(Received for publication, July 18, 1995)
From the
A silencer element (Kv1.5 repressor element; KRE) was
characterized by deletion analyses in the promoter of Kv1.5, a
voltage-gated potassium channel. The silencer element selectively
decreases expression of Kv1.5- and thymidine kinase-chloramphenicol
acetyltransferase reporter gene constructs in cell lines that do not
express Kv1.5 polypeptide. It contains a dinucleotide repetitive
element
(poly(GT)(GA)
(CA)
(GA)
),
and self-associates spontaneously in vitro to form complexes
with slow electrophoretic mobility. Deletion of the repetitive element
abolished self-association in vitro and the silencing activity
in transient transfection experiments in vivo. Electromobility
gel shift assays of KRE with GH3 cells nuclear extracts detected the
formation of a unique DNA-protein complex, which was not detectable in
Chinese hamster ovary and COS-7 cells. This complex does not react with
an antibody against nonhistone high mobility group 1 protein, which
binds KRE in gel retardation assays. These observations establish that
a dinucleotide tandem repeat sequence, capable of self-association,
forms part of a cell-specific silencer element in a mammalian gene.
The molecular basis of controlling cell excitation is dependent
on pre- and post-translational mechanisms that include transcriptional
regulation, assembly, and various post-translational modifications of
ion channel
polypeptides(1, 2, 3, 4) .
Transcriptional regulation of mammalian voltage-gated potassium
channels plays an important role in regulating the resting potential
and the length of the action potential by determining the
cell-specificity and level of expression of these polypeptides in
cardiac and neuronal cells(5, 6) . Many mammalian
genes encoding voltage-gated potassium channels have been isolated, and
their genomic organization has been described in detail(7) .
However, very little is known about the mechanisms that regulate the
transcription of these genes. The existence of multiple K channel genes, each of which codes for a channel protein that is
highly conserved and similar to several other channels, creates an
interesting problem in terms of structural organization,
transcriptional regulation, and tissue-specific
expression(8, 9, 10, 11, 12) .
We recently characterized the promoter of Kv1.5, a voltage-gated
delayed rectifier potassium channel, and showed that it lacks a
canonical TATA box, has several transcription start sites, and contains
a cAMP response element located at the 5`-noncoding sequences that can
modulate the cAMP-induced transcriptional response of Kv1.5-CAT
reporter gene constructs(2) . We also showed that cAMP
and KCl depolarization regulate the expression of Kv1.5 in a
cell-specific manner. These results highlighted the fact that cells
maintain tight control of the genes that regulate membrane potential
and cell excitation at the transcriptional level, and that
transcriptional control represents an important mechanism for modifying
cell excitability.
For most eukaryotic genes, the tissue specificity and the level of expression are determined by cis-acting elements located within the 5` flanking sequences(13) . In this paper we investigate the cis-regulatory sequences that determine the cell-specific expression of Kv1.5 gene. We have chosen GH3 cells, a clonal pituitary cell line, which is the only known cell line that expresses Kv1.5 gene(14) . Kv1.5 is also expressed in normal rat pituitary cells in vitro and in vivo(14) . Our results document that a DNA element containing a dinucleotide repetitive sequence (Kv1.5 repressor element; KRE) represses the expression of Kv1.5-CAT and TK-CAT reporter gene constructs in transient transfections of COS-7 and CHO cell lines, while expression of these constructs in GH3 cells is not affected. A fragment of 160 bp that contains the repetitive element (102 bp) and the 5` flanking sequences (58 bp) is necessary and sufficient to confer the silencing effect to an heterologous promoter. The double-stranded DNA containing the repetitive element self-associates spontaneously to form large complexes with slow electrophoretic mobility that can be detected by polyacrylamide and agarose gel electrophoresis. Electromobility gel shift assays (EMSA) with GH3 and control nuclear extracts show that KRE forms a unique DNA-protein complex. In addition, nonhistone high mobility group 1 protein (hHMG1) also binds to KRE in EMSA(15) . Collectively, these experiments indicate that a repetitive sequence may play an important role in regulating the cell-specific expression of Kv1.5.
