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(Received for publication, July 28, 1995; and in revised form, August 21, 1995) From the
The sequence encoding the vesicular acetylcholine transporter
(VAChT) has recently been localized within the first intron of the
choline acetyltransferase (ChAT) gene in various species. In rat, we
previously identified a class of VAChT mRNAs that may originate from
the same promoter region as two ChAT mRNAs. Here, we demonstrate by a
detailed analysis of the 5`-noncoding region of the VAChT gene, that
two specific VAChT promoters lie within the first intron of the ChAT
gene. Two VAChT mRNAs are generated from these promoters. These results
demonstrate that the promoter regions of these two genes are
intermingled, which highlight the unique organization of the ChAT/VAChT
gene locus.
Functional presynaptic expression of the neurotransmitter
acetylcholine (ACh)
Figure 1:
Schematic representation of the rat
VAChT gene and of the R- and V-type VAChT mRNAs. Black and white boxes indicate coding and noncoding sequences,
respectively. Position 1 corresponds to the translation
initiation codon. Vertical bars represent two donor splice
sites in the exon R (-1435 and -1357) and an acceptor
splice site(-309) used to generate the R-type VAChT
mRNAs(4) . Vertical dotted lines represent the
positions of the 5` end of a VAChT cDNA (-856, (5) ) and
of a primer extension product (-426, (4) ). VAChT mRNAs
not subjected to the splicing of the genomic region between the
acceptor and donor splice sites indicated above are designated as
V-type.
In this study, we demonstrate that the first intron of
the ChAT gene contains specific VAChT promoter regions. These regions
give rise to two V-type VAChT mRNAs of 2.6 and 3 kb, previously
detected by Northern blotting(4) . We report a detailed
analysis of the 5` molecular diversity of VAChT mRNAs. These data
clarify the transcription pattern of the rat ChAT/VAChT gene locus.
Figure 2:
RNase protection analysis of the 5` region
of the rat VAChT gene. A, schematic representation of the rat
VAChT gene as in Fig. 1. Additional vertical bars represent the regions containing the 5` ends of V2
(-888/-863) and V1 (-426/-402) mRNAs and the 3`
end of the VAChT mRNAs (+1998). Abbreviations: B, Bsu36I; H, HindIII. B,
representation and positions of the cRNA probes (A1, A2, B1, B2, C1)
and of the amplified DNA fragments (A, B, C) from which these probes
were produced (see ``Experimental Procedures''). The
sequences of the PCR primers used are: AF, 5`-CATCCTGGGCGCATCTCAGAAG;
AR, 5`-ACGGCCTCTCTGCACCGCAG; AF`, 5`-GAGACTCACCGCGTCATA; BF, 5`-TGCCAAG
ACTTTCTGCCTAAGGGC; BR, 5`-GTTCCTCCCACTGCTCAGCCATC; BF`, 5`-TTGCGT
GCGCTGTGCCT; CF, 5`-CAGAGGCTGATCTGTTCAGCCTGT; CR, 5`-CCTCCTCTCA
GTCCTCATACCCTC. C, RNase protection analysis. Total RNA from
spinal cord (SC) or, as a negative control, liver (L), where VAChT mRNAs could not be
detected(4, 6) , was hybridized with the probes A1,
A2, B1, B2, C1 and tested for protection from RNases. The amounts of
RNA hybridized to the probes are indicated at the top of each lane.
Note that the probe A1 was hybridized with up to 50 µg of liver
RNA. Pr+RNases, probe with RNases (subjected to each
experimental step); Pr-RNases, probe without RNases. Open arrows indicate the fragments completely protected by the
probe. Filled arrows show the other fragments specifically
protected in spinal cord. Sizes were determined by comparison with
known sequencing reaction products electrophoresed in separate lanes of
the same gel.
Figure 3:
Northern blot analysis. A,
schematic representation of the rat VAChT gene (see Fig. 2) and
of the probes used. Probes 1 and 4 were restriction fragments HindIII (H)-PvuII (P) for 1 and SmaI (S)-EcoRI (E) for 4. Probes 2
and 3 were obtained by amplification with the primers 2F/2R and 3F/3R,
respectively, whose sequences are: 2F, 5`-CCGAAGTCCAGGCTGAGGAGGA; 2R,
5`-CAAGTGGAGGGAGAAAGAAA; 3F, 5`-TGGAGGAAGAGGCAAGAGCGGA; 3R, identical
to primer AR. B, Northern blotting was performed with
brainstem, spinal cord, or, as a negative control, liver
poly(A)
Figure 4:
Identification of two VAChT promoters. A, structure of the VAChT gene as described in Fig. 2.
