD (L), MIC (M) and Fkh (F). The expression such as P,M,L or M,L indicates the overlapping of the binding sites for these factors. The open triangle indicates the TATA-box.
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2.2. Construction of the reporter plasmids Appropriate regions of the ser1 promoter were amplified by PCR with the primers listed in Supplementary Table 1 and inserted between the SmaI and XhoI sites of the Photinus luciferase vector PGV-Basic (Toyo Ink). The series of ser1 gene reporter plasmids was referred to as PGV-ser1. To construct PGV-ser1 (70M2) and PGV-ser1 (70M4) with mutations at the 70 region of the ser1 promoter, the promoter region between 1602 and þ42 was divided into two parts at the HindIII site at 54, and the upstream region of the HindIII site was amplified with primers incorporating each mutation. This upstream fragment was joined with the downstream fragment and inserted into the PGV vector. PGV-fibH (5661/þ10) carrying both the UEEs and promoter-proximal elements of the fibH gene from 5661 to þ10 was constructed by insertion of the PCR fragment between the SmaI and NheI sites of PGV-Basic. Construction of pRL-A3 (128/þ28) involved insertion of the PCR fragment of the Bombyx actin A3 gene promoter between the EcoRI and XhoI sites of the Renilla luciferase vector pRL-null (Promega) for use as an internal control (Nishita and Takiya, 2009).
Fig. 2. Binding of MIC to promoter-proximal regions. The oligonucleotides Sr-320, Sr180, Sr-140, and SrCP (Supplementary Table 2) were labeled and used as probes for EMSAs. Silk gland extracts were prepared from the entire MSG (MSG) or PSG (PSG) at the fourth molt (IVm) or on day 2 of the fifth instar (V), and the posterior region of MSG (MSG-P in B) on day 2 of the fifth instar. In those lanes indicated with þPOU.C, EMSA was conducted in the presence of a POU-M1-specific competitor.
Fig. 3. Binding of MIC to the 70 region of the sericin-1 gene. The Sr-70 oligonucleotide was labeled and used as a probe for EMSA (A). Competition experiments were conducted using the Sr-70S oligonucleotide and its mutants (B). The effect of two basepair changes in Sr-70S M3 was weak. Nucleotide sequences of the Sr-70 probe and competitors are shown below the panels. The putative MIC-binding element (see Fig. 9A) is indicated by a line over the nucleotide sequence of Sr-70. (C) PGV-ser1 (1602/þ42): WT, or its mutant constructs in the 70 region PGV-ser1 (70M2) and PGV-ser1 (70M4) were introduced into the silk glands and treated for the luciferase assay. The activity was normalized to activity of the internal control pRLA3(128/þ28). Error bars represent S.D. The p value indicated with * is <0.02, and with ** is <0.005.
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2.3. Luciferase reporter assay The reporter plasmids were introduced into silk glands from 1-day old larvae of the fifth instar with a gene gun (BioRad) according to Shimizu et al. (2007) with several modifications. Tangsten particles (25 mg) were mixed with PGV-ser1 or PGV-fibH DNA (250 mg) and pRL-A3 internal control DNA (25 mg) and treated according to the manufacturer’s instructions. A 70-cm Tefzel tube (BioRad) was coated with the tangsten particles coated with DNA and cut into 12-mm pieces. The particles were introduced into silk glands on day 1 of the fifth instar using the pressure of N2 gas at 100 psi. The silk glands were then transplanted into larvae at the same stage.
595
Each reporter plasmid, together with the internal control, was introduced into three silk glands, and luciferase activity was measured individually at 2 days after the transplantation with a Dual luciferase assay kit (Promega). Silk glands that appeared to be unhealthy were omitted from the assay. These experiments were repeated at least three times. The Photinus luciferase activity was normalized to the Renilla luciferase activity. 2.4. Preparation of the silk gland extracts Stage- and territory-restricted silk gland extracts were prepared as previously described (Tsuda and Suzuki, 1981; Takiya et al., 1990). On day 2 of the fifth instar, ordinary MSG extracts were prepared
Fig. 4. Competition of MIC-binding with the mutant oligonucleotides of Sr-320 and Sr-180. The competitive effects of the Sr-320 short oligonucleotide Sr-320S and its mutants on MIC-binding to Sr-320 (A) and Sr-70 (B) and the effects of Sr-180S and its mutants (C) were examined. (D) Nucleotide sequences of the oligonucleotides used as competitors in (A), (B), and (C). Putative MIC-binding elements are indicated with lines and the POU-HD-binding element is indicated with a broken line over the sequences.
