(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) 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.
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) 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.
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].