(Received for publication, November 15, 1995)
From the
We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5`-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5`-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment.
Muscarinic acetylcholine receptors (mAChR) ()are the
members of the superfamily of G-protein-coupled receptors, which are
characterized by the presence of seven putative transmembrane domains.
mAChRs are widely expressed in the central and the peripheral nervous
system. In the brain, mAChRs are thought to play critical roles in
higher functions, including attention, regulation of movement,
learning, and memory(1, 2) . Five subtypes of mAChR
(m1-m5) have been identified by molecular cloning (3) .
Each subtype of mAChRs shows a distinct and complicated distribution in
peripheral tissues and brain, suggesting subtype-specific
functions(4, 5, 6, 7) . Although the
coding region of each mAChR subtype has been cloned and sequenced, the
precise structures of their 5`-noncoding regions and regulatory regions
have not been reported. Cloning and analysis of the genetic regulatory
elements of these genes should yield important insights into the
mechanisms that underlie the tissue- and site-specific expression of
each subtype of mAChR.
In this study, we focused on defining the sequences that regulate m4 mAChR gene expression. In mammals, the m4 mAChR gene is expressed predominantly in the central nervous system, although its expression has been detected in some peripheral tissues such as rabbit lung (but not human or rat lung)(8, 9) . In rat brain, m4 mRNA is present in the cerebral cortex, striatum, olfactory bulb, and pyramidal cell layer of the hippocampus(4, 7, 10, 11) . m4 mAChR has been suggested to function not only postsynaptically but also presynaptically in some regions(12, 13) . As a first step toward elucidating the regulation mechanism of m4 mAChR gene expression, we have characterized the rat m4 mAChR gene and identified its regulatory region.
Transfection was performed using
Lipofectamine reagent (Life Technologies, Inc.),
essentially as recommended in the manual. Cells were seeded in Corning
24-well microtiter plates (0.7
10
cells per well
for NG108-15, 0.2
10
cells per well for L6
and 3Y1B) or 12-well microtiter plates (1.6
10
cells/well for PC12D) and were cultured for 1 day before
transfection. 0.45 or 0.3 µg of plasmid DNA and 3 or 0.7 µl of
Lipofectamine
reagent were used per each well for PC12D
cells or other cells, respectively. Cells were incubated with the DNA
mixture for 5 h (NG108-15, L6) or overnight (PC12D, 3Y1B). Cells
were harvested 3 days after transfection. Enzyme assays were performed
using the Promega Luciferase assay system. The luciferase activity was
measured using a scintillation counter (Packard, Tri-Carb 1500) as
described in the manual supplied by Promega. We measured the activity
of pGL2-Basic vector itself, which contains no insert, to determine
background activity. Transfection efficiency was determined by
cotransfection of pEF-CAT construct. The CAT activity was measured as
described by Nordeen et al.(20) .
Figure 1: Schematic representation of the rat m4 mAChR gene (the phage clone JH1411) and isolated cDNAs. Exons are indicated by boxes (open boxes represent the coding sequence). cDNAs 1 and 2 were isolated by single-strand ligation to single-stranded cDNA-PCR using primers complementary to sequences within the coding region. cDNA 3 was isolated by reverse transcription-PCR using primers complementary to sequences within exon 1 (5`-CTCTGGCTTGTTCCGCCGTCT-3`) and exon 2 (5`-ATCTCCCGGCTCCTCCACGTCT-3`). Splicing patterns for each cDNA and predicted structures of each mRNA are shown. A, ApaI; H, HindIII; K, KpnI; P, PstI; Sc, SacI; Sl, SalI; Sm, SmaI restriction cutting sites.
