From the
The regulatory region of the neuronal nicotinic acetylcholine
receptor
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
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
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
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) .
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
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
The activation of the neuronal nicotinic acetylcholine
receptor
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
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
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
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
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
We thank Tarik Möröy and Thomas Theil for
the gift of the Brn-3 expression vectors.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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).
2
subunit gene via the 11-base motif and perhaps produce the observed
highly restricted expression pattern characteristic of this gene.
-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.
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).
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) .
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.
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.
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.
-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.
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) .
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).
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.