Figure 5:
Self-association of KRE. A, P-labeled KRE (both strands) was preincubated for 4 h at
37 °C and then subjected to electrophoresis on a 4% polyacrylamide
gel without further treatment (lane 1), after heating at 90
°C for 5 min (lane 2), or after mixing with 1 ng (lane
3) or 10 ng (lane 4) of unlabeled KRE. The resulting
products were visualized by autoradiography. A 100-bp ladder was
labeled with
P and used as molecular size markers. B, I,
P-labeled KRE was electrophoresed
on a 8% polyacrylamide gel. The monomeric probe was excised, eluted,
and incubated for 4 h at 37 °C (lanes 5-8). Prior to
loading each sample was preheated at 50 °C (lane 6), 70
°C (lane 7), or 90 °C (lane 8) for 5 min. The
resulting products were resolved on a 4% polyacrylamide gel and
visualized by autoradiography. In lanes 1-4, the eluted
probe was incubated at 37 °C with preheated (H) or
phenol-denatured (P) nuclear extracts from GH3 or COS-7 cells
as indicated. B, II, a 2-day exposure of lanes
1-4 demonstrating a band that migrates above the 800-bp
molecular size marker. C, a 172-bp
P-labeled DNA
fragment, excised from the somatostatin promoter (both strands), was
preincubated for 4 h at 37 °C and then subjected to electrophoresis
on a 4% polyacrylamide gel, without further treatment (lane
1), after heating at 90 °C for 5 min (lane 2), or
after mixing with 40 ng (lane 3) of unlabeled fragment. The
resulting products were visualized by autoradiography. D, a
P-labeled 2.6-kb fragment containing KRE was preincubated
for 4 h at 37 °C in the presence or absence of 2 M NaCl
and then subjected to electrophoresis on a 3% agarose gel without
further treatment (lanes 1 and 5), and after the
addition of excess cold probe (lanes 2-4 and 6-8). Lanes 2 and 6 represent
preheating at 90 °C for 5 min before loading. The resulting
products were visualized by autoradiography.
Figure 1: Multiple transcription start sites of Kv1.5 gene in GH3 cells and heart. A, schematic representation of the 5`-flanking and non-coding regions of Kv1.5 gene. The numbers indicate distance in base pairs upstream from the translation start site (ATG). The asterisk denotes +1 transcription start site in the heart, as described by Mori et al.(2) . The arrows denote transcription start sites confirmed by the primer extension assay and RPA (panels B and C). The RNA probe used contains sequences from -476 to -17. All major protected fragments obtained in the RNase protection analysis (panel C) and the primer extension products (panel B) are schematically illustrated below the probe as solid bars. B, Primer extension analysis. An oligonucleotide complementary to the nucleotides -120 to -150 of Kv1.5 noncoding region was hybridized to 50 µg of total cellular RNA from GH3 cells. Control primer extension analyses were performed with 50 µg of liver RNA or tRNA. A sequence reaction of a known template was used as molecular size marker. The arrows with the asterisks denote the specific bands that were confirmed both by RNase protection and primer extension assays. C, RNase protection analysis. 20 µg of total cellular RNA from two different samples of GH3 cells were hybridized to the riboprobe (panel A). After treating with ribonucleases A and T1, the protected fragments were analyzed on a polyacrylamide/urea gel. Control RNase protection analyses were performed with 20 µg of total cellular liver RNA or tRNA. The lengths of the protected fragments were determined by using the sequencing reaction of a known template as a molecular size marker. Similar results were obtained in four independent experiments. The arrows with the stars denote the specific bands that were confirmed both by RNase protection and primer extension assays.
