©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Regulatory Element of a Nicotinic Acetylcholine Receptor Gene Interacts with a DNA Binding Activity Enriched in Rat Brain (*)

(Received for publication, December 14, 1994)

Minjie Hu (1) (2) Catherine B. Bigger (1) Paul D. Gardner (1)(§)

From the  (1)Center for Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245-3207 and the (2)Biochemistry Program, Dartmouth Medical School, Hanover, New Hampshire 03755

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nicotinic acetylcholine receptors are ligand-gated ion channels that play a critical role in signal transmission in the nervous system. The genes encoding the various subunits that comprise functional acetylcholine receptors are expressed in distinct temporal and spatial patterns. Studies to understand the molecular mechanisms underlying the differential expression of the receptor subunit genes have led to the identification, in this report, of a 19-base pair cis-acting element that is required for transcriptional activation of the rat beta4 subunit gene. Screening of computer data bases with the 19-base pair element revealed the sequence to be unique among known transcriptional regulatory elements. Loss of this element resulted in drastically reduced beta4 promoter activity in transfected cholinergic SN17 cells. Furthermore, this element specifically interacts with nuclear proteins prepared from both SN17 cells and adult rat brain. UV cross-linking experiments indicated the presence, in SN17 nuclear extracts, of a prominent protein species (approximately 50 kDa) that interacts specifically with the 19-base pair element. These results lead us to hypothesize that interactions between the 50-kDa protein and the novel 19-base pair element are necessary for transcriptional activation of the beta4 subunit gene.


INTRODUCTION

Characterization of the genetic events leading to the expression of specific neurotransmitter receptors is necessary for a thorough understanding of the complex communication mechanisms within the central nervous system. Much of the knowledge gained in this respect comes from studies of the nicotinic acetylcholine (ACh) (^1)receptor gene family and, in particular, from studies of the ACh receptors expressed in skeletal muscles. Expression of ACh sensitivity is fundamental to the formation of neuromuscular synapses, and it has been shown that the increases in ACh sensitivity that occur in muscles following innervation are consequences of changes in the abundance and distribution of ACh receptors (reviewed in (1) ). In addition, the postsynaptic response to ACh is further modified by subsequent changes in the functional properties of ACh receptors(2) . These studies have clearly demonstrated that regulation of ACh receptor expression is critical for proper formation and function of neuromuscular synapses. It is likely this is the case for cholinergic synapses in the central nervous system as well.

The accessibility of neuromuscular synapses has provided a model system for the study of synaptogenesis. However, unlike their neuromuscular counterparts, cholinergic synapses in the central nervous system have proven much more difficult to analyze, particularly at a molecular level. Recently though, molecular cloning approaches have led to the identification of a family of genes encoding neuronal nicotinic ACh receptors (reviewed in (3) ). Members of this family include alpha2-alpha9(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and beta2-beta4(4, 7, 10, 16, 17, 18, 19) . Reconstitution studies have demonstrated that different combinations of alpha and beta subunits leads to the formation of functionally distinct classes of ACh receptors in the Xenopus oocyte system(5, 7, 8, 20, 21, 22) . In addition, the ability of the alpha7, alpha8, and alpha9 subunits to form functional homomeric receptors has been demonstrated(13, 14, 15) , and recent in vivo studies of the chick ACh receptor family indicate that the alpha5 subunit can assemble with multiple ACh receptor subunits to form distinctive receptor subtypes in brain(23) . It is likely, then, that the functional diversity exhibited by the neuronal ACh receptor family results from the differential expression and incorporation of different subunits into mature receptors. In support of this hypothesis, in situ hybridization and immunohistochemical studies have demonstrated that each of the ACh receptor subunit genes exhibits distinct, yet overlapping, temporally and spatially restricted patterns of expression in the peripheral and central nervous systems(3, 11, 12, 15, 16, 17, 18, 24, 25, 26, 27) . These patterns of expression are consistent with the proposed heteromeric compositions of neuronal ACh receptors in vivo(3, 21, 24, 28, 29) and, together with the functional studies, suggest specific molecular mechanisms for generating functional diversity in terms of cholinergic signal transmission within the nervous system. Despite these recent advances, the molecular details underlying the differential expression of the neuronal ACh receptor subunit genes remain largely unknown.

