Involvement of an Upstream Stimulatory Factor as Well as cAMP-responsive Element-binding Protein in the Activation of Brain-derived Neurotrophic Factor Gene Promoter I*

Antibodies

Antibodies used for the supershift assay were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). These were as follows: rabbit polyclonal antibody against ATF2 (N-96), C/EBPβ (C-19), C/EBPδ (C-22), the CREB/CREM family (X-12) (which partially cross-reacts with other ATF/CREB and CREM families), c-Fos (D-1), Fra1 (N-17), USF1 (C-20), and USF2 (N-18) and mouse monoclonal antibody against ATF1 (C41–5.1) (which specifically reacts with ATF1, but not with other ATF/CREB transcription factors). Additionally, mouse monoclonal antibody against the CREB family (25C10G) (not specific for ATF1) reacts with ATF1, CREB and CREM; furthermore, mouse monoclonal antibody against CREB (24H4B) specifically reacts with CREB, but not with other ATF/CREB transcription factors. Normal IgG was also used as a control.

Cell Culture

Primary cultures of rat cortical neurons were prepared from the cerebral cortexes of 17–18-day-old rat (Sprague-Dawley) embryos as described previously (18). Briefly, small pieces of cerebral cortex were dissected by enzymatic (DNase I (Sigma) followed by trypsin (Sigma)) treatment and mechanical dissociation, and the cells were seeded at 5 × 10⁶ cells in a 60-mm culture dish (Iwaki). The cells were grown for 48 h in Dulbecco's modified Eagle's medium (Nissui) containing 10% fetal calf serum, and then the medium was replaced with serum-free Dulbecco's modified Eagle's medium containing glucose (4.5 mg/ml), transferrin (5 μg/ml), insulin (5 μg/ml), sodium selenite (5 μg/ml), bovine serum albumin (1 mg/ml), and kanamycin sulfate (100 μg/ml) (TIS medium). Cytosine arabinoside (Sigma) was also added at 2 μm to prevent the proliferation of glial cells. The medium was replaced with fresh TIS medium, but devoid of cytosine arabinoside, 2 h before DNA transfection.

RNA Isolation and Reverse Transcription (RT)-PCR

Total RNA was extracted from the cultured cells using ISOGEN (NipponGene). RT-PCR was performed as described previously (19). Briefly, total RNA (1 μg) was reverse transcribed into cDNA in 20 μl of 1× first strand buffer containing 0.5 μm oligo(dT)15 (5′-AAGCTTTTTTTTTTV-3′) as a primer, 200 units of SuperScript II reverse transcriptase (Invitrogen, Lifetech Oriental), 400 μm dNTPs, and 10 units of RNase inhibitor (Invitrogen, Lifetech Oriental) as recommended by the manufacturer. After reverse transcription, the reaction mixture was treated with 1.1 units of RNase H (Invitrogen) at 37 °C for 20 min and used for PCR as cDNA solution. PCR was performed in 50 μl of 1× PCR buffer containing 1 μl of cDNA solution, 1.25 units of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences), 1.5 mm MgCl₂, 200 μm dNTPs, and 0.5 μm primer pair. To distinguish four alternative exons (exon I, exon II, exon III, and exon IV) of the rat BDNF gene, E-I (5′-ACTCAAAGGGAAACGTGTCTCT-3′), E-II (5′-CGGTGTAGGCTGGAATAGACT-3′), E-III (5′-CTCCGCCATGCAATTTCCACT-3′), E-IV (5′-GTGACAACAATGTGACTCCACT-3′), and E-Vas (5′-GCCTTCATGCAACCGAAGTA-3′) were used. For the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified using GAPDH sense (5′-TCCATGACAACTTTGGCATTGTGG-3′) and antisense (5′-GTTGCTGTTGAAGTCGCAGGAGAC-3′) primers. For amplification of BDNF cDNA, the PCR conditions, after preheating at 95 °C for 10 min, were as follows: denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1.5 min for 32 (exon I), 31 (exon II), 26 (exon III), and 29 (exon IV) cycles, and a final extension at 72 °C for 10 min. GAPDH amplification was carried out for 31 cycles under the same conditions. PCR products were separated by electrophoresis on 2% agarose gels, and the densities of the DNA bands stained with ethidium bromide were analyzed using a Bit-Map loader (ATTO, Japan) and software (NIH Image 1.52).

Reporter Vectors Used for Promoter Analysis and Overexpression Experiments

For the promoter analysis, the luciferase reporter vector, pGL3-BDNF promoter I (pBDNFpI), was constructed by ligation of pGL3-Basic vector (Promega) with the region (−528 to +138) of rat BDNF gene promoter I. The procedure for plasmid construction was described previously (18). As an internal control vector against the luciferase experimental vector, we used Renilla luciferase vector carrying the human elongation factor 1α promoter (pRL-EF1α) (7). For the overexpression experiments shown in Fig. 9, we used β-galactosidase vector carrying the human elongation factor 1α promoter (EF1α-β-gal) as an internal control because pRL-EF1α itself could interfere with the assessment of firefly luciferase gene induction (data not shown).

