Transcriptional Regulation of Neuronal Nicotinic Acetylcholine Receptor Genes
A POSSIBLE ROLE FOR THE DNA-BINDING PROTEIN Puralpha *

(Received for publication, March 13, 1997, and in revised form, March 31, 1997)

Qun Du , Alan E. Tomkinson and Paul D. Gardner Dagger

From the Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245-3207

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Nicotinic acetylcholine receptors constitute a multigene family (alpha 2-alpha 9, beta 2-beta 4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta 4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis revealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Puralpha . Electrophoretic mobility shift assays indicated that Puralpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Puralpha monoclonal antibody revealed the presence of Puralpha , or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Puralpha and the 19-bp element is critical for proper expression of the beta 4 subunit gene.


INTRODUCTION

One of the key events that takes place during development of the nervous system is the formation of synapses. Numerous lines of evidence indicate that expression of neurotransmitter sensitivity is central to synaptogenesis (reviewed in Ref. 1). It is also clear that changes in sensitivity to neurotransmitters are due to changes in the expression of neurotransmitter receptors. Recent advances toward a molecular understanding of the formation of nicotinic cholinergic synapses in the nervous system were made with the isolation of a family of genes (alpha 2-alpha 9 and beta 2-beta 4) encoding subunits of the nicotinic receptors for acetylcholine (nACh1; reviewed in Ref. 2). Functionally diverse nACh receptors can be generated by distinct combinations of the subunits in vitro (3-5). In addition, each of the subunits exhibits discrete temporally and spatially restricted expression patterns in vivo (2). Despite these important advances, however, the cellular and molecular mechanisms controlling the expression of the nACh receptor genes are relatively obscure, although several recent studies suggest that both positive and negative transcriptional regulatory mechanisms are involved in receptor gene expression (6-16).

Three of the nACh receptor genes, those encoding the beta 4, alpha 3, and alpha 5 subunits, are tightly linked within the rat genome, spanning only approximately 60 kilobase pairs (17). This genomic organization, coupled with the high nucleotide sequence similarities between the three genes, suggests that the genes arose through tandem duplication of a common ancestral gene as has been suggested previously (17). On the other hand, the genomic organization may also reflect a regulatory mechanism that ensures co-expression of the genes in the appropriate cell types at the appropriate developmental time. Such a mechanism would be consistent with recent data indicating the presence, in vivo, of receptors consisting of beta 4, alpha 3, and alpha 5 subunits (18). However, the expression of these genes is not completely overlapping (2) and, thus, some other mechanism must account for this disparity. One possibility is that while each of the subunit genes is independently regulated transcriptionally, they share some common regulatory features, which are active in the appropriate developmental and cellular environments. Several laboratories, including ours, have characterized distinct promoter regions for the beta 4 (10, 11, 14) and alpha 3 (9, 15, 16) subunit genes. Within these regions, several transcriptional regulatory elements have been identified. Thus far, the only regulatory element common to both the beta 4 and alpha 3 genes is a consensus Sp1-binding site, which appears critical for transcription of the two genes (9, 14). Our analysis of the rat beta 4 subunit promoter region led to the identification of three elements, E1-E3, which may play roles in beta 4 gene expression (14). E2 is the aforementioned Sp1-binding site, E3 shares some sequence homology with the consensus AP1-binding site, and E1 is a novel 19-bp regulatory element (14). E1, characterized by three CCCT repeats, forms specific complexes with proteins present in nuclear extracts prepared from neuronal cells (11, 14). We have now used E1 as an affinity ligand in a purification scheme to isolate, from bovine brain nuclear extracts, the nuclear proteins which bind specifically to this element. In the most highly purified fraction, four polypeptides with apparent molecular masses of 31, 43, 65, and 114 kDa were detected. We termed these E1-binding proteins, neuronal nACh receptor promoter-binding proteins (NARP31, -43, -65, and -114). Peptide sequence analysis indicated that NARP43 is the bovine homologue of Puralpha , a DNA-binding protein previously shown to stimulate transcription of the myelin basic protein gene in oligodendrocytic cells (19). Electrophoretic mobility shift assays (EMSA) demonstrated that Puralpha is present in nuclear extracts prepared from brain and binds directly and specifically to E1, raising the possibility that this interaction plays a role in the expression of the beta 4 subunit gene.


