Brain-derived Neurotrophic Factor Expression in Vivo Is under the Control of Neuron-restrictive Silencer Element*

T<A><AC>o</AC><AC>&cjs1171;</AC></A>nis TimmuskDagger §parallel , Kaia PalmDagger §, Urban Lendahl**, and Madis MetsisDagger Dagger Dagger §§¶¶

From the Dagger  Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden, the § Department of Developmental Neuroscience, Biomedical Center, Uppsala University, S-751 23 Uppsala, Sweden, the ** Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden, the Dagger Dagger  Center for Gene Technology, Tallinn Technical University, and the §§ Institute of Chemical Physics and Biophysics, Tallinn EE0026, Estonia

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Neuron-restrictive silencer element (NRSE) has been identified in multiple neuron-specific genes. This element has been shown to mediate repression of neuronal gene transcription in nonneuronal cells. A palindromic NRSE (NRSEBDNF) is present in the proximal region of brain-derived neurotrophic factor (BDNF) promoter II. Using in vitro binding assays, we establish that the upper half-site is largely responsible for the NRSEBDNF activity. To delineate the in vivo role of NRSE in the regulation of rat BDNF gene, promoter constructs with intact and mutated NRSEBDNF were introduced into transgenic mice. Our data show that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung, i.e. in the tissues in which the intact reporter gene and endogenous BDNF mRNAs are expressed. Mutation of NRSEBDNF did not lead to the ectopic activation of the reporter gene in any other nonneural tissues. In the brain, NRSEBDNF is involved in the repression of basal and kainic acid-induced expression from BDNF promoters I and II in neurons. However, NRSEBDNF does not control the activity of the BDNF gene in nonneuronal cells of brain.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Neurotrophins (NTs)1 are secreted polypeptides that regulate the survival of selective populations of developing neurons and maintenance of the characteristic functions of mature neurons (1-3). The family of NTs includes nerve growth factor, brain-derived neurotrophic factor (BDNF), NT-3, and NT-4/5. Each factor is differentially involved in neurogenesis as well as in neuronal adaptive responses, implying well controlled regulatory mechanisms underlying their expression. Multipromoter structure, common to NT genes (4-8), has apparently evolved to confer complex regulation of their expression. We have previously shown that four promoters direct tissue-specific expression of the rat BDNF gene (8). Promoter IV is active in nonneural tissues (lung, heart, and muscle), whereas promoters I, II, and III are predominantly used in the brain and regulated by glutamatergic and GABAergic neurotransmitter systems (8, 9). Recent studies have shown that changes in BDNF expression are linked to synaptic plasticity during development (10, 11) and also to the process of memory and learning (12-15). Mice heterozygous for BDNF gene null-mutation have severe impairment in hippocampal long term potentiation (16, 18) and in normal spatial learning (19).

An inverted repeat within the first intron of rat BDNF gene shows substantial similarity to the neuron-restrictive silencer element (NRSE). NRSE, also known as repressor element-1 (RE1), has been defined as a negative-acting DNA regulatory element to prevent the expression of neuronal genes in nonneuronal cell types or in inappropriate neuronal subtypes (20-22). The first evidence that NRSE is able to mediate repression of transcription came from the analysis of the promoters of rat SCG10 (21) and rat type II sodium channel (NaChII) (20) genes. To date, several studies have proposed that NRSE-like sequences, present in the regulatory regions of multiple neuronal genes, are important for their neuron-specific expression (23-29). The identification of a NRSE-binding protein revealed that the neuron-restrictive silencer factor (NRSF) (22), also known as RE-1 silencing transcription factor (REST) (30), is a novel member of the Krüppel family of zinc-finger transcription factors. The same factor, called as X2-box repressor, was identified to bind to the X2-box of the immune system-specific major histocompatibility complex class II gene, DPA, and repress its activity in the terminally differentiated B-cell lineage (31). REST/NRSF/XBR mRNA is expressed at high levels in most nonneural tissues throughout development (22, 30, 31). In the nervous system, REST/NRSF/XBR mRNA levels are high in the undifferentiated neuronal progenitors (22, 30) and decrease during development. Our previous data revealed that REST/NSRF/XBR mRNA expression proceeds at varying levels also in the neurons of adult brain (32).

The present study explores molecular mechanisms underlying BDNF gene expression, focusing on the function of the palindromic NRSE (NRSEBDNF) in the proximal region of promoter II. We have previously shown that BDNF promoter-reporter gene construct, covering promoter I, promoter II, and flanking regions, recapitulates endogenous BDNF expression in transgenic mice (33). Currently, we have generated transgenic mice carrying the BDNF promoter construct with mutations in the NRSEBDNF sequence, and our results strongly suggest that the transcriptional regulation of BDNF gene in vivo is under the control of NRSEBDNF.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
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References

General Methods-- RNA isolation, chloramphenicol acetyltransferase (CAT) assay, RNase protection assay (RPA), and in situ hybridization were performed as described (8, 33, 34).

