Materials

Thrombin, ECLplus Western blotting detection reagent and [γ-³²P] ATP (>1,000 Ci/mmol) were from Amersham Pharmacia Biotech ( www.apbiotech.com). Heparin agarose was from Sigma ( www.sigmaaldrich.com). Calmodulin-dependent protein kinaseII and PKA were from New England BioLab,  www.neb.com. Goat peroxidase-conjugated anti mouse IgG was from Cell Signaling Technology, Inc. ( www.cellsignal.com) PKC was from Upstate Biotechnology ( www.upstate.com). Ni-NTA superflow resin was from Qiagen ( www.qiagen.com). The other chemicals were of the purest grade commercially available.

Construction of expression plasmid and expression in E. coli

The cDNA fragments containing the entire coding sequence of bMBD 2/3 were kindly provided by Dr. Mita of the National Institute of Entomological and Sericultural Science. Sequence analysis was done using an ABI sequencer 377 ( www.appliedbiosystems.co.jp). Sequence homologies were done by BLAST search (Altschul et al. 1990). The cDNA fragments containing the entire coding sequence of bMBD2/3 were amplified by PCR with primers containing BamHI or EcoRI. The amplified fragments were digested with BamHI and EcoRI. The digested fragment was inserted into the BamHI and EcoRI sites of an expression vector, pET32a. This cDNA was transformed into E. coli strain BL21. Transformed E. coli cells (BL21) were incubated overnight in LB medium. The medium was diluted to 1:100 and incubated for 3 h. Expression of thioredoxine-fusion protein was then induced by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG), followed by additional incubation for 24 h at 16 °C. The cells were collected by centrifugation at 5,000 g for 5 min, and then stored at −80 °C.

Purification of bMBD2/3 protein

All procedures were carried out at 4 °C. The frozen cells (26.5 g) were suspended in phosphate-buffered saline (PBS) [140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ (pH 7.3)], disrupted by sonication and then cleared by centrifugation at 12,000 g for 30 min. The fusion protein was bound to a Ni-NTA superflow resin equilibrated with buffer A (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole; pH 8.0). After washing, proteins were eluted with a step-wise increase in imidazole. The 0.15 M imidazole eluate was dialyzed against PBS. To remove His-tagged thioredxin, the protein was incubated with thrombin at 22 °C for 16 hr. After centrifugation the supernatant was applied to a Ni-NTA column equilibrated with buffer A. The passed-through fraction was applied to a heparin agarose equilibrated with 25 mM Hepes-NaOH (pH 7.6). After washing, proteins were eluted with a step-wise increase in NaCl. The 0.3 M NaCl eluate was recovered. The sample was stored at −80 °C. Proteins were determined using BSA (Fraction V, Sigma) as the standard by the method of Lowry et al. (1951).

SDS–PAGE

SDS–polyacrylamide gel analysis was performed according to the method of Laemmli (1970), using a 4.5% stacking gel and a 15% separating gel, at a constant current of 16 mA. The proteins in a gel were visualized with Coomassie Blue stain.

Electrophoretic mobility shift assay (EMSA)

The sequence of oligonucleotides used in EMSA is 5′-cc^mggcc^mggcc^mggcc^mgg-3′ as the methylated DNA probe and 5′-ccggccggccggccgg-3′ as the unmethylated DNA probe. The oligonucleotides were labeled with ³²P at their 5′ ends and gel purified prior to EMSA. The purified protein was incubated in 10 mM Tris HCl (pH 8.0) containing 3 mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40, 5% glycerol, 0.4 mg/ml BSA and one pico mole of radiolabeled probe. Samples were incubated for 30 min at 25 °C. Thirty picomoles of competitor DNA were used per binding reaction. To separate the protein-DNA complexes, the reaction mixtures were loaded onto a running nondenaturating 6% polyacrylamide gel. Electrophoresis was carried out. Gels were dried and band patterns were analyzed using a bioimaging analyzer (BAS 1000; FUJIX).