We reasoned that deletion analyses of the promoter would enable us
to identify specific cis-regulatory sequences that interact with
trans-acting factors and regulate the GH3 cell-specific expression of
Kv1.5 gene. A series of 5` deletion mutants of Kv1.5 promoter fused to
the chloramphenicol acetyltransferase gene (pKvCAT) were transfected
using the Ca/PO precipitation method into GH3 cells (2) (Fig. 2, A and B). COS-7 cells,
which do not express Kv1.5,
were used as control (Fig. 2B). To control for transfection efficiency, a
plasmid containing the
-galactosidase gene was co-transfected into
the same cells. Transfection of these deletion mutants containing 3100
to 1071 bp 5` to the translation start site into GH3 and COS-7 cells
showed a marked discrepancy in CAT expression between the two cell
lines (Fig. 2B). pKvCAT-induced CAT activity in GH3
cells was up to 18-fold higher than background activity of a
promotorless CAT construct, pBs-CAT. In contrast, transient
transfection of the same reporter gene constructs into COS-7 cells
resulted in CAT activity that was similar or up to 2-fold higher than
background pBsCAT activity (Fig. 2B). Remarkably,
deletion of the segment between 1071 and 894 bp upstream from the
translation start site (KRE) resulted in a marked increase in CAT
activity in COS-7 cells, to a level similar to that of GH3 cells (Fig. 2C). Transient transfection of p1071KvCAT and
p894KvCAT reporter gene constructs into several other non-GH3 cells
such as HeLa, H9C2, and CHO cells resulted in a similar pattern of CAT
expression; CAT activity induced by p894KvCAT was 3-7-fold higher
than that induced by p1071KvCAT in all cell lines tested (Fig. 2C). In contrast, there was no significant change
in the level of CAT activity expressed in GH3 cells. Thus, deletion of
178 bp from the 5` end of p1071KvCAT resulted in a significant increase
of CAT activity in multiple cell lines, possibly by the deletion of a
cis-regulatory element that represses the expression in non-GH3 cells.
The start sites of the transfected constructs were confirmed by RNase
protection analysis of total RNA derived from COS-7 cells transfected
with p648KvCAT. The results showed several protected bands, five of
which were identical to the ones described in GH3 cells. A
COS-7-specific potential start site was detected, located 5` to the
ones described in GH3 cells (data not shown). Control assays with tRNA,
liver, and non-transfected COS-7 cells did not detect Kv1.5-specific
transcripts (data not shown).
Figure 2:
Identification of GH3 cell-specific
cis-regulatory elements in Kv1.5 gene. A, schematic maps of
deletion mutants of Kv1.5 gene-CAT reporter constructs. The arrows represent the transcription start sites identified by primer
extension and RNase protection assays. The plasmids are named according
to the distance in base pairs (bp) of the 5` flanking region from the
translation start site. pBs-CAT, a promoterless CAT gene cloned into
pBS. B, a comparison of CAT expression of successive 5`
deletion mutants of Kv1.5 gene transiently transfected into COS-7 and
GH3 cells using the Ca/PO4 precipitation method(2) . Cells were
harvested 2 days after transfection for the CAT assay. Relative CAT
activities were calculated by comparing the activities of the
transfected plasmids with that of pBs-CAT, correcting for transfection
efficiency (-gal) and for number of cells (protein concentration).
Values are presented as mean ± S.D. of at least three
experiments. C, a comparison of CAT expression of p1071KvCAT
and p894KvCAT transiently transfected into five different cell lines.
Transfections, CAT assays, and expression of results were as described
in panel A.
We next determined whether KRE was sufficient for conferring the silencing activity to a heterologous promoter. KRE was cloned 5` and 3` to the thymidine kinase CAT reporter gene (TK-CAT) in both orientations (Fig. 3A). Transient transfection experiments showed that cloning of the KRE 5` to the TK promoter in both orientations resulted in a 50% inhibition of TK-driven CAT activity in COS-7 and CHO cells (p < 0.05), with no significant effect on the level of expression in GH3 cells (pKRE(F)- or pKRE(R)-TK-CAT; Fig. 3B). Cloning of the same fragment 3` to the CAT gene in both orientations (pTK-CAT-KRE (F or R)), resulted in a 20% inhibitory effect on TK-CAT expression in COS cells that did not reach statistical significance (p > 0.05), while pTK-CAT-KRE(F) transfected into CHO cells caused a 30% inhibition of CAT activity. Taken together, these results suggest that the silencer activity of KRE is essentially orientation-independent, while position may modify the silencing effect.
Figure 3: KRE regulates the expression of an heterologous promoter. A, schematic maps of TK-CAT reporter gene constructs with the KRE. The constructs contain the KRE fragment cloned either 5` (KRE-TK-CAT) or 3` (TK-CAT-KRE) to the TK-CAT reporter gene construct in both orientations (F, forward; R, reverse). B, a comparison of CAT expression of control TK-CAT and TK-CAT reporter gene constructs containing the KRE in GH3 cells and control cell lines. Transfections and CAT assays were as in Fig. 2C. Results are expressed as percent change from the expression of control pTK-CAT reporter gene. Values are presented as mean ± S.D. of at least three experiments. Asterisk denotes a significant inhibition of CAT activity (p < 0.05).