Construction of BDNF I+IImutCAT Construct-- A 0.3-kb XbaI/HindIII fragment encompassing BDNF exon II and its flanking regions was cloned into pBSSK vector (Stratagene), and a single-stranded uracil-rich template was prepared using Escherichia coli strain CJ 236. Site-directed mutagenesis was performed using the Kunkel method (35). Mutation of NRSE in the 0.3-kb XbaI/HindIII fragment was carried out in three steps using the following synthetic oligonucleotides. Mutated nucleotides are shown in lowercase letters. NRSEmut1, 5'-CGAGCAGAGTCCATTCAGCAgaTTttACAGAGCCAGCGGATTTGT-3'; NRSEmut1+2 , 5'-ACAGAGCCAGCGGATTTGTttGAcaTGGTAGTACTTCATCCAG-3'; NRSEmut 5'-GGGCGAGCAGAGTCCgggacGCAgaTTtACAGAGCCAGCGGATTTGT-3'.

Finally, the 0.3-kb XbaI/HindIII fragment carrying the mutated NRSE (NRSEmut) sequence was used to generate BDNFI+IImutCAT construct applying the same cloning strategy as earlier for constructing BDNFI+IICAT (33).

Transgenic Mice-- Transgenic mice were generated and analyzed for transgene integration as described previously (33). Transgenic founders were analyzed using dot blot analysis with a CAT-specific probe (33).Transgene copy number was estimated using BDNF exon I-specific probe (8) by comparing the intensity of BDNF exon I-specific signal in the wild-type animals to that in the transgenic animals. Quantification was performed with PhosphorImager using ImageQuant software (Molecular Dynamics).

Pharmacological Treatments-- Adult male and female transgenic animals (body weight, 20-30 g) were used in all experiments. KA (20 mg/kg in saline buffer) was injected intraperitoneally, and the animals were sacrificed at 3, 6, or 24 h after the injections. All animal experiments were approved by the local ethical committee.

Electromobility Shift Assay-- Cellular extracts of thymus and hippocampus were prepared as described (36). Mobility shift assays were performed as described (37). Oligonucleotides corresponding to the AP1 (Promega, Madison, WI) and cAMP response element (Promega) sequences, and to the NRSE derived of rat type II sodium channel gene (20) and rat SCG10 gene (21) were used as positive controls. In competition studies, the amount of added unlabeled oligo-competitor is presented as molar excess. In the supershift assay, c-Fos supershifting antibody was used (sc-447; Santa Cruz Biotechnology, Santa Cruz, CA).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The Inverted NRSE Repeat in the First Intron of BDNF Gene Controls the Activity of BDNF Promoters I and II in the Brain, Thymus, and Lung-- The inverted repeat (NRSEBDNF) in the proximal region of BDNF promoter II is composed of two NRSEs. The 5' element has 89% identity with the NRSE consensus sequence (29), whereas the 3' element shows only 57% identity, suggesting it to be an atypical NRSE. The results of previous deletion and mutation analyses have established that not all 21 residues of the NRSE consensus are critical for NRSE-mediated negative regulatory effects on transcription (29). Mutations introduced to certain positions of the consensus sequence have been suggested to be important for the silencing activity of NRSE (20, 21). In the upper element of NRSEBDNF, the nucleotides in the critical positions are identical with the consensus sequence, and in the lower element, they show 80% identity, suggesting that both elements in NRSEBDNF have the potential to mediate repression. To determine whether this inverted repeat-like element participates in the regulation of BDNF gene, mutations at potentially relevant residues were made (mut-NRSEBDNF) (Fig. 1) and analyzed in transgenic mice.


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Fig. 1.   Schematic representation of the BDNF-CAT fusion constructs that were used to generate transgenic mice in relation to the rat BDNF gene structure. In the gene structure, the nomenclature for the exons originates from the study by Timmusk et al. (8). The structure of the rat BDNF gene is shown at the top with exons indicated as boxes and introns as lines. UTRs of the exons are indicated as open boxes, and the region of exon V encoding the preproBDNF is indicated by the filled box. Broken lines show the regions of the BDNF gene that are included in BDNFI+IICAT and BDNFI+IImutCAT fusion genes. BDNFI+IICAT is identical to BDNFI+IImutCAT, except that the intact NRSEBDNF sequence in the proximal region of BDNF promoter II, shown as a filled oval, is mutated to mut-NRSEBDNF, shown as an open oval, using in vitro mutagenesis. The sequences of intact and mutated NRSEBDNF are as follows: NRSEBDNF, 5'-TCCATTCAGCACCTTGGACAGAGCCAparallel GCGGATTTGTCCGAGGTGGTAGTACTT; mut-NRSEBDNF, 5'-TCCgggacGCAgaTTttACAGAGCCAparallel GCGGATTTGTttGAcaTGGTAGTACTT. Residues that have been established to be critical for NRSE silencing activity and are conserved in NRSEBDNF as compared with the consensus sequence (29) are underlined. The bar symbol (parallel ) identifies the border between the 5' and the 3' elements in the NRSEBDNF repeat. Lowercase letters in the mut-NRSEBDNF sequence mark the base substitutions introduced to NRSEBDNF by in vitro mutagenesis. The predicted CAT transcripts with BDNF exon I and exon II are shown at the bottom of the figure. Thin lines indicate the regions that are spliced out of the primary transcripts.