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To analyze the promoter activity of the ser1 gene in vivo, we first cloned the 50 upstream and core promoter regions from 1602 to þ47 into a luciferase reporter vector, introduced the reporter construct into silk glands with a gene gun, and analyzed the luciferase activity. As shown in Fig. 1A, PGV-ser1 (1602/þ47) was expressed efficiently and specifically in the MSG. The activity was comparable to that of the fibH reporter construct PGV-fibH (5661/þ10) containing the UEEs in the PSG. The 50 deletion series resulted in a three-step decrease in promoter activity in the MSG (Fig. 1B). The deletion from 1362 to 1250 decreased the activity to approximately 40% of PGV-ser1 (1602/þ47), and the deletion from 201 to 116 diminished almost all of the activity. However, the residual activity of PGV-ser1 (115/þ42) was still significant compared to PGV-ser1 (36/þ42), which included the core promoter (refer to the insert graph in Fig.1B).
between 115 and 37 (Mach et al., 1995; Takiya et al., 2003), a candidate element for POU-M1 binding was found near the 1350 region (Fig. 1B). However, we reinvestigated the binding of factors to the upstream region of ser1 because the region where the POU-M1 gene is expressed does not correspond to the region where ser1 is expressed (Kokubo et al., 1997; Matsunami et al., 1998). The ser1 gene is expressed in the middle and posterior parts of the MSG at the fifth instar, while the POU-M1 gene is strongly expressed in the ASG and anterior part of the MSG. We synthesized oligonucleotides including or excluding the binding elements for POU-M1, Fkh and LIM-homeodomain (LIMHD) proteins of the ser1 promoter (Supplementary Table 2), and used them as probes and competitors for EMSA. Several probes gave specific patterns with MSG extracts from the fifth instar, but not with MSG extracts from the fourth molt larvae or PSG extracts (Fig. 2A). Using ordinary extracts prepared from the entire MSG, a few vague bands were observed, but we could not examine the binding specificity of the factors responsible for such vague bands. We prepared a new extract exclusively of the region where ser1 is expressed. The posterior section of the MSG on day 2 of the fifth instar was collected and treated for the preparation of tissue extracts, as previously described (Takiya et al., 1990). We designated this extract “MSG-P extract”. EMSAs of MSG-P extracts generated specific patterns with the Sr-320 and Sr-180 probes (Fig. 2B). Competition experiments using oligonucleotides containing POU-M1-binding elements showed that these complexes were different from POU-M1 (see þPOU.C lanes in Fig. 2B). These complexes were named MSGeintermolt-specific complex-A (MICA) for Sr-180 and -B (MIC-B) for Sr-320.
3.2. Binding of MSG-specific factors to the promoter-proximal elements
3.3. The region around 70 of the sericin-1 gene was indispensable for promoter activity in vivo
In addition to two POU-M1-binding elements between 201 and 116 (Matsuno et al., 1990), and one Fkh-binding element
The Sr-180 oligonucleotide contained the typical POU-M1binding element ATGAATAA, and the Sr-320 oligonucleotide
from the entire middle silk glands, and MSG-P extracts were prepared only from the posterior region of the middle silk gland. 2.5. Electrophoretic mobility shift assay The electrophoretic mobility shift assay (EMSA) was carried out as previously described by Matsuno et al. (1989) and Takiya et al. (1997, 2005) using the oligonucleotides shown in Supplementary Table 2 as the probes and competitors. The proteineprobe complexes were separated on 7% polyacrylamide gels. For the competition experiments, a 100-fold molar excess of unlabeled oligonucleotides was added to the reaction. 3. Results 3.1. Deletion assay of the sericin-1 gene promoter in vivo
Fig. 5. EMSAs using the probes Sr-1350, Sr-340, and Sr-310. (A) The binding factors in each extract shown above the panel were analyzed using the Sr-1350, Sr-340, and Sr-310 oligonucleotides as probes. MIC-A detected with Sr-310 probe and factor B detected with Sr-340 probe (see Fig. 9B) are marked. (B) Competition of MIC-A detected with the Sr310 probe with the Sr-70S and its mutant oligonucleotides.
