(Received for publication, August 3, 1995)
From the
Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5` deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.
A defining feature of neurotransmitter systems is the
coordinated use of disparate molecular elements to achieve a specific
functional goal. Cholinergic neurons exemplify this by utilizing
nicotinic and muscarinic receptors and the enzymes choline
acetyltransferase and acetylcholinesterase (AChE, ()EC
3.1.1.7) to modulate the effects of the neurotransmitter acetylcholine.
AChE is a serine hydrolase that is located in a variety of tissue
environments by linkage of diverse anchoring subunits or
carboxyl-terminal sequences to the constant catalytic domain of the
enzyme (reviewed in Massoulie et al., 1993; Taylor and Radic,
1994). In humans AChE activity is encoded by a single gene, ACHE, located within 6 kilobases of DNA sequence on chromosome
7q22 (Getman et al., 1992; Ehrlich et al., 1992).
Multiple forms of AChE protein arise from alternative splicing of exons
found at the 3` end of the open reading frame (Li et al.,
1991). The alternative splicing gives rise to carboxyl termini which
differ in their capacity to disulfide bond with other catalytic and
structural subunits or associate with the plasma membrane (cf.
Massoulie et al., 1993; Taylor and
Radic, 1994).
The necessity for coordinated expression of each component of cholinergic neuronal systems for the maintenance of homeostasis is illustrated by the loss of receptor function and skeletal muscle tone in myasthenia gravis and the profound symptoms associated with AChE inhibition from organophosphate poisoning. Many components of cholinergic neural systems have not been investigated at the level of the gene, and the nature of regulatory mechanisms which foster coordinated expression of cholinergic macromolecules remains obscure.
One approach useful for investigating gene structure and expression relies on interspecies comparison of a single genetic locus. The genomic structure, alternative RNA splicing, and amino acid sequence of genes encoding AChE have been investigated in a wide variety of species (Rachinsky et al., 1990; Li et al., 1991, 1993). Promoter elements controlling expression of AChE genes in mammalian cells have been identified and studied functionally in Torpedo (Ekström et al., 1993) and mouse muscle (Mutero et al., 1995). Partial sequences of the human ACHE 5`-flanking region have been reported (Ben Aziz-Aloya et al., 1993), but little is known about functional promoter elements or transcriptional regulation of ACHE in mammalian cells. We have cloned and characterized putative promoter sequences for the human ACHE gene and have determined the transcription initiation sites. From functional studies we find that GC-rich sequence elements containing consensus binding sites for Sp1 are essential for transcription of ACHE, while the transcription factor AP2 acts as a repressor of human ACHE and mouse Ache transcription in vitro.
Figure 1: Comparison of human ACHE and mouse Ache DNA sequences encompassing exon 1. RNA cap sites for mouse Ache are indicated by the dashed line between arrows. Underlined sequence corresponds to exon 1, the first untranslated exon of Ache. The mouse sequence has been reported previously (Li et al., 1993).
Since the NT2/D1 cells displayed greater AChE enzyme activity and promoter activity of ACHE upstream sequences relative to other human cell lines, this cell line was chosen for defining the location of the promoter region of the ACHE gene. Given the sequence similarity between the human and mouse genes upstream of exon 1, and the known promoter activity of the mouse Ache gene in this region (Li et al., 1993), we constructed 5` deletion mutants by PCR amplification of construction A to analyze the promoter potential of this region. The locations of the upstream primers used for amplification of constructions B through E were chosen so that the Sp1 consensus sites in the putative promoter region upstream of exon 1 would be sequentially deleted (Fig. 2). An additional 5` deletion mutant (construction F) was prepared by restriction digestion of construction A with Bsu36I. This resulted in the removal of the GC-rich region and most of the sequence similar to mouse exon 1. Transient transfection of constructions A through F in NT2/D1 cells revealed no significant difference in promoter activity between constructions A, B, and C. However, construction D was almost 5-fold lower in promoter activity than A and B, suggesting that the second upstream Sp1 consensus sequence (Fig. 1) is essential for activated transcription of ACHE.
Figure 2: Promoter activity of ACHE-luciferase (Lucif) gene chimeras transiently transfected into NT2/D1 teratocarcinoma cells. Transfection efficiencies were normalized by co-transfection with the lacZ gene driven by the CMV early promoter. Data represent the means and standard errors of three separate experiments, each performed in triplicate.
