©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Neuronal Nicotinic Acetylcholine Receptor 2 Subunit Gene Promoter Is Activated by the Brn-3b POU Family Transcription Factor and Not by Brn-3a or Brn-3c (*)

Nathaniel G. N. Milton (1), Alain Bessis (2), Jean-Pierre Changeux (2), David S. Latchman (1)(§)

From the (1)Medical Molecular Biology Unit, Department of Molecular Pathology, University College London Medical School, the Windeyer Building, Cleveland Street, London W1P 6DB UK and the (2)Laboratoire de Neurobiologie Moléculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The regulatory region of the neuronal nicotinic acetylcholine receptor 2 subunit gene, which contains six copies of the octamer-related sequence CCCCATGCAAT, is activated by the Brn-3b POU family transcription factor but not by the closely related factors Brn-3a and Brn-3c. This effect is in contrast to the previously documented inhibitory effect of Brn-3b on octamer-containing promoters that are activated by Brn-3a and Brn-3c. Activation of the 2 gene by Brn-3b requires that both the POU domain and other N-terminal sequences are derived from Brn-3b and is dependent on the intactness of the 2 gene regulatory region, being lost in truncated derivatives containing one, two, or four copies of the octamer-related sequence. Surprisingly, however, these truncated derivatives are activated by Brn-3c. These effects are discussed in terms of both the influence of the target sequence and its context in the promoter on activation by the various forms of Brn-3 as well as of the processes that restrict expression of the 2 subunit gene to a few cells in the nervous system.


INTRODUCTION

The tissue-specific regulation of gene expression is regulated by a balance between positively and negatively acting cellular transcription factors that modulate gene activity in opposite directions (for review see Ref. 1). In the case of genes expressed specifically in neuronal cells, negative regulation appears to play a critical role with many neuronally expressed genes, such as those encoding SCG10 and the type II sodium channel possessing a silencer element that inhibits gene expression in non-neuronal cell types (for review see Ref. 2). Such negative regulation is also observed in the case of the gene encoding the neuronal nicotinic receptor 2 subunit, which has the most restricted expression of all the nicotinic receptor subunits in both rats (3) and chickens, where it is expressed exclusively in the spiriformis lateralis nucleus of the diencephalon (4).

Thus, the regulatory region of this gene contains an inhibitor element that reduces the activity of the promoter itself and also has this effect when linked to the heterologous promoter of SV40(5) . This regulatory region contains six copies of the sequence CCCCATGCAAT, which appear to be responsible for its inhibitory effect. Thus the inhibitory effect of the regulatory region in transfection experiments can be relieved by including an oligonucleotide containing the 11-base pair motif, suggesting that it binds an inhibitory factor that can be titrated off the promoter by excess oligonucleotide(5) .

The 11-base pair motif shows strong homology to the octamer consensus sequence ATGCAAAT, which is bound by members of the POU family of transcription factors (for reviews see Refs. 6 and 7). As many of the members of this family are specifically expressed in the nervous system, we wished to test the possibility that members of this family could regulate the 2 subunit gene via the 11-base motif and perhaps produce the observed highly restricted expression pattern characteristic of this gene.

In particular, we wished to investigate the effect on this gene of the three members of the Brn-3 POU family Brn-3a (also known as Brn-3 or Brn-3.0)(8, 9, 10) , Brn-3b (also known as Brn-3.2)(9, 11, 12, 13) , and Brn-3c (also known as Brn-3.1)(9, 10, 14) . Thus these three factors are specifically expressed in neuronal cells in the peripheral and central nervous systems with each factor having a distinct expression pattern(8, 9, 10, 11, 12, 13, 14) . Moreover, although all these factors can bind to the octamer consensus sequence (15, 16) and other related motifs(10, 12) , they have distinct effects on gene expression. Thus, Brn-3a and Brn-3c can transactivate an artificial promoter containing a consensus octamer motif (15, 16) or the promoter of the gene encoding the neuronal intermediate filament protein -internexin(17) . In contrast, Brn-3b represses both of these promoters and can also interfere with their transactivation by Brn-3a(15, 17) . We have therefore investigated the effects of each of the forms of Brn-3 on the 2 subunit gene promoter as well as the dependence of these effects on the 11-base pair octamer-related motif.