Our previous studies established that a 9.5-kb genomic fragment including BDNF promoters I and II (BDNFI+IICAT) confers the appropriate tissue-specific pattern of the reporter gene expression (33). In the present study, we replaced the intact NRSEBDNF in the BDNFI+IICAT construct with the mut-NRSEBDNF and introduced the mutant construct (BDNF I+IImutCAT) (Fig. 1) into mice. Altogether, nine founders were obtained, and six of them were bred to obtain F1 transgenic lines for further analyses. In all six lines, transgene activity was the highest in the brain, whereas outside the central nervous system, high levels of CAT activity were observed in thymus and lung (Fig. 2). Analysis of CAT activity in different brain regions revealed slight variations in the pattern of expression from one founder line to another; however, the highest levels of CAT activity were always detected in the hippocampus, midbrain, and thalamus. As the spatial expression pattern of the reporter gene in BDNFI+IImutCAT mice was indistinguishable from the transgene expression pattern in BDNFI+IICAT animals, we concluded that mutation of NRSEBDNF is not sufficient to allow ectopic expression from BDNF promoters I and II in tissues that do not express the intact promoter construct and also endogenous BDNF mRNA.


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Fig. 2.   NRSEBDNF mediates repression of the reporter gene in the adult brain, thymus, and lung. Mean levels of CAT activity measured in different brain regions, in thymus, and in lung of six transgenic founder lines carrying BDNFI+IImutCAT (filled circles) are compared with the mean levels of CAT activity measured in these structures of nine founder lines with BDNFI+IICAT (open circles). Relative CAT activity values are expressed as the percentage of [14C]chloramphenicol conversion per mg of protein. The mean and the S.E. of the mean of the relative CAT values are presented in the logarithmic scale. ctx, cerebral cortex; hc, hippocampus; cbl, cerebellum; str, striatum; tha, thalamus; mid, midbrain; ste, brainstem; thy, thymus; lun, lung.

However, quantitative differences in the levels of reporter gene expression were seen in transgenic mice with the mutated promoter construct as compared with the animals carrying the intact promoter construct. In the brain, thymus, and lung, total CAT activity and relative CAT activity per transgene copy number was significantly higher in all six transgenic lines carrying mutated transgene as compared with CAT levels in any of the nine founder lines possessing intact reporter construct (Fig. 2). The levels of CAT activity in the thymus and lung of individual BDNFI+IImutCAT lines were on average about 50-fold higher as compared with the levels of CAT activity in thymus and lung of mice carrying BDNFI+IICAT transgene (Fig. 2). In the brain, all the brain regions showed elevated levels of CAT activity in transgenic animals with BDNFI+IImutCAT as compared with CAT activity levels in mice expressing BDNFI+IICAT (Fig. 2). In the hippocampus, which exhibited the highest levels of the reporter gene expression in the brain, CAT activity was on average 15-fold higher in transgenic animals with BDNFI+IImutCAT than in animals with BDNFI+IICAT (Fig. 2). The differences in CAT activity in the hippocampus ranged to more than 100-fold between the individual transgenic founder lines with BDNFI+IImutCAT as compared with the individual lines carrying BDNFI+IICAT. These data indicate that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung.

Quantitative RPAs were performed to establish whether NRSEBDNF located 0.7 kb downstream of BDNF exon I and 120 bp upstream of BDNF exon II could affect the activity of BDNF promoter I, promoter II, or both. The analysis revealed that in the hippocampus, CAT mRNAs with BDNF exon I (exon I-CAT) were on average about 15-fold higher, and CAT mRNAs with BDNF exon II (exon II-CAT) about 12-fold higher as compared with the levels in the animals with intact promoter construct (Fig. 3). Upon mutation of NRSEBDNF, the levels of exon I-CAT mRNA in thymus and lung were on average about 40-fold higher, and the levels of exon II-CAT mRNA were on average about 50-fold higher as compared with the levels in mice carrying BDNFI+IICAT transgene (Fig. 3). These data demonstrate that disruption of NRSEBDNF affects the activity of both BDNF promoter I and promoter II and leads to the enhanced expression from both promoters.


