Brain-derived Neurotrophic Factor Expression in Vivo
Is under the Control of Neuron-restrictive Silencer Element*
T
nis
Timmusk
§¶
,
Kaia
Palm
§¶,
Urban
Lendahl**, and
Madis
Metsis

§§¶¶
From the
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

Center for Gene Technology, Tallinn
Technical University, and the §§ Institute of
Chemical Physics and Biophysics, Tallinn EE0026, Estonia
 |
ABSTRACT |
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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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).
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RESULTS |
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'-TCCATTCAGCACCTTGGACAGAGCCA GCGGATTTGTCCGAGGTGGTAGTACTT;
mut-NRSEBDNF,
5'-TCCgggacGCAgaTTttACAGAGCCA 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 ( ) 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.
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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.
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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
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
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
2-subunit (nACh
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 nACh
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 (nACh
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 nACh
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.
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.
 |
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