Previous investigations indicated that regulation at the level of transcription plays a key role in the differential expression of ACh receptor subunits in the nervous system(30, 31, 32, 33, 34) . These reports demonstrated that both positive and negative regulatory mechanisms are involved in the expression of neuronal receptor genes; however, specific transcriptional regulatory elements and associated factors have not been identified conclusively for any of the mammalian neuronal ACh receptor subunit genes. In our earlier studies aimed at identifying regulatory elements required for basal level and induced expression of the rat nicotinic ACh receptor beta4 subunit gene, we reported the identification of cis-acting sequences within the beta4 5`-flanking region capable of activating transcription of a reporter gene in transiently transfected nerve growth factor-stimulated PC12 cells(33) . Here we report the identification of a 19-base pair (bp) element in the beta4 promoter region that, when deleted, resulted in a significant loss of reporter gene expression following transfection of a cholinergic cell line. Site-directed mutagenesis experiments of the 19-bp element were consistent with the transfection data and demonstrated that this element is necessary for high level expression of the beta4 subunit gene. The 19-bp sequence specifically interacts in electrophoretic mobility shift assays (EMSA) with proteins present in nuclear extracts prepared from either a cholinergic cell line or adult rat brain. The DNA binding activity is also present, albeit in significantly lower abundance, in adult rat liver, lung, and kidney, while being completely absent in heart. Furthermore, ultraviolet (UV) cross-linking experiments indicated that the 19-bp element interacts with a DNA-binding protein with an apparent molecular size of 50 kDa. Interestingly, computer analysis of the 19-bp sequence suggests this to be a novel transcriptional regulatory element. Interactions of this novel element with its unidentified binding protein are potentially important for the establishment or maintenance of the cholinergic phenotype.


EXPERIMENTAL PROCEDURES

Generation of Plasmids and DNA Probes

We previously reported the generation of a rat beta4/luciferase expression plasmid, pX1B4FH, containing a 226-bp FokI/HindIII fragment (spanning nucleotides -89 to +137, relative to the beta4 transcription initiation site; (33) ). The insert of this construct was excised and subcloned into Bluescript SK II (Stratagene) to yield pBLSKB4FH. Exonuclease III was used to generate two 5`-deletions of the FokI/HindIII fragment in pBLSKB4FH, and the resulting fragments were subcloned into the promoterless luciferase expression vector pXP1 (35) to yield pX1B4D4 and pX1B4D18. pX1B4D4 contains a 200-bp fragment spanning nucleotides -63 to +137 while pX1B4D18 contains a 219-bp fragment spanning nucleotides -82 to +137 (sequences shown in Fig. 1).


Figure 1: Nucleotide sequence of the 5`-flanking region of the rat nicotinic ACh receptor beta4 subunit gene. The transcription initiation site is labeled +1. The 5` ends of the deletions used in transfection experiments (Fig. 2) are indicated by arrows adjacent to the names of the deletions. The 3` terminus of each deletion is the indicated HindIII site. The boxed sequence corresponds to the synthetic oligonucleotide used for the EMSA ( Fig. 3and Fig. 4) and UV cross-linking experiments (Fig. 6).




Figure 2: A 19-bp region of the beta4 5`-flanking DNA contains positively acting transcriptional regulatory sequences. SN17 cells were transiently transfected with pXP1, pX1B4FH, pX1B4D18, and pX1B4D4 (see Fig. 1). pXP1 is a promoterless luciferase expression vector and serves as a negative control(35) . To correct for differences in transfection efficiencies between dishes, the luciferase activity in a cell extract was normalized to the beta-galactosidase activity in that same sample (see ``Experimental Procedures''). Error bars represent S.E.




Figure 3: SN17 nuclear extracts contain a DNA binding activity that interacts specifically with the beta4 19-bp region. EMSA with SN17 nuclear extract and radiolabeled beta4 19-bp double-stranded oligonucleotide. The first lane (Unbound Probe) is an EMSA in the absence of nuclear extract. The next three lanes are EMSA using 1, 4, and 16 µg of SN17 nuclear extract, respectively. Competition experiments were carried out with 16 µg of SN17 nuclear extract and 60- or 240-fold molar excess of nonradiolabeled competitor oligonucleotides.