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Fig. 3.   NRSEBDNF represses the activity of both BDNF promoter I and promoter II. RPA analysis of exon I-CAT and exon II-CAT mRNA expression levels in the hippocampus of brain, and thymus, and lung of transgenic animals carrying reporter genes with the intact or mutated NRSEBDNF. The values represent the mean levels of exon I-CAT (filled triangles) or exon II-CAT (filled squares) mRNA expression in six transgenic lines carrying BDNFI+IImutCAT transgene and the mean levels of exon I-CAT (open triangles) or exon II-CAT mRNAs (open squares,) in nine founder lines carrying BDNFI+IICAT reporter gene. The mean of the CAT mRNA values and the S.E. of the mean are presented in the logarithmic scale. hc, hippocampus; thy, thymus; lun, lung.

NRSEBDNF Is Involved in the Repression of the Basal and KA-induced Expression from BDNF Promoter I and Promoter II in the Neurons of Brain-- Earlier studies suggested that NRSE is required to prevent expression of neuronal genes in nonneural tissues and cell populations (38). Next we examined whether high levels of CAT mRNA expression and CAT activity detected in the brain of BDNFI+IImutCAT animals resulted from the ectopic activation of the transgene in nonneuronal cells. Using in situ hybridization, CAT mRNA expression was seen in only three of nine founder lines with the intact promoter construct. In contrast to this, the expression of CAT mRNA was detected in all six founder lines with mutant promoter construct (Fig. 4). In all transgenic lines with BDNFI+IImutCAT, CAT mRNA expression was the highest in the neurons of the hilar region and CA3 pyramidal layer of hippocampus. As CAT mRNA was expressed in the same neuronal subpopulations in mice carrying the intact or the mutated promoter construct, we concluded that NRSEBDNF is not restricting the transgene expression to specific subsets of neurons (Fig. 4 and data not shown). Mutation of NRSEBDNF did not lead to the ectopic expression of transgene in nonneuronal cell populations of brain.


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Fig. 4.   NRSEBDNF is involved in the repression of the basal and KA-induced expression from BDNF promoter I and promoter II in certain neuron populations of the brain. Dark-field photomicrographs of autoradiograms of in situ hybridization showing the expression of CAT mRNA at the level of dorsal hippocampus of control (contr.) and kainic acid-treated (+KA) transgenic mice. P4 and Q4, founder lines with BDNFI+IICAT; m1, and m26, founder lines with BDNFI+IImutCAT; CA3, the pyramidal layer CA3 of hippocampus; hi, hilar region; dg, dentate gyrus; contr., untreated transgenic mice; +KA, transgenic animals sacrificed at 3 h following KA treatment.

Our previous data revealed that the neurons of hippocampus exhibit altered levels of BDNF expression from promoters I and II following KA-induced seizures. BDNF transcripts with exon I were induced more than 50-fold, whereas transcripts with exon II were increased about 5-fold, at 3 h following KA treatment (9). We have also shown that BDNFI+IICAT recapitulates the KA-induced expression from promoters I and II (33). These data suggested that kainate responsive cis-acting elements are present in the 9.5-kb BDNF genomic region of BDNFI+IICAT. To determine whether NRSEBDNF may be involved in the KA-induced regulation of BDNF gene, we studied the expression levels of CAT mRNA in the hippocampus of transgenic mice with the intact or mutated promoter constructs at different time points following KA administration. Using in situ hybridization, a marked increase in transgene expression was detected in the pyramidal neurons of CA3 and in the neurons of hilar region in three out of nine founder lines with BDNFI+IICAT and in all six analyzed lines with BDNFI+IImutCAT at 3 h following KA treatment (Figs. 4 and 5). In the dentate gyrus, no alterations in transgene expression levels were detected in any of BDNFI+IICAT animals; however, significantly increased levels of CAT mRNA were seen in scattered neurons in three of six founder lines with BDNFI+IImutCAT, at 3 h after KA administration (Fig. 5). Quantitative RPA analysis revealed that in the hippocampus of mice with the intact promoter construct transgene expression levels increased on average 4-fold, whereas in the mutant animals the levels increased on average 15-fold following 3 and 6 h of KA treatment (Fig. 6 and data not shown). As established by in situ hybridization, these higher induction levels resulted from the increased CAT mRNA expression per neuron and also from the increased number of neurons expressing CAT mRNA. At 24 h after drug injection, CAT mRNA levels had returned back to control levels in the hippocampus of both, BDNFI+IICAT and BDNFI+IImutCAT mice, as measured by RPA (data not shown). These data suggest that in the neurons of hippocampus, the induction level of BDNF mRNA transcribed from promoters I and II following KA treatment is under the control of NRSEBDNF.