The boxes represent the regions inserted into the luciferase
expression plasmid. B, transient luciferase assay. Triplicate
transfections were carried out for each experiment. Results are
expressed as the luciferase activity of the tested constructs as a
multiple of that obtained with the promoterless plasmid. Values
represent the mean of two (293, PC-G2) or three (PC-12) independent
experiments performed with two different plasmid
preparations.
RNase protection experiments were performed to localize the
transcription initiation site(s) of the V-type VAChT mRNA(s). Two
transcription start sites at positions -402 and -426 (Fig. 2A) were mapped with both radiolabeled antisense
riboprobes A1 and A2 (Fig. 2B). In spinal cord extracts
both probes yielded a similar pattern of protected fragments (Fig. 2C). First, a cluster of up to 7 protected
fragments of 115-121 nucleotides, whose sizes differ by one
nucleotide, was clearly revealed. The 5` ends of these fragments lie
between positions -425 and -431, within an A-rich sequence
(-433, 5`-AAAGAAAAAAAAA-3`, -421). The presence of these A
residues may explain why the RNase protection assay gave multiple bands
rather than a single band. The 5` end of the amplified primer extension
product obtained previously ((4) , position -426) maps to
this region, confirming that it corresponds to the same 5` end of a
VAChT mRNA. A less intense 93-nucleotide fragment was also protected,
demonstrating an additional 5` end at position -402. Thus, these
two sites are used to generate a first type of V-VAChT mRNA designated
as V1. In addition, both A1 and A2 probes were completely protected,
indicating that a mRNA extending further upstream was also present. The
transcription initiation site for this transcript was determined with
the riboprobes B1 and B2 (Fig. 2B). Several identical
protected fragments were visualized with both probes (Fig. 2C). Three major fragments of 223, 231, and 248
nucleotides were obtained, indicating three prevalent transcription
initiation sites at positions -863, -871, and -888,
respectively. Thus, the cDNA isolated previously (5) was nearly
full-length, and the corresponding mRNA is referred to as V2.
Therefore, two clusters of transcription start sites separated by about
450 bp are used for V-type VAChT mRNA synthesis, confirming a diversity
in the 5` region of the V-VAChT mRNAs. Surprisingly, the probes B1 and
B2 were also completely protected from RNases, evidence for an
additional mRNA species. Using the riboprobe C1 (Fig. 2B), which covers a sequence further upstream, as
far as exon R, a single protected fragment was detected, corresponding
to the complete protection of the probe (Fig. 2C).
Thus, the sequence of this last mRNA, designated V3, extends at least
to 16 nucleotides from the 3` end of the exon R. These results show
the existence of several V-type VAChT mRNAs. In addition, for VAChT
mRNAs no diversity in the 3` region was detected (data not shown). A
single 3` end, corresponding to position +1998, was found, which
suggests that they derive from the use of a specific polyadenylation
signal located 18 bp upstream. This indicates that VAChT mRNAs differ
only by the length of their 5`-noncoding sequences.
These results show that the VAChT mRNAs of 3 and 2.6 kb correspond
to the V2- and V1-type mRNAs, respectively, which is in accordance with
the sizes calculated from their sequences. Moreover, the V3-type mRNA
encodes VAChT. This mRNA is probably produced from the same promoter as
the R-type mRNAs and results from the nonexcision of the region between
the exon R and the acceptor splice site at position -309.
Both promoter regions were highly active
in the noncholinergic cell lines. Therefore, they lack the regulatory
motifs responsible for the tissue-specificity of VAChT gene expression.
These sequences may be located in the upstream R-type promoter. Indeed,
a rat genomic sequence upstream from the R-type exon has been shown to
confer cholinergic specificity to the R-type promoter in
vitro(12) . Moreover, this region directs the in vivo expression of a heterologous downstream promoter in the
cholinergic neurons(13) . These results raise the question of
whether this region controls the tissue-specific expression of both the
ChAT and the VAChT genes. However, this remains to be analyzed. Fig. 5shows the sequence of the genomic region containing the
VAChT specific promoters. The sequences in and around the transcription
initiation sites lack TATA and CAAT boxes or an ``initiator''
motif, which can promote basal transcription of genes without a TATA
box(14) . The sequences of both HX and XS promoter regions are
GC-rich (63 and 67% GC, respectively) and contain several consensus Sp1
binding sites. These promoter regions thus have the characteristics
consistent with multiple transcription initiation sites (14, 15) . In addition, consensus sequences for a
glucocorticoid response element, conserved in human (see the sequence
in (5) ), and for a E-box are present in the upstream region of
V1 mRNA. Whether or not these potential cis-acting elements
are functional remains to be determined.
Figure 5:
Nucleotide sequence of the VAChT promoter
regions. The sequence is numbered relative to the translation
initiation codon. The V2 and V1 transcription initiation sites are
indicated by filled and open bent arrows,
respectively. The putative regulatory elements, Sp1,
glucocorticoid-responsive element(16) , and E2 box (17) are highlighted with open, broken-line,
and shaded boxes, respectively.