MATERIALS AND METHODS

Plasmid DNA

The 2 receptor constructs were prepared as described previously(5) . The 2.6 Sil()constructs contain the promoter region from -810 to +81 relative to the transcriptional start site (containing six copies of the 11-base pair motif) upstream of the SV40 promoter and the luciferase gene. The remaining constructs contain the regions from -810 to -41 with four copies of the motif (2.4. Sil), from -810 to -80 with two copies of the motif (2.2 Sil), from -810 to -118 with one copy of the motif (2.1 Sil), and from -810 to -144 lacking any copies of the motif (2.0 Sil).

The Brn-3a, Brn-3b, and Brn-3c expression vectors contain full-length cDNA clones for each of these proteins (9) cloned under the control of the Moloney murine leukemia virus promoter in the vector pLTRpoly, which has been modified by deletion of a cryptic splice site in the simian virus 40 3`-untranslated region(9, 15, 16) . In the case of Brn-3a, an expression vector containing cDNA derived from the spliced form of the Brn-3a mRNA was used. The Brn-3b vector contains a full-length cDNA derived from the intronless RNA that encodes Brn-3b, whereas the Brn-3c vector similarly contains a full-length cDNA derived from the mRNA encoding Brn-3c, which is produced from the primary transcript by the removal of a single intron(9) .

Transfection

Transfection of DNA was carried out according to the method of Gorman(18) . Routinely 1 10 BHK-21 cells (19) were transfected with 10 µg of the reporter plasmid and 10 µg of the expression vector. In all cases cells were harvested 72 h later. The amount of DNA taken up by the cells in each case was measured by slot blotting the extract and hybridization with a probe derived from the ampicillin resistance gene in the plasmid vector(20) . This value was then used to normalize the values obtained in the luciferase assay as a control for differences in uptake of plasmid DNA in each sample.

Luciferase Assays

Assays of luciferase activity were carried out with extracts that had been equalized for protein content according to the method of Bradford(21) .


RESULTS

In initial experiments we transfected BHK-21 fibroblast cells (20) that lack any endogenous Brn-3 (11) with plasmids expressing each of the forms of Brn-3 (9) and a reporter plasmid containing the entire 2 gene inhibitory region with six copies of the inhibitory motif upstream of the SV40 promoter driving the luciferase gene (2.6)(5) . In these experiments (Fig. 1), the reporter gene construct was strongly transactivated (approximately 18-fold) by co-transfection with the Brn-3b expression vector compared with the level of promoter activity when co-transfected with the expression vector lacking any insert. In contrast, neither the Brn-3a or Brn-3c constructs had any effect on the activity of the 2.6 construct. The effect of Brn-3b on the 2.6 construct was dependent on the region containing the six copies of the 11-base pair motif because deletion of this region, leaving sequences further upstream (from -810 to -144) in the 2 promoter intact, in the construct 2.0 abolished induction by Brn-3b (Fig. 1).


Figure 1: Luciferase activity in BHK-21 cells transfected with the intact 2.6 construct or the 2.0 construct lacking all copies of the 11-base pair motif and either expression vector alone or the same vector expressing the indicated Brn-3 form. Values are expressed as percentages of the activity of the 2.6 or 2.0 constructs alone and are the averages of four determinations. The standard error of the mean is indicated by the bars.



These experiments showing specific activation of the 2 regulatory region by Brn-3b are in contrast to our previous experiments in which a test promoter containing an octamer motif (15, 16) or the -internexin promoter (17) were transactivated by Brn-3a and Brn-3c and inhibited by Brn-3b. Indeed, Brn-3b was able to inhibit transactivation by Brn-3a when the two factors were co-transfected with either of these promoters(15, 17) . We therefore compared the effect of co-transfecting the 2.6 construct with combinations of two different Brn-3 factors with that observed with a single factor alone (together with the empty expression vector to ensure that the amount of DNA transfected in each case was the same). In these experiments (Fig. 2) Brn-3b was able to transactivate the 2.6 construct in either the presence or the absence of Brn-3a or Brn-3c, although the degree of activation was somewhat reduced in the presence of Brn-3a.


Figure 2: Luciferase activity in BHK-21 cells transfected with the intact 2.6 construct and the indicated combinations of the expression vector lacking any insert or containing an insert derived from each of the forms of Brn-3. Values are expressed as percentages of the activity of the 2.6 construct alone and are the averages of four determinations. The standard error of the mean is shown by the bars.