Figure 4: The DNA binding activity is enriched in adult rat brain. Nuclear extracts prepared from SN17 cells and the indicated adult rat tissues were used in EMSA with the radiolabeled beta4 oligonucleotide. The first lane (Unbound Probe) is an EMSA in the absence of nuclear extract. Three amounts of each nuclear extract (1, 4, and 16 µg) were used while the amount of radiolabeled beta4 oligonucleotide remained constant.




Figure 6: The 19-bp region interacts with a DNA-binding protein with an apparent molecular size of 50 kDa. The beta4 oligonucleotide was cross-linked to proteins present in an SN17 nuclear extract using ultraviolet irradiation for the indicated periods of time. The proteinase K treatment and the competition experiment were done as described under ``Experimental Procedures.'' The molecular weight estimate was made by comparison of the mobility of the cross-linked species (arrow) to the mobilities of ^14C-labeled protein standards whose sizes are listed on the left of the figure.



Synthetic oligonucleotides used in the EMSA and UV cross-linking experiments were obtained from a commercial source (Cruachem, Inc.). The 19-bp complementary oligonucleotides (sequence shown in Fig. 1) were annealed and then radioactively labeled with [-P]ATP using T4 polynucleotide kinase.

Cell Culture and Transfections

SN17 cells (36) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Approximately 24 h prior to transfection, cells were plated onto 60-mm culture dishes at a density of 10^5 cells/ml. Cells were transfected with 10 µg of test plasmid (beta4 subunit promoter fragments fused upstream of the luciferase gene) and 5 µg of a beta-galactosidase expression vector, pCH110 (Pharmacia Biotech Inc.). Transfections were performed using a modified calcium phosphate co-precipitation method(37) . Forty-eight hours following transfection, cells were harvested and assayed for luciferase activity using a commercially available kit (Promega Corp.) and an AutoLumat LB953 luminometer (EG& Berthold). All transfections were done a minimum of three times with at least two preparations of plasmid DNA. To correct for differences in transfection efficiencies between dishes, luciferase activity in each sample was normalized to the beta-galactosidase activity in that same sample. beta-Galactosidase activity was measured using a commercially available kit (Galacto-Light; Tropix, Inc.).

Preparation of Nuclear Extracts

Various tissues were dissected from adult female Sprague-Dawley rats (Charles River Laboratories) and immediately used for preparation of nuclear extracts. SN17 cells were grown to 70% confluence in 150-mm dishes before harvesting. Nuclear extracts were prepared according to the procedure of Christy et al.(38) and stored at -80 °C. Protein concentrations were determined by the method of Bradford(39) .

Electrophoretic Mobility Shift Assays

EMSA (40) were performed according to the method of Christy et al.(38) . Radioactively labeled double-stranded oligonucleotide probes (15 fmol) were incubated with various amounts of nuclear extract (as indicated in the figure legends) in the presence of 2 µg of poly(dIbulletdC). Competition experiments were done with a 5-min preincubation of unlabeled, competitor, double-stranded oligonucleotides with nuclear extracts prior to the addition of labeled oligonucleotide. Electrophoresis of binding reactions was done through 6% polyacrylamide gels in low ionic strength buffer.

Site-directed Mutagenesis

Oligonucleotide site-directed mutagenesis of wild type pX1B4FH was accomplished by polymerase chain reaction (PCR) amplification using a commercially available kit (Perkin-Elmer Cetus). The sequence of the 5`-mutagenic oligonucleotide (nucleotides -82 to -64) was 5`-TTTGGATCCCTCTCAGACCCGACACGGCACGTTGA-3` (top strand), and the sequence of the 3` primer was 5`-GTTGTTGTCGACCCAGCTTCTGTG-3` (bottom strand). The mutated bases are shown in bold type. The 5` primer contains a BamHI site, and the 3` primer contains a SalI site, which were used for directional cloning following PCR. Extra bases were added to the 5` end of each primer to facilitate restriction enzyme digestion. The pBLSKB4FH plasmid was used as the template for the PCR amplification reactions. The PCR products containing mutations were purified, digested with BamHI and SalI, and ligated into the BamHI/SalI sites of the pXP1 vector to create pX1B4FHM. The mutations in pX1B4FHM were confirmed by DNA sequence analysis.

