From the Department of Biochemistry, Sciences II, University of Geneva, 1211 Geneva 4, Switzerland
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ABSTRACT |
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Within the chick central nervous system,
expression of the 3 nicotinic acetylcholine receptor gene is
restricted to a subset of retinal neurons, the majority of which are
ganglion cells. Transient transfection in retinal neurons and in neural
and non-neural cells from other regions of the chick embryo allowed the
identification of the cis-regulatory domain of the
3 gene. Within
this domain, a 75-base pair fragment located immediately upstream of
the transcription start site suffices to reproduce the neuron-specific
expression pattern of
3. This fragment encompasses an E-box and a
CAAT box, both of which are shown to be key positive regulatory
elements of the
3 promoter. Co-transfection experiments into
retinal, telencephalic, and tectal neurons with plasmid reporters of
3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic
helix-loop-helix transcription factors indicate that the cis-regulatory
domain of
3 has the remarkable property of discriminating accurately
between related members of the basic helix-loop-helix protein family.
The sequence located immediately 3' of the E-box participates in this
selection, and the E-box acts in concert with the nearby CAAT box.
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INTRODUCTION |
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In vertebrates, both negative and positive regulations play an important role in determining neuronal gene expression. Negative regulation (reviewed in Refs. 1 and 2) is best exemplified by REST/NRSF, a factor that represses transcription of the SCG-10 and type II Na+ channel genes in non-neuronal cell-types, whereas most neurons lack REST/NRSF and thus express these two genes (3, 4). Conversely, the Olf-1 transcription factor positively regulates several genes (e.g. the olfactory neuron-specific G protein, the type III adenylyl cyclase) specifically expressed in olfactory neurons (5). Vertebrate homologs of the basic helix-loop-helix (bHLH)1 factors involved in Drosophila neurogenesis (6) act as positive or negative regulators in the acquisition of neuronal identity. For instance, the atonal- and achaete-scute-related activators are transiently expressed in parts of the central and peripheral nervous system during early development, and their null mutation or ectopic expression profoundly influences neurogenesis (reviewed by Lee et al. (7)). However, the direct regulation by bHLH proteins of genes that define neuronal identity has never been documented.
Several neuronal nicotinic acetylcholine receptor (nAChR) genes are expressed early in neural development (8-12), and, since they encode transmembrane sensors capable of fluxing Ca2+ and other cations upon stimulation (reviewed in Ref. 13), an understanding of their regulation should help explain how the genetic program puts together the mechanisms needed for epigenetic environmental cues to participate in development. This is illustrated in a recent report by Shatz and associates (14) showing that, in the developing retina, cholinergic synaptic transmission between newly generated amacrine and ganglion cells is responsible for the propagation of spontaneous waves of action potentials that may be critical for the establishment of visual system circuitry.
Several nAChR subunit genes, including 4,
2,
3, and
4, are
expressed in the chick retina
(10),2 and expression of
3
is confined to ganglion cells and amacrine neurons (15). Forsayeth and
Kobrin (16) have shown that the
3 subunit co-assembles in
vivo with the
4,
2, and
4 subunits to form a functional
nicotinic receptor endowed with distinctive properties. We have
isolated a short 5'-sequence of the
3 gene containing promoter
elements that are sufficient to target reporter gene expression to
those retinal neurons that normally express
3 in vivo
(11, 15). The stringent neuronal specificity of the
3 promoter and
its activation during the period of neuronal fate determination make it
an attractive system in which to study the functional interactions
between transcription factors and cis-acting regulatory elements that
help establish the diverse neuronal phenotypes.
In this report, we carry out a functional analysis of the
cis-regulatory domain, establishing that transcription of the 3 gene
is under the direct control of bHLH factors. Moreover, we show that the
3 promoter is able to discriminate accurately between related
members of the bHLH family, thereby effecting the stringent neuron-specific regulation of the gene.
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EXPERIMENTAL PROCEDURES |
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DNA Constructions--
Standard molecular biology techniques
were used (17) unless otherwise stated. Construction of the reporter
plasmids 3RS-CAT and
3RS-lacZ and of the
reference plasmids SV-CAT and SV-lacZ was
described by Hernandez et al. (15). Point mutations in the
3RS sequence (Fig. 1A) were introduced by polymerase
chain reaction and checked by sequencing. The mutant DNA fragments were
cloned at the SmaI site of the pCAT00 plasmid (18). SF-E and
SF-3E were obtained by ligation of PvuII linkers (CCAGCTGG;
New England Biolabs) to the 5'-end of fragment SF (Fig. 2A).
