Departments of Anesthesiology and Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2520
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ABSTRACT |
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Karadsheh, Michael F. and
E. Delpire.
Neuronal Restrictive Silencing Element Is Found in the KCC2 Gene:
Molecular Basis for KCC2-Specific Expression in Neurons.
J. Neurophysiol. 85: 995-997, 2001.
KCC2
is one of four known isoforms of the K-Cl cotransporter with an
expression pattern restricted to neurons. It mediates efflux of
Cl across neuronal membranes and plays an
important role in GABAergic and glycinergic neurotransmission. To
understand the molecular basis for neuronal specificity of KCC2
expression, we isolated and sequenced portions of the KCC2 gene,
including some of its 5' flanking (control) region. We found a 21-bp
sequence, within intron 1, that shares 80% homology to the consensus
site for neuronal-restrictive silencing factor binding. We demonstrated
that this specific sequence of the KCC2 gene promotes transcriptional
regulation by showing that nuclear proteins isolated from a mouse
neural progenitor cell line interact with this 21-bp element and by
establishing that this element silences reporter gene expression in
nonneuronal cells.
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INTRODUCTION |
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GABA and glycine are
inhibitory neurotransmitters in the adult CNS. However, they produce
excitatory responses in young immature brain (Ben-Ari et al.
1989; Ehrlich et al. 1999
). The conversion of
the GABA effect from excitatory to inhibitory occurs shortly after
birth and is related to the intracellular Cl
concentration in neurons (Ehrlich et al. 1999
;
Owens et al. 1996
). Immature neurons have an
intracellular Cl
concentration higher than
predicted for a passive distribution (Ehrlich et al.
1999
; Owens et al. 1996
). On activation of
GABAA receptor, there is an outward movement of
Cl
that leads to membrane depolarization
(excitation). During postnatal development, the intracellular
Cl
concentration decreases to values below
equilibrium and GABA induces Cl
influx
resulting in membrane hyperpolarization (inhibition).
The expression of the Na-K-2Cl cotransporter, NKCC1, decreases
(Plotkin et al. 1997), whereas expression of KCC2, an
isoform of the K-Cl cotransporter, increases (Clayton et al.
1998
; Lu et al. 1999
) during postnatal
development. These changes in cotransporter expression are consistent
with a decrease in intracellular Cl
concentration and with the switch from depolarizing to hyperpolarizing GABA currents.
Analysis of KCC2 expression by Northern blot, in situ hybridization,
and immunofluorescence has revealed that KCC2 expression is restricted
to neurons (Lu et al. 1999; Payne et al.
1996
). To study the neuronal specificity of KCC2 expression, we
isolated and examined the gene encoding this cotransporter. In the
course of identifying and isolating exon 1 and the upstream regulatory region of the gene, we discovered a 21-bp motif downstream of exon 1 that resembles the consensus sequence of the known neuronal-restrictive silencing element (NRSE). We report here that this KCC2 element binds
to nuclear proteins and inhibits the transcription of a reporter gene
in C17 nonneuronal cells.
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METHODS |
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Genomic library
A Lamda FIXII mouse genomic library (Stratagene, La Jolla, CA)
was screened using the first 280-bp fragment of the rat KCC2 cDNA
(Payne et al. 1996). Five positive clones were isolated. After secondary screening, two single positive clones were grown in
liquid culture, and phage DNA was isolated and mapped with a panel of
restriction enzymes. Southern blot analysis was performed to identify
exon 1. A 12-kb EcoRI-NotI fragment was subcloned into the vector Bluescript, and 1.6 kb at the 5' end was sequenced.
Electromobility shift assay
C17 progenitor cells from eight confluent 10-cm culture dishes
were trypsinized, washed, and spun for 5 min at 1,500 rpm. Nuclear
proteins were isolated as previously described (Shelton et al.
1992) and quantitated using the Bradford assay (Bradford 1976
). Two complementary neuronal-restrictive silencing element (NRSE) oligonucleotides were end-labeled with
32P-dATP using T4 polynucleotide kinase,
annealed, and purified by phenol:chloroform extraction. Protein-DNA
binding was achieved by incubating 7 µg nuclear protein with 2 µl
probe (4 pmols) in a 25-µl reaction containing (in mM) 100 KCl, 5 MgCl2, 1 EDTA, 0.5 DTT, 0.1 ZnCl2, 10% glycerol, 0.05% Nonidet P-40, and 10 HEPES pH 7.7 for 15 min at room temperature. The reactions were then separated on 4% polyacrylamide gel.
PGL3 constructs and luciferase assays
A 1.5-kb EcoRI-XhoI fragment of the mouse
KCC2 gene containing some 5' flanking sequence, the putative minimal
promoter and some 5' untranslated region was ligated at the
EcoRI and XhoI sites of the promoterless
luciferase reporter gene vector pGL3-basic (Promega). This new vector
was designed to test the activity of the KCC2 promoter. Two synthetic
complementary oligonucleotides containing the 21-bp KCC2 NRSE sequence
flanked by EcoRI sites were thenligated at the
EcoRI site upstream of the KCC2 promoter. The three vectors
(pGL3-basic, pGL3-KCC2, and pGL3-NRSE-KCC2) were transfected in C17
progenitor neural cells for promoter assay, together with a pSVgal
vector to correct for transfection efficiency. C17 cells were
transfected using 2.5 µg construct, 1.5 µg
-galactosidase, and 3 µg/ml lipofectamine (Gibco). After 36 h, the cells were washed
and lysed with 300 µl lysis buffer (Promega).