UV Cross-linking

Radioactively labeled double-stranded oligonucleotides (10^5 cpm) were incubated with SN17 nuclear extract in the presence of 2 µg of poly(dIbulletdC) for 15 min at room temperature. Samples were irradiated with UV light in a Stratalinker (Stratagene) for the indicated amounts of time. Proteinase K treatment was done with 1 µg (0.02 milliAnson unit) of enzyme at 37 °C for 15 min following irradiation. Competition experiments were done with a 200-fold molar excess of unlabeled beta4 oligonucleotide in the binding reaction prior to irradiation. The cross-linked species were subjected to SDS-polyacrylamide gel electrophoresis (12.5% resolving gel), and the gel was dried and exposed to x-ray film.


RESULTS

Functional Analysis of the beta4 Promoter Region

We previously described the isolation of 226 bp of the 5`-flanking region of the rat beta4 subunit gene and demonstrated the ability of this region to activate the expression of a luciferase reporter gene in transiently transfected PC12 cells(33) . To further localize important transcriptional regulatory sequences within the beta4 5`-flanking DNA, additional 5`-deletional analysis was carried out using the 226-bp FokI/HindIII genomic fragment present in pX1B4FH (see ``Experimental Procedures''). The sequences of two 5`-deletions, pX1B4D4 and pX1B4D18, are shown in Fig. 1. To determine the effect of these deletions on beta4 promoter activity, transient transfections of the cholinergic cell line, SN17, were carried out (Fig. 2). SN17 cells were chosen for subsequent promoter studies as they are more receptive to CaPO(4)-mediated transient transfection relative to PC12 cells. Deletion of 7 bp from the 5` end of the 226-bp FokI/HindIII fragment (to yield the construct, pX1B4D18) resulted in an approximately 30% drop in luciferase activity (compare the activities of pX1B4FH and pX1B4D18 in Fig. 2). Deletion of another 19 bp (to yield the construct, pX1B4D4) led to a 90% decrease in luciferase activity when compared to the full-length clone (see Fig. 2). These results indicate the presence of positively acting sequences within the 26 bp between the 5` ends of pX1B4FH and pX1B4D4. Present within these 26 bp is a 5-bp element, CCCCT, directly repeated 2 times in the 19 bp between the 5` ends of pX1B4D18 and pX1B4D4 (see Fig. 1). Since loss of this region resulted in a substantial decrease (90%) in reporter gene activity, the possible interaction of this 19-bp sequence with cellular factors was investigated.

The 19-bp Element Interacts with DNA-binding Proteins Present in Nuclear Extracts Prepared from Cell Lines and Rat Tissues

EMSA were used to determine whether nuclear extracts prepared from SN17 cells contain proteins that interact with the 19-bp element. Incubation of a radioactively labeled double-stranded oligonucleotide corresponding to the 19-bp sequence with an SN17 nuclear extract resulted in the formation of one prominent protein-DNA complex (arrow B in Fig. 3) and two less prominent complexes (arrows A and C in Fig. 3). Importantly, the amount of each complex increased with increasing protein concentration (Fig. 3), and specificity for the 19-bp sequence was demonstrated by EMSA in the presence of either unlabeled oligonucleotides corresponding to the beta4 19-bp sequence or to the binding sites for 3 general transcription factors, AP1, TFIID, and SP1. The results (Fig. 3) indicated that only the unlabeled beta4 19-bp oligonucleotide was capable of interfering with radioactive DNA-protein complex formation, thereby demonstrating specificity of this protein complex for the 19-bp element. Overall, these experiments, carried out with independent preparations of SN17 nuclear extracts, indicated the presence in such extracts of a DNA binding activity that interacts specifically with the beta4 19-bp element shown in transfection experiments to be required for beta4 promoter activity.

These results prompted further investigation into the possible co-expression of the beta4 gene and this newly identified promoter binding activity. Earlier anatomical studies indicated the expression of the beta4 subunit gene in discrete regions of the rat brain(18) . More recent studies document the expression of the beta4 gene in non-neural tissues such as the thymus (41) and keratinocytes(42) . Therefore, to determine whether the DNA binding activity found in SN17 nuclear extracts is present in a more physiological context (coincides with beta4 expression), EMSAs were performed using nuclear extracts prepared from a variety of adult rat tissues. DNA binding activities were found to be in brain, liver, lung, and kidney with the activity being most abundant in brain (Fig. 4). There does not appear to be any DNA binding activity in heart nuclear extract. Interestingly, nuclear extracts from kidney contain a DNA binding activity that results in a distinct pattern of complexes when incubated with the beta4 oligonucleotide (Fig. 4). This result has been seen in EMSA carried out with independent preparations of kidney nuclear extract; however, the significance of this observation remains to be elucidated.