1KK encompasses nucleotides 151-334 of the chick
1 nAChR
promoter (19, 20) (GenBankTM accession number M15307),
flanked by KpnI restriction sites that were used for
subcloning into pCAT00. The
1/
3 hybrid promoter was obtained by
ligation of the 5'-end of
1KK with the 3'-end of
3RS at their
shared PvuII restriction site (Fig. 7A). The expression plasmid (pEMSV) encoding MyoD was kindly provided by the
Weintraub laboratory (Fred Hutchinson Cancer Research Institute, Seattle), the CTF-4 cDNA by J. Schmidt (SUNY, Stony Brook), and the
CASH-1 cDNA by T. Reh (University of Washington, Seattle).
Molecular Cloning of the Chicken NeuroD and NeuroM cDNAs-- The cloning of chicken NeuroD (GenBankTM Y09596) and chicken NeuroM (GenBankTM Y09597) is described by Roztocil et al. (21).
Expression and Purification of the CTF-4 Protein--
A
HindIII cDNA fragment encoding the bulk of the CTF-4
protein (22) was cloned in phase in the appropriate pDS bacterial expression vector (23). High level accumulation of His-tagged fusion
protein was achieved by incubating Escherichia coli
transformants at 37 °C for 2 h with 2 mM
isopropylthiogalactoside in rich broth. Purification of the fusion
protein was performed on Ni2+-nitrilotriacetic acid-agarose
(Qiagen) in denaturing conditions. The eluted protein (48.5 kDa) was
renatured from 8 M urea by serial dialysis and stored at
20 °C in 10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 1 mM
-mercaptoethanol.
Isolation of Nuclear Proteins and Gel Mobility Shift
Analysis--
Retinae and optic tecta were dissected in cold
phosphate-buffered saline, and nuclei were isolated as described by
Matter-Sadzinski et al. (24). Nuclear proteins were obtained
by adding NaCl to the suspension of nuclei to a concentration of 1 M, in the presence of the protease inhibitors leupeptin,
phenylmethylsulfonyl fluoride (both at 0.7 µg/ml), and Trasylol
(1%), incubating for 15 min on ice, and centrifuging for 45 min at
50,000 rpm (Beckman TLA 100). The proteins in the supernatant were
quantified (Bio-Rad protein assay) and stored at 70 °C. The probes
were 35-bp double-stranded fragments (underlined in Fig.
1A) end-labeled by fill-in of 5'-overhangs with Klenow
enzyme in presence of [
-32P]dATP. For band shifts,
bovine serum albumin was added to nuclear proteins to keep the total
protein load at 6 µg/lane. Poly(dI-dC) (2.5 µg), used as
nonspecific competitor DNA, was mixed with the probe (20,000 cpm) in FT
buffer (25 mM HEPES, pH 7.6, 5 mM
MgCl2, 40 mM KCl), proteins were added, and the
mixture (20 µl) was incubated for 15 min on ice. The samples were
then loaded on a 7.5% polyacrylamide gel containing 2.5% glycerol.
After a 2.5-h run at 200 V, the gel was dried and exposed overnight at
70 °C with an intensifying screen.
Cell Cultures, Transfection, and CAT and -Galactosidase
Assays--
All tissue culture reagents except as indicated were
purchased from Life Technologies, Inc. Plasticware was from Nunc. Chick embryos were staged according to Hamburger and Hamilton (25). Cells
from different regions of the CNS were prepared as described previously
(11, 24). Neuroretina, pigment epithelium, optic tectum, telencephalon,
and cerebellum were dissected from E4-E13 embryos, collected in
Ca2+- and Mg2+-free Hanks' balanced salt
solution and incubated with 0.05% trypsin for 10 min (E4-E8) or with
0.1% trypsin for 20 min (E9-E13). Desoxyribonuclease I (Boehringer
Mannheim) was added to 30 µg/ml for 5 min, and then trypsin was
inactivated by adding fetal calf serum to 5%. Cells were pelleted,
rinsed in Opti-MEM medium, resuspended in Opti-MEM at densities as
indicated below, and subjected to the transfection procedure. Plasmid
DNA (5 µg in 100 µl of Opti-MEM) was mixed with Lipofectin reagent
(20 µg diluted in 200 µl of Opti-MEM), and the transfection
solution was added to 200 µl of cell suspension containing 4-6 × 106 cells. In co-transfection experiments with two
constructs, 5 µg of reporter plasmid was mixed with 3.5 µg of
expression vector. In all instances, the ratio of DNA to Lipofectin was
4/1. Glial cells and primary or secondary cultures of CEFs were
prepared and transfected as described by Matter-Sadzinski et
al. (24). 24 or 48 h after transfection, cells were collected
and processed for CAT assay. The amount of acetylated
14C-labeled chloramphenicol was determined by scintillation
counting and/or by scanning the chromatogram with a Linear Analyzer LB 284/285 (Berthold). In each experiment, an aliquot of cells was transfected with pSV-CAT, and the resulting CAT activity was
arbitrarily set at 100. The activities obtained in parallel with other
constructs were calculated relative to this value. 100-150 µg of
cytosolic proteins were used in CAT assays such that the proportion of
acetylated [14C]chloramphenicol in cells transfected with
pSV-CAT did not exceed 70%. The activity of the SV40
promoter was similar in different neuronal and non-neuronal cell-types
at different embryonic stages (24). The means and S.D. values were
calculated with data obtained in at least five independent
experiments.