Figure 3: In vitro transcription of ACHE gene 5`-flanking fragments in HeLa cell nuclear extracts. A, representative autoradiograph of one in vitro transcription experiment. B, map of DNA templates used for in vitro transcription experiments. ACHE mRNA levels were normalized to a transcript produced from a DNA fragment containing the CMV promoter included in each reaction. Data represent the means and standard deviations of three separate in vitro transcription experiments.
The size of the RNA transcript produced in these in vitro transcription reactions suggested that, unlike mouse Ache RNA transcripts isolated from myoblast and erythroleukemia cell lines, a dominant transcription start site is used by human ACHE. The approximate location of this cap site corresponds to the most 3` site used by mouse Ache and coincides with a consensus DNA sequence for an initiator (Inr) element similar to one found in the terminal deoxynucleotidyl transferase gene promoter (Smale et al., 1990). To confirm the location of the mRNA cap site used by ACHE, RNase protection analysis was performed with RNA isolated from NT2/D1 cells. A 750-bp antisense cRNA probe was generated by transcribing a human DNA fragment containing sequences corresponding to exon 1. This probe was then used to protect NT2/D1 total cellular RNA. Two closely spaced bands, 73 and 76 bases in length, were protected from digestion (Fig. 4A). The size of these bands is consistent with RNA transcripts resulting from the use of two different cap sites at the 5` end of exon 1. Similar to the ACHE in vitro transcription product seen in Fig. 3A, the 5` end of these two bands coincides with the terminal deoxynucleotidyl transferase Inr element consensus site present at the 5` end of exon 1. However, the in vitro transcription assays revealed only one band, suggesting a single cap site was used in the HeLa cell nuclear extract environment. To resolve this apparent discrepancy, ACHE RNA products from in vitro transcription reactions with HeLa cell nuclear extract were electrophoresed on a higher percentage polyacrylamide gel. Under these conditions, the band representing the ACHE-specific transcript resolved into two bands as well (Fig. 4B). Thus evidence from experiments using distinct methodologies and two different human cell lines indicates that two mRNA cap sites are present at the 5` end of exon 1 in ACHE and that these sites reside within a consensus sequence for an initiator element (Fig. 4C).
Figure 4: Determination of transcription start sites of ACHE. A, RNase protection analysis of NT2/D1 cell total RNA. The antisense cRNA probe consisted of 750 bases extending from the KpnI site in the 1-2 intron to base -318 upstream of exon 1. Lane 1, NT2/D1 total RNA; lane 2, tRNA; lane 3, digested probe; lane 4, undigested probe. B, in vitro transcription of ACHE in HeLa nuclear extract. a, CMV template; b, ACHE template; c, CMV transcript; d, ACHE transcripts. Reaction was as in Fig. 3, except RNA transcripts were separated on an 8% polyacrylamide gel. C, location of cap sites at the consensus Inr element of human ACHE.
In order to ascertain
the importance of the initiator element consensus site in ACHE transcription, three bases within this site were deleted by
digesting construction B with Bpu1102I, blunting the ends with
S1 nuclease, and ligating the ends. This resulted in the removal of
three bases, TCA, in the putative Inr sequence GGCTCAGCC, yielding the
Inr element deletion mutant B3. Insertion of this mutant Inr
construction, which was otherwise identical to construction B, into the
luciferase reporter vector and transfection into NT2/D1 cells revealed
that these three bases are essential for promoter activity. The B
3
construction displayed less than 2% of the promoter activity compared
to the wild-type construction B (Table 2). This result is in
accord with the RNase protection data described above which tentatively
placed the two mRNA cap sites for ACHE at base 3 (C) and base
6 (A) of this putative Inr element.
To examine the
possibility that increased levels of AP2 protein expressed in the
transfected cells were repressing ACHE promoter function by
competition with endogenous transcription factors (squelching), an
amino-terminal deletion mutant of the AP2 open reading frame was
cotransfected into untreated NT2/D1 cells with construction B. This AP2
mutant, N165AP2, lacks the transcriptional activation domain of
the wild-type factor but retains the dimerization and DNA-binding
domains.