EXPERIMENTAL PROCEDURES

Buffers

Buffer C consisted of 25 mM HEPES (pH 7.8), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 20% glycerol. Buffer D consisted of 50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol. Buffer E consisted of 20 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 10% glycerol. Binding buffer consisted of 20 mM HEPES (pH 7.4), 5 mM DTT, 1 mM MgCl2, 50-100 mM KCl, and 6% glycerol. Buffer Z consisted of 25 mM HEPES (pH 7.6), 10 mM MgCl2, 1 mM DTT, 20% glycerol, and 0.1% Nonidet P-40.

Substrate Preparation for Electrophoretic Mobility Shift Assays

The sequence of E1 used as a probe and competitor in EMSA is shown in Fig. 1 along with the sequence of a mutated form of the element used as a competitor in EMSA. The E1 complementary oligonucleotides (from Cruachem, Inc.) were annealed and then radioactively labeled with [gamma -32P]ATP (DuPont NEN) using T4 polynucleotide kinase (Promega). The specific activities of the probes were typically between 14,000 and 17,000 cpm/fmol. Competitor oligonucleotides were prepared by annealing equal amounts of non-radioactive complementary oligonucleotides.


Fig. 1. Structural features of the promoter region of the rat nACh receptor beta 4 subunit gene. The arrow indicates the initiation site (+1) and direction of transcription of the beta 4 gene. E1, E2, and E3 are three regulatory elements believed to be important for beta 4 gene expression (see the Introduction). The sequences of the top strands of the wild type and mutant E1 oligonucleotides are indicated. Mutated bases are underlined. These mutations, when introduced into the beta 4 promoter, resulted in an approximately 70% reduction in promoter activity (11).
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Preparation of Nuclear Extracts

Nuclear extracts were prepared as described by Christy et al. (20). Bovine brains were obtained within 1 h of death from a local slaughterhouse (Kiolbassa Provision Company), while fresh rat brains were from adult female Harlan Sprague Dawley animals (Charles River Laboratories). The final rat brain nuclear extract pellet was resuspended in buffer C, while the final bovine brain nuclear extract pellet was resuspended in buffer D in preparation for phosphocellulose chromatography (see below). The nuclear extracts were dialyzed overnight in the buffer in which they were resuspended. Insoluble material was removed by centrifugation.

Protein Assays

During phosphocellulose and DNA-cellulose chromatography, protein was monitored by measuring absorbances at 280 nm. Protein concentrations were determined by using bicinchoninic acid (Pierce).

Electrophoretic Mobility Shift Assays

E1 binding activity was detected using EMSA as described previously (11). To demonstrate sequence specificity of the DNA-protein complexes, competition experiments were done with a 15-min preincubation of unlabeled double-stranded oligonucleotides prior to the addition of labeled oligonucleotide. The amount of DNA present in specific DNA-protein complexes was quantified using a Molecular Dynamics PhosphorImager and Imagequant software. Antibody supershift experiments were performed by incubating 1 µg of a monoclonal antibody directed against Puralpha (9C12, Ref. 24) or a nonrelated preimmune serum with 0.5 µg of bovine brain nuclear extract for 10 min at 37 °C before the addition of specific probe. Following a 1-h incubation at 4 °C, the binding reactions were analyzed by electrophoresis through 6% native acrylamide gels and visualized by autoradiography.