Northern Blot Analysis--
48 h after transfection, the cells
were rinsed twice with ice-cold phosphate-buffered saline and lysed in
guanidine thiocyanate (17). Total RNA was isolated, gel-fractionated,
and hybridized as described (10, 15). We used as probe a
32P-labeled 3 genomic fragment containing 250 bp of exon
5 (encoding amino acids Met308-Gln391) and 240 bp of intron 5 (15).
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RESULTS |
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Identification of Regulatory Elements in the 3 Promoter--
In
the chick CNS, the
3 nAChR gene is selectively expressed in the
neuroretina. We have previously shown that essential neuron-specific promoter elements are located in a short
EcoRI-SphI DNA fragment, 143 bp in length and
located just upstream of the transcription start site (11, 15). This
3RS fragment contains several putative binding sites for
transcription factors: CACCC and CAGCTG (E-box) motifs, a CAAT box, and
two TATA-like motifs located at
56 bp and
30 bp relative to the
transcription initiation site (Fig. 1A). To investigate how these
different sites contribute to promoter activity, point mutations were
introduced by polymerase chain reaction into each site, and the mutated
fragments were fused to the CAT reporter gene. The
constructs were transfected into neurons freshly dissociated from E5
chick neuroretina (24), and 48 h later the transfected cells were
processed for CAT activity. Mutations in the E-box and in the CAAT box
produced spectacular effects, completely abolishing promoter activity
in retinal cells. In contrast, mutations in the other motifs had no
significant impact on promoter activity (Fig. 1B).
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The E-box Is a Key Element of the 3 Promoter--
Deleting the
5'-end of
3RS, which contains the CACCC and E-box motifs (SF; Fig.
2A), resulted in the complete
inactivation of the promoter in retinal cells (Fig. 2B).
Since mutating the E-box in the full-length
3RS fragment
(E-box*, Fig. 2A) was sufficient to obtain a
similar effect, we tested whether it is possible to reactivate the
truncated SF fragment by adding one or several E-boxes at its 5'-end
(constructs SF-E and SF-3E; Fig. 2A). The addition of one
E-box restored promoter activity in retinal cells to a level comparable
with that of the complete
3RS fragment, and three E-boxes allowed
activity levels consistently higher than
3RS (Fig. 2, B
and C). The tissue specificity of the reactivated promoters
was examined by transfection into cells in which the
3 promoter is
normally silent, namely cells from the optic tectum and telencephalon,
CEFs or glial cells (Fig. 2C). No promoter activity was
detected in any of them, indicating that SF-E and SF-3E are regulated
as specifically as the
3RS fragment. Thus, the addition of one E-box
is sufficient to reconstitute a promoter with the same specificity and
activity as the wild-type fragment, demonstrating that the E-box is a
key regulator of the
3 nAChR gene.
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Precise Positioning of the E-box and CAAT Box Is Required for Promoter Activity-- Mutation of the CAAT box, which is located 9 bp downstream of the E-box, abolishes promoter activity in retinal cells (Fig. 1B), suggesting that direct interactions between proteins bound to these two elements may take place. Due to the helicity of DNA, the addition or deletion of base pairs in the intervening sequence should disrupt the alignment of bound factors, thereby decreasing promoter activity. Several mutants were constructed by the addition or deletion of nucleotides between the E-box and the CAAT box, and they were tested for promoter activity in E5 retinal cells (Fig. 3). Reducing the distance by 1, 2, and 3 bp was sufficient to decrease promoter activity 5, 8, and 10-fold, respectively. In contrast, the addition of 1-4 bp had either a modest effect (Fig. 3) or no effect at all (SF-E, Fig. 2), depending on the particular additional base pairs. This suggests that steric hindrance between bound factors contributes to the decrease in promoter activity when the distance between the two motifs is reduced.