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Fig. 5.   CAT mRNA expression in the hippocampus of the founder lines carrying the BDNF promoter construct with mutated NRSEBDNF at 3 h following KA treatment. Bright-field photomicrographs showing labeled neurons of hippocampus in untreated animals with BDNFI+IImutCAT (A and B) and at 3 h following KA-induced seizures (C-F). Labeling is restricted to weakly stained large cells (neurons). Shown are the cellular magnifications of the hilar region of two different transgenic founder lines m1 (A) and m17 (B) carrying the BDNFI+IImutCAT transgene. Cellular magnification views of CAT mRNA expression in the hippocampus of BDNFI+IImutCAT animals at 3 h following KA treatment are depicted in C-F, where C and D show the CAT mRNA expression in the hilar region of m26 transgenic line and E and F show the CAT mRNA expression in the dentate gyrus cells of m41 line. Note the strong increase in the hybridization density at 3 h following KA-induced seizures in the majority of neurons of the hilar region (C and D) versus a few neurons of the dentate gyrus region (E and F). D and F are higher magnification images of C and E, respectively. Scale bar, 12 µm in A, B, D, and F; 100 µm in C and D.


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Fig. 6.   NRSEBDNF mediates repression of the KA-induced transgene expression in the neurons of hippocampus. Expression of CAT mRNA and endogenous BDNF mRNA in the founder lines carrying BDNFI+IICAT (Q4 and P4) and BDNFI+IImutCAT (m1 and m26). The increases of CAT mRNA at 6 h following KA treatment were normalized relative to the increases of endogenous BDNF mRNA and to the level of GAPDH mRNA. c., total RNA derived from the hippocampus of untreated transgenic animals; +KA, total RNA derived from the hippocampus of transgenic mice at 6 h following KA treatment; tRNA, yeast tRNA.

Palindromic NRSE of BDNF Gene Forms Unique Complexes with Cellular Proteins of Hippocampus and Thymus-- Finally, we examined whether both elements of NRSEBDNF are required for its functional activity within and outside the nervous system. EMSA analysis revealed several unique NRSEBDNF-protein complexes that exhibited tissue-specific pattern of migration (Fig. 7). Competition experiments showed that the thymus-specific complex could be disrupted with a 50-fold excess of unlabeled oligonucleotides corresponding to the upper element (NRSEBDNF11) (Fig. 7, left). The lower element (NRSEBDNF21) appeared to be a less effective competitor. The same amount of unlabeled NRSEBDNF21 oligos was not sufficient to disrupt this complex (Fig. 7, left) and a 250-fold excess of unlabeled NRSEBDNF21 was needed to abolish the thymus-specific complex (data not shown). These data reveal that thymus-specific factors discriminate between the two elements and suggest that the upper element of NRSEBDNF is critical for its activity in thymus. Previous studies have shown that the NRSE-binding transcription factor REST/NRSF/XBR is predominately expressed in nonneural tissues (22, 30-32). Next, we examined whether REST/NRSF/XBR-like activity is present in the large complex detected from thymus extracts. A 50-fold excess of unlabeled oligonucleotides containing the NRSE sequences of NaChII (NRSENaChII) (Fig. 7, left) and SCG10 genes (data not shown) resulted in the disappearance of the thymus-specific complex formed on NRSEBDNF, suggesting that these NRSEs compete for the same factor as does the upper element of NRSEBDNF. Several other studies have shown that NRSEBDNF11, NRSENaChII, and NRSESCG10 bind REST/NRSF/XBR protein and form a complex with slow migration in EMSA analysis (22, 29, 30, 32). Accordingly, we suggest that the NRSEBDNF-specific complex detected from thymus extracts contains REST/NRSF/XBR. The mutant NRSEBDNF (mut-NRSEBDNF) oligonucleotide, as expected, did not form any specific DNA-protein complexes with thymus extracts because excess of any of the specific unlabeled competitors failed to affect the weak unspecific complexes detected (Fig. 7, left).


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Fig. 7.   Palindromic NRSE of BDNF gene forms unique complexes with cellular proteins of hippocampus and thymus. NRSEBDNF interactions with the cellular proteins of thymus (left) and hippocampus (middle and right) were analyzed by EMSA. Left, mutations introduced to NRSEBDNF affect its ability to bind the cellular proteins of thymus. Middle and right, NRSEBDNF forms two distinct complexes in the binding reactions with cellular extracts of hippocampus. In the left and middle panels, competition experiments a 50-fold excess of different unlabeled oligonucleotides was included. In the competition experiments shown in the right panel, a 10-fold excess of unlabeled oligonucleotide competitors, as indicated above the lanes, has been added to the binding reactions. Note that the mutations introduced have specifically affected the ability of mut-NRSEBDNF oligo to form slowly migrating complexes with the protein extracts of thymus and hippocampus. EMSAs were carried out in the conditions where the concentration of labeled NRSEBDNF and mut-NRSEBDNF oligos in the binding reactions was increased 100-fold as compared with the EMSAs on B. wt, NRSEBDNF; mut, mutated NRSEBDNF; 11, the 5' half of NRSEBDNF; 21, the 3' half of NRSEBDNF; NaChII, NRSE derived from rat sodium channel type II; -, no oligocompetitor added; C1 and C2, complexes 1 and 2 of hippocampus.