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contained the typical LIM-HD-binding element CAATTA. We performed additional EMSAs using probes including a core sequence (TAAT) recognized by homeodomain (HD) proteins or probes including AT-rich cis-elements. It was found that MIC-A bound strongly to the Sr-70 probe (Fig. 3A and Supplementary Fig. 1), although this probe contained neither typical POU-M1-binding elements nor LIM-HD-binding elements. MIC-A on the Sr-70 probe competed effectively with the oligonucleotides Sr-320, Sr180, and Sr-70 itself, and partly with Sr-1350 (Supplementary Fig. 2). Competition experiments with mutant oligonucleotides of Sr-70 clearly demonstrated the sequence specificity of MIC-A binding (Fig. 3B). The Sr-70S M1, M2, M3, and M5 mutants did not compete with the MIC-A binding to the Sr-70 probe, whereas the Sr-70S M4 competed as efficiently as the wild type oligonucleotides. We constructed reporter plasmids, PGV-ser1 (70M2) and PGVser1 (70M4), with the same two base-pair mutations as Sr-70S M2 and M4 oligonucleotides, respectively. These plasmids were introduced into silk glands and analyzed for promoter activity. As shown in Fig. 3C, PGV-ser1 (70M2) lost almost all promoter activity, while PGV-ser1 (70M4) indicated the same activity as the wild type construct. Thus, the region around 70 of ser1 was indispensable for promoter activity in vivo.
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3.4. MIC-B contained MIC-A and had the same DNA-binding specificity The oligonucleotide, Sr-320, effectively competed with MIC-A binding to the Sr-70 probe (Supplementary Fig. 2). We further analyzed the relationship between MIC-A and MIC-B by examining their binding specificity with various mutant oligonucleotides as competitors (Fig. 4). MIC-A with the Sr-70 probe and MIC-B with Sr320 were similarly competed with the Sr-320S mutant oligonucleotides (compare Fig. 4A with Fig. 4B). Sr-320S M3 and M4 lost the ability to compete with both MIC-A and MIC-B, and the effects of other mutants were partial. The wild type and mutant Sr-180 oligonucleotides also competed with both MIC-A and MIC-B with similar efficiency (Fig. 4C), to the extent that DNA-binding specificity of MIC-A and MIC-B was essentially identical. During the course of this study, we found that the Sr-320 oligonucleotide contains an atypical LIM-HD-binding element CATAAT around 340 (Supplementary Fig. 3). Two LIM-HD-binding elements around 340 and 310 within the Sr-320 probe were separated into the oligonucleotides, Sr-340 and Sr-310, and these oligonucleotides were used for EMSAs as probes. As shown in Fig. 5, these probes detected different complexes; the first one was denoted as MIC-A for the Sr-310 probe and the other one was
Fig. 6. Competition of MIC-binding to Sr-70 or Sr-320 probes with Sr-1350 region. Sr-1350S and its mutants were used as competitors for the binding of MIC to Sr-70 (A) or Sr-320 (B). Effects of Sr-340 and Sr-750 oligonucleotides on MIC-binding to Sr-70 were also examined. (C) Nucleotide sequences of the competitors. The putative MIC-binding element and LIM-HD-binding elements are shown with lines.