Purification of E1-binding Proteins

Crude nuclear extract from bovine brain (365 mg) was applied directly onto a 1.6 × 50-cm P11 phosphocellulose column (Whatman) equilibrated with 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl. After washing with three column volumes of the same buffer, bound proteins were eluted with a 1-liter linear gradient of NaCl from 50 mM to 800 mM in 50 mM Tris (pH 7.5). Approximately 9-ml fractions were collected and assayed for E1 binding activity using EMSA as described above. Fractions with binding activity (11 mg) were pooled, concentrated by ultrafiltration, dialyzed against buffer E, and then loaded onto a native calf thymus DNA-cellulose column (10 ml), which was equilibrated with buffer E. After washing with three column volumes of the same buffer, bound proteins were eluted with a 100-ml linear gradient of NaCl from 50 mM to 1.0 M in buffer E. Fractions containing E1 binding activity (2.5 mg) were pooled, and dialyzed against buffer Z in preparation for DNA sequence-specific affinity chromatography. Preparation and regeneration of the E1 affinity column was done essentially as described by Kadonaga and Tjian (21). Two 23-base complementary oligonucleotides containing the E1 sequence (see Fig. 1) were synthesized and purified by polyacrylamide gel electrophoresis. Complementary 4-base overhangs (5'-GATC-3') were added to the E1 sequences to increase the efficiency of concatenation. The oligonucleotides were annealed, phosphorylated, concatenated, and subsequently coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech Inc.). Dialyzed, pooled fractions from the DNA-cellulose column were incubated for 10 min at 4 °C with 2 mg of poly(dI-dC)·poly(dI-dC). Insoluble material was removed by centrifugation. The poly(dI-dC)·poly(dI-dC)-protein mixture was applied to the affinity column equilibrated with buffer Z containing 0.1 M KCl. The column was washed with buffer Z containing 0.1 M KCl, followed by elution of E1 binding activity using a linear gradient of KCl from 0.2 M to 1.0 M (in buffer Z). Fractions containing E1 binding activity were pooled, dialyzed against buffer Z containing 0.1 M KCl, reabsorbed to the regenerated affinity resin, and eluted as described above.

SDS-PAGE and Peptide Sequencing

Protein samples were separated on 10% SDS-polyacrylamide gels as described by Laemmli (22) and stained with silver (Bio-Rad). Polypeptides were transferred to a polyvinylidene difluoride membrane in buffer containing 12.5 mM Tris (pH 8.3), 96 mM glycine and 10% methanol. The membrane was stained with 0.1% Amido Black in 10% acetic acid. After destaining, NARP43 was excised, washed with HPLC-grade H2O, dried, and subjected to amino acid microsequencing (Harvard Microsequencing Facility) as described previously (23).

Expression and Purification of GST-Puralpha Fusion Proteins

A glutathione S-transferase-Puralpha fusion construct, GST-PUR-(1-322), was generously provided by Drs. Phang-Lang Chen and Wen-Hwa Lee (University of Texas Health Science Center at San Antonio) (24). Expression and purification of GST and the GST-Puralpha fusion proteins was carried out as described (24). The GST moiety was removed from the GST-Puralpha fusion protein (0.5 mg) by cleavage with thrombin (1,500 units). The GST moiety was removed from the reaction mixture by the addition of glutathione-Sepharose beads followed by centrifugation. Sample stability, the efficiency of thrombin cleavage, and the purity of the released Puralpha were analyzed by SDS-PAGE (data not shown).


RESULTS AND DISCUSSION

We previously identified several transcriptional regulatory elements, E1-E3 (Fig. 1), in the rat beta 4 subunit gene (14). One of these elements, E2, is a binding site for the regulatory factors, Sp1 and Sp3 (14),2 while the identities of the proteins interacting with E1 and E3 are unknown. The study described below focuses upon E1 and the proteins with which it interacts.

A DNA Binding Activity That Interacts with E1 Is Conserved between Rat and Bovine

Our earlier analyses of the beta 4 subunit gene focused upon the rat gene. As there are advantages for using bovine brain versus rodent brain as a source of nuclear extract, we determined whether nuclear extracts from both species yielded similar electrophoretic mobility shift patterns when incubated with E1. As shown in Fig. 2, this is indeed the case. Incubation of radioactively labeled E1 with brain nuclear extracts prepared from both species resulted in the formation of three prominent DNA-protein complexes (Fig. 2). Competition experiments using either unlabeled oligonucleotides corresponding to the beta 4 E1 sequence or to the binding sites for the general transcription factors Sp1, TFIID, or AP1 indicated that only the unlabeled beta 4 oligonucleotide was capable of interfering with radioactive DNA-protein complex formation. Similar results have been obtained from several independent preparations of nuclear extracts from each species (data not shown). These results indicated that bovine brain is a suitable source of nuclear extracts from which to purify the DNA-binding factors that interact with E1. We describe below experiments resulting in a highly purified preparation of complex A. 