Using cell extracts of hippocampus, EMSA analysis detected two NRSEBDNF-specific complexes. Complex 1 is fast migrating, whereas complex 2 migrates similarly to the complex from the thymus extracts (Fig. 7). Competition studies of complex 1 revealed that this complex could be disrupted with the addition of 50-fold excess of unlabeled oligos containing the 3' element of NRSEBDNF and not with the similar excess of unlabeled NRSEBDNF11, NRSENaChII, and NRSESCG10 oligos. A 250-fold excess of unlabeled NRSEBDNF11 oligos was needed to disrupt this complex (Fig. 7, middle, and data not shown). Complex 1 was also detected using protein extracts from other regions of brain (cerebral cortex, striatum, olfactory bulb, thalamus, and brainstem) (data not shown). These data suggest that complex 1 is a brain-specific complex that forms preferentially on the lower element of NRSEBDNF and most likely does not contain REST/NRSF/XBR, because several unlabeled NRSE oligos could not disrupt this complex by competing with the wild-type NRSEBDNF oligo (see Fig. 7, middle). Subsequent studies of the composition of the complex 1 revealed that the 3' element of NRSEBDNF is predominately recognized by AP-1-like transcription factors, because a 50-fold excess of unlabeled AP1 oligonucleotide was sufficient to disrupt the formation of complex 1 on the lower half-site of NRSEBDNF (Fig. 7, middle). c-Fos antibody, which is reactive also with other members of c-Fos family, did not recognize this complex in our EMSA analysis. This suggest that some of the c-Fos family members, such as c-Fos, FosB, Fra-1, and Fra-2 are most likely not involved in the formation of complex 1 on NRSEBDNF (data not shown). Using protein extracts from hippocampus and other brain regions, mut-NRSEBDNF could target only a single complex with identical mobility and specificity to complex 1 (Fig. 7, middle, and data not shown). These results show that no new binding sites had been created in mut-NRSEBDNF as compared with the NRSEBDNF. Furthermore, a 50-fold excess of unlabeled mut-NRSEBDNF efficiently competed with the wild-type NRSEBDNF for the binding of complex 1 specific proteins from hippocampus extracts. These data suggest that mutations introduced to the NRSEBDNF sequence have no substantial effect on the formation of complex 1.

We have previously shown that REST/NRSF/XBR mRNA expression proceeds at relatively low levels in the neurons of hippocampus of adult brain (32). It is possible that complex 1-specific proteins are abundant in hippocampal extracts and/or bind the NRSE sequence with high affinity and that makes minor or weaker complexes difficult to detect. Using hippocampus extracts under conditions where the concentration of the labeled NRSEBDNF was raised 100-fold and about 90% of the oligo was bound to complex 1, we started to detect a slowly migrating complex, complex 2. Further studies revealed that addition of a 10-fold excess of unlabeled NRSEBDNF11 oligo was sufficient to disrupt the formation of complex 2 and not complex 1. A similar excess of unlabeled NRSEBDNF21 diminished the formation of both complexes (Fig. 7, right). We have previously shown that the 5' NRSE of NRSEBDNF could efficiently compete with the full NRSEBDNF palindrome for the binding of REST/NRSF/XBR (32). Accordingly, our current data suggest that the slowly migrating NRSEBDNF-specific complex, complex 2, which is detected from the extracts of hippocampus and is competed out by the upper half of NRSEBDNF and other NRSE sequences (Fig. 7, right, and data not shown), contains REST/NRSF/XBR. An excess of unlabeled mut-NRSEBDNF decreased the formation of complex 1 but did not affect the formation of complex 2 on NRSEBDNF (Fig. 7, right). Furthermore, EMSAs with labeled mut-NRSEBDNF oligo showed no slowly migrating complexes (Fig. 7, right). Taken together, our results suggest that the mutations introduced to the NRSEBDNF sequence specifically interfere with its REST/NRSF/XBR-like binding activity in the brain.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The purpose of this study was to investigate the role of NRSEBDNF in the regulation of BDNF gene expression. The main conclusions of our data are as follows: 1) NRSEBDNF controls the activity of BDNF promoters I and II in the brain, thymus, and lung, but not in other tissues; 2) in the adult brain, NRSEBDNF represses the activity of BDNF promoters I and II in neurons and has no effect in nonneuronal cells; 3) NRSEBDNF is involved in the repression of the KA-induced activation of BDNF promoters I and II in certain populations of neurons in the hippocampus; 4) the palindromic NRSEBDNF in the proximal region of BDNF promoter II is composed of two binding sites; however, the 5' element is largely responsible for the activity of NRSEBDNF in the thymus as well as in the brain.