Figure 2: Nucleotide sequence of the 5`-flanking and 5`-noncoding region of the rat m4 mAChR gene. Arrow heads indicate transcription initiation sites determined by primer extension analysis and S1 nuclease mapping. Numbers are relative to the upstream transcription initiation site (G). The initiation codon (ATG) is shown in bold and double-underlined. Asterisks represent the location of the 5`-end of cDNA1 and cDNA2. Consensus sequences for Sp1, AP-1, AP-2, Zif268 transcription factors, the NRSE and CAAT-box are shown in bold. Lowercase characters denote the intron sequences. Consensus sequences for splice acceptor and donor sites, three PstI sites and two SmaI sites, are underlined.
To test the possibility that PC12D cells express m4 mRNA that contains both of the two upstream exons, we carried out reverse transcription-PCR using primers specific to each exon. As shown in Fig. 1, we obtained a cDNA clone that contains sequences from both exons 1 and 2 (cDNA 3). The length of exon 2 was determined to be 158 bp, and a consensus sequence for a splice acceptor site was found next to the 5`-end of the exon (Fig. 2). This result suggests that cDNA 2 is an incompletely reverse-transcribed cDNA product derived from a mRNA variant that contains both exons 1 and 2 (mRNA 2). Exon 2 contains an in-frame upstream stop codon, and exon 1 does not contain an in-frame ATG triplet. These data indicate that each m4 mRNA variant encodes the same protein.
Figure 3:
Primer extension analysis and S1 nuclease
mapping to determine the transcription initiation sites. Panel
A, primer extension analysis. A uniformly labeled 64-bp
single-stranded DNA primer corresponding to the antisense strand of the
rat m4 mAChR gene was hybridized with 4 µg of poly(A) RNA from rat kidney (lane 1) or PC12D cells (lane
2) and then extended with reverse-transcriptase (SuperscriptII,
Life Technologies, Inc.). Lanes M, bacteriophage M13 mp18
sequence using the -40 primer. Panel B, S1 nuclease
mapping. A uniformly labeled single-stranded probe corresponding to
residues -90 to +94 was hybridized with 2 µg of
poly(A)
RNA from PC12D cells (lane 1) or rat
kidney (lane 2) and then digested with S1 nuclease. lanes
M, bacteriophage M13 mp18 sequence using the -40 primer. Panel C, strategy to map the transcription initiation sites.
The bent arrows indicate the principal transcription
initiation sites. Hf, HinfI.
The sequence of the putative promoter region of the rat m4 mAChR gene is rich in GC content (about 70% in the proximal 400 bp) and has no typical TATA-box (Fig. 2). There is a CAAT-box between bases -593 and -589 and an inverted CAAT-box ATTGG between bases -62 and -57. The putative promoter segment contains several consensus sequences for Sp1 (GGGCGG or CCCGCC) and AP-2 (CC(G/C)C(A/G)GGC) binding sites. In addition, there is a putative neural-restrictive silencer element (NRSE) in an inverted orientation between bases -857 and -837 (Fig. 4). Possible AP-1 and Zif268 binding sites are also found in the intron between exons 1 and 2.
Figure 4: Alignment of NRSEs between rat m4 mAChR, rat SCG10(35) , rat type II sodium channel (NaII)(36) , and human synapsin I (37) genes. For m4 AChR, the sense strand is written at the top and in the orientation 3` to 5`. The numbering of the bases is as described in Fig. 2. Asterisks show nucleotides that are not seen in other genes.
Figure 5: Cell type-specific expression of rat m4 mAChR-luciferase fusion genes. The left side is a schematic representation of the constructs. The right side shows the luciferase activities obtained in each cell line. All values are expressed as percent of the activities of construct A in each cell line. Luciferase activities were corrected for transfection efficiencies, as described under ``Experimental Procedures.'' The results, mean ± S.E., represent two or three transfections, each of which was performed in duplicate. The bent arrow indicates the transcription initiation sites.