Fig. 2. An E1 binding activity is conserved between rat and bovine. Nuclear extracts from rat and bovine brains were used in EMSA with 32P-labeled E1 oligonucleotide. The first lane (Unbound Probe) is an EMSA without the addition of nuclear extract. Three amounts of each nuclear extract (2.5, 5, and 10 µg) were used. Competition experiments were carried out with 100-fold molar excess of nonradiolabeled competitor oligonucleotides in the presence of 5 µg of each nuclear extract. The competitors were the E1 oligonucleotide (beta 4) and oligonucleotides corresponding to the consensus binding sites for Sp1, TFIID, and AP1. Complex A was purified as described under "Results and Discussion."
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Purification of the E1 Binding Activity

Purification of the binding activity was achieved by a combination of phosphocellulose, DNA-cellulose, and DNA sequence-specific affinity chromatographies. The E1 binding activity bound to the P-11 phosphocellulose column and eluted from the column as a broad peak between 425 and 680 mM NaCl (Fig. 3A). In contrast, a DNA-protein complex with higher electrophoretic mobility that was detected in the nuclear extract did not bind to the column (Fig. 3A). Competition experiments indicated that this faster migrating species was not DNA sequence-specific (data not shown). The origin of this nonspecific DNA-protein complex is unknown, although it is clearly not complexes B or C as they are sequence-specific and bind to the column (Fig. 3A, fraction 43). In addition, the faster migrating species was not present in all nuclear extract preparations (data not shown). An approximately 6-fold purification of the specific E1 binding activity was achieved on the phosphocellulose column.


Fig. 3. Purification of E1-binding proteins. A, phosphocellulose chromatography. The upper portion of the panel shows the protein profile as determined by monitoring the absorbances at 280 nm of every other fraction. E1 binding activity was detected by EMSA of every other fraction. The lower panel is an autoradiograph showing the E1 binding activity in the active fractions. Fractions 51-85 were pooled for further purification on DNA-cellulose. FT, flow-through. B, DNA-cellulose chromatography. The upper portion of the panel shows the protein profile as determined by monitoring the absorbances at 280 nm of every other fraction. E1 binding activity was detected by EMSA of every other fraction. The lower panel is an autoradiograph showing the E1 binding activity in the active fractions. Fractions 21-27 were pooled for further purification on the E1-affinity resin. FT, flow-through. C, E1 affinity chromatography. An autoradiograph showing the E1 binding activity in the active fractions after two rounds of affinity chromatography. Fractions 5-10 were pooled for SDS-PAGE. There is no protein profile as the quantity of protein was too low to detect by A280 readings. FT, flow-through; W, wash fraction. D, SDS-PAGE. Nuclear extract (10 µg), the phosphocellulose pool (20 µg), the DNA-cellulose pool (10 µg), and the affinity-purified E1 binding activity (~0.2 µg) were analyzed by 10% SDS-PAGE and visualized by silver staining. The positions of molecular size markers run in an adjacent lane are indicated.
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The E1 binding activity was fractionated further by native DNA-cellulose chromatography. The binding activity eluted from the column as a sharp peak between 600 and 750 mM NaCl (Fig. 3B), indicating that the protein responsible for E1 binding also binds to DNA in a sequence-independent manner. DNA-cellulose chromatography afforded an approximately 3-fold greater purification of the E1 binding activity.

The final chromatographic step was recognition site affinity chromatography on a column with covalently linked, catenated E1 oligonucleotides. The E1 binding activity bound to the affinity column and eluted at 350-500 mM KCl (Fig. 3C). Two passages of the E1 binding activity over the affinity column resulted in approximately 960-fold purification with a 40% yield in this step. A summary of the purification data is presented in Table I and indicates that, relative to the nuclear extract, the final purification was approximately 19,000-fold with a 5% recovery of activity.

Table I. Purification of E1-binding proteins


Total proteina Total activity Specific activityb Purification factor Total yield

mg units units -fold %
Nuclear extract 365 19,527 0.0535
Phosphocellulose 11 3,673 0.333 6.2 19
DNA-cellulose 2.5 2,658 1.068 19.9 13.6
E1-affinity 0.001c 1,026 1,026 19,180 5.25

a Protein concentration was determined using a bicinchoninic acid assay (from Pierce) with BSA as a standard.
b One unit of E1 binding activity corresponds to the amount of protein that will bind one fmol of 32P-labeled E1 probe in a standard EMSA. The amount of DNA present in specific DNA protein complexes was quantified using a Molecular Dynamics PhosphorImager and Imagequant software.
c Total protein was estimated by comparison of the Coomassie Brilliant Blue staining intensity of purified proteins with that of a known quantity of marker proteins.

