From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750
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ABSTRACT |
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The m2 subtype is the predominant muscarinic acetylcholine receptor subtype expressed in heart and regulates the rate and force of cardiac contraction. We have previously reported the isolation of the promoter region for the chick m2 receptor gene and defined a region of the chick m2 promoter sufficient for high level expression in cardiac primary cultures. In this manuscript we demonstrate transactivation of cm2 promoter by the GATA family of transcription factors. In addition, we define the GATA-responsive element in the chick m2 promoter and demonstrate that this element is required for expression in cardiac primary cultures. Finally, we demonstrate specific binding of both a chick heart nuclear protein and of cloned chick GATA-4, -5, and -6 to the GATA-responsive element.
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INTRODUCTION |
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The muscarinic acetylcholine receptors (mAChR)1 belong to the seven-transmembrane domain superfamily of receptors whose biological actions are elicited via activation of GTP-binding regulatory proteins (G-proteins) (1). Five different mammalian mAChR subtypes (m1-m5) and four chicken mAChR subtypes (m2-m5) have been identified and shown to be encoded by separate genes (2, 3). The m1, m3, and m5 subtypes preferentially couple to the stimulation of phospholipase C, whereas the m2 and m4 subtypes preferentially couple to the inhibition of adenylyl cyclase. The different mAChR subtypes have unique overlapping expression patterns and are found in the central nervous system, peripheral nervous system, smooth muscle, and heart (4). The m2 subtype is the main mAChR found in mammalian heart (5); however, chick heart expresses predominantly m2 with significant amounts of m4 and m3 (6). Cardiac mAChR activation causes a decrease in both the rate and force of contraction (7). These effects are mediated through the inhibition of adenylyl cyclase activity, activation of an inward-rectifying potassium channel, inhibition of a calcium channel, and inhibition of the hyperpolarization-activated pacemaker current.
Since the initial discovery of multiple muscarinic acetylcholine receptor subtypes, great effort has been made to understand the expression patterns and, ultimately, the function of the different subtypes. Although many advances have been made in determining the expression patterns of the different muscarinic receptor subtypes, little is known about the molecular mechanisms that determine these patterns. The isolation of promoter regions for the m1, m2, and m4 receptor genes has been recently reported (8-11). The mechanisms governing neural-specific expression of muscarinic receptors are beginning to be understood. For example, the m4 receptor contains a repressor element 1/neuron restrictive silencer element that plays a role in determination of neural-specific expression (10). In addition, we have demonstrated activation of the m2 promoter by the cytokines ciliary neurotrophic factor and leukemia inhibitory factor in mammalian neural cell lines. The mechanisms that regulate expression of the m2 receptor in heart are for the most part unknown. Previously, we identified regions of the chick m2 promoter necessary for basal level expression in chick heart primary cultures (9). We sought to determine if the GATA family of transcription factors plays a role in expression of the m2 receptor gene in heart.
The founding member of the GATA family of transcription factors (GATA-1) was first identified as a determinant of lineage-specific gene expression during hematopoiesis that bound to the DNA sequence (A/T)GATA(A/G) found in the promoter region of several erythroid-specific genes (12). In vertebrates, six GATA family members that contain DNA binding domains with two zinc fingers of the CX2CX17CX2C type (13) show distinct but overlapping expression patterns (for review, see Ref. 14). The GATA-1/2/3 subfamily are primarily involved with hematopoiesis, whereas members of the GATA-4/5/6 subfamily from Xenopus, mouse, and chick (15-19) play a role in expression of lineage-specific genes in a variety of tissues derived from mesoderm, including heart. Two transcripts for chick GATA-5, designated GATA-5 short and long, are produced by the use of alternative exons. The short GATA-5 isoform contains a single zinc finger and binds DNA but has decreased ability to transactivate GATA-responsive promoters (20). Several cardiac-specific genes contain functional GATA sites within their promoter regions and can be transactivated by GATA factors in noncardiac cells (21-24).