N165AP2 can therefore still homodimerize and bind to DNA
but is unable to form protein-protein interactions with other members
of the RNA polymerase transcription complex (Williams et al.,
1991). Similar to wild-type AP2, co-transfection of
N165AP2
decreases ACHE promoter activity approximately 6-fold (Table 2). These data suggest AP2 specifically represses ACHE transcription by sterically interfering either with the function
of the transcription complex at the cap sites 19 bp downstream, or with
the binding of Sp1 transcription factors 21 bp upstream.
To assess the universal nature and functional significance of AP2-mediated repression of AChE gene expression, two mouse cell lines that express AP2 were used to test the effect of this transcription factor on a DNA fragment containing the mouse Ache promoter. RNase protection experiments revealed that fibroblast cell line 10T1/2 and myoblast cell line 10TFL2-3 (a 10T1/2-derived line with transfected myogenin stably integrated into its genome) contain mRNA for AP2, indicating the factor is expressed in these cell lines (data not shown). Transient transfection of 10T1/2 and 10TFL2-3 cells with Ache promoter-luciferase reporter constructions containing either a native or mutated AP2-binding site showed that the reporter gene containing a mutated AP2 site exhibited 5-fold higher promoter activity than the wild-type promoter (Table 2). Thus mouse cell lines which express AP2 are able to repress mouse Ache expression in a manner similar to the repression of human ACHE transcription in recombinant AP2 co-transfection experiments in human NT2/D1 cells.
Figure 5:
Electrophoretic mobility shift analysis of
the mouse Ache AP2 sequence in mouse fibroblast and myotube
nuclear extracts. Double-stranded oligonucleotides containing the
wild-type (wt) or the mutated (m) AP2 site and the
flanking sequences as found in the mouse Ache promoter were
end-labeled with [P]dCTP and incubated with
nuclear extracts prepared from undifferentiated 10T1/2 (1/2)
fibroblasts (lanes 2, 4, 6-8) and
10TFL2-3 (TF) myotubes (lanes 3 and 5), or with purified recombinant AP2 protein. A specific
anti-AP2 antibody was used for supershift experiments (lanes
1, 4, 5, and 10). Competition with
unlabeled oligonucleotides containing the AP2 consensus sequence (lane 7) and a mutated AP2 sequence (lane 8) are also
shown.
Figure 6: Repression of in vitro ACHE transcription by AP2. Representative autoradiograph of an in vitro transcription experiment with AP2 protein. Recombinant AP2 protein was incubated with ACHE construction B` in HeLa cell nuclear extracts. The ratios of ACHE RNA transcripts to control CMV promoter-driven RNA transcripts are as follows: lane 1, 1.0; lane 2, 0.82 ± 0.04; lane 3, 0.67 ± 0.06; lane 4, 0.32 ± 0.05. Data represent the means and standard deviations of four separate experiments.
Figure 7: DNase footprint analyses of AP2 and Sp1 binding to the ACHE promoter region. A, protection of essential Sp1 consensus binding sites by Sp1 protein. B, protection of the distal and proximal AP2 consensus binding sites by AP2 protein. C, effect of prior incubation on binding of AP2 and Sp1 to the ACHE promoter region. Circled symbols indicate the designated transcription factor was added first, followed by the addition of the second factor.
Labeling of the sense or antisense strand of B` and incubation with AP2 protein yielded two footprints that correspond to the distal and proximal AP2 consensus binding sites downstream of the Sp1 sites in the ACHE promoter (Fig. 7, B and C). AP2 also protected a region of exon 1 from DNase digestion, indicating that AP2 binding may occur 45 bp downstream of the transcription cap sites. However, evidence from transfection experiments indicates this downstream site is not involved in AP2-dependent transcriptional repression of ACHE. ACHE promoter constructs of smaller size were produced by digesting construction B with the restriction enzyme EclXI. This resulted in the removal of all sequences downstream of base +20 in exon 1, including the downstream AP2-binding site identified in the footprinting experiments. When this construction was cloned into the luciferase reporter vector and co-transfected into NT2/D1 cells with the AP2 expression vector, promoter activity was decreased approximately 70% compared to cultures that were co-transfected with the expression vector alone (data not shown). This result is consistent with the ability of AP2 to repress promoter activity of the full-length constructs in transient transfection and in vitro transcription experiments, indicating the AP2-binding site present in exon 1 is not involved in the observed repression of ACHE promoter activity.