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denoted as factor B for the Sr-340 probe. MIC-A detected with the Sr-310 probe showed identical DNA-binding specificity as those detected with the Sr-70 and Sr-180 probes (Fig. 5B and Supplementary Fig. 4). 3.5. MIC-A bound to the region around 1350 of the sericin-1 gene When the Sr-1350 oligonucleotide was used as a probe in EMSA, a vague but specific band that was detected in MSG-P extracts appeared around the MIC position (Fig. 5A and Supplementary Fig. 1). The affinity of MIC-binding to the Sr-1350 probe was not strong enough to directly examine the binding specificity. Competition experiments were conducted with its mutant oligonucleotides as competitors. Competition for MIC-A binding to Sr-70 with the Sr-1350 and Sr-1350S oligonucleotides was partial, but sequence specificity for the competition was clearly observed (Fig. 6A and C). Sr-1350S M3 and M4 could not compete with MIC-A binding. The effects of these mutant oligonucleotides on MIC-B were the same as on MIC-A (Fig. 6B). The results indicate that the 1350 region of ser1 is a MIC-A binding site. 3.6. The region around 1350 of the sericin-1 gene enhanced the promoter activity in MSG The region around 1350 of ser1 contains overlapping sequence elements recognized by many HD proteins, such as LIM-HD, POU-HD, Antp, Ubx, Dfd, and Ftz (Fig. 7). We analyzed function of the region around 1350 using a deletion mutant PGVser1(1340/þ42). The deletion from 1362 to 1340, lacking all the HD-protein-binding sequences, decreased the promoter activity to the same level as PGV-ser1 (1249/þ42) (Fig. 7). Thus, the 1350 region maintained a tissue (MSG)-specific enhancerlike activity.
Fig. 7. Enhancement of the expression by the 1350 region of the sericin-1 gene. PGVser1 (1602/þ47), (1362/þ47), (1340/þ47), (1249/þ47), and (201/þ47) were introduced into the silk glands, and the luciferase activity was measured after separating the MSG and PSG. The activity is indicated relative to the activity of PGVser1(1602/þ47) in the MSG. The p value indicated with * is <0.05 and with ** is <0.02. The nucleotide sequence around the 1350 region is shown above. HD-binding elements overlapping around 1350 are shown with thick bars below the sequence. The deletion positions of PGV-ser1 (1362/þ47) and PGV-ser1 (1340/þ47) are indicated with arrows over the sequence.
3.7. Coexistence of oligonucleotides, including LIM-HD-binding elements, affected MIC-A binding to the ser1 promoter The Sr-70 oligonucleotide did not contain either typical (CAATTA) or atypical (CATAAT) LIM-HD elements; however, the oligonucleotides, Sr-1350, Sr-320 and Sr-180 contained the typical LIM-HD element. We investigated the effects of oligonucleotides containing the LIM-HD-binding elements on MIC-A binding to the Sr-70 probe. Addition of the Sr-340 oligonucleotide to the reaction weakened the MIC-A band on Sr-70. The intensity of the MIC-A band was recovered with a mutation of the LIM-HD element in Sr-340 (Fig. 6A). On the other hand, Sr-750, including both typical and atypical LIM-HD elements (Fig. 6C), stimulated MIC-A binding to the Sr-70 probe, and its mutant lost such ability. Similar phenomena were clearly observed with the Sr-320 probe (Fig. 8A). In the presence of the Sr-340 oligonucleotide, a weak but sharp MIC-A band was detected instead of a MIC-B band. In the presence of the Sr-750 oligonucleotide, a highly strengthened MIC-A band was seen, and mutations of the LIM-HD-binding elements in Sr-750 diminished the effect. Because a clear and strong MIC-A band was observed with the Sr-320 probe in the presence of the Sr-750 oligonucleotide, the effects of the Sr-1350 and its mutant oligonucleotides on MIC-A on the Sr-320 probe were examined in the presence of Sr-750. The binding specificity of MIC-A to the Sr-1350 region was reconfirmed. The mutants, M3 and M4 did not compete MIC-A binding, and M5 and M6 competed partially (Fig. 8B).
3.8. Consensus sequence recognized by MIC-A MIC-A seemed to bind elements recognized by HD proteins. We selected several sequence elements from the intron of ser1 (Supplementary Table 2) and examined the ability of the oligonucleotides to compete with MIC-binding. Of these oligonucleotides, the Srþ1380 and Srþ2440 competed with MIC-binding almost completely, while Srþ1890, containing a typical LIM-HD element,
Fig. 8. Effects of LIM-HD-binding elements on MIC-A-binding to the Sr-320 probe. (A) Competition effects of the LIM-HD-binding elements on the binding of MIC to Sr-320 were examined in EMSAs with the oligonucleotides Sr-340, Sr-340S, Sr-750 and Sr750M as competitors. (B) Effects of Sr-1350S and its mutant oligonucleotides on MIC-binding to the Sr-320 probe in the presence of Sr-750. In addition to the competitors, all EMSA reactions contained a 100-fold excess amount of Sr-750.