Analysis of the protein content of the E1 binding activity by silver staining after SDS-PAGE revealed four major polypeptides with molecular masses of 31, 43, 65, and 114 kDa (Fig. 3D). We refer to these proteins as neuronal nACh receptor promoter-binding proteins, or NARP (i.e. NARP31, NARP43, etc.; Ref. 14).

NARP43 Is the Bovine Homologue of Puralpha

In several preparations of the E1 binding activity, NARP43 was the most abundant species; therefore, it was the first NARP to be subjected to amino acid sequence analysis. Following electrophoresis, NARP43 was electroblotted onto a polyvinylidene difluoride membrane and digested with trypsin. The cleavage products were separated by high performance liquid chromatography, and four peptides were sequenced. The four amino acid sequences of these peptides were identical to sequences in the previously described Puralpha sequence (Fig. 4; Ref. 25). Puralpha is a sequence-specific DNA-binding protein that has recently been implicated in cell type-specific transcriptional regulation of myelin basic protein in oligodendrocytes (19). Its recognition element is purine-rich (25), as is the sequence of E1 (Fig. 1).


Fig. 4. Identification of NARP43 as Puralpha . The four peptide sequences of NARP43 were compared with the primary sequence of Puralpha (25) and shown to be identical to the regions indicated. Puralpha consists of 322 amino acids containing three repeats of a 23-amino acid motif (class I repeats) and two repeats of a 26-amino acid motif (class II repeats) as shown.
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Puralpha Interacts Specifically with E1

To determine whether Puralpha does in fact interact with E1, EMSA were carried out with a GST-Puralpha fusion protein (24). As shown in Fig. 5, GST-Puralpha forms two major complexes with the radiolabeled E1 oligonucleotide, the larger of which is specific in that it can be competed away by an excess of unlabeled wild type E1 oligonucleotide but not by an excess of a mutant E1 oligonucleotide (see Fig. 1 for the sequences). GST alone does not form any specific complexes with E1 (Fig. 5). To confirm that Puralpha interacts specifically with E1, the GST moiety was cleaved from the GST-Puralpha fusion protein, the GST moiety was removed by thrombin cleavage (see "Experimental Procedures"), and the recovered Puralpha protein was used in EMSA. As shown in Fig. 5, purified Puralpha interacts specifically with E1 forming one major complex that can be competed by an excess of wild type E1 oligonucleotide but not by mutant E1 oligonucleotide.


Fig. 5. Puralpha binds specifically to E1. Prokaryotically produced GST, GST-Puralpha , and Puralpha from which the GST moiety had been removed were used in EMSA with radiolabeled E1 oligonucleotide. The first lane (Probe) is an EMSA without the addition of any protein. Two amounts of GST or GST-Puralpha (1 and 2 µg) and Puralpha (0.15 and 0.3 µg) were used. Competition experiments were carried out with 100-fold molar excess of nonradiolabeled competitor oligonucleotides in the presence of either 2 µg of protein (GST and GST-Puralpha ) or 0.3 µg of protein (Puralpha ). See Fig. 1 for the oligonucleotide sequences. WT, wild type E1 competitor oligonucleotide; Mut, mutant E1 competitor oligonucleotide.
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Puralpha or an Antigenically Related Protein Is Present in Brain Tissue

