© 1999 American Heart Association, Inc.
Original Contribution |
Purification and Preliminary Characterization of a Cardiac Kv1.5 Repressor Element Binding Factor
From the Cardiovascular Division, Brigham and Women's Hospital, Boston, Mass.
Correspondence to Dr Gideon Koren, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail koren{at}calvin.bwh.harvard.edu
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Abstract |
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Abstract—We have previously demonstrated that the cell-specific expression of Kv1.5 promoter is regulated by a silencer (Kv1.5 repressor element; KRE) containing a dinucleotide-repetitive element, (GT)19(GA)1(CA) 15(GA)16. Electromobility gel shift assays (EMSAs) of KRE with GH3 nuclear extracts detected a unique DNA-protein complex, which was not detectable in Chinese hamster ovary or COS-7 cells. We further delineated KRE and determined that a 52-bp fragment that contained a (GT)10(GA)1(CA)10 dinucleotide-repetitive element was sufficient for silencer activity. EMSAs using nuclear extracts isolated from the heart and from GH3 cells demonstrated that the 52-bp element formed specific and identical gel shift effects. These complexes were not detectable in EMSA experiments with liver nuclear extracts. Magnetic DNA affinity purification and UV cross-linking experiments identified a 27-kDa KRE binding factor (KBF) in GH3 cell nuclear extracts. Purified KBF reacted specifically with double-stranded KRE, abolishing the formation of multimeric KRE-DNA complexes. Thus, the interaction between KRE and KBF may play an important role in regulating the GH3- and cardiac-specific expression of Kv1.5.
Key Words: Kv1.5 • transcription • silencer • heart • K+ channel
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Introduction |
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Silencers are cis-acting regulatory DNA elements that downregulate gene transcription. They generally exhibit their activity in an orientation- and position-independent manner.1 2 In some cases, these silencer elements may serve to modulate the extent of expression of their respective genes in different cells or tissues.3 4 Such silencers would function as governors restricting fluctuations in gene activity, thereby preventing deleterious consequences of overexpression.4 Other silencer elements control cell type–specific gene expression, such as in the cases of the cardiac myosin light chain 2,5 type II Na+ channel,6 M4 muscarinic acetylcholine receptor,7 osteocalcin,8 and Kv1.5 genes.9 Although some silencer elements do not interact with DNA binding proteins,10 others appear to function through DNA-protein interactions.11 12
DNA in general and dinucleotide-repetitive elements in particular are conformationally active molecules that are capable of adopting several types of conformations as dictated by their sequence, DNA binding proteins, and various ions.13 14 15 The DNA motif (CA)n is the most frequent tandem dinucleotide repeat in the mammalian genome.16 This repeat can respond to ionic conditions or negative supercoiling with a transition from the conventional B-DNA to a non–B-DNA forms.15 Some types of non–B-DNA conformation include left-handed Z-DNA, which occurs principally at segments of alternating C and G (or A) residues; cruciform DNA, which occurs at inverted repeat sequences; and bent DNA, which occurs at repeating A tracts.13 17
The mechanisms that account for transcriptional regulation in mammalian voltage-gated potassium channels are poorly understood. Several DNA elements that are important in the transcriptional regulation of Kv1.5,9 Kv1.4,18 and Kv3.19 genes have been identified. Of special interest for this report is the Kv1.5 repressor element (KRE) located in the 5' flanking region of the cardiac Kv1.5 potassium channel gene.9 Deletion of KRE resulted in the loss of the cell-specific expression of a reporter gene containing the Kv1.5 promoter linked to chloramphenicol acetyltransferase (CAT). KRE acts as a silencer in cell lines that do not express Kv1.5 and has no effect on the reporter gene expression assessed in transfected GH3 cells (a pituitary cell line that expresses Kv1.5). KRE contains a dinucleotide-repetitive element that is necessary but not sufficient for mediating silencer effects in cells that do not express Kv1.5. Double-stranded KRE self-associates in vitro to form DNA-DNA complexes with slow electrophoretic mobility. Furthermore, incubation of KRE with GH3 cell nuclear extracts results in a specific gel shift effect. Here, we report a more detailed characterization of KRE and the detection of the binding activity in heart cell nuclear extracts. A fragment of 52 bp that contains 42 bp of the repetitive elements and 10 bp of the 5' flanking sequence is sufficient to confer the silencing effect to a heterologous promoter and binding to GH3 cell nuclear extracts in electromobility gel shift assays (EMSAs). Magnetic DNA affinity purification and UV cross-linking experiments resulted in the identification of a 27-kDa KRE binding factor (KBF) present in GH3 cell nuclear extracts. EMSA competition experiments showed that KBF inhibits self-association and that an excess of KRE promotes self-association even in the presence of KBF. Collectively, these experiments suggest that KBF may regulate the cell-specific expression of Kv1.5 by abolishing the silencer effects of KRE.