To determine if binding of AP2 and Sp1 proteins to the ACHE promoter is mutually exclusive, both proteins were assayed together in footprinting experiments after each factor was incubated alone with the DNA template end-labeled in the sense strand. Subsequent addition of Sp1 protein to the reaction had no effect on AP2 binding, nor did AP2 alter binding of Sp1 (Fig. 7C, lanes 4-6). These experiments show AP2 does not influence binding of Sp1 to the essential second upstream Sp1-binding site. Interestingly, AP2 and Sp1 also prevented DNase I digestion of a small region upstream of the Sp1 sites (bases -136 to -118), but only when present together in the footprinting reaction. Neither protein alone protected this region. As the DNA base sequence of this region does not correspond to any known binding determinant for either of these transcription factors, the significance of this finding remains to be assessed.
Other gene promoter regions with GC-rich characteristics have formerly been regarded as housekeeping entities, whereby gene products involved in maintaining the economy of the cell are constitutively expressed. However, a number of closely regulated genes have been found to possess promoters with a high frequency of G and C residues (Lusky et al., 1987; Mumula et al., 1988), thus weakening an artificial promoter classification system based solely on DNA base sequence. One consistent finding in studies of genes with GC-rich promoters has been the use of multiple transcription start sites, presumably due to the variability in binding sites available to proteins involved in activating the RNA polymerase II transcription complex (Kollmar et al., 1994; Lu et al., 1994). Mouse Ache is representative of this, where transcription start sites of the gene in murine erythroleukemia and myoblast cell cultures range over a 20-bp region upstream of the Inr element. Evidence presented here shows that human ACHE transcription in NT2/D1 and HeLa cells differs from mouse Ache by having only two closely spaced RNA cap sites, both of which originate at the Inr element. Given the high degree of similarity between the promoter and Inr element regions of the two genes, preference for cap site utilization may be tissue-specific. Future investigation into ACHE cap site usage in human muscle and hematopoietic cell lineages should be informative in this regard.
Evidence from both transfection and in vitro transcription
experiments shows the second upstream Sp1 site 71 bp 5` of the first
cap site in ACHE is essential for activated transcription.
Analysis of deletion constructs revealed this single site with
attendant downstream sequences confers promoter activity equivalent to
that observed from over 1 kilobase pair of sequence upstream of exon 1.
Sp1 protein also protects this site from DNase I digestion in
footprinting experiments. These results suggest the presence of a
compact locus for regulating activated transcription of ACHE,
wherein EGR-1, Sp1, and other factors might compete for overlapping
binding sites located within a limited portion of the promoter region
of ACHE. Although the effect of EGR-1 on human ACHE transcription is unknown, the overlapping Sp1 and EGR-1 consensus
binding sites in the mouse Ache promoter foster competition
for binding between EGR-1 protein and Sp1 protein (Mutero et
al., 1995). Similarly, the NF-B site present in the intron
region downstream of exon 1 of ACHE appears to be non-functional.
Co-transfection of expression vectors containing the p50 and p65
subunits of this transcription factor had no effect on construction A
in NT2/D1 cells (data not shown).
Functional AP2-binding sites which mediate transcriptional
activation are often found upstream of core promoter regulatory
regions, as has been reported for human genes encoding collagenase,
growth hormone, keratin K14, metallothionein IIA, and proenkephalin,
and for the murine major histocompatibility complex H-2 k gene and the SV40 virus regulatory region (Haslinger and Karin,
1985; Lee et al., 1987; Hyman et al., 1989; Williams et al., 1988; Leask et al., 1991; Mitchell et
al., 1987). The presence of two consensus sites for AP2 located
between the Sp1 sites and the Inr element of the ACHE promoter
region is unusual and suggests a novel role for AP2 in transcriptional
regulation.