Inspection of KRE sequence
revealed that it contains a
poly(GT)(GA)
(CA)
(GA)
dinucleotide repetitive element. To further characterize the silencer
element and determine the sequences essential and sufficient for its
activity, the KRE element was divided into three fragments (Fig. 4A). Fragment A contains 58 bp 5` to the
repetitive sequence, fragment B contains the 102-bp repetitive element,
and fragment C contains 18 bp 3` to the repetitive element. Cloning
elements A or B in front of the TK-CAT reporter gene (pA- or pB-TK-CAT; Fig. 4B) or the Kv1.5 promoter (pA or pB-478KvCAT; Fig. 4C) did not result in a significant inhibitory
effect in COS-7 cells. However, when fragments A and B (pA+B) were
cloned 5` to either the TK-CAT (Fig. 4B) or to
p478KvCAT (Fig. 4C) reporter gene constructs,
transfection into COS-7 cells resulted in a 50-70% inhibitory
effect on CAT activity, suggesting that fragments A and B are
sufficient for the silencing activity. By contrast, there was no
significant effect on CAT activity when the same constructs were
transfected into GH3 cells. Taken together, these results show that DNA
sequences in the repetitive element and in fragment A are essential and
sufficient for conferring silencer activity to an heterologous
promoter, and that the silencer element represses expression in COS-7
and CHO cell lines.
Figure 4: Mapping segments of KRE that are important for silencer activity. A, schematic representation of the KRE. The KRE was arbitrarily divided into three fragments: fragment A, 58 bp 5` to the repetitive sequence; fragment B, 102 bp containing the dinucleotide repetitive element; fragment C, 18 bp 3` to the repetitive element. B, a comparison of CAT expression of TK-CAT reporter gene constructs containing either fragments A, B, or A+B of KRE cloned 5` to the promoter and transiently transfected into either GH3 or COS-7 cells. Transfections, CAT assays, and expression of results were as in Fig. 3B. Values are presented as mean ± S.D. of at least three experiments. Asterisk denotes significant inhibition of CAT activity (p < 0.05). C, comparison of CAT expression of p478Kv1.5-CAT reporter gene constructs containing either fragment A, B, or A+B of KRE cloned 5` to the promoter and transiently transfected into either GH3 or COS-7 cells. Transfections, CAT assays, and expression of results were as in Fig. 3B. Values are presented as mean ± S.D. of at least three experiments. Asterisk denotes significant inhibition of CAT activity (p < 0.05).
To confirm that the KRE also can induce formation of
putative multimeric complexes of longer DNA strands, we next tested
whether a linear DNA fragment of about 2 kb would associate to form
complexes of slower mobility. The results show that incubating a
2600-bp linear DNA containing the 178-bp KRE fragment at 37 °C with
an excess of unlabeled fragment in high ionic strength (2.5 M
NaCl) resulted in the formation of new complexes that migrated near the
5.0-kb marker in agarose gel electrophoresis (Fig. 5D, lanes 5 and 7). The intensity of the band increased
with excess of the 2.6-kb unlabeled fragment (Fig. 5D, lane 8). Preheating resulted in the dissociation of the high
molecular weight complex (Fig. 5D, lane 6). At
low ionic strength, this band was not detectable even in the presence
of excess of unlabeled fragment (Fig. 5D, lanes
1, 3, and 4). Control experiments with a 2.9-kb
linearized pBluescript DNA fragment did not result in the formation of
new complexes with low electrophoretic mobility (data not shown). The
structure of the complexes formed by KRE will have to be defined.
However, these results resemble the pattern of multi-strand complexes
recently reported by Gaillard and Strauss, who showed that a 2390 bp
DNA fragment containing a (CA) repetitive element
dimerized to form a complex that migrated slowly in agarose
gel(18) . Using electron microscopic imaging, they demonstrated
that these fragments self-associated to form X-shaped structures,
congruous with the association of two linear double-stranded DNA
fragments to form four-stranded complexes.