Many neuronal and a few nonneuronal genes contain NRSE-like sequences in their regulatory regions (29), suggesting a common mechanism underlying their expression. Most previous studies have used transient expression assays in different cell lines and demonstrated that NRSE acts as a silencer in nonneuronal cell types (20, 21, 23-25, 27, 28, 39, 40). So far, molecular mechanisms of NRSE-mediated silencing are largely unravelled. A recently isolated protein REST/NRSF/XBR, which belongs to the family of zinc finger transcription factors, and shows a predominantly nonneural expression pattern, has been demonstrated to bind to NRSE sequences and mediate repression in transient expression assays (22, 30-32).

Our transgenic studies aimed to examine the role of NRSEBDNF in the regulation of BDNF gene in vivo. Our current data show that in nonneural tissues, mutation of NRSEBDNF resulted in significantly increased levels of the transgene expression in thymus and lung, where endogenous BNDF promoters I and II, also, are active (33). Activation of reporter gene expression was not detected in any other nonneural tissues. Accordingly, we suggest that NRSEBDNF contributes to the regulation of BDNF gene specifically in thymus and lung. However, mutation of this element alone is not sufficient to activate the expression from BDNF promoters I and II in other nonneural tissues. This suggestion implies that the silenced state of BDNF gene in most tissues is accomplished through the utilization of different or more complex sets of regulatory elements. All transgenic studies published so far have reported the complexity of NRSE-mediated effects on the tissue-specific gene regulation. Mutation of NRSE in the rat Na,K-ATPase alpha 3 promoter led to aberrant expression of the transgene only in a few nonneural tissues (41). Recent transgenic studies of the regulation of the mouse cell adhesion molecule L1 gene revealed that in some nonneural tissues, NRSE was rather controlling than preventing ectopic expression from L1 promoter during embryonic development (42, 43). In conclusion, these data, together with our observations indicate that in many cases, NRSE alone is not sufficient to silence the locus of a neuronal gene in the full set of nonneural tissues.

The results of our study also reveal that NRSEBDNF controls gene expression in neurons, because mutation of NRSEBDNF led to significantly increased levels of transgene expression in the neurons of brain, particularly in the region of hippocampus, thalamus, and midbrain. These results are unexpected and do not support the role of this element as a neuron-restrictive silencer. NRSE-mediated effects on gene expression within the nervous system have also been reported by all recent transgenic studies. For example, transgenic studies of the rat Na,K-ATPase alpha 3 promoter revealed that replacement of NRSE with a random sequence led to the increased expression of the transgene in the brain (41). A transgenic study of the neuronal nicotinic acetylcholine receptor beta 2-subunit (nAChbeta 2) promoter showed that mutation of a few residues in the NRSE sequence was sufficient to silence or enhance transgene expression in different structures of the developing central nervous system (44). Transgenic analysis of mouse L1 promoter revealed that NRSE-mediated effects within the central nervous system could be linked to a developmental stage and a certain neural structure. For example, in the adult brain, deletion of the NRSE led to reduction of L1 promoter activity in cortex, striatum, and hippocampus, whereas in the thalamus, L1 promoter activity was enhanced (43). These data together with our findings clearly demonstrate that NRSE is critical for the basal expression of several neuronal genes within the nervous system and that NRSE-elicited effects are dependent on the cellular context.

In this study, we show that disruption of the integrity of NRSEBDNF did not activate transcription from BDNF promoters I or II in the nonneuronal cells of brain. In good agreement with our study, mutation of NRSE in the nAChbeta 2 promoter did not lead to the ectopic reporter gene expression in glia or other nonneuronal cells of brain (44). In contrast, deletion of NRSE from L1 promoter resulted in the ectopic reporter gene expression in the meninges surrounding the cerebellum and in the choroid plexus (42). The ability of NRSE to repress nonneural expression of some (L1) and not other (nAChbeta 2 and BDNF) neuronal genes suggests that NRSE participates in various regulatory modules and that the interplay with other regulatory elements is required for NRSE to elicit its effects on gene regulation in these cells. Alternatively, regulation of the nAChbeta 2 and BDNF genes in nonneuronal cells is under the control of different cis-elements that provide binding sites for other transcriptional repressors. For example, putative mammalian homologues of Drosophila tramtrack (45) or Caenorhabditis elegans lin-26 (46) transcription factors could be good candidates to control neuronal genes in nonneuronal cells and prevent nonneuronal cells from expressing neuronal fates.

