©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
GH3 Cell-specific Expression of Kv1.5 Gene
REGULATION BY A SILENCER CONTAINING A DINUCLEOTIDE REPETITIVE ELEMENT (*)

(Received for publication, July 18, 1995)

Yasukiyo Mori Eduardo Folco Gideon Koren (§)

From the Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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)(1)(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.


INTRODUCTION

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^1 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.


MATERIALS AND METHODS

Plasmid Constructions

p3100KvCAT, p2009KvCAT, p1573KvCAT, and p1144KvCAT constructs correspond to the previously described p2300KVCAT, p1215KVCAT, p779KVCAT, and p350KVCAT constructs, respectively(2) . Initially(2) , the numbers marked the distance from the transcription start site. Here, the numbers mark the distance from the translation start site. All constructs contain most of the 5` noncoding region, with the exception of the 20 bp immediately 5` to the ATG. p1071KvCAT, p648KvCAT, p478KvCAT, and p79KvCAT were prepared by using PCR with four forward primers (primers 1-4) and a reverse primer (primer 5) or the T7 primer. p1144KvCAT was used as a template, and the PCR products were cloned as ClaI-HindIII fragments into pBs-CAT (pBs = Bluescript Ks(-), Stratagene; pBs-CAT ((2) )). The sequences of the primers were: 1) 5`-GGGCCATCGATGGTCTCAGTCTCCTGCC-3`; 2) 5`-GGAAGATCGATTCTAAAAGAAACATAG-3`; 3) 5`-TTCTCATCGATTTGCCCTGGGAGTTTCA-3`; 4) 5`-CCTACGCGAGGCTTGGA-3`; 5: 5`-GCCAAGCTT-CCATGGTGCGCCCCAGAC. p894KvCAT was generated by deleting a XhoI-PstI fragment from p1144KvCAT, followed by blunt-ending and ligation. p284KvCAT was prepared by cloning a SacI-SacI fragment, which includes the CAT gene from p3100KvCAT into pBs. The KRE fragment was prepared by PCR, using primers 1 and 5, and cloned into pBs as a ClaI-PstI fragment (pBs-KRE). pTK-CAT constructs containing the KRE fragment at both sides and in both orientations were generated by cloning a KRE fragment from pBs-KRE into pTK-CAT using HindIII and SalI sites 5` to the TK-CAT promoter (for pKRE-TK-CAT) or by cloning the TK promoter 5` to CAT-KRE construct (for pTK-CAT-KRE). The TK promoter in TK-CAT contains 160 bp (-109 to +51) linked to CAT gene(17) .

Transfection Experiments and CAT Assays

Transfection experiments and CAT assays were performed as described by Mori et al.(1993). Briefly, 15 µg of each plasmid were cotransfected with 1 µg of pCMV-betagal. CAT activity was determined by a dual-phase diffusion assay (2) and was normalized for transfection efficiency by beta-galactosidase activity and for cell density by protein concentration. All cells used in this study were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.

RNase Protection Analyses and Primer Extension

Total RNA was isolated by guanidinium isothiocyanate/cesium chloride centrifugation and subjected to RNase protection and primer extension analyses, as described previously(2) . For RNase protection analysis, a riboprobe containing the sequences corresponding to the fragment between -476 and -17 bp from the translation start site of Kv1.5 gene cloned in pBs was generated using the T7 promoter. For primer extension analysis, an oligonucleotide primer complementary to the sequence from -120 to -150 relative to the translation start site of Kv1.5 was used (5`-CCCTCGTCCCCGTCGTCGAAGAACTGCAGT--3`).

Electromobility Gel Shift Assays

Nuclear microextractions were carried out essentially as described by Therrien et al.(17) from 2 times 10^7 cells. For EMSA, 2 µg of nuclear extract were incubated at room temperature for 15 min with 2 µg of poly(dI-dC) (Sigma) in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, after which 0.5 ng of P-labeled DNA probe was added(2) . The mixtures (20 µl) were incubated at room temperature for 30 min, and the DNA-protein complexes were resolved on 4% native polyacrylamide gels and visualized by autoradiography. The DNA fragments used as probes were labeled to a specific activity >0.5 times 10^6 cpm/ng by [alpha-P]dCTP using Klenow reaction. EMSA assays with hHMG1 were done in the absence of poly(dI-dC) since it inhibits the binding of hHMG1 to KRE.