Figure 6:
Electromobility gel shift assays with KRE. A, P-labeled KRE, or different portions of it
(see Fig. 4A), were incubated with buffer
alone(-) or with nuclear extracts from GH3, COS-7, or CHO cells
as indicated. The resulting DNA-protein complexes were resolved on a 4%
polyacrylamide gel and visualized by autoradiography. The arrow denotes a GH3 cell-specific gel shift complex. B,
P-labeled KRE was incubated with nuclear extracts from GH3
cells. The specificity of binding was determined by competition assay
with increasing amounts of KRE (lanes 3-5) or an
unrelated DNA fragment (a 162-bp HindIII fragment derived from
CRE modulator protein, CREM-
(2) ; lanes
8-10). The competitors were used at 100-, 300-, and 600-fold
molar excess over the probe. C,
P-labeled
A+B fragment was incubated with buffer alone (lane 1),
with hHMG1 (lanes 2-4), or with GH3 cell nuclear
extracts (lanes 5-7). Lanes 3 and 6 contain 150-fold molar excess of cold A+B fragment. Lanes
4 and 7 contain 1 µl of anti-hHMG-1 antiserum
(supershift assay). The resulting DNA-protein complexes were resolved
on a 4% polyacrylamide gel and visualized by autoradiography. The arrows denote specific DNA-protein
complexes.
We next tested whether high
mobility group 1 protein (hHMG1; (19) ), a nonhistone
chromatin-associated protein that selectively recognizes either
multistrand complexes, including poly(CA) or cruciform
DNA, would bind to KRE. EMSA using fragment A+B as a probe with
hHMG1 resulted in a gel shift and the formation of a DNA-protein
complex (Fig. 6C, lane 2). Competition with
150-fold molar excess of A+B abolished the gel shift effect (lane 3). Addition of anti-hHMG1 antibody resulted in a
supershift effect, and the complex migrated more slowly than the
control complex did (lane 4)(19) . This complex
differs in its migration pattern from the one formed by nuclear
extracts of GH3 cells (lane 5). Competition with a 150-fold
excess of cold probe resulted in a moderate decrease in the amount of
DNA-protein complex formed (lane 6), and the addition of
anti-hHMG1 antibody did not result in a supershift effect (lane
7), suggesting that the KRE-binding factor does not contain HMG1
protein.
Excitable cells must maintain tight control of the number and diversity of ion channels expressed in the cell membrane. However, very little is known about the transcription factors that control the cell specificity and the level of expression of voltage-gated channels in general and potassium channels in particular. Recently, we showed that cAMP and depolarization regulate the expression of Kv1.5 in a cell-specific manner(2) . In primary cardiocytes, cAMP and depolarization increase the steady-state levels of Kv1.5 transcript. In GH3 cells, cAMP reduces the steady-state levels of Kv1.5 transcript. A cAMP response element (CRE) located at the 5` noncoding region specifically binds the CRE-binding protein and the CRE modulator protein(2) . The promoter of Kv1.5 contains a putative glucocorticoid response element(2) . Recently, Takimoto et al.(14) showed that glucocorticoids increase the steady-state levels of Kv1.5 transcript and protein in GH3 cells and in normal rat pituitary cells. Nuclear run-on experiments suggested that the effect is at the transcriptional level. Taken together, our work and that of Takimoto et al.(14) show that Kv1.5 can rapidly respond to various hormonal stimuli and that the hormonal response in GH3 cells reflects the response of rat pituitary cells in vivo.
Here we described a DNA sequence that functions as
a cell-specific silencer, as evidenced by the selective suppression of
the Kv1.5- and TK-CAT reporter gene constructs in several cell lines
with no apparent effect on their expression in GH3 cells. However, the
level of suppression of Kv1.5-CAT-reporter's activity in COS-7
cells was up to 90% compared with 50-70% inhibition of KRE-TK-CAT
activity. Thus, it is likely that the cell-specific expression of Kv1.5
promoter is determined by additional cis-regulatory elements located
either 3` or 5` to the repetitive sequence. The silencer contains a
dinucleotide repetitive element
(poly(GT)(GA)
(CA)
(GA)
)
that associates spontaneously in vitro to form complexes with
slow electrophoretic mobility. The repetitive element and 58 bp of 5`
flanking sequences are necessary and sufficient for its function.