-galactosidase activity was obtained by incubating 100 µl lysate to 100 µl 2 × ONPG buffer [(in mM) 120 Na2HPO4, 80 NaH2PO4, 2 MgCl2, 100
-mercaptoethanol, and 4.4 o-nitrophenyl
-D-galactopyranoside] at
37°C for 24 h, followed by the addition of 334 µl 1 M
Na2CO3, and measurement of
optical density at 420 nm. Luciferase activity was measured in a EG and
G Autolumat luminometer (Model LB953) by injecting 100 µl luciferase
reagent (Promega) to 40 µl lysate. Results were expressed as ratios
between luciferase and
-galactosidase activities.
-galactosidase
activity was determined to control for changes in transfection efficiency.
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RESULTS AND DISCUSSION |
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Negative transcriptional regulation of neuronal genes in
nonneuronal cells was first described for the SCG10 and type II
Na+ channel genes (Kraner et al.
1992; Mori et al. 1990
). Since these original
reports, transcription of many genes expressed specifically in neurons
have been shown to be regulated through this mechanism. These genes
contain in their promoter or introns a specific sequence NRSE that
constitutes the binding site for a protein expressed in nonneuronal
cells (NRSF or REST) and inhibits their transcription. This negative
transcriptional regulation is also critical in the development of the
nervous system. Most genes expressed in differentiated or mature
neurons are not found in precursor and immature cells. Up-regulation of
these neuronal specific genes coincides with the down-regulation of
NRSF expression (Schoenherr and Anderson 1995
).
Importance of NRSF regulation is also evidenced by the multiple
malformations observed by Chen and coworkers in different nonneural
tissues and by the embryonic lethality of complete inactivation of NRSF
in the knockout mouse (Jones and Meech 1999
).
Out of four genes that encode K-Cl cotransporter, KCC2 has been shown
to be neuronal specific with potential relevance in CNS development and
intracellular Cl homeostasis (Clayton et
al. 1998
; Lu et al. 1999
). Previous work has
determined that KCC2 expression is low at birth and increases during
postnatal development (Clayton et al. 1998
; Lu et
al. 1999
). The KCC2 gene is therefore a very good candidate for
regulation by NRSF. To understand the molecular basis for the
restrictive expression of KCC2 in neurons, we isolated the promoter
region of the KCC2 gene. A mouse lambda phage genomic library was
screened with a probe consisting of the first 280-bp fragment of the
rat KCC2 cDNA (Payne et al. 1996
). A ~18-kb genomic
clone was obtained and analyzed by restriction digest and Southern blot
analysis. This genomic clone contains ~7 kb of 5' flanking region,
exon 1, and ~11 kb of the downstream sequence. The 5' flanking region (1.5 kb of it), exon 1, and a short portion of intron 1 were sequenced. We did not characterize the 5' boundary nor the 3' end of the gene. The
entire sequenced fragment was analyzed using the transcription factor
database available at http://www@genome.ad.jp. As expected, we found in
intron 1 a 21-bp sequence with high sequence homology (81%) to
the known consensus sequence of NRSE (Fig.
1). This consensus was derived from 19 sequences for which NRSF binding was experimentally determined
(Schoenherr et al. 1996
). Out of the four mismatched nucleotides, three are located at positions most frequently modified in
functional NRSEs and subsequently demonstrated to be nonessential of
the NRSF binding (Schoenherr et al. 1996
).
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To demonstrate that the 21-bp fragment of the KCC2 gene is involved in the regulation of KCC2 expression, we first examined possible interaction of this fragment with nuclear proteins isolated from C17 nonneuronal cells. Two complementary synthetic oligonucleotide primers were annealed, labeled with 32-P, and incubated with nuclear proteins isolated from mouse progenitor C17 neural cells. Interaction of the probe with nuclear proteins was demonstrated by acrylamide gel electrophoresis. As demonstrated in Fig. 2A, the mobility of the probe (lane 1) was retarded in the presence of nuclear proteins (lane 2), indicating protein-DNA interaction. The protein-DNA complex was completely displaced by a cold NRSE fragment (Fig. 2B, lane 2) but not by an unrelated DNA fragment of the same size and same G + C content (Fig. 2B, lane 3).
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Using a luciferase gene reporter assay, we examined the effect of the 21-bp KCC2 NRSE fragment on gene transcription. A 1,500-bp EcoRI-XhoI fragment consisting of the most proximal 5' flanking region of the KCC2 gene, and possibly containing the minimal promoter (see Fig. 1), was inserted upstream of the promoterless luciferase gene. Two complementary synthetic oligonucleotides consisting of the 21-bp KCC2 NRSE sequence and flanking EcoRI sites were annealed and ligated upstream of the 1,500-bp fragment. The three vectors (pGL3 basic, pGL3-KCC2, pGL3-NRSE-KCC2) were transfected in C17 cells and assayed for luciferase activity. As shown in Fig. 3, pGL3-basic had little luciferase activity, pGL3-KCC2 yielded significant luciferase activity, while the addition of the NRSE sequence (pGL3-NRSE-KCC2) inhibited completely the KCC2 promoter-induced luciferase signal as indicated by a return to baseline levels.
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These results demonstrate that the KCC2 NRSE-like 21-bp sequence is capable of silencing transcription of the luciferase reporter gene in C17 nonneuronal cells. These are pluripotent, undifferentiated, neuronal or glial-precursor cells. Taken together, these experiments suggest an important role for this putative element in regulating KCC2 gene expression.
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ACKNOWLEDGMENTS |
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E. Delpire is an Established Investigator from the American Heart Association.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36758.
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FOOTNOTES |
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Address for reprint requests: E. Delpire, Anesthesiology Research Div., Dept. of Anesthesiology, T-4202 Medical Center North, Nashville, TN 37232-2520 (E-mail: eric.delpire{at}mcmail.vanderbilt.edu).
Received 6 March 2000; accepted in final form 28 September 2000.
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REFERENCES |
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