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 9

Effects of overexpression of dominant negative CREB or USF on the activation of BDNF promoter I. The dominant negative expression vectors of CREB M1 (A), K-CREB (B), A-CREB (C), and A-USF (D) were co-transfected with firefly luciferase reporter vectors carrying BDNF promoter I and E. coli β-galactosidase internal control vectors carrying EF1α promoter into rat cortical neurons. Empty vectors were co-transfected as a control. Forty hours after transfection, cells were stimulated with 25 mm KCl (+) or vehicle (−) and incubated for another 6 h. Cell lysates were then extracted for measuring luciferase activity. The value for the transcriptional activation of pBDNFpI(full) obtained with 25 mm KCl was standardized to 100%. The variability in the basal transcriptional activities is probably caused by an experimental flexibility, which could be caused mostly by culturing conditions. The data represent the mean ± S.E. from experiments performed in triplicate. The same result was obtained from three independent experiments.

Plasmid pBDNFpI(Δ488–344) was generated by double digestion of pGL3-BDNFpI with BglII and SpeI, and blunted with T4 DNA polymerase (TaKaRa DNA blunting kit). The digested plasmid was then self-ligated. pBDNFpI(Δ378–349) was first generated by PCR with primers (forward A: 5′-GGATCCTCCCCTCCTAGCCTAT-3′; reverse: 5′-GCGACCGCGGCTTGAGTTGAATGAA-3′) to produce the fragment that corresponded to the upstream region from −528 to −379. After the fragment was ligated into the SmaI site of pGL3-Basic vector (this plasmid was termed the up-vector), another PCR fragment amplified with primers (forward: 5′-TATCCGCGGCAACTAGTGGCTCGCCC-3′; reverse B: 5′CGCAAGCTTCCTGGGGCTGTGGCAAAGAT-3′) that corresponded to the downstream region from −348 to +138 was inserted into the SacII and Hind III sites of the up-vector. In the manner described above, a series of internal deletion mutants was then constructed: pBDNFpI(Δ357–331) (forward A, reverse: 5′-CGACCGCGGTGTAAGCCAAGCTCTC-3′; and forward: 5′-TATCCGCGGGTGCCTCTCGCCTAGTC-3′, reverse B), (Δ335–309) (forward A, reverse: 5′-CGCGATGGCCACTAGTTGCCCACAGGAA-3′; forward: 5′-CAATATGGCCAGTCCCTAAGAGGAAAAGGG-3′, reverse B), (Δ345–184) (forward A, reverse: 5′-ATTATGGGCCCACAGGAACCGGTG-3′; and forward: 5′-CGCGCTGGGCCCTAGAACAAGTCACTCC-3′, reverse B), (Δ277–184) (forward A, reverse: 5′-CGCGATGGCCACAACTTTCCCTT-3′; and forward: 5′-CAATATGGCCACGTCCGCTGGAGACCCTT-3′, reverse B), (Δ277–205) (forward A, reverse: 5′-CGCGATGGCCACAACTTTCCCTT-3′; and forward: 5′-CAATATGGCCATTTGATCATCACTCACGACC-3′, reverse B), (Δ277–223) (forward A, reverse: 5′-CGCGATGGCCACAACTTTCCCTT-3′; and forward: 5′-CAATATGGCCATCACCCCTCCCCCCC-3′, reverse B), (Δ288–245) (forward A, reverse: 5′-GCTAGAGGCCTTTTCCTCTTAGGGACTGAT-3′; and forward: 5′-CATATAGGCCTAGAACAAGTCACTCCTGCT-3′, reverse B), (Δ173–154) (forward A, reverse: 5′-CGACCGCGGACGTGGTCGTGAGTGA-3′; and forward: 5′-TATCCGCGGGGGAGGGGCACGAACTTT-3′, reverse B), (Δ151–114) (forward A, reverse: 5′-GCTATGGCCACCATGACTAAGGG-3′; and forward: 5′-TGCGCTGGCCAAGGGAGTCACAGTGAGTT-3′, reverse B), (Δ107–31) (forward A, reverse: 5′-GCGCGAGGCCTTGGTAAAAAGGA-3′; and forward: 5′-CATATAGGCCTCGCGCTGCGCTTTTCTGG-3′, reverse B), and (Δ107–65) (forward A, reverse: 5′- GCGCGAGGCCTTGGTAAAAAGGA-3′ and forward: 5′-CATATAGGCCTGGCCCCCTCCCC-3′, reverse B). The site-directed mutagenesis for the substitution of a few bases or for the deletion of 3–7 bases within pBDNFpI was performed using a QuikChange site-directed mutagenesis kit (Stratagene). CRE2m, TRE1m, TRE3m, CRE2Δ, TRE1Δ, TRE2Δ, and TRE3Δ were generated, using as forward primers, 5′-CAGTGAGTTGGTCACGGACCTGGCTCAGAGAGGCTG-3′, 5′-CCTTTTTACCAAGGGAGGACCAGTGAGTTGGTCACGTAAC-3′, 5′-GGTCACGTAACTGGCGACGAGAGGCTGCCCTGG-3′, 5′-GGGAGTCACAGTGAGTTGGTCTGGCTCAGAGAGGCTGCCCTG-3′, 5′-GAAGTTTCCTTTTTACCAAGCAGTGAGTTGGTCACGTAACTGGC-3′, 5′-CCAAGGGAGTCACAGTGAGCGTAACTGGCTCAGAGAGGCTGC-3′, and 5′-CAGTGAGTTGGTCACGTAACGAGAGGCTGCCCTGGCCCC-3′, respectively, and the complementary primer pair (underlined bases are substituted nucleotide bases). TRE2/CRE2Δ was generated by further deletion of 5 bases from CRE2Δ, using 5′-CCAAGGGAGTCACAGTGAGCTGGCTCAGAGAGGCTGCCC-3′ and the complementary primer.