Analyses of KRE Complexes by Electrophoresis

The DNA fragments containing KRE or parts of it were prepared as described above. A 2.6-kb fragment with KRE was generated as a SalI-EcoRI fragment from p1144KvCAT (the SalI is located in the polylinker of pBs, while the BamHI is located in the CAT gene). After labeling with [alpha-P]dCTP using the Klenow reaction, the DNA fragments were ethanol-precipitated in the presence of 2 M ammonium acetate to remove the unincorporated nucleotides. In some experiments (Fig. 5B), further purification of the labeled DNA was done by electrophoresis in 8% polyacrylamide gel. The appropriate fragments were localized by autoradiography and excised from the gel, and the DNA was eluted overnight at 37 °C into 400 µl of TE buffer. The fragments were phenol-extracted, ethanol-precipitated, and then resuspended in 20 µl of TE buffer. Each DNA fragment (10^4 cpm) was incubated in the indicated condition. The reaction was ethanol-precipitated, resuspended in 20 µl of EMSA binding buffer, and immediately loaded onto a 4% native polyacrylamide gel. The gel was dried and exposed. A 100-bp DNA marker (Life Science) was used after labeling by [-P]ATP and T(4) polynucleotide kinase.


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.




RESULTS

Characterization of a Silencer Element in Kv1.5 Gene

The tissue-specific expression of many eukaryotic genes is regulated by cis-acting sequences located near the promoter. In order to analyze the mechanism of cell-specific expression of Kv1.5 gene, we selected as a model system GH3 cells, a clonal pituitary cell line that expresses Kv1.5 polypeptide(14) . We first defined the transcription start site(s) of Kv1.5 promoter in GH3 cells using primer extension assays and RNase protection analyses. We expected multiple start sites, since Kv1.5 promoter lacks a canonical TATA box. Indeed, primer extension assays (n = 3) revealed multiple identical start sites in the heart and in GH3 cells (Fig. 1B). The top two products (700 bp), detectable in RNA derived from GH3 cells and from the heart, correspond to the transcription start sites previously described by Mori et al.(1993). The bottom three products (92-102 bp) detectable in GH3 cells and heart, correspond to transcription start sites located 212-222 bp 5` to the translation start site. A relatively minor product (242 bp), which was detected in GH3 cells, corresponds to a start site located 362 bp 5` to the translation start site. Long exposure revealed that this product was also detectable in the heart (data not shown). The third group of products (317-321 bp) corresponds to a potential start site located 437-441 bp 5` to the translation start site. RNase protection assays (RPA) were used to confirm the location of the various start sites (Fig. 1C). Using a probe derived from a Kv1.5 genomic clone (Fig. 1A), the RPA revealed five protected bands ranging in size from 185 to 387 bp. The bottom bands confirmed the start sites located 212-222 bp 5` to the translation start site, and the band marked by the asterisk (Fig. 1C) confirmed the start site located 362 bp 5` to the translation start site. The potential start sites located 437-441 bp 5` to the translation start site, could not be confirmed by RPA. Long exposure revealed a product that corresponds to the fully protected probe (data not shown), most likely originating from the start sites described by Mori et al.(2) and confirmed by the primer extension assay. RPA protected three additional bands (247, 312, and 387 bp) that could not be confirmed by the primer extension assays. It seems plausible that high GC content (2) may have resulted in local RNA secondary structure, causing differences in the apparent transcription start sites. Control RPA and primer extension assays using RNA derived from liver and tRNA were negative. Despite these caveats that reflect the limitations of the assays used, taken together, these results indicate that the transcription start sites in the heart and in GH3 cells are identical. Thus, GH3 cells can be used as a system to analyze the cell-specific expression of Kv1.5 gene.


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(4) precipitation method into GH3 cells (2) (Fig. 2, A and B). COS-7 cells, which do not express Kv1.5,^2 were used as control (Fig. 2B). To control for transfection efficiency, a plasmid containing the beta-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 (beta-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)(1)(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).