The results presented thus far strongly implicate Puralpha as playing a role in regulating the expression of the beta 4 subunit gene via interactions with E1. To determine whether Puralpha (or an immunologically related protein) is present in a more physiological context, we carried out supershift EMSA using a monoclonal antibody against Puralpha and crude nuclear extracts prepared from bovine brains. The extracts were incubated with one of three radiolabeled E1 probes (the coding strand, the noncoding strand, or double-stranded E1) and an anti-Puralpha monoclonal antibody (24) prior to electrophoresis (see "Experimental Procedures"). Both single- and double-stranded probes were used to gain more insight into the interactions between Puralpha and E1; Puralpha has been shown to bind to both single- and double-stranded DNA targets (19). As shown in Fig. 6, incubation of the coding strand probe (ssc) with nuclear extract led to the appearance of a single DNA-protein complex (lower arrow). The mobility of this complex was unaltered by the addition of anti-Puralpha antibody or a preimmune serum. Similarly, incubation of the noncoding strand probe (ssnc) or the double-stranded probe (ds) with nuclear extract led to the appearance of one major DNA-protein complex (Fig. 6, upper arrow). These complexes migrated more slowly than that seen with the coding strand probe raising the possibility that different proteins can interact with the two strands. In support of this hypothesis is the observation that the addition of anti-Puralpha antibody led to a slight decrease in the mobilities of the complexes formed with the noncoding and double-stranded probes (Fig. 6, arrowhead). Although the decrease seen following addition of anti-Puralpha antibody was not as large as might be expected given the size of the antibody, it may be a consequence of the overall electrostatic charge of the DNA-Puralpha -anti-Puralpha antibody complex, such that the binding of antibody may not greatly change the overall charge of the DNA-protein complex. Given that separation through native acrylamide gels is based on charge rather than size, the small change in overall charge upon binding of antibody may not result in a large decrease in mobility. Nonetheless, these data suggest the presence of Puralpha , or an antigenically related protein, in bovine brain and further suggest that different proteins are capable of interacting with the coding and noncoding strands. It is also possible that the proteins which interact with the coding strand also interact with the noncoding strand and double-stranded DNA but in conjunction with Puralpha , resulting in a slower migrating DNA-protein complex. Alternatively, different proteins may interact with the two strands and the affinity of the protein for the noncoding strand is higher than the affinity of the protein for the coding strand such that when the double-stranded probe is used, it forms a complex with the noncoding-strand-binding-protein. Further analyses should help resolve these questions.


Fig. 6. Puralpha , or an antigenically related protein, is present in brain nuclear extracts. Single-stranded, radiolabeled probes corresponding to either the coding (ssc) or noncoding (ssnc) strand of E1, and double-stranded, radiolabeled E1 (ds) were incubated with 0.5 µg of bovine brain nuclear extract alone, in the presence of a nonrelated preimmune serum (Preimmune) or in the presence of a monoclonal antibody directed against Puralpha (Anti-Puralpha ) prior to electrophoresis. The lower arrow indicates the coding strand DNA-protein complex, while the upper arrow indicates the noncoding strand DNA-protein and double-stranded DNA-protein complexes. The arrowhead indicates the supershifted noncoding strand DNA-protein and double-stranded DNA-protein complexes.
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In summary, we used a combination of phosphocellulose, DNA-cellulose, and DNA sequence-specific affinity chromatographies to purify the E1 binding activity approximately 19,000-fold from bovine brains. There are four major polypeptides in the most purified fraction. One of the proteins has been identified as the bovine homologue of the transcriptional regulatory factor, Puralpha . The identities of the other three proteins remain unknown but are the focus of current efforts. In addition, the functional role Puralpha plays in beta 4 subunit gene expression is under investigation. In this regard, it is interesting to note that Puralpha has been shown to interact with the retinoblastoma protein, an important regulatory factor known to be involved in cell cycle regulation and the expression of specific genes required for cellular differentiation (26). The molecular weight of the retinoblastoma protein is approximately 110 kDa, raising the possibility that NARP114 may be the bovine retinoblastoma protein. Finally, we previously showed that Sp1 and Sp3 are involved in beta 4 gene expression via their interactions with E2, which is located immediately downstream of E1 (14).2 We hypothesized that Sp1 and Sp3 may interact with the proteins that bind to E1 (14).2 With the identification of NARP43 as Puralpha , we are now in a position to begin testing that hypothesis.


FOOTNOTES

*   This work was supported by grants (to P. D. G.) from the National Institutes of Health (NS30243), the Council for Tobacco Research, U.S.A., and the Smokeless Tobacco Research Council, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245-3207. Tel.: 210-567-7251; Fax: 210-567-7247; E-mail: gardner{at}uthscsa.edu.
1   The abbreviations used are: nACh, nicotinic acetylcholine; NARP, neuronal nACh receptor promoter-binding protein; bp, base pair(s); EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
2   C. B. Bigger, I. N. Melnikova, and P. D. Gardner, unpublished results.

ACKNOWLEDGEMENTS

We thank Drs. Wen-Hwa Lee and Phang-Lang Chen for the GST-Puralpha construct, Dr. Edward M. Johnson for anti-Puralpha antibodies, Drs. William Lane and John Neveu of the Harvard Microchemistry Facility for amino acid sequencing, the folks at Kiolbassa Provision Company for bovine brains, and our colleagues in the Department of Molecular Medicine (in particular, Drs. Catherine Bigger, Steve Britt, Eileen Lafer, Irena Melnikova, Eduardo Montalvo and Kondury Prasad) for many useful discussions and reagents.


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