Preparation of brain crude homogenate from Bombyx mori

Crude protein kinase fraction was obtained from the brain of Bombyx mori. First, the brain was rapidly dissected out of 3-day-old larvae of 5th instar in Ringer's solution. The brain was homogenized in the homogenization buffer (25 mM Tris-HCl [pH 7.5], 50 mM mercaptoethanol, 2 mM EGTA, 1mM PMSF, and 0.25 M sucrose). The homogenate was used as the brain extract.

Protein kinase assay

The typical reaction mixture with a total volume of 20 ul contained 50 mM Tris-HCl (pH 7.5), 6.25 mM MgCl₂, 0.125 mM CaCl₂, 2 mM dithiothreitol, 0.1 mM EDTA, 0.1 ug bMBD2/3 protein, 50 units protein kinase and 50 µM [γ-³²P] ATP. After incubation at 25 °C for 25 min, the samples were treated with SDS–sample buffer. After electrophoresis, the gel was stained with Coomasie Brilliant Blue and dried. The band patterns were analyzed using a bioimaging analyzer. The position of bMBD2/3 protein on a gel was identified by Coomasie Brilliant Blue staining. Protein kinase C was assayed by addition of phosphatidyl serine, and dioleine. Ca/CaM dependent protein kinase II was assayed by addition of 1.2 µM calmodulin.

Western blotting

The recombinant protein was dialyzed against distilled water and freeze-dried. The protein was mixed with complete adjuvant and immunized into the rabbit. After boost injection, the serum was isolated. The antiserum was subjected to immunoaffinity chromatography with bMBD2/3 immobilized on the affinity column (2-fluoro 1-methyl pyridinium toluene-4-sulfonate activated cellulofine; Seikagaku [ www.seikagaku.com]). The tissue was homogenized in the homgenization buffer [10 mM Hepes (pH 7.6), 10% glycerol, 0.1 M KCl, 3 mM, MgCl₂, 0.1 mM EDTA, 1 mM Na₃VO₄, and 1 mM DTT]. After SDS–PAGE, the proteins were transferred to a nitrocellulose membrane (Advantec,  www.advantecmfs.com). After blocking, the primary antibody was incubated (1:5000). After washing, goat peroxidase-conjugated anti rabbit IgG (H+L) was incubated as secondary antibody (1:5000). After washing, protein bands were visualized using ECLplus Western blotting detection reagent and analyzed by Typhoon 9400 (Amersham Pharmacia Biotech).

West-western blotting

The brain was homogenized as described above. After SDS–PAGE, the protein was transferred to a nitrocellulose membrane. After blocking, the purified bMBD2/3 protein (10 ug/ml) was incubated in 10 mM sodium phosphate buffer (pH 7.4) containing 0.1M NaCl, 0.2mM, NaN₃, and 0.2 mM EGTA. After washing, the primary antibody was incubated (1:5000). After washing, goat peroxidase-conjugated anti rabbit IgG (H+L) was incubated as secondary antibody (1:5000). After washing, protein bands were visualized using ECLplus Western blotting detection reagent and analyzed by Typhoon 9400 (Amersham Pharmacia Biotech).

Molecular cloning of bMBD2/3

Multiple EST clones were identified by Blast search using the MBD2b sequence. One of the clones, bMBD2/3, originating from a Baculovirus-infected Bombyx mori fat body cDNA library was identified and sequenced (Figure 1). Its insert is 2.4 kbp long, including the poly A tail. The carboxyl terminal non-coding region was 1.5 kbp long. bMBD2/3 bears an open reading frame corresponding to aa 249. In insects, two MBDs (from A. domesticus [aMBD 2/3] and D. melanogaster [dMBD2/3]) were previously isolated and cloned. The expressed recombinant proteins were characterized (Ballestar et al. 2001; Roder et al. 2000; Tweedie et al. 1999). bMBD2/3 showed 67.6 and 53.7% similarity to aMBD2/3 and dMBD2/3, respectively. The methyl-CpG-binding domain of bMBD2/3 closely resembles that of aMBD 2/3 but differed from that of dMBD2/3 (Figure 2). However, the carboxyl-terminal domain was almost conserved among them. The carboxyl-domain is a transcriptional repression domain, which changes the local chromatin structure, mainly through recruitment of histone deacetylase. bMBD was suggested to bind DNA and regulate the transcription of B. mori.