We have determined the structure of 5`-noncoding region of the rat m4 mAChR gene. Two 5`-noncoding exons are located approximately 5 kb upstream from the coding exon. PC12D cells express at least two alternatively spliced variants of mRNA (mRNAs 1 and 2 in Fig. 1, the latter is predicted based on the splicing patterns of cDNAs 2 and 3), which originate from these two upstream exons. Also, we have obtained cDNAs that correspond to cDNAs 1 and 2 (Fig. 1) using RNA from rat brain by reverse transcription-PCR (data not shown). Previous studies have shown that genes encoding rat m1, m3, m5, and porcine m2 mAChR contain at least one intron in their 5`-noncoding regions and that m4 mAChR gene also has a potential splice acceptor site in its 5`-noncoding region(21, 22) . There are no introns in the coding or the 3`-noncoding regions of these genes(3) . The porcine m2 mAChR gene has at least two alternatively spliced 5`-noncoding exons, although its precise gene structure is not known(23) . Another group has reported that two 5`-noncoding exons are located about 5.5 kb upstream from the coding exon in the rat m4 gene(24) .
The 5`-flanking region
of the rat m4 mAChR gene lacks a TATA-box, is GC-rich, and contains
several potential Sp1 binding sites. These characteristics are typical
of promoters for housekeeping genes, which are constitutively
expressed. However, recent studies have demonstrated that some highly
regulated genes, including immediate early genes, developmentally
regulated genes, and tissue-specific genes, also have promoters that
lack TATA boxes(25) . Genes encoding several other
G-protein-coupled receptors, including
D(26, 27) , D
(28) ,
and D
(29) dopaminergic and
(30) and
(31, 32) adrenergic receptors, also have GC-rich
promoters that lack TATA boxes. These promoters also contain several
putative Sp1 binding sites, although the requirement of Sp1 binding for
transcription of these genes is yet to be determined. In some genes
that have promoters lacking TATA boxes, binding of Sp1 has been shown
to play a critical role in transcription
initiation(33, 34) .
In mammals, the m4 mAChR gene
is expressed primarily in neurons. We assessed whether or not the
5`-flanking region of the m4 mAChR gene functions as a cell
type-specific promoter by transient expression assays using
non-neuronal cell lines and m4 mAChR-expressing neuronal cell lines.
The fragment containing 1073 bp of the 5`-flanking region (Fig. 5, construct B) was found to be sufficient for cell
type-specific expression of the m4 mAChR-luciferase fusion gene.
Deletion from the 5` side to base -435 did not result in a loss
of promoter activity but rather in a loss of specificity of expression,
indicating that the region -1073 to -435 represses the
expression of m4 mAChR gene in cell lines where the m4 receptor is not
expressed. The existence of a putative NRSE between bases -857
and -837 is consistent with the repression of the m4
mAChR-luciferase fusion gene expression in non-neuronal cell lines,
although the involvement of the NRSE in the neuron-specific expression
of the m4 mAChR gene remains to be proven. NRSE is a silencer element
that is known to regulate neuron-specific expression of several genes
including the rat SCG10(35) , rat type II sodium
channel(36) , and human synapsin I genes(37) . However,
m4 mAChR is not expressed in all neurons and shows a unique,
site-specific expression pattern in the brain. If we assume that NRSE
is involved only in determining the neuron-specific expression of the
m4 mAChR gene, an additional mechanism may be required to restrict the
expression to only a subset of neurons. It remains to be determined
whether the 5`-flanking region we have isolated contains some elements
regulating the specificity of expression among different kinds of
neurons. The mouse serotonin 2 receptor (5-HT) gene is
known to possess repressor domains that regulate its neuron-specific
expression and an activator domain that allows gene expression in a
glial cell line that expresses the 5-HT
receptor(38, 39) . The m4 mAChR is also known to
be expressed in some glial cell lines (40) , although there is
no direct evidence for expression of the m4 mAChR in glia cells in
vivo.
In summary, we have determined the structure of the rat m4 mAChR gene and identified its promoter region. It has two 5`-noncoding exons, from which at least two alternatively spliced variants of mRNA originate in PC12D cells. The fragment containing 1073 bp of the 5`-flanking region is sufficient for cell type-specific (at least neuron-specific) expression.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D78484[GenBank], D78485[GenBank].