Expression vectors, A-CREB and A-USF, were generously provided by Dr. C. Vinson (National Cancer Institute, National Institutes of Health, Bethesda, MD).

DNA Transfection, Luciferase, and β-Galactosidase Assays

DNA transfection was carried out over 3 days in culture and using calcium phosphate/DNA precipitation as already described (18). Briefly, the calcium phosphate/DNA precipitates were prepared by mixing one volume (100 μl) of plasmid DNA (6 μg, pBDNFpI:pRL-EF1α = 10:1) in a 250 mmCaCl₂ solution with an equal volume of 2× HEPES-buffered saline (50 mm HEPES-NaOH (pH7.05), 280 mm NaCl, 1.5 mm Na₂HPO₄), and added to each 60-mm dish. For overexpression experiments, we used plasmid DNA (6 μg, pBDNFpI:EF1α-β-gal:expression vector = 5:1:30). The dish was washed three times with phosphate-buffered saline and fresh TIS medium added. After 40 h, the transfected cells were stimulated with 25 mm KCl (25 mm KCl stimulation) or vehicle for 6 h and cell lysates were prepared.

For the dual (firefly and Renilla) luciferase assay, cell lysates were extracted with passive lysis buffer (Promega) and used as described previously (7). For the measurement of β-galactosidase activity as an internal control, cell lysates were extracted with 250 μl of cell lysis buffer containing 1 mm potassium phosphate (pH 7.8), 1 mm dithiothreitol, and 0.5% Triton X-100; 30 μl of the lysate was used for the chemiluminescence-based β-galactosidase assay (CLONTECH), and 20 μl for the firefly luciferase assay.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)

Forty hours after the medium exchange at 3 DIV, cells were stimulated with 25 mm KCl (25 mm KCl stimulation) or vehicle and incubated for 6 h. Then, nuclear extracts were prepared as described with minor modifications (20). EMSAs were carried out using probes corresponding to the BDNF-PI sequence (these are indicated in Fig. 4) as reported previously (21). Briefly, end-labeling of DNA probes was performed at 37 °C for 20 min in 10 μl of reaction mixture (6.6 mmTris-HCl, pH 7.4, 50 mm NaCl, 6.6 mmMgCl₂, 0.5 mm dATP, 0.5 mm dGTP, 0.5 mm dTTP, 1 mm dithiothreitol) containing 200 ng of DNA, 2 μl of [³²P]dCTP, and 1 unit of Klenow fragments. Then, DNA probes were recovered with a Sephadex G-50 column. The DNA-protein binding reaction was carried out at 25 °C for 15 min in 20 μl of binding buffer (20 mm HEPES-NaOH, pH 7.9, 80 mm NaCl, 0.3 mm EDTA, 0.2 mm EGTA, 0.2 mm phenylmethanesulfonyl fluoride, 10% glycerol, 2 μg of poly(dI-dC), 0.2–0.4 ng of ³²P-labeled DNA probes, and 5 μg of nuclear extract). Then, DNA-protein complexes were separated on 4% polyacrylamide gel at 132 V for 2.5 h. The protein concentration was determined by the method of Lowry. For supershift EMSA, a series of antibodies (2 mg/ml) was used at a dilution of 1:20.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 4