In previous experiments using chimeric constructs containing different regions of Brn-3a and Brn-3b, we have shown that different domains of Brn-3a are required for activation depending on the target promoter involved (Fig. 3). Thus activation of the -internexin promoter requires the N-terminal domain of Brn-3a(17) . In contrast, activation of a test promoter containing a consensus octamer motif requires the DNA binding POU domain of Brn-3a(15) , and this domain can act as an independent activation domain because it can transactivate this promoter when expressed in isolation independent of the rest of Brn-3a(17) .


Figure 3: Summary of gene transactivation data with the Brn-3a (A) and Brn-3b (B) vectors and chimeric proteins containing different subregions of each factor. Data are summarized for constructs containing a consensus octamer motif cloned upstream of a test thymidine kinase promoter (tK-oct) (15), the -internexin promoter (17), and the 2 promoter (Fig. 4). The division of Brn-3a and Brn-3b subdomains I, II, III, and IV is as follows: Brn-3a: subdomain I, amino acids 1-40; subdomain II, amino acids 41-108; subdomain III, amino acids 109-267; subdomain IV (POU domain), amino acid 268 to the end; Brn-3b: subdomain II, amino acids 1-92; subdomain III, amino acids 93-169; subdomain IV (POU domain), amino acid 170 to the end.



We therefore tested the effect of these chimeric constructs and the isolated POU domains of Brn-3a and Brn-3b on the 2.6 construct. In these experiments (Fig. 4), none of the chimeric constructs and neither of the isolated POU domains were able to activate the 2.6 constructs. Because the chimeric construct 4 contains all of Brn-3b except the POU domain, this indicates that the POU domain of Brn-3b and upstream regions of the molecule are both required for activation of the 2.6 molecule. Moreover, the 2.6 construct was not activated by co-transfection with a combination of the chimeric construct 4 and the Brn-3b POU domain construct, indicating that these two regions must be present on the same protein molecule for activation to occur.


Figure 4: Effect on the 2.6 construct of co-transfection with vectors expressing each of the indicated forms of Brn-3, the isolated POU domains of Brn-3a (POU-A) or Brn-3b (POU-B), the chimeric constructs coded as indicated in Fig. 3 (1-4), or the Brn-3b POU domain vector together with chimera 4 (POU-B+4). Values are expressed as percentages of the activity of the 2.6 construct and are the averages of four determinations. The standard error of the mean is shown by the bars. VEC, vector.



In previous work with the 2 gene, it has been shown that the inhibitory effect of the region containing six copies of the 11-base pair element is disrupted when two or more of these motifs are deleted with constructs containing the region of the promoter with one, two, or four motifs actually having an enhancing effect on gene expression(5) . In view of the complex activity of this region, we tested the effect of the Brn-3 expression vectors on constructs containing the region of the 2 gene with one, two, or four copies of the 11-base pair motif. In these experiments (Fig. 5A), Brn-3b was unable to transactivate any of these constructs and indeed had an inhibitory effect on their expression so that activity was reduced below that observed upon co-transfecting these constructs with expression vector alone.


Figure 5: Effect of the different forms of Brn-3 on the activity of the 2.0 (A-2.0), 2.1 (A-2.1), 2.2 (A-2.2), 2.4 (A-2.4), and 2.6 (A-2.6) constructs containing the region of the 2 promoter with 0, 1, 2, 4, and 6 copies, respectively, of the 11-base pair motif. A, the luciferase activity in each case is expressed as a percentage of the activity of each reporter plasmid when transfected alone. VEC, vector. B, the basal activity of each promoter alone is shown to indicate that the differences observed are not due to gross differences in the basal activity. In all cases, the values are the means of four experiments, and the standard error of the mean is shown by the bars.



Interestingly, however, all three of the truncated constructs were strongly activated by Brn-3c, although this was not observed for the 2.0 construct lacking any copies of the 11-base pair motif (Fig. 5A) or for the intact construct containing all six copies (Fig. 5B). Hence Brn-3b and Brn-3c have different effects on the 2 promoter constructs depending on the number of 11-base pair motifs that they contain, with the intact region behaving differently from its truncated derivatives.

These differences were not dependent on differences in the basal activity of these constructs that might result, for example, in a particular construct being maximally active and hence not being activable by a specific POU factor. Thus, the 2.4 and 2.6 constructs showed similar basal activity (Fig. 5B), but one was transactivated by Brn-3c and not Brn-3b, while the other was activated by Brn-3b and not Brn-3c.