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Materials and Methods |
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Polymerase Chain Reaction (PCR) and Reporter Gene Constructs
The KRE sequence was divided into 3 fragments (A, B, and C) as previously described.9 Deletion mutants of KRE shown in Figure 1
(except
A29B1 and A10B1, in which
29 and 10 refer to length of fragment A in bp) were obtained by
PCR with forward primers containing a HindIII restriction
site (underlined), 5'-TTCAGGGGTGCAAGCTTGTCTCAGTCT-3' (for
ABC), 5'-CCTGCCTTTTGATAAGCTTGTAGGCAAA-3' (for
A29BC, A29B,
A29B1B2, and KREM99),
5'-GGCAAATAGGAAGCTTTTCATGTGTGTG-3' (for
A10BC, A10B, and
A10B1B2), and
5'-GGTAGAGTTTCAAAGCTTTGTGTGTGTG-3' (for BC), and reverse
primers containing a BamHI restriction site (underlined),
5'-TTAAAGGATCCGGGAGTAGTGAACTCTC-3' (for ABC,
A29BC, and A10BC),
5'-GTAGTGAAGGATCCCTCTCTCTCTCTCT-3' (for
A29B and A10B), and
5'-TCTCTCTGGATCCGTGTGTGTGTGTGTG-3' (for
A29B1B2, A10B1B2, and
KREM99). In all cases,
p3100KvCAT9 was used as a template. A neutralizing
monoclonal antibody to the DNA polymerase of Thermus thermophilus
(Clontech) was used to reduce nonspecific amplification products
and primer-dimer artifacts. The PCR products were gel purified
using a glass-based purification system (Pharmacia), digested with
HindIII and BamHI, and subcloned 5' to thymidine
kinase (TK)–CAT. The clone KREM99 (Figure 1A) was obtained when the PCR reaction was carried out in the
absence of the antibody.
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The clone KREM80 was made by PCR with the forward primer, 5'-GGCAAAAGCTTAGAGTTTCAT(GT)19, containing a HindIII site (underlined) and the reverse primer, 5'-TCTCTCTGGATCC(TG)15(TG)-3', containing a single base substitution in the B1 domain (G, in italics) and a BamHI site (underlined). The PCR product was then digested with HindIII and BamH1 and subcloned 5' to TK-CAT.
The clones A29B1, A10B1, KREM72, KREM52, KREM42a, and KREM42b were generated by annealing oligonucleotides containing HindIII and BamHI restriction sites (underlined) and then subcloning 5' to TK-CAT. For example, M42a was made with the forward primer 5'-AGCTTAGAGTTTCAT(GT)5(GA)1(CA)10G-3' and the reverse primer 5'-ATCTCAAAGTA(CA)5(CT)1(GT)10CCTAG-3'. Clones were sequenced after their subcloning into TK-CAT using the Applied Biosystems model 377 automated sequencer.
Cell Lines and Transfection Experiments
All cells used in this study were maintained in DMEM containing
10% FCS. Transient transfections were carried out with a
liposome-based method (Life Technologies, Inc) following the
manufacturer's instructions. Forty-eight hours after transfection, the
cells were harvested and assayed for protein concentration,
ß-galactosidase, and CAT activities as described by Mori et
al.20 CAT activity values were normalized for transfection
efficiency by ß-galactosidase activity and for cell density by
protein concentration as described by Mori et al.9
Relative CAT activity was calculated by comparing the activities of
TK-CAT plasmids containing KRE deletion mutants with the activity of
control TK-CAT plasmid (100%). Values are presented as
mean±SD for a minimum of 3 determinations, each of which were
performed in either duplicate or triplicate. Statistical significance
was determined by 1-factor ANOVA, with P<0.05 considered
significant.