Our data also establish that NRSEBDNF, which is located 700 bp downstream of BDNF exon I and 120 bp upstream of BDNF exon II, could suppress the activity of both BDNF promoter I and promoter II. Studies of major histocompatibility complex class II genes have revealed that mutations within the X2 box, which shares sequence similarity with NRSE, also lead to suppressed activity of the promoter, if the element (X2 box) is located in close proximity to the transcription initiation site (31, 47) On the other hand, it has been suggested that NRSE functions as an enhancer when located less than 50 bp upstream or downstream from the TATA box, but acts as a silencer when located further upstream (44). The recent study showed that the NRSE does not necessarily have to be close to the promoter to function as an enhancer, because a NRSE present 10 kb downstream of the L1 promoter can still function as an enhancer in certain cell populations (43). Taken together, these data clearly demonstrate that NRSE-mediated effects depend largely on the promoter identity.

BDNF, like other NTs, is known to be produced in limited amounts during development, and small changes in its expression levels are deleterious (48, 49). There is abundant evidence that BDNF expression in the hippocampus is modulated by glutamatergic activation (14, 15, 50). Because increases in BDNF expression in hippocampal and cortical neurons after brief periods of seizure activity are associated with long term potentiation (reviewed in Ref. 51), neuroprotection, or epilepsy (reviewed in Ref. 52), we next asked whether NRSEBDNF is a module within the BDNF gene that could contribute to any of these neural activity-involved processes. Our present study established that NRSEBDNF is involved in the repression of the seizure activity-dependent activation of BDNF promoters I and II in neuronal populations of the hippocampus. Several studies have approached the molecular mechanisms underlying BDNF gene regulation by glutamatergic activation. Genomic regions have been identified that mediate KA-induction of BDNF expression in the neurons of brain in vivo (33) and in the neuronal cultures in vitro (53). Calcium-responsive regions involved in the induction of BDNF gene in C6 glioma cells have also partially been characterized (54). Calcium/calmodulin-dependent protein kinases II and IV have been shown to be involved in the regulation of the seizure-induced expression of BDNF mRNA in vivo (55). Recently, it was identified that a cAMP response element and a juxtapositioned novel calcium response element within the proximal region of BDNF promoter III mediate the Ca2+-dependent transactivation of BDNF gene in hippocampal and cortical neurons after membrane depolarizations (56, 17). The present study is the first to demonstrate that another specific element within the BDNF gene (NRSEBDNF) is implicated in the regulation of appropriate responsiveness of BDNF promoters I and II to the changes in neuronal activity.

NRSEBDNF consists of two NRSEs arranged as an inverted repeat. Previous studies have shown that NRSEBDNF and its upper half alone could efficiently bind in vitro translated REST/NRSF/XBR (22, 32). The results of this study suggest that the 5' half is largely responsible for the NRSEBDNF activity in the thymus and brain, where it most likely binds REST/NRSF/XBR and/or REST/NRSF/XBR-like factors, and this complex mediates repression of transcription from BDNF promoters I and II. Our in vitro binding data suggest that the 3' half-site is a preferential target of brain-specific proteins that also have the AP-1-sequence binding activity. The role of the 3' element in the regulation of BDNF gene in vivo still remains to be established. Taken together, NRSEBDNF could integrate different regulators and various regulation pathways to mediate repression and ensure appropriate levels of expression from BDNF promoters I and II.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Toomas Neuman for the critical reading of the manuscript. We thank Erik Nilsson and Annika Ahlsén for technical assistance.

    FOOTNOTES

* This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council for Engineering Sciences, and the Swedish Medical Research Council; funds from the Karolinska Institute; Karolinska Institute's co-operation program with Baltic countries; the Tore Nilsson Foundation for Medical Research; the Lars Hierta Foundation; and the Åke Wiberg's Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Contributed equally to this work.

parallel Supported by Swedish Medical Research Council. To whom correspondence should be addressed. Tel.: 46-18-4714-384; Fax: 46-18-559-017; E-mail: tonis.timmusk{at}mun.uu.se.

¶¶ Partially supported by an International Research Scholars' Award from Howard Hughes Medical Institute.

The abbreviations used are: NT, neurotrophin; BDNF, brain-derived neurotrophic factor; bp, base pair(s); kb, kilobase pair(s); NRSF, neuron-restrictive silencer factor; EMSA, electrophoretic mobility shift assay; nACh, nicotinic acetylcholine receptor; NaCh, sodium channel; mut, mutant; CAT, chloramphenicol acetyltransferase; RPA, RNase protection assay; KA, kainic acid; RE, repressor element; REST, RE-1 silencing transcription factor.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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