(Received for publication, December 14, 1994)
From the
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
4 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
4 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
4 subunit gene.
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) ()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
2-
9(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and
2-
4(4, 7, 10, 16, 17, 18, 19) .
Reconstitution studies have demonstrated that different combinations of
and
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
7,
8, and
9 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
5 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 4 subunit gene, we
reported the identification of cis-acting sequences within the
4 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
4 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
4 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.
Figure 1:
Nucleotide sequence of the 5`-flanking
region of the rat nicotinic ACh receptor 4 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 4 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
-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 4 19-bp region. EMSA with SN17 nuclear
extract and radiolabeled
4 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
4 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
4 oligonucleotide remained
constant.
Figure 6:
The 19-bp region interacts with a
DNA-binding protein with an apparent molecular size of 50 kDa. The
4 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
C-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.
These results prompted further investigation into the possible
co-expression of the 4 gene and this newly identified promoter
binding activity. Earlier anatomical studies indicated the expression
of the
4 subunit gene in discrete regions of the rat
brain(18) . More recent studies document the expression of the
4 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
4 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
4 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.
Figure 5:
Site-directed mutagenesis of the 19-bp
region results in significantly lower transcriptional activity of the
4 promoter. The indicated sequence changes were introduced into
the
4 insert in pX1B4FH (see Fig. 1). SN17 cells were
transiently transfected with the wild type (pX1B4FH) and mutated
(pX1B4FHM)
4/luciferase expression constructs. Transfection
controls were carried out as described in the legend for Fig. 2. Error bars represent S.E.
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 2 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
2 (31) and
7 (32) subunit genes and the
rat
3 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
4 subunit gene we have
isolated does not exhibit significant homology with any of these
positively acting transcriptional regulatory regions nor to the
2
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 4 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
4 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
4 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
4 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
4/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
4 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
4, 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 4 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 4 subunit gene expression was initially
thought to be limited to specific neurons(18) . However, recent
reports suggest a more ubiquitous expression pattern for the
4
gene with
4 mRNA being detected in cells such as thymocytes (41) and keratinocytes(42) . Thus, it appears that the
4 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
4
promoter and that cell type-specific expression of the
4 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
4 subunit gene. This element, as determined by 5`-deletional
analysis and site-directed mutagenesis, is necessary for full
transcriptional activation by the
4 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
4 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
4
subunit gene expression.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L22646[GenBank].