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indispensable for promoter activity of ser1 in the MSG. The 70 region contains the core sequence (TAAT) recognized by many HD proteins, whereas the 1350 and 180 regions contain sequences recognized by LIM-HD proteins. To investigate whether there is a factor that specifically binds to these elements, a new MSG extract (MSG-P extract) was prepared from the posterior region of MSG. With this MSG-P extract, tissue- and stage-specific binding factors (MIC-A and -B) were clearly detected. The factors were related to each other in their DNA-binding specificity and were bound to the 1350, 320, 180, and 70 regions with slightly different EMSA patterns. MIC-B was first detected with the Sr-320 probe was thought to contain at least two DNA-binding factors or factor complexes (Fig. 9B): one (factor B) bound to the sequence element CATAAT around 340, and the other (MIC-A) bound to the 10-bp AT-rich element around 310, overlapping with the LIM-HDbinding element CAATTA. MIC-A bound to the 10-bp region which might be divided into two overlapping sequence elements recognized independently by factors A and A0 , representing different surfaces of the protein. Mutations at the overlapping nucleotides inhibited the binding of MIC-A completely, while mutations at the nucleotides recognized by either A or A0 inhibited it partly. Therefore, MIC-A was competed partially even with the mutant oligonucleotides at the 10-bp MICA-binding sequence. The element recognized by factor A0 around 1350 was likely destroyed in part by the T nucleotide, shown in Fig. 9, and the intensity of the MIC band with the Sr-1350 probe was low. The broad band (MIC-B) observed with the Sr-320 probe seemed to be due to interactions of multiple factors. The band (MIC-B)
did not (Supplementary Fig. 5). Comparison of the nucleotide sequences of the MIC-binding elements and other LIM-HD-binding elements indicated that the 10-bp sequence occasionally overlapped with the LIM-HD-binding sequence CAATTA, and the region extended 5-bp from the side of the CAATTA element (Fig. 9A). 4. Discussion Genes introduced transiently into cultured cells were reported to be modified and to form a chromatin structure similar to the endogenous genes within several hours (Agelopoulos and Thanos, 2006). The in vivo expression system for analyzing promoter activity using a gene gun and organ transplantation in the silkworm B. mori has led to the identification of enhancer and promoter elements necessary for the PSG-specific expression of fhx and fibH (Horard et al., 1994, 1997; Nony et al., 1995; Shimizu et al., 2007). Moreover, the promoter elements of a Bombyx wing cuticle protein gene were recently identified using a transient assay system with the gene gun (Wang et al., 2009). We applied this method to the ser1 gene expressed specifically in the MSG. The region from 1602 to þ47 of ser1 was sufficient for MSG-specific transcription, and the promoter elements that had not been found in vitro were detected. Although the efficiency was as high as that of the fibH gene carrying UEEs under the conditions used in this study, we do not exclude the possibility that additional enhancer elements exist in the further upstream region and intron of the ser1 gene. A series of deletions in the ser1 promoter showed that at least three regions around 1350, 180, and 70 were important for specificity and efficiency in vivo. The 70 region was particularly
A
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B
Fig. 9. Consensus sequence found in MIC-binding elements. (A) Nucleotide sequences of the 6 MIC-binding sites and 4 MIC-unbound but putative binding sites for LIM-HD are shown. The elements corresponding to the consensus sequence for MIC-binding are boxed, and putative binding sites for POU-M1 and LIM-HD are indicated with lines. The dotted T nucleotide at 1341 is probably not part of the consensus sequence and weakens the binding of MIC to the 1350 region. Because the mutant oligonucleotide Sr-320 M4 competed almost completely with MIC-binding to the Sr-320 probe (Supplementary Fig. 3), a change of the A nucleotide at position 2 to the G nucleotide is probably occurring, but we did not find an endogenous MIC-binding element of which the nucleotide at position 2 is G. (B) A model for MIC-binding to the ser1 promoter. MIC-A has two DNA-binding surfaces or is composed of at least two factors A and A0 and binds to the elements around 1350, 320, 180, and 70, containing the consensus sequence. Factors A and A0 independently recognize overlapping half sites of the consensus sequence, and MIC-A can bind weakly to the half sites. Factor B, recognizing the element CATAAT, forms MIC-B with MIC-A and likely stabilizes MIC-A-binding; thus, MIC-B was easily detected with the Sr-320 probe. Factor C, recognizing CAATTA, disturbs the binding of MIC-A to the 1350, 320, and 180 regions. Competition of factor C with Sr-750 enhanced the binding of MIC-A to these sites.