DNA binding activities in the proximal region and their sensitivities to CRE . A, DNA binding activities using four kinds of DNA probes covering the proximal region.a, nucleotide sequences covering the proximal region and position of DNA probes for EMSA used here. Four native promoter I DNA probes (npIPb-1, -2, -3, and -4) were designed to cover the proximal region from -−109 to −62 of BDNF promoter I. b, EMSA for detecting transcription factors which bind to the proximal region of BDNF promoter I. Rat cortical neurons were stimulated with 25 mm KCl and incubated for 6 h. Nuclear extracts were then prepared and subjected to EMSA after DNA-protein binding reaction. Inhibitors APV (200 μm) and nicardipine (Nic.; 5 μm) were added 10 min before the 25 mm KCl stimulation. B, competitive effect of consensus CRE (consCRE), npIPb-3, and its mutant (npIPb-3m1) on the DNA binding activity to npIPb-3. a, nucleotide sequences of competitors. Bold letters indicate the position of consensus CRE, CRE-PI, and mutated CRE-PI.Dotted gray letters indicate the substituted bases. b, using a labeled npIPb-3, a competitive binding reaction was performed with a 1-, 10-, and 50-fold molar excess of cold competitors. Arrows indicate the doublet of bands, the upper and the lower band, and asterisks nonspecific bands. F means free probes. The experiments were done twice, and the same results were obtained.

Production of BDNF Transcripts Containing Exons I, II, III, and IV Induced via Membrane Depolarization

Although the complicated structure of the BDNF gene is suggested to give rise to at least eight species of BDNF transcript (4), it has already been reported that the exon III-containing transcript is the major form, the expression of which is controlled by BDNF-PIII in response to the Ca²⁺signals evoked via membrane depolarization in rat cortical neurons (8,9). In addition, we have also reported that BDNF-PI is activated by membrane depolarization, the basal promoter activity being ∼8-fold less than that of BDNF-PIII (7, 18). To confirm whether endogenous 5′-exon specific BDNF transcripts, particularly those containing exon I, are induced by membrane depolarization or not, we examined the increase in each transcript by RT-PCR. To distinguish each transcript, we designed a forward primer corresponding to exon I, II, III, or IV and a reverse primer corresponding to the common exon V (Fig.1 A). As shown in Fig.1 B, the expression of three transcripts containing exons I, II, and III was up-regulated by the exposure of cells to 25 mm KCl in medium (25 mm KCl stimulation), whereas the expression of the exon IV-containing transcript remained constant even after the stimulation. The minimum cycle numbers required for detecting transcripts by RT-PCR were 32 and 26 cycles for exon I and III, respectively (see the legend of Fig. 1), indicating that the expression level of the exon I-containing transcript is lower than that of the exon III-containing transcript. These findings indicate that BDNF-PI is activated by membrane depolarization in neurons although the expression level of the exon I-containing transcript is quite low, compared with that of the exon III-containing transcript.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 1

Expression of 5′-exon-specific BDNF transcripts stimulated by membrane depolarization. A, structure of the BDNF gene and location of primers for RT-PCR. The BDNF gene accommodates four alternative promoters, BDNF-PI, -PII, -PIII, and -PIV, which reside upstream of exons I, II, III, and IV, respectively (4). Gray boxes indicate 5′-noncoding exons I, II, III, and IV; the closed box the region coding prepro-BDNF mRNA in exon V; and the open box the 3′-noncoding region.Arrows indicate the positions of primers used for RT-PCR.EI, EII, EIII, and EIV are specific forward primers corresponding to exons I, II, III, and IV, respectively, and the reverse primer (EV) is located in the coding region. B, rat cortical neurons (5 DIV) were stimulated with 25 mm KCl (25 mm KCl stimulation), and total RNA was isolated at the times indicated (0, 6, and 12 h) and subjected to RT-PCR. The relative intensities of amplified DNA fragment containing each exon were analyzed with an Imaging scanner and plotted. The minimum cycle numbers required for detecting the amplified DNA were 32, 31, 26, and 29 for exons I, II, III and IV, respectively. The data represent the mean ± S.E. from the experiment performed in triplicate, and the same tendency was obtained from at least two separate experiments.