Expression and purification of the recombinant protein

To characterize bMBD2/3, cDNA for bMBD2/3 was expressed in E.coli and purified using affinity chromatography. The cDNA for bMBD2/3 was inserted into an expression vector pET32a and expressed in E. coli as a His–tagged thioredoxin fusion protein. The fusion protein recovered in the soluble fraction was bound by a Ni-superflow column. The fusion protein was eluted after washing. After dialysis, the fusion protein was cut to remove thioredoxin and the soluble fraction was recovered. This fraction was then applied to a heparin agarose. SDS–PAGE analysis showed that the purified protein was homogeneous (Figure 3). The evaluated molecular mass on SDS–PAGE was 26 kDa. About 0.1 mg protein was recovered from 10 liter culture of E. coli.

EMSA of bMBD2/3

Whether the purified bMBD2/3 specifically binds methyl DNA or not was examined. As shown in Figure 4, lane 5, and lane 6, bMBD2/3 did not bind non-methyl-DNA, but did bind to methyl-DNA (Figure 4, lane 1 and lane 2). The DNA binding was inhibited by methyl DNA, but not inhibited by non-methyl DNA (Figure 4, lane 3, and lane 4). This result indicates that bMBD2/3 specifically bound methyl DNA.

Western blotting of bMBD2/3

The availability of an antibody against dMBD2/3, which shows weak methyl DNA binding, was shown, but the identification of aMBD2/3 using immuno blotting, which shows a strong binding, was not done. To ascertain the distribution of bMBD2/3 in the tissues of B. mori, immunoblotting of several tissues was done using the purified antibody as the primary antibody (Figure 5). One band around 27 kDa was detected in almost all tissues examined including the brain and silk gland (lanes 4 and 8).

Immunodetection of dMBD2/3 in D. melanogaster revealed two signals, one at Mr 30,000 and one at Mr 25,000, which represent the long and the short isoforms of the protein, respectively (Marhold et al. 2002). Early embryonic stages contained only the long isoform while late embryonic stages contained only the short isoform. Both proteins were present during embryogenesis. As described above, D. melanogaster genomic methylation is restricted to the early stage. It is apparent that the methyl DNA-related protein is expressed during embryogenesis in D. melanogaster.

In B. mori, methyl DNA was contained in DNA of larval silk glands (Patel et al. 1987). The DNA CpG methylation site of B. mori was recently identified using PCR (Tatematsu et al. 2003). We will try to clarify the relationships between bMBD2/3 and methylated CpG islands in the B. mori genome. Immunoblotting demonstrated that bMBD2/3 was expressed in the brain (Figure 5, lane 8). Methyl DNA binding protein plays important roles in brain development (Armstrong 1997). Further Blast search indicated that partial sequences for DNA methyl transferase were isolated from the brain cDNA library of B. mori (unpublished observations). bMBD2/3 and DNA methyl transferase may regulate DNA methylation and the transcription of neuropeptides in the brain of B. mori because mammalian methyl DNA binding protein regulates the transcription of brain-derived neurotrophic factor (BDNF) (Chen et al. 2003).