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Functional Analyses of Neuronal bHLH Proteins--
Since the E-box
is a binding site for transcription factors of the bHLH family, we
postulated that expression of the 3 gene is under the control of
such a factor in the neuroretina. Several bHLH genes are sequentially
expressed in the developing chick CNS. While CASH-1 is
expressed in proliferating cells (26), NeuroM is transiently
expressed in cells that have withdrawn from the mitotic cycle but have
not yet migrated in the outer layers, whereas NeuroD labels
neurons that are migrating and differentiating (21). CTF-4
(22) is widely distributed in the nervous system, but its expression is
significantly enhanced in the
retina.3 Since
3
expression at early stages of retina development coincides with
expression of these different bHLH proteins, we tested if any of them
was able to trans-activate the
3RS promoter. Co-transfections with
expression vectors encoding CASH-1, NeuroM, NeuroD, or CTF-4 into E8
tectal and E9 telencephalic neurons revealed that none of these
proteins was able to trans-activate the
3RS promoter ectopically,
although all four were functional transcription factors capable of
strongly enhancing activity of the
E-box-driven promoter of the muscle nAChR
1 subunit (
1KK
fragment, Fig. 4A, Table I).
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Ectopic Activation of the 3 Promoter by a bHLH
Protein--
Because functional domains are well conserved among
members of the bHLH protein family, we asked if MyoD, a transcriptional activator of muscle-specific genes that binds to the CAGCTG motif (29),
was capable of replacing endogenous neuronal bHLH proteins. Co-transfections with a MyoD expression vector revealed that ectopic expression of MyoD was sufficient to trans-activate the
3RS promoter in neurons from the optic tectum, telencephalon, and cerebellum that do
not normally express
3 (Fig. 5,
A and B). In parallel experiments, telencephalic
or tectal cells were co-transfected with MyoD and
3RS-lacZ constructs, and trans-activation was observed by
X-gal staining in about 20% of transfected cells (Table I). This
activation is mediated by the E-box, since MyoD failed to activate the
promoter bearing a mutant E-box (Fig. 5A). Moreover, as
determined by Northern blot analysis with RNA obtained from transfected
tectal cells, MyoD was also able to trans-activate the
endogenous
3 gene (Fig. 5C),
clearly indicating that the transfected
3RS promoter behaves in
neurons much as the native promoter does.
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Influence of MyoD on 3 Promoter Activity in the Developing
Retina--
We investigated the effect of MyoD on
3RS promoter
activity within the domain of
3 expression. Transcription of
3 in
neuroretina is first detected on E4, whereupon activity of the promoter
rapidly increases and peaks on E5, decreasing later to relatively low levels (11). We wanted to determine whether ectopic MyoD could activate
the
3 promoter earlier in development, increase the peak value at
E5, or maintain a high level of activity at later stages. The MyoD
expression vector was co-transfected with
3RS-CAT or with
3RS-lacZ into retinal cells isolated at different stages between E4 and E13. MyoD had no influence on promoter activity at early
stages of development, but it enhanced CAT synthesis in the developed
retina (E8-E13) without modifying the proportion of
-galactosidase-positive cells (Fig. 6;
Table I). The levels of endogenous
3 mRNA were affected in the
same way, with a MyoD-induced increase on E13 but not on E5 (data not
shown). In contrast, MyoD strongly stimulated promoter activity of the
1KK fragment both in E5 and E13 retinal cells, indicating that
functional MyoD was indeed synthesized in these cells (Table I). We
interpret these observations as suggesting that endogenous neuronal
bHLH protein(s) for which MyoD can substitute are not limiting in early
retina, whereas later in development, ectopic expression of MyoD
compensates for decreased amounts of the endogenous bHLH
protein(s).
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A Hybrid 1/
3 Promoter Behaves Like the
3
Promoter--
Our experiments highlight the remarkable capacity of the
3RS promoter to discriminate between different members of the bHLH family, in striking contrast to the
1KK promoter, which was
activated by all five of the bHLH proteins we tested. Because sequences in the vicinity of the E-box may play an important role in the recognition of a specific bHLH factor, we constructed a hybrid promoter
from portions of the
1KK and
3RS fragments. The
1KK promoter
contains two E-boxes. The distal
1 E-box and the
3 E-box have the
same sequence (CAGCTG), and we took advantage of the fact that it is a
PvuII restriction enzyme recognition site to fuse the two
promoters at this level (Fig.