Self-association of KRE

Recently, Gaillard and Strauss (18) showed that a DNA fragment containing a poly(CA) dinucleotide repetitive element self-associated to form four-strand structures detectable by electron microscopy and gel electrophoresis. We therefore decided to test whether KRE, which also contains poly(CA)-poly(GT) elements, would associate spontaneously to form stable multimeric DNA complexes in vitro. The approach used was similar to the one described by Gaillard and Strauss(18) . Electrophoresis of KRE in non-denaturing gels after incubation for 4 h at 37 °C showed, in addition to the predominant band that corresponds to the double-stranded probe, multiple bands with slower electrophoretic mobility (Fig. 5A, lane 1). Preheating of the probe at 90 °C resulted in heat dissociation of the double-stranded probe and the putative multimeric complexes with slower electrophoretric mobility to single strands that migrated above the double-stranded probe (Fig. 5A, lane 2). The bands formed an irregular ladder, indicating that the putative multimeric complexes may contain a varying number of strands with different conformation, resulting in marked variability in electrophoretic mobility. Adding an excess of unlabeled KRE fragment did not change the pattern of migration or the intensity of the bands (Fig. 5A, lanes 3 and 4). Size selection by excision of the double-stranded probe from a polyacrylamide gel and incubation at 37 °C for four hours resulted in the formation of a regular ladder with an intense lower band corresponding to the labeled double-stranded fragment, and two additional bands of slower mobility. The middle band migrated just above the 500-nucleotide marker (Fig. 5B, I, lanes 1-7), while the top band migrated near the 1-kb marker (Fig. 5B, II, lanes 1-4). Incubation of the probe with heat-denatured (H) or phenol-denatured (P) nuclear extracts derived from COS-7 or GH3 cells did not change the pattern of migration of these complexes in native gels (Fig. 5B, I and II, lanes 1-4). Thus, denatured proteins derived from nuclear extracts did not modify the mobility of these complexes, supporting the hypothesis of self-association of KRE. Taken together, these results strongly suggest that this DNA element forms complexes with substantially reduced electrophoretic mobility in native gels. The stability of these complexes as a function of temperature was tested (Fig. 5B, lanes 5-9). When heated to 90 °C in a buffer of low ionic strength, the multimeric complexes dissociated to single strands that migrated more slowly than the native double-stranded probe (lane 8). Electrophoresis of a control 172-bp HindIII fragment excised from the somatostatin promoter showed a predominant band corresponding to the double-stranded probe, with no evidence for the formation of complexes with slower electrophoretic mobility (Fig. 5C, lane 1). Adding unlabeled fragment did not change the pattern of migration (Fig. 5C, lane 3). Preheating the probe at 90 °C resulted in heat dissociation of the double-stranded probe to single strands that migrated more slowly than the native double-stranded probe (Fig. 5C, lane 2). Thus, a DNA fragment excised from the somatostatin promoter, which lacks a dinucleotide repetitive element, did not associate to form multi-strand complexes.

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.

Identification of a Silencer Binding Protein

To identify a protein or proteins that may interact with KRE, a series of electromobility gel shift assays were carried out using nuclear extracts derived from GH3, COS-7, and CHO cells and the three KRE fragments as probes (Fig. 6A). The results showed that during incubation in the EMSA buffer, the probe formed complexes that migrated more slowly than the double-stranded probe (lane 1). The top two bands in this lane can be also seen in lanes 2-4. Incubation of GH3 cells nuclear extract with KRE resulted in a gel shift and in the formation of a complex migrating slightly above the probe (lane 2, arrow). An indistinguishable sized complex could also be detected by EMSA, using fragment A+B as a probe (lane 6). Heat inactivation or phenol denaturation of the extracts abolished the formation of this complex (data not shown). EMSA using fragments B alone, B+C, or A as probes (lanes 10, 14, and 18, respectively) did not result in a significant gel shift effect, suggesting that sequences in fragments A and B are essential and sufficient for the detection of this binding activity. EMSA with nuclear extracts derived from COS-7 or CHO cells resulted in a gel shift and the formation of a complex with identical mobility in both cell lines that differed from the GH3 complex (lanes 3 and 4). EMSA using fragments A+B, B, B+C, or A as probes resulted in the formation of similar-sized complexes in both cell lines (lanes 7 and 8, 11 and 12, and 15 and 16), suggesting that the complex may contain a non-sequence-specific DNA-binding protein. Heat inactivation abolished the formation of these complexes (data not shown). The specificity of interactions between KRE and protein(s) present in GH3 nuclear extract was tested in a competition assay with unlabeled DNA fragments (Fig. 6B). The binding of KRE fragments to protein(s) could be competed out by an excess of unlabeled KRE, and not by an unrelated DNA fragment of similar size.


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-alpha(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.


DISCUSSION

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)(1)(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 10^5 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 46005 (to G. K.) and by an American Heart Association Fellowship Award (to Y. M.) and Establish Investigator Award (to G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-6728; Fax: 617-732-5132.

(^1)
The abbreviations used are: CAT, chloramphenicol acetyltransferase; KRE, Kv1.5 repressor element; bp, base pair(s); kb, kilobase pair(s); CHO, Chinese hamster ovary; EMSA, electromobility gel shift assay; hHMG1, nonhistone high mobility group protein group 1; RPA, RNase protection assay; TK, thymidine kinase.

(^2)
Y. Mori and G. Koren, data not shown.


ACKNOWLEDGEMENTS

We thank Dr. T. Michel for critical reading of the manuscript. We thank Dr. Robert G. Roeder for providing hHMG1 cDNA and anti-hHMG1 polyclonal antibody.


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