Calcium-responsive Elements Located in Two Distinct Regions of BDNF Promoter I

To assign the calcium (Ca²⁺)-responsive elements in BDNF-PI, we constructed a series of firefly luciferase reporter vectors containing various lengths of the promoter I region with internal deletions (see Fig. 2) for the measurement of promoter activity. Each vector was transfected withRenilla luciferase control vector driven by EF1α promoter (pRL-EF1α) into rat cortical neurons at 3 DIV. We previously reported that the luciferase activity derived from the vector carrying the region from −528 to +138 increased in response to the Ca²⁺influx through L-VDCC, which was caused by 25 mm KCl stimulation, suggesting the presence of Ca²⁺-responsive elements within the region up to nucleotide position −528 on BDNF-PI (18). As shown in Fig. 2, the luciferase activities of plasmid vectors pBDNFpI(Δ378–349), -(Δ357–331), -(Δ335–309), -(Δ288–245), and -(Δ151–114) were nearly as inducible as those of pBDNFpI(full). Although the use of pBDNFpI(Δ488–344) decreased the luciferase activity, the deletion of the 5′-flanking region up to −310 (pBDNFpI(−311 to +138)) did not decrease the luciferase activity, compared with that of pBDNFpI(full) (data not shown), suggesting that the upstream region up to −310 is not so largely involved in the Ca²⁺ signal-mediated activation of BDNF-PI. In contrast, the use of plasmid vectors pBDNFpI(Δ345–184), -(Δ377–184), -(Δ377–205), and -(Δ377–223) resulted in a decrease in Ca²⁺ responsiveness of ∼40–60%, suggesting that this region includes the Ca²⁺-responsive elements. In addition, more marked decreases in Ca²⁺ responsiveness were observed with the deletion mutants pBDNFpI(Δ107–31) and -(Δ107–65), also suggesting a localization of elements responsible for a strong Ca²⁺ response in this region. We called these two regions the distal and proximal region, respectively. On the other hand, deletion of DNA sequences from −173 to −154 (pBDNFpI(Δ173–154)) rather increased the luciferase activity, indicating that DNA elements which function as silencers could be located within this region.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 2

Effect of internal deletion on transcriptional activation of BDNF promoter I. Firefly luciferase reporter vectors carrying various lengths of BDNF promoter I with an internal deletion were transfected into rat cortical neurons with pRL-EF1α control vector at 3 DIV. Forty hours after DNA transfection, cells were stimulated with 25 mm KCl (+) or vehicle (−), and incubated for 6 h. Then, cell lysates were extracted and used for luciferase reporter assay. The structures of a series of deletion constructs used here are illustrated on the left. Anarrow indicates the transcriptional initiation site (+1). The open box indicates the region of exon I (+1 ∼ + 138). Luciferase in the gray box indicates the firefly luciferase gene. The deleted regions in the BDNF promoter I are indicated by a bent line with the nucleotide positions. pBDNFpI(full) means the wild-type construct without any deletion. The value of KCl-stimulated transcriptional activation of pBDNFpI(full) was indicated as 100%. The data represent the mean ± S.E. from at least four independent experiments.

Critical Role of DNA Sequences Including the CRE-like Element and a Few Upstream Sequences in the Activation of BDNF-PI

As shown in Fig. 2, the proximal region from −107 to −65 was extremely effective in the activation of BDNF-PI. Therefore, we focused on the proximal region and investigated which DNA sequences within this region contribute to the activation of BDNF-PI. Using the TFSEARCH program (www.rwcp.or.jp/papia/) (22), we found that this region contained several candidate DNA-binding sites for transcription factors, such as CRE- and 12-O-tetradecanoylphorbol-13-acetate -responsive element (TRE)-like sequences (Fig.3 A). To investigate the role of these sites in the activation of BDNF-PI, we constructed a series of plasmid DNAs having internal mutations and deletions at the sites and transiently transfected them into rat cortical neurons. As shown in Fig. 3 B, mutation and deletion of the downstream TRE-like sequence (TRE3m and TRE3Δ) did not affect the luciferase activity, whereas a series of mutants of the CRE-like sequence (CRE2m, CRE2Δ) showed a marked decrease in luciferase activity. Deletion of the middle TRE-like sequence which partially overlapped with CRE (TRE2Δ) resulted in the same decrease as with the CRE2 mutants (CRE2m, CRE2Δ). On the other hand, mutants of the upstream TRE-like sequence (TRE1m, TRE1Δ) were also effective in decreasing the transcriptional activity. However, the mutation combined with the deletion of CRE (TRE1/CRE2Δ) decreased the BDNF-PI activation to almost the same level as CRE2Δ, suggesting a minor contribution of upstream TRE to the activation of BDNF-PI. Among the mutant plasmid vectors constructed, plasmid TRE2/CRE2Δ with an 11-base pair deletion containing middle TRE- and CRE-like sequences was most effective in reducing the level of BDNF-PI activity, indicating that the DNA containing the CRE-like sequence and a few bases upstream of this sequence are mainly responsible for the activation of BDNF-PI.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 3

Assignment of Ca²⁺-responsive element in the proximal region of BDNF promoter I. A, the DNA sequences covering from nucleotide position −109 to −62 and the location of TRE- and CRE-like elements (designated by TRE and CRE, respectively) indicated by the TFSEACH program are shown.Dotted bold letters indicate the bases substituted by mutagenesis, asterisks the deleted bases, anddashed lines the unchanged bases. B, mutations or deletions were introduced into the region from −109 to −62 of BDNF-PI, and the transcriptional activity was measured by transient DNA transfection. C, the construct accommodating the deletion of the distal region covering from −227 to −223 is shown as Δdistal, and that accommodating both the deletion and mutation (CRE2m) is shown as Δdistal/CRE2m. DNA transfection and 25 mm KCl stimulation were carried out by the same experimental schedule as described in the legend of Fig. 2. The gray ellipse indicates the CRE-like sequence (CRE-PI) and the gray rectangular box, the TRE-like sequences. The capital letter X in the ellipse and rectangular box indicates destruction of the CRE- or TRE-like sequence by mutagenesis. The value of KCl-stimulated transcriptional activation of pBDNFpI(full) was indicated as 100%. The data represent the mean ± S.E. from at least three independent experiments.