EMSAs
Nuclear extractions from cultured cells were carried out
essentially as described by Therrien and Douin.21 Nuclear
extractions from rat heart or liver were performed as described by Thai
et al.22 EMSA experiments were performed as described by
Mori et al.9 20 EMSA competition experiments with
unlabeled DNA were carried out following the same protocol, except that
the appropriate amount of unlabeled competitor fragments was added 30
minutes before the labeled probe. Complex bands were quantified by
densitometry with the NIH Image software program.
Label Transfer by UV Cross-Linking
Probes were generated by PCR. The PCR mixtures contained
bromodeoxyuridine triphosphate and dCTP at 40 µmol/L each; dATP,
dGTP, [
-32P]dATP, and
[-32P]dGTP at 20 µmol/L each; and
[-32P]dCTP at 10 µmol/L. Purified
protein (10 ng) was preincubated with 10 ng of poly(dI-dC) for 15
minutes at room temperature. Then, 105 Cerenkov
cpm of the 32P-labeled probe was added to the
binding reaction mixture in either the presence or the absence of
unlabeled competitor. After 30 minutes of incubation at room
temperature, the mixtures were irradiated in a UV Stratalinker 1800
(Stratagene) with UV light to a total dose of 0.6 J. Probe DNA was then
digested for 10 minutes at 37°C after the addition of 1 U of DNase I,
1 U of micrococcal nuclease, 33 mmol/L
MgCl2, and 33 mmol/L
CaCl2. After the addition of EDTA to 30
mmol/L, the mixtures were resolved by SDS-PAGE.
Purification of KBF
All buffers contained 0.5 mmol/L phenylmethylsulfonyl
fluoride, 2 µg/mL pepstatin, 2 µg/mL aprotinin, and
1 mmol/L DTT. KBF was purified using the magnetic DNA affinity
purification method developed by Gabrielsen and Huet.23
Briefly, double-stranded DNA was biotinylated by a dTTP analog with
biotin covalently attached to the pyrimidine ring (Clontech) using a
Klenow reaction. Then, the biotinylated probe was bound to streptavidin
M-280 Dynabeads (Dynal) following the manufacturer's recommendations.
The protein binding was performed in 1x EMSA buffer (in mmol/L,
HCl [pH 7.5] 10, NaCl 50, and EDTA 1, and 5% glycerol) supplemented
with 0.5 to 1 µg of poly(dI-dC) per microgram of nuclear extract used
as starting material. Then, the mixtures were washed 2 to 4 times in
1x EMSA buffer, and the bound protein was eluted with 1x EMSA buffer
supplemented with 0.5 mol/L NaCl. SDS-PAGE was performed according to
the method of Laemmli.24 Gels were stained with the Silver
Stain Plus kit (Bio-Rad) or Coomassie brilliant blue (Sigma). Purified
samples were desalted and concentrated with microcon-10 or centricon-10
(Amicon) depending on the volume of sample. Protein concentration from
purified samples was determined by a microbicinchoninic acid
method (Pharmacia) and by absorbance at 280 nm.25
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Results |
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KRE Core Structure Definition
To define the sequences of KRE that are essential for its silencer activity, we created serial deletions at either the 3' or 5' end of KRE (Figure 1A
). The KRE sequence was divided into 3 fragments as
previously described.9 The repetitive element (fragment B;
Figure 1A) was further analyzed by subdividing it into 3
domains: B1, B2, and B3 (Figure 1A). Each KRE deletion mutant
was cloned 5' to the TK-CAT promoter and tested for silencer activity
using transient transfections of Chinese hamster ovary (CHO), COS-7,
and GH3 cell lines. Serial deletion in fragment A revealed that 10 bp
of fragment A (A10, abutting the B1 domain
[Figure 1A]) were essential for silencer activity. Thus, all 3
deletions, including ABC, A29BC, and
A10BC functioned as effective silencers when
cloned 5' to the TK-CAT promoter (Figure 1B). However, deletion
of the last 10 bp of fragment A (A10 and cloning
of BC in front of the TK-CAT promoter [BC]) did not reduce expression
of CAT activity in COS-7 and CHO cell lines, thus abolishing silencer
activity (Figure 1B). In contrast, deletion of fragments C
(A29B and A10B) and B3
(A29B1B2 and A10B1B2) did
not affect silencer activity. However, replacement of part of the B1
domain with (CA)17 (KREM99)
or deletion of B2 (A29B1 and
A10B1) abolished silencer activity. Thus, both B1
and B2 seem to form part of the core element of the silencer. None of
the KRE deletion mutants mediated silencer activity in GH3 cells
(Figure 1B).