We can now propose a model
for the transcription of the rat ChAT/VAChT gene locus. First, VAChT
and ChAT genes may be transcribed from a promoter region located
upstream from exon R. VAChT mRNAs are then generated when the
transcription is stopped at position +1998. These VAChT mRNAs
either are spliced to generate the R-type mRNAs, or remain unspliced
(V3-type). Alternatively, transcription of the VAChT/ChAT genes may
continue until the ChAT polyadenylation signal. R-ChAT mRNAs are then
obtained by splicing out a 7-kb fragment containing the VAChT open
reading frame. Second, the VAChT gene may be transcribed from the two
promoters localized in the first intron of the ChAT gene to produce the
VAChT mRNAs V1 and V2. Finally, the ChAT gene may be transcribed from
promoters located downstream from the VAChT gene, giving rise to the
ChAT N- and M-type mRNAs. Note that all VAChT mRNAs we detected have
the same 3` end, and thus there appears to be no bicistronic mRNA. To conclude, we provide evidence that promoter regions are contained
within the first intron of the ChAT gene. The organization of the rat
ChAT/VAChT locus is both complex and unusual, with the promoters of
these two genes intermingled. The analysis of this organization may
reveal novel mechanisms involved in the expression of eukaryotic genes.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U34796[GenBank].
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24654-24657
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in cholinergic neurons requires the
activity of particular proteins: (i) a high affinity choline
transporter on the plasma membrane, which controls the supply of
extracellular choline; (ii) a vesicular acetylcholine transporter
(VAChT), which translocates cytoplasmic ACh to the interior of synaptic
vesicles; and (iii) choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), which synthesizes ACh from
choline and acetyl coenzyme A. cDNAs encoding ChAT have been cloned and
this led to the subsequent isolation and characterization of the ChAT
gene (for review see (1) ). In rodent, ChAT is encoded by
several mRNAs with different 5`-untranslated sequences. They are
generated by differential promoter utilization and alternative splicing
events(2, 3) . Recently, cDNA and genomic sequences
encoding VAChT have been identified and the VAChT gene was thereby
localized to the first intron of the ChAT
gene(4, 5, 6, 7) . This gene
organization is well conserved between nematode and mammals, including
man(4, 5, 7) , and thus may have functional
significance. Both ChAT and VAChT genes are in the same transcriptional
orientation and both are required to express the cholinergic phenotype.
To our knowledge, this organization is unique in mammals. There are two
classes of VAChT mRNAs in the rat, encoding the same VAChT protein,
that we designate as R- and V-types. First, the R-type VAChT mRNAs
contain common 5`-noncoding sequences (exon R) with two ChAT mRNAs and
may therefore be transcribed from the same promoter ( (4) and Fig. 1). In nematodes, this seems to be the only mechanism for
generating VAChT mRNAs(7) . Second, V-type mRNA species differs
from the R-type mRNAs by the 5`-noncoding sequences ( (4) and (5) , Fig. 1). However, the molecular mechanisms by
which the V-type mRNA species is produced has not been clearly
elucidated.
RNA Isolation
Total and poly(A) RNAs were purified as described
previously(8, 4) .
Ribonuclease (RNase) Protection Assays
cRNA probes
were synthesized from DNA templates obtained by amplification of
particular gene regions. Sequences were amplified with primer pairs
A
/A
, B/B, or
C/C (Fig. 2B). The
corresponding PCR products A, B, and C, were subcloned (TA cloning,
Invitrogen) downstream from the SP6 promoter. Linear templates for cRNA
synthesis (Fig. 2B) were prepared from the subcloned
PCR products either by digestion (Bsu36I for the B2 probe, HindIII for the C1 probe) or by amplification with a reverse
SP6 primer and a forward specific primer (AF`, AF, and BF` for the
probes A1, A2, and B1, respectively). Antisense cRNA probes were
synthesized (9) using SP6 RNA polymerase (Promega) in the
presence of [
-P]GTP (3000 Ci/mmol, Amersham
Corp.). After digestion with DNase, full-length cRNAs were purified by
electrophoresis on a 4% polyacrylamide, 8.3 M urea gel. RNase
protection assays were performed as described(9) , except that
RNAs were hybridized to cRNA probes for 16 h at 63 °C and digested
with 25 µg/ml RNase A and 1 µg/ml RNase T1 at 30 °C for 50
min.
Northern Analysis
Northern blot experiments were
performed as described(10) . DNA probes (see Fig. 3A) were P-labeled by nick
translation (Life Technologies, Inc., probe 3) or random priming
(Amersham, other probes).