Additionally, we constructed a plasmid vector, pBDNFpI (Δdistal/CRE2m), with both a deletion of the distal region (Δ277–223) and mutation of CRE2 (Fig. 3 C). Compared with the luciferase activity of wild-type pBDNFpI(full), the activity of Δdistal was low, which was consistent with the data shown in Fig. 2. On the other hand, Δdistal/CRE2m showed a greater decrease in luciferase activity than Δdistal, the level of which was almost the same as that of CRE2m (Fig. 3 C). These results indicate that the proximal region, rather than the distal region, mainly contributes to the activation of BDNF-PI, induced by the Ca²⁺ signals in rat cortical neurons.

Transcription Factors That Bind to the Proximal Region

We next investigated the transcription factors that could bind to the proximal region. To do this, we designed four kinds of native promoter I DNA probes (npIPb-1, -2, -3, and -4) to entirely cover the proximal region, and we performed EMSA (Fig.4 A, a). Among these four kinds of DNA probes, DNA binding activity was obvious with npIPb-2 and -3 probes (Fig. 4 A, b). For npIPb-1 and -4 probes, however, shifted bands were only faint. An increase in DNA binding activity induced by 25 mm KCl was observed with npIPb-1, -2, and -3, but the increase obtained with npIPb-3 was very weak. Addition of APV, an antagonist for theN-methyl-d-aspartate receptor (NMDA-R), or nicardipine, an antagonist for L-VDCC, tended to reduce the DNA binding activity of npIPb-1, -2, and -3 probes, but the reduction was very weak with npIPb-3. The doublet of shifted bands were observed only with probe 3.

To investigate whether the binding to npIPb-3 probe involves the CRE-like sequence, we examined the competitive effect of a consensus CRE. As shown in Fig. 4 B (b), both the upper and lower bands were gradually erased when an excess of not only the cold npIPb-3 but also consensus CRE competitors was added. The mutation of two bases introduced into the CRE-like sequence (npIPb-3m1) failed to affect the DNA binding (Fig. 4 B, a andb). These results indicate that the CRE-like sequence in the proximal region functions as the CRE. The competitors containing a consensus TRE failed to compete with the binding to npIPb-3 probe, indicating that the TRE-like sequence on npIPb-3 was unable to function as the TRE. However, the binding to npIPb-2 probe was reduced by an excess of consensus TRE (data not shown), indicating that the TRE-like sequence on npIPb-2 probe functions as the TRE. These findings indicate that the CRE-like sequence in the proximal region works as the CRE and the proteins binding to npIPb-3 probe were different from those binding to the npIPb-2 probe.

Binding of CREB Family Transcription Factors to the CRE of BDNF Promoter I

To identify the DNA-binding proteins that can bind to the CRE-like sequence of BDNF-PI (CRE-PI), we performed EMSA using npIPb-3 probe, in which antibodies against transcription factors were added prior to the start of the DNA-protein binding reaction. When the anti-CREB family or anti-CREB specific antibody was added to the mixture containing npIPb-3 probe with nuclear extracts prepared from 25 mm KCl-stimulated cortical neurons, supershifted bands were observed but faintly (Fig. 5 A,lanes 2 and 3). When anti-ATF1, anti-ATF2, anti-c-Fos, or anti-C/EBPβ specific antibody was added to the mixture, no shifted band was observed (Fig. 5 A,lanes 4–7). When the native promoter III DNA probe (npIIIPb) containing CRE-PIII that was previously identified as a calcium-responsive element (CaRE3) (8, 9) was used, stronger shifted bands than those with npIPB-3 probe were observed in the presence of anti-CREB family or anti-CREB specific antibody (Fig. 5 B,lanes 2 and 3). Anti-phosphorylated CREB specific antibody was also effective in shifting the bands upon EMSA (data not shown). This supershift of bands caused by anti-CREB family or anti-CREB specific antibody was clearly observed with consensus CRE probe (Fig. 5 C, lanes 2and 3). To test further whether the CRE-PI in the proximal region could solely function as the CRE, we used DNA probes, pICRE and pIIICRE, in which the CRE-like sequence of BDNF-PI or -PIII was surrounded by the same sequences as surrounded the consensus CRE. As shown in Fig. 5, D and E (lanes 2 and 3), supershifted bands were observed in the presence of anti-CREB family or anti-CREB specific antibody, in which the supershift of the bands was observed at almost the same level as that of pIIICRE probe. Thus, the CRE-PI in the proximal region of BDNF-PI functions as the CRE by itself, which is able to introduce CREB or CREB family proteins for binding, and its effectiveness seems to be equal to that of BDNF-PIII. As described above, however, the level of supershift of the bands formed on npIPb-3 probe in the presence of anti-CREB family or anti-CREB antibody appeared to be lower than that formed on native pIII DNA probe (Fig. 5, A andB).