West-western blotting analysis using bMBD2/3

In mammals, some functional proteins that interact with methyl DNA binding protein have been identified using a two hybrid system (Kokura et al. 2001; Screaton et al. 2003; Lembo et al. 2003). But there are no reports of such proteins in insects. We tried a west-western blotting analysis to determine if interacting proteins for bMBD2/3 exist in B. mori. In the mammalian brain, methyl DNA binding proteins play important roles in gene expression (Chen et al. 2003). So brain extracts were used as proteins to interact with bMBD2/3. As shown in Figure 6, 10 protein bands interacting with bMBD2/3 were detected. Their molecular weights were 25, 29.5, 30, 31, 33, 41, 46, 55, and 65, respectively. One protein band was detected around 27 kDa. This protein was suggested to be bMBD2/3 because western blotting showed the band at about 27 kDa (Figure 6, lane 2). These results suggest that proteins that interact with bMBD2/3 exist in the insect brain.

Phosphorylation of bMBD2/3 in vitro

DNA binding proteins such as nuclear receptors, are well known to be phosphorylated by protein kinase such as PKC, cdk/cyclin, MAPK and PKA (Rochette-Egly 2003). Phosphorylation changes DNA binding, ligand binding, recruitment of chromatin remodellers and modifiers to decompact repressive chromatin. Finally phosphorylations can regulate transcription positively or negatively. Recently, methyl DNA binding protein was reported to be phosphorylated by protein kinase (Sakai et al. 2002). MeCP2, which is abundantly expressed in the central nervous system, was phosphorylated by calcium-dependent protein kinase (Chen et al. 2003). We examined the phosphorylation of bMBD2/3 by protein kinase and brain extract in vitro. The brain extract from the B. mori exhibited protein kinase activity of bMBD2/3 (Figure 7, lane 5). Using mammalian protein kinase, PKC and CaM kinase phosphorylated bMBD2/3 (Figure 7; lanes 3 and 4).

What is the significance of the phosphorylation of bMBD2/3? Phosphorylation may change the affinity for the methylated DNA and interactions with proteins. Phosphorylation of the DNA binding region may produce the release of bMBD2/3 from methylated-DNA and activate transcription. Actually, mammalian MeCP2 was phosphorylated in the brain and released from the promoter of BDNF. The determination of phosphorylated amino acid residues of bMBD2/3 must be determined. Further, in vivo phosphorylation of bMBD2/3 must be defined using radiolabeled insect brain.

MBD protein, except MBD4, interact to the various transcriptional regulatory protein such as histone deacetylase, ATPase, and DNA methyl transferase (Tatematsu et al. 2000; Brehm et al. 2000). Activated PKC was known to enter the nucleus from the cytosol and function. Phosphorylation of bMBD2/3 in the nucleus may change the interaction of these proteins. Regulatory proteins such as ATPase and deacetylase have been shown to be present in insects by enzymatic assays (Tweedie et al. 1999). As described above, bMBD2/3 was distributed in almost all tissues of adult B. mori (Figure 5) and some proteins that interact with bMBD2/3 in the insect tissues were detected using west-western blotting analysis (Figure 6). So it would be possible to identify and purify the regulatory proteins that interact with bMBD2/3 directly, using bMBD-bound affinity chromatography. Such information may clarify the relationship between the phosphorylated bMBD2/3 and the regulatory proteins.

Methylation of DNA has been reported in various insects. Insect MBD protein was reported to be only one type, although vertebrates have five types. The mammalian proteins each have specific characters. Mutations in MeCP2 are the cause of Rett syndrome, an X-linked progressive neurological disorder, and MeCP2 functions as a selective regulator of neuronal gene expression (Armstrong 1997; Chen et al. 2001). MBD3 plays an important role in embryo development and MBD2 influences some forms of behavior (Jung et al. 2003; Hendrich 2001). MBD4 regulates the removal of thymine or uracil from a mismatched CpG site (Hendrich et al. 1999). Recently, proteins that specifically interact with MBD were identified using a two hybrid system (Kokura et al. 2001; Screaton et al. 2003; Lembo et al. 2003). These interacting proteins probably determine the physiological specificity of MBD proteins as described above. Identification of interacting proteins with bMBD2/3 will help to clarify the physiological role of bMBD2/3 in the insect cell and discover new functions specific to insect MBD.