Site-directed Mutagenesis of the 19-bp Element Leads to Significantly Lower beta4 Promoter Activity

To confirm the functional significance of the 19-bp element, site-directed mutagenesis was performed. A mutant beta4 promoter region was constructed in the context of the 226-bp FokI/HindIII fragment in which 9 of the 19 bp were altered (see ``Experimental Procedures''). Transient transfections of SN17 cells with the wild type (pX1B4FH) and mutated (pX1B4FHM) promoter constructs indicated that mutation of the 19-bp element had a drastic effect on beta4 promoter activity resulting in an approximately 70% decrease in activity (Fig. 5). Taken together, these results strongly suggest that the 19-bp region is critical for wild type beta4 transcriptional activity, and, furthermore, that this region most likely interacts with transcriptional regulatory factors present in SN17 cells and various rat tissues.


Figure 5: Site-directed mutagenesis of the 19-bp region results in significantly lower transcriptional activity of the beta4 promoter. The indicated sequence changes were introduced into the beta4 insert in pX1B4FH (see Fig. 1). SN17 cells were transiently transfected with the wild type (pX1B4FH) and mutated (pX1B4FHM) beta4/luciferase expression constructs. Transfection controls were carried out as described in the legend for Fig. 2. Error bars represent S.E.



The 19-bp Element Interacts with a Protein with an Apparent Molecular Size of 50 kDa

UV cross-linking experiments (43) were carried out to estimate the size(s) of the protein(s) interacting with the beta4 19-bp sequence. SDS-polyacrylamide gel electrophoresis of irradiated binding reactions revealed the presence of a single major cross-linked species with an apparent molecular size of 50 kDa (Fig. 6). The cross-linked protein was not observed when unlabeled specific beta4 competitor was included in the binding reaction nor when the irradiated reaction mixture was treated with proteinase K prior to electrophoresis (Fig. 6). In addition, cross-linked species were not observed when nuclear extract was omitted from the binding reaction or when the binding reaction was not subjected to irradiation (Fig. 6). These data suggest that a 50-kDa protein present in SN17 nuclear extracts is capable of specifically interacting with the beta4 19-bp sequence and further raise the possibility that this interaction is involved in the transcriptional activation of the beta4 subunit gene.


DISCUSSION

Although the analysis of transcriptional regulation of the neuronal nicotinic ACh receptor gene family is still in its infancy, the presence of both positive and negative regulation in the expression of the receptor subunit genes is clear(30, 31, 32, 33, 34) . For example, Bessis et al.(30) recently reported the identification of a silencer element in the chick alpha2 subunit gene. This element is composed of six tandem repeats of an Oct-like motif (CCCCATGCAAT) but does not appear to interact with any member of the Oct family(30) . Interestingly, if the tandem nature of the repeats was disrupted, the silencer activity was lost, and, further, if two, four or five of the motifs were deleted, the element actually enhanced transcription. The silencer element was shown by EMSA to specifically interact with at least one, and possibly two, nuclear proteins, the identities of which remain to be elucidated(30) . Similarly, positive regulatory regions have been identified in the chick alpha2 (31) and alpha7 (32) subunit genes and the rat alpha3 subunit gene(34) ; however, specific DNA-protein interactions have not been demonstrated in any of these cases. The 19-bp regulatory element of the rat beta4 subunit gene we have isolated does not exhibit significant homology with any of these positively acting transcriptional regulatory regions nor to the alpha2 silencer element, and therefore represents, to the best of our knowledge, the first characterized positive regulatory element within the mammalian neuronal nicotinic ACh receptor gene family.

Computer-assisted comparisons of the beta4 19-bp element with binding sites for known transcription factors did not reveal any significant homologies other than a 90% identity with the consensus binding site (``GC-box'') for the zinc finger transcription factor SP1(44) , raising the possibility that SP1 may be involved in beta4 subunit gene expression. However, our EMSA data (Fig. 3) demonstrate that an oligonucleotide corresponding to the SP1 consensus binding site does not compete with the beta4 19-bp element for binding to proteins in SN17 nuclear extract, thus indicating this to be unlikely, at least in terms of the 19-bp sequence.