7A). In the hybrid promoter,
termed
1/
3, the 56 bp upstream of the reconstituted CAGCTG E-box
come from the
1 promoter, while the 69 bp downstream of it come from
the
3 promoter. Promoter activity of the hybrid was compared with
that of
1 and
3 in myotubes and in CEFs (Fig. 7C). As
expected, the
1 promoter had a strong activity in myotubes and was
weak in CEFs. In contrast, the
3 and
1/
3 promoters were
completely silent in both cell types. In co-transfections, MyoD
consistently and strongly enhanced activity of the
1 promoter but
did not influence the
1/
3 promoter. In tectal and telencephalic neurons, the
3 and
1/
3 promoters were completely silent and could not be trans-activated by CASH-1, NeuroM, NeuroD, or CTF-4 (Fig.
4 and data not shown). The hybrid promoter was only found to be active
in retinal cells, where it reached an activity level somewhat lower
than
3 (Fig. 7B). It is known from a previous study that
the distal E-box of the
1 promoter is sufficient to drive reporter
gene transcription in myotubes (30), yet when it is flanked in 3' by
sequences from the
3 promoter, its activity becomes restricted to
retinal cells. Indeed, the hybrid promoter behaves much like the
3RS
or SF-E fragments (Fig. 2), suggesting that the 69-bp sequence located
downstream of the
3 E-box contains essential elements that act in
concert with the E-box to confer stringent specificity upon the
3
promoter.
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DISCUSSION |
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A short fragment, 75 bp in length and located immediately upstream
of the transcription start site, is sufficient to generate the
neuron-specific expression pattern of the 3 nAChR gene. Inquiring into the underlying mechanisms, we present evidence that the
3 promoter is positively regulated by an E-box acting in concert with a
neighboring CAAT box. The
3 promoter appears to have a simple
structure and be devoid of the regulatory complexities resulting either
from inhibitory DNA elements that prevent expression in non-neuronal
cell types (reviewed by Schoenherr and Anderson (2)) or from
multipartite elements whose active combinations vary in the course of
the development (31). Very few neuron-type specific promoters have been
characterized in detail, and we do not know whether the simple
organization of the
3 cis-regulatory domain is a common feature of
genes whose expression is confined to restricted subsets of
neurons.
The finding that an E-box is a key regulator of the 3 gene
emphasizes the role of bHLH factors in neural transcriptional control.
Several members of the bHLH family are transiently expressed in the
developing CNS. ASH-1 is widely expressed in proliferating precursor cells (26). ASH-1 and neurogenin-1
(ATH-4C) exhibit complementary domains of expression in the
neuroepithelium, suggesting that these early bHLH genes might be
associated with specification of cell identity (32). The widespread
expression of NeuroM and NeuroD in postmitotic
cells at distinct times in neural development suggests that they do not
define functionally distinct neuronal phenotypes but rather successive
stages of a cell's life course (21, 33). Transcription of the
3
gene is activated in a small subset of proliferating retinal cells, and
then it is continuously expressed during cell differentiation and in
the mature ganglion and amacrine cells (11). Although ASH-1,
NeuroM, and NeuroD are expressed in subsets of
retinal cells and instances of overlapping expression between these
factors and
3 have been detected, we found that the
3 promoter
was also active in cells that do not express these bHLH
genes.4 Co-transfection experiments have confirmed that the
ASH-1, NeuroM, and NeuroD proteins do not control transcription of the
3 gene. None of the known neuronal bHLH proteins is present
throughout the period of
3 expression, and, although we cannot rule
out the possibility that
3 is sequentially regulated by distinct bHLH proteins during development, we favor the idea that it is regulated by a particular, unidentified bHLH protein whose expression is associated with specific neuronal phenotypes.