DISCUSSION

The activation of the neuronal nicotinic acetylcholine receptor 2 region by Brn-3b reported here is the first time that the transactivation of a natural promoter has been observed with this factor. Indeed, in previous experiments with Brn-3b(15, 16, 17) , this factor had an inhibitory effect on the activity both of an artificial test promoter containing an octamer motif and of the -internexin promoter and was also able to inhibit the ability of Brn-3a to activate such promoters.

Although the form of Brn-3b that we have used in our experiments represents the major form of Brn-3b in the spinal cord(9) , a longer form has been detected in both the retina (13) and the central nervous system(12) , although it is absent in spinal cord(9) . This form contains additional N-terminal sequences that are homologous to the N-terminal region of Brn-3a. Interestingly, the equivalent region of Brn-3a is lacking in a naturally occurring short isoform of this factor (9) and is required for activation of the -internexin promoter(17) . In agreement with this homology, the long form of Brn-3b is able to activate a test promoter containing a binding site derived from the corticotrophin-releasing hormone promoter linked to a test prolactin promoter. Hence it appears that, as well as the existence of three different Brn-3 factors encoded by different genes (22) and with different activities, these factors also have different isoforms with different expression patterns and with potentially different effects on target promoters.

Interestingly, however, the test construct containing the corticotrophin-releasing hormone promoter binding sites linked to the prolactin promoter was activated by Brn-3a as well as by the long form of Brn-3b(12) . Hence the 2 promoter not only is the first example of activation by the short form of Brn-3b but also represents the first case of specific activation by Brn-3b and not by Brn-3a or Brn-3c. It is likely that these differences in the effects on specific promoters of the different forms of Brn-3 and their various isoforms arise from differences in the precise binding site in each promoter and its context relative to other sequences. Effects of this type have previously been observed with other transcription factors. Thus, for example, the glucocorticoid receptor can activate gene expression by binding to a glucocorticoid response element but represses gene expression following binding to a distinct sequence element known as the negative glucorticoid response element(23) . Similarly, the YYI factor can activate the human papilloma virus 18 promoter by binding to its target sequence when an adjacent switching element is also present, but the binding of YYI to its target sequence represses gene expression when this switching element is deleted(24) .

Thus the differences in the nature of the binding site for the Brn-3 factors in the different promoters used thus far as well as the context of the binding site relative to binding sites for other factors are likely to account for the differences in the effects observed. Indeed the influence of binding site sequence and promoter architecture are well illustrated by the results described here because the 11-base pair motif in the 2 regulatory region differs from the octamer(15, 16) or corticotrophin-releasing hormone (12) binding sites used by us previously or by others. Moreover Brn-3b-mediated activation via this motif is dependent on the presence of the entire inhibitory region containing all six copies of the motif and is not observed with the constructs containing one, two, or four copies. However, these constructs but not the construct containing the intact region are transactivated by Brn-3c, the first constructs to be activated by Brn-3c and not Brn-3a (see for comparison Refs. 15 and 17).

In addition to illustrating the importance of binding site sequence and context in the response to Brn-3 factors, these findings also indicate the role of the entire regulatory region in producing the appropriate response of the 2 promoter. Such an effect is important because it provides a molecular framework for understanding why, in previous work with this region, its inhibitory effect in a range of cell types was dependent on the intact region with progressive deletion producing an enhancer activity in constructs with one, two, or four motifs(5) . Thus it is likely that an inhibitory factor exists whose effect, like activation by Brn-3b, is dependent on the intactness of the region with its six copies of the 11-base pair motif that can act as a target site for POU family transcription factors.

Further studies will be required to identify this putative factor as well as to indicate whether Brn-3b is expressed in the restricted regions of the nervous system that express the 2 subunit and may therefore play a role in its activation in vivo. It is already clear, however, that the short form of Brn-3b can activate a specific promoter in a manner that indicates the importance of the intactness of the 2 promoter regulatory region for mediating its response to specific transcription factors.


FOOTNOTES

*
This work was supported by the Medical Research Council. 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.

The abbreviation used is: Sil, silencer.


ACKNOWLEDGEMENTS

We thank Tarik Möröy and Thomas Theil for the gift of the Brn-3 expression vectors.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.