Interestingly, the 5`-CCTCCCCTCCC-3` sequence of the beta4 19-bp element is found within the promoter regions of several eukaryotic genes encoding proteins known to play a variety of roles in neuronal development and differentiation. These genes include those encoding the subunit of the neurotrophic factor, nerve growth factor (NGF; (45) ), as well as the proto-oncogenes, c-myc(46) and c-fos(47) . The binding of NGF to its high affinity receptor and the subsequent activation of a cellular program leading to neuronal differentiation has been well documented (see (48) for review). Indeed, we have previously shown that NGF increases the transcriptional activities of beta4/luciferase fusion genes in transfected PC12 cells (33) . One of the earliest events in the NGF-induced cascade are increases in the transcriptional activities of the c-myc and c-fos proto-oncogenes(48) . The products of these two genes are involved in the expression of a number of genes leading ultimately to neuronal differentiation. It is interesting to speculate as to whether the 50-kDa polypeptide we have shown to interact with the beta4 19-bp element also interacts with the NGF, c-myc, and c-fos promoter regions, and, consequently, is involved in the NGF-mediated regulatory cascade. As the nicotinic ACh receptor genes are members of the ``late'' class of NGF-induced genes(47) , some mechanism must account for the differential temporal patterns of expression of the beta4, NGF, c-myc, and c-fos genes. Further studies should help elucidate this mechanism and determine whether the CCTCCCCTCCC sequences in the NGF, c-myc, and c-fos genes are involved in transcriptional activation.

The data presented in this report clearly indicate the presence in nuclear extracts prepared from SN17 cells, adult rat brain, liver, lung, and, to a much lesser extent, kidney, of a DNA binding activity that interacts with the beta4 19-bp element. As UV cross-linking experiments revealed the existence of a single protein species interacting with the 19-bp element, it is reasonable to propose that the multiple shifted complexes observed in the EMSA are consequences of interactions between the 19-bp sequence and the 50-kDa protein in either a homomeric or heteromeric manner. The identities of the 50-kDa protein and any possible binding partner(s) remain unknown but are the focus of current studies.

The presence of DNA binding activities in non-neural tissues was, at first glance, surprising given that beta4 subunit gene expression was initially thought to be limited to specific neurons(18) . However, recent reports suggest a more ubiquitous expression pattern for the beta4 gene with beta4 mRNA being detected in cells such as thymocytes (41) and keratinocytes(42) . Thus, it appears that the beta4 gene may not be expressed exclusively within the nervous system. This leads us to speculate that the DNA binding activity we observed in the EMSA may represent the binding of a more general transcription factor (as opposed to a neuronal-specific factor) to the beta4 promoter and that cell type-specific expression of the beta4 gene product may result from the interaction of this potentially general transcription factor (the 50-kDa protein) with a non-DNA-binding heterodimer partner expressed in a more specific fashion. This possibility awaits further investigation.

In summary, we have identified a novel 19-bp, positively acting, transcriptional regulatory element within the 5`-flanking region of the rat nicotinic ACh receptor beta4 subunit gene. This element, as determined by 5`-deletional analysis and site-directed mutagenesis, is necessary for full transcriptional activation by the beta4 promoter. Specific DNA-protein interactions between this element and proteins present in nuclear extracts prepared from a variety of sources were detected in EMSA. Importantly, a nuclear protein with apparent molecular size of 50 kDa was demonstrated to bind the 19-bp sequence, thus raising the possibility that interactions between the 19-bp element and the 50-kDa polypeptide are necessary for complete transcriptional activation of the beta4 subunit promoter. Further characterization of the binding protein(s) that interact with the 19-bp element should provide considerable insight into the molecular mechanisms governing beta4 subunit gene expression.


FOOTNOTES

*
This work was supported by Grant NS30243 from the National Institutes of Health. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L22646[GenBank].

§
To whom correspondence and reprint requests should be addressed: Center for Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245-3207. Tel.: 210-567-7251; Fax: 210-567-7277.

(^1)
The abbreviations used are: ACh, acetylcholine; bp, base pair(s); EMSA, electrophoretic mobility shift assay(s); PCR, polymerase chain reaction; NGF, nerve growth factor.