Another mammalian transcriptional cis-regulatory sequence that contains
a dinucleotide repetitive element is the skeletal muscle tropomyosin
(TM) enhancer(20) . This enhancer has a modular structure and
contains a 40-bp poly(CA) element, the deletion of which reduces the
level of expression of tropomyosin-CAT reporter gene constructs. To the
best of our knowledge, KRE is the first example of a dinucleotide
repetitive DNA sequence forming part of a cell-specific silencer.
Tandem repeat sequences of 2-5 reiterated nucleotides are
frequently found in eukaryotic genome. The most common type is a
dinucleotide CA repeat that can appear in up to 10 different locations, each of which contains up to 60 base
pairs(21) . Their varying length provides a useful system for
the generation of genetic markers that can be used for mapping and
linkage analyses(22) . These elements can induce a
conformational change from right-handed B-DNA to left-handed Z-DNA and
negative supercoiling(23) . It is not clear whether these
conformational changes play a role in transcriptional regulation,
either as part of an enhancer or as part of a silencer. Another role of
these sequences is to induce recombination between DNA strands in both
bacteria and eukaryotes(24) . The polymorphic nature of
repetitive elements in the mammalian genome may result in the existence
of a shorter, transcriptionally inactive, repetitive element in Kv1.5
gene of other species or even in different individuals within the same
species.
A 120-bp double-stranded DNA fragment containing a 60-bp
tract of poly(CA) was recently shown to associate
spontaneously, forming multimeric complexes in
vitro(18) . Insertion of the 60-bp tract into a 2390-bp
plasmid resulted in the formation of stable multimeric complexes that
appeared to form four-strand X-shaped-like structures. Using
poly(CA)
as a probe and nuclear extracts derived from
cultured monkey cells, EMSA revealed that the probe was specifically
recognized by high mobility group proteins (HMG) group 1 and group 2.
KRE contains a stretch of GT and CA repetitive elements and forms in vitro complexes electrophoretically similar to the ones
described by Gaillard and Strauss(18) . Thus, it is likely that
the complexes formed by the 2.6-kb fragment contain four-strand
complexes. In addition, KRE binds recombinant HMG1 protein, which is
known to bind to cruciform DNA and probably to four-way
strands(25) . Recently, hHMG1 was shown to interact with TATA
box-binding protein in the presence of oligonucleotides containing TATA
boxes(19) . The TATA box-binding protein-hHMG1 complex
prevented the formation of preinitiation complex by binding to TFIIB,
and in an in vitro transcription assay HMG1 was capable of
inhibiting RNA polymerase II activity by about 30-fold. Moreover, a Drosophila HMG-box containing a protein called DSP1 was
described as capable of converting Dorsal and NfkB from transcriptional
activators to repressors. This effect requires a sequence termed a
negative regulatory element. Another protein, HMGI(y), stimulates NfkB
activity(26) . We speculate that one possible mechanism for KRE
to suppress transcription may involve the binding of HMG1 protein and
inhibition of the formation of preinitiation complex. EMSA revealed
that the KRE forms a unique complex with protein(s) derived from GH3
cells nuclear extracts. We speculate that the factor(s) present in GH3
cells may abolish the inhibitory effect of HMG1, promoting the
expression of Kv1.5 in these cells. These factors may interact with the
nuclear factors that control the cAMP transcriptional response and with
glucocorticoid receptors.
We believe that KRE represents an important example wherein a dinucleotide repetitive element within the context of a mammalian promoter plays a role in regulating the level of gene expression in a cell-specific manner. We speculate that polymorphic changes in the primary structure of dinucleotide repetitive elements in the mammalian genome may render them transcriptional inactivators (silencers), thus modifying the level of expression of mammalian genes on an individual basis. This phenomenon may depend on the primary structure of the dinucleotide repetitive element, its location relative to the promoter, and the contextual sequences.
Excitable cells maintain a tight control on the expression of genes
that modify cell excitation. Both pre- and post-translational processes
regulate the expression and gating of voltage-gated K channels(1, 27, 28, 29) . We
described here a novel silencer that participates in restricting the
expression of Kv1.5 reporter gene constructs to GH3 cells. However, the
mechanisms that control the expression of Kv1.5 in the heart and other
tissues remain to be elucidated.