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 5

Analysis of transcription factors binding to the proximal region using supershift EMSA. Nuclear extracts were prepared from the cells stimulated with 25 mm KCl and were subjected to supershift EMSA after DNA-protein binding reaction. Antibodies (0.08 μg/ml) against the CREB family, CREB, ATF1, ATF2, c-Fos, C/EBPβ, and normal IgG were added to the reaction mixture before the DNA-protein binding reaction was started at 25 °C (see “Experimental Procedures”). S indicates the positions of supershifted bands. The experiments shown in A andB were performed using the DNA probes derived from BDNF promoter I (npIPb-3) and from BDNF promoter III including the CRE of BDNF-PIII (from bp −36 to −29) (npIIPb), respectively. Experiments inC–E were performed using the DNA probes containing the consensus CRE (C), and the CRE-like sequence of BDNF-PI (CRE-PI) (D) and of BDNF-PIII (CRE-PIII) (E), surrounded by DNA sequences that were randomly chosen from the nucleotide sequences of pBR322. The bold lettersin the DNA probes indicate the position of the consensus CRE, CRE-PI, and CRE-PIII. The experiments were done twice, and the same results were obtained.

Binding of USF to a Site Overlapping CRE

Searching other binding sites for known transcription factors in the proximal region of BDNF-PI using the data base TFSEARCH, we found that the USF-binding site (5′-TGGTCACGTAACTG-3′) was located and overlapped the CRE-PI (Fig.7 A). Therefore, we next investigated whether USF could be included in the DNA-protein complex formed on the npIPb-3 probe. As shown in Fig. 6 B, addition of anti-USF1 or -2 specific antibody prior to the DNA-protein binding reaction resulted in a complete disappearance of the lower band of doublet (lanes 4 and 5). Furthermore, addition of anti-USF1 but not anti-USF2 antibody partially erased the upper band and slightly shifted its position downward (lane 4). The partial disappearance and downward shift of the upper band were also observed with the consensus CRE probe (Fig.6 C, lane 4), although only the upper band was obtained with this probe. When anti-CREB family antibody was added, supershifted bands appeared faint with npIPb-3 probe but were clear with consensus CRE probe (Fig. 6, B and C,lanes 2), which was consistent with the results shown in Fig. 5 (A and C). Furthermore, using the npIPb-3 probe or consensus CRE, anti-c-Fos antibody did not give rise to the shifted band (Fig. 6, B and C,lane 3). Thus, the upper band of the doublet formed by npIPb-3 probe, at least in part, consists of CREB family transcription proteins or, at least, CREB and the lower band of the doublet consists of USF1/USF2 only.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 7

Effects of mutations in CRE-PI and the USF-binding site on DNA binding activity. EMSA was performed with the same experimental schedule as described in the legend of Fig. 4.A, nucleotide sequences of five DNA probes. Nucleotide bases in open boxes are substituted. In npIPb-3m1 and -3m4 probes, both CRE-PI and USF-binding sequences were destroyed by mutations. In npIPb-3m2 and -3m3, the USF-binding site but not CRE-PI was lost. B, the cells were treated with 25 mmKCl (+) or vehicle (−), and the nuclear extracts were prepared for EMSA after DNA-protein binding reaction. Arrows indicate the upper and the lower bands and asterisks nonspecific bands.F indicates free probes. The specific radioactivity of every mutant DNA probe was almost the same (data not shown). The experiments were done twice, and the same results were obtained.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 6

Binding of USFs to the proximal region.Supershift EMSA was performed according to the same experimental schedule as described in the legend of Fig. 5. A, the DNA sequences of the probes used here, consensus CRE and native promoter I DNA probe (npIPb-3), are shown and the underlined bold letters indicate the consensus CRE and CRE-PI. B and C, USF1 andUSF2 indicate antibodies acting specifically against USF1 and USF2, respectively. Antibodies against the CREB family and c-Fos were also added. The experiments were done three times, and the same results were obtained.