We next correlated the silencer activity with the ability of these
fragments to form DNA-protein complexes when incubated with GH3 nuclear
extracts. EMSA using 32P-labeled KRE deletion
mutants indicated that all KRE deletion mutants that exhibited silencer
activity in non-GH3 cells (Figure 1B) formed DNA-protein
complexes with GH3 cell nuclear extracts (Figure 2). By contrast, the KRE deletion mutants
that did not exhibit silencer effects (BC,
KREM99, A29B1, and
A10B1) did not bind to GH3 nuclear extracts
(Figure 2). These results indicated that the B1 and B2 domains
of KRE and the 10 bp of fragment A are essential for mediating both the
silencer effect in non-GH3 cells and the GH3 cell–specific binding
activity. Interestingly, under the conditions optimized for EMSA, both
ABC and A10BC probes could form
multimeric DNA complexes with slow electrophoretic mobility
that migrated slower than the double-stranded probe but faster than
DNA-protein complexes (Figure 2, lanes 1 and 5).
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To further refine the core elements of KRE, series of short deletions
and point mutations were created (Table 1). Point mutation of
(GA)1 to (CA)1
(KREM80) did not affect silencer activity or
binding. We then created successive deletions of the
(GT)n and (CA)n repeats and
compared their silencer and binding activities with that of
A10B1B2 deletion mutant (Table 1). The
results showed that KRE deletion mutants with at least 10
(GT)n and 10 (CA)n repeats
mediated silencer effects in non-GH3 cells and bound to GH3 cell
nuclear extracts. However, the mutants KREM42a
and KREM42b, which contained 5
(GT)n and 5 (CA)n,
respectively, lost their silencing activity and gel shift effects
(Table 1). Therefore, 10 repeats were sufficient for silencer
activity. Taken together, these results suggest that the minimum number
of (GT)n and (CA)n repeats
necessary for maintaining silencer activity may range from 6 to 10
repeats.
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To further test the core element defined in the transfection and
gel-retardation assays, we used nuclear extracts derived from tissue
that expresses Kv1.5 (rat heart) and control tissue (rat liver) that
does not express the protein. EMSAs revealed that incubation of
KREM52 with heart nuclear extracts resulted in a
gel shift with a complex of a size indistinguishable from that
obtained by reacting GH3 nuclear extracts with the same probe (Figure 3). By contrast, liver nuclear extracts
did not contain any gel shift activity.
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Identification and Purification of a KBF in GH3 Cells
To identify the KRE binding proteins present in GH3 cells,
nuclear extracts from this cell line were submitted to a magnetic DNA
affinity purification step as described by Gabrielsen and
Huet.23 The experiments were carried out at 2 different
ratios of poly(dI-dC) (to modulate the stringency of binding) and in
the presence of protease inhibitors (to avoid the
degradation of the purified protein). The same procedure was used with
nuclear extracts derived from COS-7 cells to determine whether the
silencer effects in non-GH3 cells involve any DNA-protein interaction
that cannot be detected by EMSA. Silver staining of the proteins eluted
from GH3 cells, at low concentration of poly(dI-dC), revealed a
prominent 27-kDa band (Figure 4A).
Two additional minor bands (28 and 30 kDa) were detectable in the
original silver staining but are too light to be clearly discernible in
Figure 4A. By contrast, no detectable protein was eluted when
COS-7 nuclear extracts were submitted to the same procedure (Figure 4A). Supplementation of the binding reaction with the same ratio
of protein to poly(dI-dC) as in EMSA experiments (1:1) resulted in the
detection of a single 27-kDa band in experiments carried out with GH3
cell nuclear extracts. This protein, present in GH3 nuclear
extracts that interacts with KRE, was named KBF. EMSAs using GH3
nuclear extracts and purified KBF resulted in an identical gel shift
effect, which indicates that KBF most likely represents the
binding activity present in GH3 cell nuclear extracts (Figure 4B). The DNA binding activity of the purified protein resulted
in a 50% yield (Table 2). Thus, with a
single purification step we obtained a 1400-fold purification. Taken
together, these results suggest that double-stranded KRE interacts with
a 27-kDa protein present in GH3 but not in COS-7 nuclear
extracts.