Our results demonstrate that MyoD is able, upon ectopic expression in
central neurons, to induce transcription of both transfected and
endogenous 3 promoters. MyoD itself is absent from the nervous system, but expression in the developing brain of several other myogenic regulatory genes such as mef-2 and myf-5
suggests that interesting parallels may exist between muscle and neuron
differentiation (34, 35). Although MyoD acts as an activator of
3 in
subsets of central neurons, it is incapable of inducing transcription of this gene in non-neuronal cells, suggesting that induction requires
additional co-activators that are exclusively expressed in neurons. In
early retina, MyoD has no influence on
3 promoter activity,
presumably because the bHLH protein, which controls
3 transcription
(and for which MyoD can substitute), is available in nonlimiting
amounts. Since ectopic expression of MyoD does not increase the
proportion (about 15%) of retinal cells expressing
3, we propose
that the endogenous bHLH protein must, like MyoD, synergize with
neuronal co-activators that, in the retina, are confined to the subset
of neurons forming the domain of
3 expression. However, these or
similar co-activators are also present in other subsets of neurons
elsewhere in the developing CNS, as demonstrated by MyoD's ability to
transactivate
3 in a fraction of neurons from different regions of
the CNS (Table I). Thus,
3 expression depends on the presence in the
same neuron of both the appropriate bHLH protein and the appropriate
co-activators. In the retina, such a combination is predicted to occur
in ganglion and amacrine cells.
The ability of the 3 promoter to distinguish between different
members of the bHLH family determines its stringent neuron-type specificity. The mechanism by which E-boxes discriminate in
vivo between related bHLH proteins is poorly understood. We have
shown that although CTF-4 binds to the
3 E-box with high affinity
in vitro, it is not competent to activate
3 transcription
in vivo. Our experiments did not provide evidence that the
base pairs flanking the
3 E-box constitute part of a negative
cis-acting element that may prevent ASH-1, NeuroD, NeuroM, and CTF-4
from functioning as activators. Thus, the selection mechanism enabling
3 to discriminate between different bHLH proteins is probably
different from the mechanism proposed by Weintraub et al.
(28) for the regulation of the IgH gene. To locate the sequences that
confer specificity to the E-box in
3, we constructed a hybrid
1/
3 promoter where the E-box was flanked in 5' with sequence from
1. Whereas the activity of the
1 promoter is enhanced in neurons
by the four different neuronal bHLH proteins we have tested, the hybrid
does not respond to them, behaving like the
3RS promoter and thus demonstrating that the 3'-flanking sequence is sufficient to confer upon the E-box an ability to discriminate between bHLH factors. Moreover, we have shown that the CAAT box in
3 is an essential positive regulatory element and that shortening the distance between the E-box and CAAT box by 1, 2, or 3 bp strongly decreases promoter activity in retinal cells. The E-box presumably cooperates with the
CAAT box and, in order to allow appropriate protein-DNA interactions, the distance between the two motifs should be no shorter than 9 bp.
Mobility shift experiments indeed suggest that nuclear proteins specifically interact with a very short fragment of the
3 promoter that encompasses the E-box and the CAAT box, supporting the view that
the
3 promoter utilizes a cooperative mechanism consisting of at
least two juxtaposed protein-DNA complexes as components of the
transcriptional activation process. The different neuronal bHLH
proteins we have tested perhaps fail to activate
3 because of their
inability to interact with a neighboring complex. Thus, the finely
tuned regulation of the
3 gene depends on the availability of the
appropriate bHLH factor and co-activators. The identification of other
genes regulated by members of the extending family of neuronal bHLH
proteins should help determine whether the regulatory mechanism used by
the
3 gene is shared by other neuron-specific genes.
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ACKNOWLEDGEMENTS |
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We thank Christine Alliod for expert
technical assistance, Sabine Couturier for the EMSA protocol, and
Kerstin Johansson for constructing the 1/
3 hybrid promoter.
We are indebted to Thomas Reh, Jacob Schmidt, and the Weintraub
laboratory for cDNA clones.
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FOOTNOTES |
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* This work was supported by the Swiss National Science Foundation and the State of Geneva.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Sciences II, 30 quai Ernest-Ansermet, 1211 Genève 4, Switzerland. Tel.: 41-22-702-6492; Fax: 41-22-702-6483; E-mail:
jean-marc.matter{at}biochem.unige.ch.
1
The abbreviations used are: bHLH, basic
helix-loop-helix; nAChR, nicotinic acetylcholine receptor; CAT,
chloramphenicol acetyltransferase; X-gal, 5-bromo-4-chloro-3-indoyl
-D-galactosidase; CEF, chick embryonic fibroblast;
E4-E13, embryonic days 4-13; bp, base pair(s); CNS, central nervous
system.
2 J.-M. Matter, unpublished data.
3 T. Roztocil, unpublished observation.
4 L. Matter-Sadzinski, M. Ballivet, and J.-M. Matter, manuscript in preparation.
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REFERENCES |
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