Changes in DNA Binding Activities Induced by Mutations of CRE-PI and/or the USF-binding site

Because we found that the CRE-PI and USF-binding site overlapped in the proximal region, we next investigated which DNA sequences are preferentially or commonly used for the binding of CREB family proteins or USF. For this purpose, we introduced several substitutions of nucleotide bases in the CRE-PI and USF-binding site by constructing four mutant DNA probes for EMSA (Fig.7 A). The npIPb-3m1 and -3m4 probes were designed to destroy both CRE-PI and the USF-binding site. On the other hand, the npIPb-3m2 and -3m3 were designed for destruction of the USF-binding site only. Comparing the intensities of the upper and lower bands formed on the npIPb-3 probe, as shown in Fig.7 B, the upper band was found to disappear with npIPb-3m1 and -3m3 but remain with npIPb-3m2 and partially so with -3m4, whereas the lower band tended to remain with npIPb-3m1 and -3m3 but disappear with npIPb-3m2 and -3m4. Mutation of npIPb-3m3 probe, even though it is located outside the CRE-PI, markedly decreased the upper band, the DNA-protein complex of which should include the CREB family. Mutation of npIPb-3m2 probe, introduced at the 5′ outside of the CRE-PI, selectively removed the lower band but not the upper band. Taken together, the formation of the upper band tended to be dependent upon the 3′ side sequences, and that of the lower band upon the 5′ side sequences within this region.

Transcriptional Activity of BDNF Promoter I Carrying the Mutated CRE/USF-binding site

Because substitutions of CRE-PI and/or the USF-binding site affected the binding activity of CREB family proteins and USF (Fig. 7), we constructed plasmid vectors in which the same substitutions were introduced into the proximal region of BDNF-PI and, then, measured transcriptional activity using transient DNA transfection. The luciferase activity of vector pBDNFpI-3m1 (also termed pBDNFpI-CRE2m in Fig. 3, A and B) decreased by approximately 50% in terms of Ca²⁺responsiveness (Fig. 8), which was consistent with the data shown in Fig. 3. Mutation of npIPb-3m2, which preferentially removed the lower band but not the upper band (Fig.7 B), resulted in a decrease of ∼20% in Ca²⁺responsiveness. In contrast, mutation of npIPb-3m3, by which both the upper and the lower bands were erased (Fig. 7 B), resulted in the greatest decrease in Ca²⁺ responsiveness. The luciferase activity of pBDNFpI-3m4 was almost the same as that of pBDNFpI-3m1, whose mutation resulted in the disappearance of both the upper and lower bands but the upper band tended to be stronger than that for mutation of npIPb-3m3. Thus, the Ca²⁺responsiveness of four reporter mutant vectors showed a good correspondence with the preferential binding of DNA probes into which the same mutation was introduced (Figs. 7 and 8), indicating an involvement of the transcription factors that preferentially bind to CRE-PI and the USF-binding site in the activation of BDNF-PI.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 8

Effect of mutation within CRE-PI and/or the USF-binding site on the activation of BDNF promoter I. We constructed reporter vectors carrying the same substitutions in CRE-PI and the USF-binding site as were introduced into the DNA probes for EMSA (Fig. 7) and transfected them into rat cortical neurons for measuring transcriptional activation. Cells were stimulated with 25 mm KCl (+) or vehicle (−). Transcriptional activation was indicated as a -fold increase of the control in which pBDNFpI(full) was transfected and the 25 mm KCl stimulation was omitted. The nucleotide sequences of pBDNFpI, pI-3m1, pI-3m2, pI-3m3, and pI-3m4 were shown in Fig. 7 A. The data represent the mean ± S.E. from experiments performed in triplicate. The same tendency was obtained from two independent experiments.

Effect of Dominant Negative CREB or USF on the Ca²⁺Responsiveness of BDNF-PI

To address further the crucial role of CREB family proteins and USF in the Ca²⁺ signal-mediated activation of BDNF-PI, mutant plasmid vectors of dominant negative CREB and USF were introduced into rat cortical neurons with a reporter vector, pBDNFpI(full). Instead of the Renilla luciferase vector, we used another control vector carrying an EF1α promoter-driven Escherichia coli β-galactosidase gene. Overexpression of CREB M1, a mutant in which the serine-133 residue was converted to alanine (23), inhibited the activation of BDNF promoter I up to ∼50% of the control transfected with empty vector (Fig.9 A). Overexpression of A-CREB, a mutant with an acidic extension at the N terminus of the CREB leucine zipper domain that could interfere with the binding of DNA and transcriptional activation of wild-type CREB (24), or K-CREB, a mutant with a substitution in the DNA-binding domain that could inhibit the binding of wild-type CREB (25), decreased the activation of BDNF-PI by ∼20% (Fig. 9, B and C). On the other hand, overexpression of A-USF, a mutant with an acidic extension at the N terminus of USF (26), resulted in a marked reduction of BDNF-PI activity up to ∼60% of the control (Fig. 9 D). Combined DNA transfection of CREB M1 and A-USF, however, tended to induce the death of cortical neurons transfected (data not shown).