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We then proceeded with UV cross-linking experiments to confirm that the
DNA binding activity detected by EMSA was related to the 27-kDa protein
and not to minor protein contaminants. Purified KBF (10 ng) was
incubated with A29B1B2, and the reaction mixtures
were irradiated with UV light, treated with DNase I and micrococcal
nuclease, and fractionated by SDS-PAGE. The specificity of label
transfer was tested by competition experiments with unlabeled
A29B1B2 or mutated nonfunctional KRE of identical
size (KREM99) (Figure 1A). The results
show (Figure 5) that the labeled probe
was crossed-linked to a polypeptide with an apparent molecular
mass of 
27 kDa (lane 1). The labeling of the 27-kDa protein
was completely inhibited by an excess of unlabeled
A29B1B2 (Figure 5, lane 3) but not by an
excess of the unlabeled KREM99 (Figure 5, lane 4). No protein was detected in UV cross-linking experiments in the
absence of KBF (Figure 5, lane 2) or when
A29B1B2 was incubated with nuclear extracts from
COS-7 cells (data not shown). Taken together, these results confirm
that the 27-kDa polypeptide (KBF) is the protein that interacts with
KRE.
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Binding of KRE and KBF Versus Self-Assembly of KRE
We have previously shown that the double-stranded KRE
self-associates spontaneously to form large DNA-DNA complexes with slow
electrophoretic mobility and that an excess of cold fragment promotes
these associations.9 As shown in Figure 6A, 0.5 ng of
32P-labeled A29B1B2
self-associates to form complexes with slow electrophoretic mobility in
the presence of a 100-fold molar excess of cold fragment (Figure 6A, lanes 3 and 4). These complexes had an apparent mobility of
400 and 800 bp. By contrast, the addition of an excess of
KREM99 did not promote the formation of these
complexes even in the presence of a 100-fold molar excess of unlabeled
KREM99 (Figure 6A, lanes 7 and 8).
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We next tested whether the binding of purified KBF to KRE affects
self-association of KRE. The self-association of KRE was favored by
increasing the concentration of A29B1B2 to 1 ng.
In the absence of KBF (Figure 6B, lanes 1 and 7), labeled
A29B1B2 formed DNA complexes with slow
electrophoretic mobility. Incubation of A29B1B2
with KBF resulted in a gel shift effect forming 2 DNA-protein complexes
similar to those observed with GH3 nuclear extracts (Figure 2A and 2B; Figure 6B, lanes 2 and 8). The addition of an excess of
cold probe competed with the binding in a nonlinear fashion and
promoted the reappearance of the complexes with slow electrophoretic
mobility that are identical to those formed by spontaneous DNA-DNA
interactions (Figure 6B, lanes 3 through 6). Importantly, the
appearance of self-association bands coincided with the effective
competition of the DNA-protein interactions. By contrast, an excess of
KREM99 did not compete with the binding of KBF to
KRE and did not promote the self-association of
A29B1B2 (Figure 6B, lanes 8 through 12).
These observations indicate that binding of KBF to KRE may interfere
with self-association of KRE and that an excess of KRE is necessary and
sufficient to promote self-association of KRE, even in the presence of
KBF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For most eukaryotic genes, the tissue specificity and the level of expression are determined by interactions between trans-acting factors, with cis-acting elements located within the 5'-flanking sequences.26 We have previously identified a cis-regulatory element (Kv1.5 repressor element, KRE)9 that participates in determining the cell-specific expression of Kv1.5 promoter in GH3 cells. KRE represses expression of Kv1.5-CAT and TK-CAT reporter gene constructs in transient transfections of cells that do not express Kv1.5. By contrast, expression of these constructs in GH3 cells, which express Kv1.5, is not affected.9 In the present report, we further defined the minimal sequence that is necessary and sufficient for mediating the silencer activity. Our results indicate that a 52-bp DNA fragment containing 42 bp of alternating purines and pyridines and 10 bp of 5' flanking sequence is sufficient for mediating the silencing activity. EMSA experiments revealed that this core element is also sufficient for binding to nuclear factor(s) present in GH3 cell nuclear extracts. EMSA experiments revealed that this core element is also necessary for binding to nuclear factor(s) present in GH3 cell nuclear extracts. Moreover, indistinguishable gel shift activity is also present in the nuclear extracts derived from rat heart. By contrast, this gel shift activity is not present in the liver or multiple cell lines in which Kv1.5 transcript and polypeptide are undetectable.27 The stability and specificity of interactions between KRE and the nuclear factor(s) present in GH3 cell extracts were sufficiently high to attempt a DNA affinity purification of this trans-acting factor. Indeed, a 27-kDa KBF present in the nuclei of GH3 cells was purified