ACKNOWLEDGEMENTS

We thank Kathleen Hennessy and David Hammond for their kind gift of SN17 cells and our colleagues at the Center for Molecular Medicine for useful discussions, reagents, and encouragement; in particular, Steve Britt, Bob Christy, Jim Fitzgerald, Irena Melnikova, Ed Seto, Dave Sharp, and Yufan Zhu.


REFERENCES

  1. Schuetze, S. M., and Role, L. W. (1987) Annu. Rev. Neurosci. 10, 403-457 [CrossRef][Medline] [Order article via Infotrieve]
  2. Brehm, P., and Henderson, L. P. (1988) Dev. Biol. 129, 1-11 [Medline] [Order article via Infotrieve]
  3. Sargent, P. (1993) Annu. Rev. Neurosci. 16, 403-443 [CrossRef][Medline] [Order article via Infotrieve]
  4. Nef, P., Oneyser, C., Alliod, C., Couturier, S., and Ballivet, M. (1988) EMBO J. 7, 595-601 [Abstract]
  5. Wada, K., Ballivet, M., Boulter, J., Connolly, J., Wada, E., Deneris, E. S., Swanson, L. W., Heinemann, S., and Patrick, J. (1988) Science 240, 330-334 [Medline] [Order article via Infotrieve]
  6. Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemann, S., and Patrick, J. (1986) Nature 319, 368-374 [Medline] [Order article via Infotrieve]
  7. Couturier, S., Erkman, L., Valera, S., Rungger, D., Bertrand, S., Boulter, J., Ballivet, M., and Bertrand, D. (1990) J. Biol. Chem. 265, 17560-17567 [Abstract/Free Full Text]
  8. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S., and Patrick, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7763-7767 [Abstract]
  9. Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patrick, J., and Heinemann, S. (1987) Cell 48, 965-973 [Medline] [Order article via Infotrieve]
  10. Boulter, J., O'Shea-Greenfield, A., Duvoisin, R. M., Connolly, J. G., Wada, E., Jensen, A., Gardner, P. D., Ballivet, M., Deneris, E. S., McKinnon, D., Heinemann, S., and Patrick, J. (1990) J. Biol. Chem. 265, 4472-4482 [Abstract/Free Full Text]
  11. Lamar, E., Miller, K., and Patrick, J (1990) Soc. Neurosci. Abstr. 16, 581
  12. Seguela, P., Wadiche, J., Dineley-Miller, K., Dani, J. A., and Patrick, J. W. (1993) J. Neurosci. 13, 596-604 [Abstract]
  13. Schoepfer, R., Conroy, W. G., Whiting, P., Gore, M., and Lindstrom, J. (1990) Neuron 5, 35-48 [Medline] [Order article via Infotrieve]
  14. Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M.-C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990) Neuron 5, 847-856 [Medline] [Order article via Infotrieve]
  15. Elgoyen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E., and Heinemann, S. (1994) Cell 79, 705-715 [Medline] [Order article via Infotrieve]
  16. Deneris, E. S., Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, L. W., Patrick, J., and Heinemann, S. (1988) Neuron 1, 45-54 [Medline] [Order article via Infotrieve]
  17. Deneris, E. S., Boulter, J., Swanson, L. W., Patrick, J., and Heinemann, S. (1989) J. Biol. Chem. 264, 6268-6272 [Abstract/Free Full Text]
  18. Duvoisin, R. M., Deneris, E. S., Patrick, J., and Heinemann, S. (1989) Neuron 3, 487-496 [Medline] [Order article via Infotrieve]
  19. Schoepfer, R., Whiting, P., Esch, F., Blacher, R., Shimasaki, S., and Lindstrom, J. (1988) Neuron 1, 241-248 [Medline] [Order article via Infotrieve]
  20. Patrick, J., Sequela, P., Vernino, S., Amador, M., Luetje, C., and Dani, J. A. (1993) Prog. Brain Res. 98, 113-120 [Medline] [Order article via Infotrieve]
  21. Heinemann, S., Boulter, J., Deneris, E. S., Connolly, J., Duvoisin, R. M., Papke, R., and Patrick, J. (1990) Prog. Brain Res. 86, 195-203 [Medline] [Order article via Infotrieve]