RNAs. Left panel, hybridization of 1
µg of spinal cord cytoplasmic poly(A)
RNA (a) or poly(A)
RNA (b) with probe 4. Right panel, various amounts of poly(A)
RNA
from liver (L) or brainstem (BS) were separated on
the same gel, transferred to the same nylon membrane (Hybond
N
, Amersham), and hybridized with the probes as
indicated. Autoradiograms were exposed at -70 °C. Lane
1, 5 µg of RNA, probe 1, exposed for 10 days; lane 2, 5 µg of RNA, probe 2, exposed for 10 days; lane 3, 5
µg of RNA, probe 3, exposed for 7 days; lane 4, 2 µg
of RNA, probe 4, exposed for 2 days.
Plasmid Constructions
The restriction fragments XbaI/SphI and HindIII/XbaI (Fig. 4A) were inserted into the plasmid KS Luc,
upstream from the luciferase reporter gene and downstream from a
transcription terminator. KS Luc was constructed by W. Faust and A. M.
Catherin.
Cloning junctions were verified by sequencing.
Cell Cultures and Transfection
The cell line 293
(human embryonic kidney) was maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10%
fetal calf serum. The cell lines PC-12 and PC-G2 (rat
pheochromocytomas) were grown in RPMI 1640 medium (Life Technologies,
Inc.) supplemented with 10% horse serum with 5% or 10% fetal calf
serum, respectively. Exponentially growing cells were transfected by
electroporation with a single electrical pulse at 200 V using a Bio-Rad
Gene Pulser as in (11) . Cells (10
) were
transfected with a mixture of 1 pmol of the plasmid to be tested, 1
µg of a Rous sarcoma virus-chloramphenicol acetyltransferase (CAT)
vector (used to assess the transfection efficiency) and a carrier DNA
(Bluescript) to give a total of 12 µg of DNA. Luciferase activity
was normalized with CAT activity determined in the same
extract(11) . Each transfection was carried out in triplicate.
Molecular Diversity of VAChT mRNAs
A V-type mRNA
species was previously identified by primer extension. The longest
fragment contained 426 bp of the gene sequence upstream from the
translation initiation codon(4) . A similar VAChT cDNA with a
longer 5`-noncoding sequence (856 bp) has also been
isolated(5) . Thus, several V-type VAChT mRNAs with different
5` lengths might be produced, and we analyzed this potential diversity. Northern Blot Analysis of VAChT mRNAs
Brainstem
or, as a negative control, liver poly(A) RNAs were
analyzed by Northern blot using probes specific to different regions of
the VAChT mRNAs (Fig. 3A). By hybridization with a
probe complementary to the VAChT coding region (Fig. 3A, probe 4), two mRNAs of 2.6 and 3 kb
were detected, as previously reported in spinal cord and
brainstem(4) . Moreover, a diffuse band between 3.9 and 3 kb
was also visualized (Fig. 3B, lanes b and 4). This result was confirmed in four independent experiments
performed with different RNA preparations from spinal cord or brainstem
(not shown) and by using cytoplasmic poly(A)
RNA (Fig. 3B, lane a), thus ruling out possible
contamination with nuclear primary transcripts. The hybridization
pattern obtained with probe 3 (lane 3), whose sequence is
specific to all V-type mRNAs, is identical to that of probe 4. A 7-day
exposure of the blot was required to detect in brainstem samples a weak
band near the upper point of the smear. Probe 2, specific to the V2-
and V3-type mRNAs, hybridized to the 3-kb mRNA species and also
revealed the diffuse band between 3.9 and 3 kb (lane 2). Probe
1, specific to V3-type mRNA, specifically recognized this diffuse band (lane 1). However, it is not clear if the presence of the
smear is due to poor stability of the corresponding mRNA species.
Promoter Activities and Sequence Analysis in the Region
5` to the Transcription Start Sites
V1- and V2-type mRNAs differ
by the length of their 5` sequences. They may result from transcription
either from a common promoter but initiating at different sites or from
different promoter regions. These possibilities were tested by
transient expression assays. The upstream regions of the V1 (XS) and V2
(HX) transcription initiation sites (Fig. 4A) were
cloned upstream from the luciferase reporter gene and used to transfect
three cell lines: PC-12, which expresses the endogenous VAChT
gene(5) , and PC-G2 and 293, in which no VAChT mRNA could be
detected by RT-PCR (not shown). Both constructs promoted the
transcription of the luciferase gene in all cell lines tested (Fig. 4B). Luciferase activities with XS or HX were
8-70-fold or 3-19-fold higher, respectively, than those
obtained with the promoterless plasmid, depending on the cell line.
Thus, both HX and XS fragments contain a promoter. The highest
transcriptional activity was measured with the XS promoter region,
consistent with the V1-type mRNA being more abundant than the V2-type (Fig. 3B).
))
We thank Claude Thermes,
Jean-François Julien, Annie Lamouroux, and Nicole
Faucon Biguet for helpful discussions and critical reading of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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