The ability of AP2 to repress human ACHE and mouse Ache transcription in the transfection and in vitro transcription experiments presented in this study is remarkable, since this factor has previously been described only in an activating capacity. The proximity of the AP2 consensus binding sites to the essential Sp1 site in the promoter and to the Inr element presented the possibility that the factor disrupts transcription of ACHE. DNase I protection studies showed Sp1 and AP2 are able to bind to the ACHE promoter simultaneously (Fig. 7C), indicating that AP2 does not interfere with binding of activating elements to the GC box region of the promoter.
Several lines of
evidence indicate AP2-mediated repression is due to a specific steric
interference with the basic transcription factor machinery. First,
mutation of the AP2-binding site proximal to the cap sites in the ACHE promoter eliminated AP2 repression of human ACHE promoter activity in transient transfection experiments using
human NT2/D1 cells. Similarly, mutation of the AP2 site in the mouse Ache promoter increased promoter activity in two mouse cell
lines that express AP2. These results indicate DNA binding is essential
for inhibition of transcription by AP2, a fact confirmed by band shift
analysis with nuclear extract prepared from the AP2-expressing mouse
cell lines. Second, addition of AP2 protein to in vitro transcription reactions selectively decreased transcription from
the ACHE DNA template, while the internal control template
containing the CMV immediate early promoter was unaffected (Fig. 3). If AP2-mediated repression was the result of
squelching, i.e. sequestration of components of the RNA
polymerase II transcription complex away from the DNA (Gill and
Ptashne, 1988), then transcription from both templates would have been
inhibited. Third, deletion of the activating domain of AP2
(construction N165AP2) did not alter the transcriptional
repression effect on ACHE promoter constructions. Retention of
repressor activity after removal of the protein-protein interaction
domains from the homodimer argues that AP2 sterically hinders some
aspect of RNA polymerase II-directed transcription and does not repress
transcription through either specific or non-productive protein
binding.
Together these results suggest AP2 interferes with some aspect of RNA polymerase II-mediated transcription, perhaps by blocking access of Inr binding elements or members of the RNA polymerase II transcription complex to the cap sites. This idea is supported by the fact that mutation of the proximal AP2 site alone in the human ACHE promoter is sufficient for abolishing repression by AP2 protein. AP2 bound to the second upstream binding site closer to the GC-rich region of human ACHE apparently does not interfere with the function of the transcription complex at the cap sites.
Recently,
the promoter region of the neuronal nicotinic acetylcholine receptor
3 subunit gene has been characterized and reported (Yang et
al., 1995). It is of interest to note that the promoter of this
gene is highly similar to the ACHE promoter; no TATA box is
present, an essential Sp1 site resides at -70, and AP2 binds to a
site between -22 and -30. It is not known whether AP2
functions as a repressor of the nicotinic receptor
3 subunit gene;
however, the proximity of bound AP2 to the transcription start sites of
the gene suggests that, like ACHE, the
3 subunit gene is
also susceptible to repression by AP2. This finding raises the
intriguing possibility that AP2 functions as a common regulatory
element in coordinating expression of certain members of the
cholinergic neurotransmitter system.
A steric hindrance model for
AP2-mediated repression of ACHE transcription is reminiscent
of the most frequently observed mechanism for transcriptional
repression in prokaryotes, which involves competition between
DNA-binding proteins and general transcription factors at or near gene
transcription start sites (reviewed in Levine and Manley, 1989).
Examples of eukaryotic repressors that function in a similar manner
include the repression of bovine prolactin and human glycoprotein
hormone subunit genes by glucocorticoid receptor (Sakai et
al., 1988; Akerblom et al., 1988). Repression of both
genes requires the presence of the DNA-binding domain but not the
activating domain of the receptor, indicating competition for binding
sites in the promoter is involved. Binding of SV40 large T antigen near
the transcriptional initiation site in the SV40 early promoter inhibits
transcription, a result thought to involve steric blockade of RNA
polymerase II in the region (Myers et al., 1981). In addition,
a cellular protein, LBP-1, has been shown to block transcription in
vitro from an HIV-1 long terminal repeat promoter by occlusion of
both the TATA box and transcription start sites following binding of
the factor to the DNA template (Kato et al., 1991). Thus, AP2
joins a growing family of eukaryotic proteins that serve to attenuate
gene expression by interfering with the intricate milieu fostering
transcriptional events. Future analysis of mutant ACHE transgenes will enable assessment of the physiological importance
of these interactions in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32675[GenBank].