1000-fold by
magnetic DNA affinity purification. The identification of KBF and the
absence of KRE-silencer activity in GH3 cells suggest that KBF may
abolish the silencing activity of KRE and therefore act as an
"antisilencer." Our observation in this cell line may also apply to
the regulation of Kv1.5 in vivo. Indeed, incubation of rat heart
nuclear extract with KRE resulted in a gel shift effect
indistinguishable from that of GH3 cell nuclear extract and KRE. Several trans-acting factors alter DNA conformation.28 A dinucleotide-repetitive element such as poly(GT/CA) can also change its conformation from the orthodox right-handed B-DNA to a non–B-DNA form.29 This transition can be induced by increased superhelical density,29 by 10 to 50 mmol/L of magnesium,14 and by submillimolar levels of calcium.15 Moreover, alternating purine-pyrimidine stretches can interact with each other through the formation of multistranded structures such as intermolecular triplexes or tetraplexes.17 Indeed, double-stranded DNA fragments containing poly(CA)30(TG)30 were shown to associate spontaneously in vitro to form stable 4-stranded structures that could be detected by gel electrophoresis and electron microscopy.30 Similar complexes were also formed by the self-association of double-stranded KRE9 or A29B1B2, which contain long (CA)n and (GT)n repeats. However, KREM99, which contained a short (GT)n repeat, did not form such DNA-DNA complexes or retain the silencer effect.
Our deletion analyses, the EMSA competition assays, and the UV cross-linking experiments confirm that double-stranded KRE interacts with KBF in a sequence-specific and perhaps structure-specific manner and that the DNA-protein interaction may interfere with the formation of DNA-DNA interactions in vitro and perhaps also in vivo. It is tempting to speculate that the formation of DNA-DNA interactions by (CA)n and (GT)n repetitive sequences might be crucial for the silencer effect either directly or through interactions with ubiquitous nuclear proteins. Indeed, the ubiquitous high-mobility–group (HMG)–1 and HMG-2 proteins recognize altered DNA conformations formed by (CA)n and (GT)n repetitive sequences.31 Moreover, recombinant HMG-1 protein binds to KRE in the absence of poly(dI-dC).9 KBF, by interfering with the self-association of KRE and/or the binding of HMG-1 and HMG-2, may abolish the silencer effect in GH3 cells (or heart). In cell lines (or tissues) in which KBF is not expressed, the default silencing effect would be dominant.
Several proteins, such as SWI/SNF in yeast,32 may facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The complex has been purified33 34 and shown to contain an ATP-dependent nucleosome-disruption activity. The DNA binding properties of SWI/SNF35 are similar to those of proteins containing an HMG box domain.36 Thus, the SWI/SNF complex interacts with the minor groove of the DNA helix, binds synthetic 4-way junction DNA (which is thought to mimic the topology of DNA as it enters or exits the nucleosome), and introduces positive supercoils into relaxed plasmid DNA. These properties are likely to be important in the remodeling of chromatin structure by the SWI complex35 and some HMG-box containing proteins.37 38 Members of the HMG-box family of proteins have been shown to interact functionally with other transcription factors39 40 or with other proteins that act as coactivators.41 It is also believed that members of the HMG-domain family of proteins regulate gene expression by coordinating the assembly of multiprotein complexes,39 42 thereby promoting interactions with the basic transcriptional machinery.43 The mammalian SWI/SNF–like complexes, which contain a subunit with a HMG box, may also interact with higher-order chromatin structure by 2 or more DNA binding domains, thereby mediating ATP-dependent nucleosome disruption.44
Our results suggest that the interaction between KBF and KRE may be partly responsible for the cell-specific expression of Kv1.5 in GH3 cells. Therefore, the cloning of KBF may help us to elucidate the mechanisms that control the expression of Kv1.5 in the heart and other tissues.
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Acknowledgments |
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This work was supported by a National Institutes of Health RO1 grant and by an Established Investigator Award from the American Heart Association (to G.K.). We thank Dr D. Sherris for encouragement and critical reading of the manuscript. We thank Dr J. Zhou for advice in the statistical analysis of the data.
Received November 18, 1998; accepted February 12, 1999.
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