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obtained with the Sr-320 probe became a sharp band (MIC-A) upon the addition of the oligonucleotide Sr-340 or Sr-750 as a competitor. These oligonucleotides include the sequence element, CATAAT, in Sr-340 or CATAAT and CAATTA in Sr-750 (Fig. 6C), recognized by the LIM-HD protein family but not the consensus sequence recognized by MIC-A. Furthermore, competition with the oligonucleotides, Sr-140, SC and POU.C, including the POU-M1-binding element, weakened the intensity of the MIC-B bands (Supplementary Fig. 6). Together with factors B and C, and POU-M1, might be responsible for the formation of the broad band (MIC-B). The addition of Sr-750, but not Sr-340, to the EMSA reactions with the Sr-320 probe enhanced the MIC-A band remarkably. Therefore, factor C seemed to compete for MIC-A-binding sites with factor A, and might have an inhibitory effect on ser1 gene expression. LIM-HD proteins are candidates for the identification of factor C. Effects of the Sr-340 and Sr-750 oligonucleotides were also observed on MIC-A bound to the Sr-70 probe. Factors B and C may induce their effect via the core sequence (TAAT), recognized by HD proteins, or through direct interactions with MIC-A. The Sr-180 oligonucleotide overlaps partly with the SC site of the promoter-proximal enhancing element, detected in vitro, and binds POU-M1 (Matsuno et al., 1989, 1990). In the 1350 region, many HD-binding elements, including the POU-HD-binding element, overlapped, but competition with SC oligonucleotide and POU-specific competitor POU.C did not disturb the formation of MIC-A on the Sr-70 and Sr-180 probes. Although the role of POUM1 in ser1 gene expression was not investigated in this study, we support the assumption that POU-M1 plays an inhibitory role in ser1 gene expression. The expression level of POU-M1 is complementary to that of ser1 (Kokubo et al., 1997; Matsunami et al., 1998), and POU-M1-binding sites of the POU-M1 gene promoter tend to inhibit its transcription in vitro (Xu et al., 1994). Moreover, cointroduction of a POU-M1 expression vector together with the ser1-reporter construct into silk glands suppressed ser1 gene expression (Kimoto et al., unpublished results). In contrast, MIC-A was detected in the posterior part of MSG, where ser1 is expressed efficiently, and bound to the promoter and enhancer elements indispensable for the expression of ser1 in MSG. The results suggested that MIC-A is intrinsic for tissue- and stagespecific expression of ser1 in vivo. Acknowledgments We thank Dr. Y. Nishita for the discussions and advice regarding the luciferase assay. We also deeply appreciate the anonymous reviewers for their helpful advice. Appendix. Supplementary material Supplementary material related to this article can be found at doi:10.1016/j.ibmb.2011.03.011. References Agelopoulos, M., Thanos, D., 2006. Epigenetic determination of a cell-specific gene expression program by ATF-2 and the histone variant macroH2A. EMBO J. 25, 4843e4853. Cheema, S.K., Gobin, A.S., Ohea, R., Lopez-Berestein, G., Newman, R.A., Mathur, A.B., 2007. Silk fibroin mediated delivery of liposomal emodin to breast cancer cells. J. Pharmaceutics 341, 221e229. Couble, P., Michaille, J.-J., Garel, A., Couble, M.L., Prudhomme, J.-C., 1987. Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silk glands. Dev. Biol. 124, 431e440. Fukuta, M., Matsuno, K., Hui, C.-c., Nagata, T., Takiya, S., Xu, P.-X., Ueno, K., Suzuki, Y., 1993. Molecular cloning of a POU-domain-containing factor involved in the regulation of the Bombyx sericin-1 gene. J. Biol. Chem. 268, 19471e19475. Gobin, A.S., Rhea, R., Newman, R.A., Mathur, A.B., 2006. Silk-fibroin-coated liposomes for long-term and targeted drug delivery. J. Nanomedicine 1, 81e87.
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