  22. Wada, E., McKinnon, D., Heinemann, S., Patrick, J., and Swanson, L. W. (1990) Brain Res. 526, 45-53 [CrossRef][Medline] [Order article via Infotrieve]
  23. Papke, R. L., Boulter, J., Patrick, J., and Heinemann, S. (1989) Neuron 3, 589-596 [CrossRef][Medline] [Order article via Infotrieve]
  24. Conroy, W. G., Vernallis, A. B., and Berg, D. K. (1993) Neuron 9, 679-691
  25. Wada, E., Wada, K., Boulter, J., Deneris, E., Heinemann, S., Patrick, J., and Swanson, L. W. (1989) J. Comp. Neurol. 284, 314-335 [Medline] [Order article via Infotrieve]
  26. Dineley-Miller, K., and Patrick, J. (1992) Mol. Brain Res. 16, 339-344 [Medline] [Order article via Infotrieve]
  27. Morris, B. J., Hicks, A. A., Wisden, W., Darlison, M. G., Hunt, S. P., and Barnard, E. A. (1990) Mol. Brain Res. 7, 305-315 [Medline] [Order article via Infotrieve]
  28. Mulle, C., Vidal, C., Benoit, P., and Changeux, J.-P. (1991) J. Neurosci. 11, 2588-2597 [Abstract]
  29. Luetje, C. W., and Patrick, J. (1991) J. Neurosci. 11, 837-845 [Abstract]
  30. Bessis, A., Savatier, N., Devillers-Thiery, A., Bejanin, S., and Changeux, J.-P. (1993) Nucleic Acids Res. 21, 2185-2192 [Abstract]
  31. Daubas, P., Salmon, A. M., Zoli, M., Geoffroy, B., Devillers-Thiery, A., Bessis, A., Medevielle, F., and Changeux, J.-P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2237-2241 [Abstract]
  32. Matter-Sadzinski, L., Hernandez, M., Roztocil, T., Ballivet, M., and Matter, J. (1992) EMBO J. 11, 4529-45389 [Abstract]
  33. Hu, M., Whiting-Theobald, N. L., and Gardner, P. D. (1994) J. Neurochem. 62, 392-395 [Medline] [Order article via Infotrieve]
  34. Yang, X., McDonough, J., Fyodorov, D., Morris, M., Wang, F., and Deneris, E. S. (1994) J. Biol. Chem. 269, 10252-10264 [Abstract/Free Full Text]
  35. Nordeen, S. K. (1988) BioTechniques 6, 454-457 [Medline] [Order article via Infotrieve]
  36. Hammond, D. N., Lee, H. J., Tonsgard, J. H., and Wainer, B. H. (1990) Brain Res. 512, 190-200 [CrossRef][Medline] [Order article via Infotrieve]
  37. Van der Eb, A. J., and Graham, F. L. (1980) Methods Enzymol. 65, 826-839 [Medline] [Order article via Infotrieve]
  38. Christy, R. J., Yang, V. W., Ntambi, J. M., Geiman, D. E., Landschulz, W. H., Friedman, A. D., Nakabeppu, Y., Kelly, T. J., and Lane, M. D. (1989) Genes & Dev. 3, 1323-1335
  39. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  40. Fried, M., and Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505-6525 [Abstract]
  41. Mihovilovic, M., Denning, S., Berrih-Aknin, S., and Roses, A. D. (1993) Soc. Neurosci. Abstr. 123, 13
  42. Pereira, E. F. R., Albuquerque, E. X., George, P. G., Horton, R. M., Conti-Tronconi, B. M., and Grando, S. A. (1994) Soc. Neurosci. Abstr. 468, 12
  43. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology , John Wiley and Sons, Inc., New York
  44. Evans, B. A., and Richards, R. I. (1985) EMBO J. 4, 133-138 [Abstract]
  45. Watt, R., Nishikura, K., Sorrentino, J., Ar-Rushdi, A., Croce, C. M., and Rovera, G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6307-6311 [Abstract]
  46. Treisman, R. (1985) Cell 42, 889-902 [Medline] [Order article via Infotrieve]
  47. Halegoua, S., Armstrong, R. C., and Kremer, N. E. (1991) in Current Topics in Microbiology and Immunology , pp. 119-170, Springer-Verlag, Heidelberg
  48. Greenberg, M. E., Greene, L. A., and Ziff, E. B. (1985) J. Biol. Chem. 260, 14101-14110 [Abstract/Free Full Text]

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