In this manuscript we demonstrate 1) transactivation of m2 reporter constructs by the GATA family of transcription factors, 2) determination of the GATA-responsive element in the m2 promoter, 3) binding of a chick heart nuclear protein to the GATA-responsive element, and 4) regulation of basal level expression of m2 in chick heart primary cultures by the GATA-responsive element.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Cell Transfection--
Primary chicken heart
cultures were prepared from 9-day embryos as described and grown in
either defined media or medium M199 supplemented with 5% fetal bovine
serum and 1% penicillin, streptomycin at 37 °C in a humidified 5%
CO2 environment (25). For transfections, cells were plated
out into 24-well plates and transfected 60 h later with either
Transfectam reagent (Promega), for cells grown in defined media, or by
the calcium phosphate method as described (9, 26). For Transfectam
transfections, the transfection mixture contained 100 ng/well
pSV--galactosidase (Promega) to correct for minor differences in
transfection efficiency and 0.037 pmol/well reporter construct
(117-134 ng) brought to a final total DNA concentration of 234 ng/well
with pGEM4 (Promega) carrier DNA. The calcium phosphate transfection
mixture contained 100 ng/well pSV-
-galactosidase (Promega) and 0.048 pmol (153-183 ng) of reporter construct brought to a final amount of
283 ng with carrier DNA. Cells were lysed 32 h after the addition
of transfection mixture. The human choriocarcinoma cell line JEG-3 (American Type Culture Collection, Rockville, MD) was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin at 37° in a humidified 10% CO2 environment. Cells were plated at a density of
20,000/well into 24-well plates, transfected 48 h after plating,
and harvested 48 h later as described (27). The transfection
mixture contained 177 ng/well reporter construct and varying amounts of
GATA factor expression plasmids. A plasmid containing the
lacZ gene driven by the Rous sarcoma virus promoter
(RSV-
-galactosidase) was included to correct for minor differences
in transfection efficiencies (28). The total DNA concentration was kept
constant by the addition of empty expression vector. COS-7 cells
(American Type Culture Collection, Rockville, MD) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 1% penicillin, streptomycin at 37° in a humidified
10% CO2 environment. Transfections were performed by the
calcium phosphate method. Briefly, calcium phosphate precipitates
containing 17 µg of either GATA factor expression constructs or empty
expression vector were added to 10-cm dishes grown to approximately
70-90% confluency.
Determination of Luciferase and -Galactosidase
Activity--
Luciferase activities were determined using a Berthold
AutoLumat LB 953 as described (27).
-Galactosidase activity was determined using the o-nitrophenyl
-D-galactopyranoside colorimetric assay for cells
transfected by the calcium phosphate method and the Galactolight
chemiluminescent assay (Tropix) for cells transfected with Transfectam
(27). The luciferase activity was normalized to
-galactosidase
activity and in some cases expressed as -fold above the
luciferase/
-galactosidase activity of promoterless luciferase vector
(pGL3 Basic).
Preparation of Extracts and Electrophoretic Mobility Shift
Assays--
Heart cell nuclear extracts were isolated from 9-day
embryonic chick heart primary cultures grown in defined media 96 h
after plating as described (29). Whole cell extracts were prepared from
COS-7 cells 36 h after the addition of transfection mixture as
described (30). Electrophoretic mobility shift assays (EMSA) were
performed as described (30). Briefly, 10 fmol of double-stranded oligonucleotides labeled with [-32P]ATP using T4
polynucleotide kinase were used as the probe. Experiments were
performed with either 1.5-2.5 µg of nuclear extracts isolated from
9-day embryonic chick heart primary cultures or 2.2 µl of whole cell
mini-extracts isolated from COS-7 cells. Binding reaction components
were added in the following order: 1) buffer, salts, and nonspecific
DNA; 2) protein extracts; 3) cold competitor DNA; and 4) probe.
Antibody, either 5 µg of anti-HA or 100 ng of anti-myc, was added 10 min after the addition of probe for supershift experiments. The binding
reaction was incubated on ice for 45 min and separated on a 5%
polyacrylamide 0.25× Tris borate-EDTA gel prerun for 2 h at
4 °C. The dried gel was exposed to Kodak X-Omat film at
70 °C with two Dupont lightning intensifying screens for the indicated time. Double-stranded oligonucleotides used were: 3'-GATA,
5'-GATCATGGAAGAGAAAGATAAAGCGGCTGCC-3'; 3'-GGTA,
5'-ATGGAAGAGAAAGGTAAAGCGGCTGCC-3'; MID-GATA,
5'-CTAGTAAGCTAGGTTTGATAGCTCTGTGTAT-3'; 5'-GATA,
5'-CTAGGAAATATGAATTATCATCGGCTCACTT-3'. Synthetic GATA site,
5'-TGCGGATAGATAAGGCCGGAATTCGGATC-3'.
DNA Constructs--
The constructs pNMR26-29 containing 789 bp,
2 kbp, 3.3 kbp, and 8 kbp fragments, respectively, of the m2 promoter
cloned into the pGL3 Basic (Promega) luciferase reporter gene vector
have been previously described (9). The m2 789-bp reporter construct was used as a template for polymerase chain reaction overlap
site-directed mutagenesis (31) using Pfu polymerase
(Stratagene). All polymerase chain reaction constructs were sequenced
with the Applied Biosystems Taq Dye Terminator Sequence kit
to ensure the GATA to GGTA change and the absence of secondary
mutations. The plasmid pAP/GATA was constructed by placing a synthetic
GATA, 5'-TGCGGATAAGATAAGGCCGGAATT-3', site upstream of the 44 to +71
region of the human liver/bone/kidney alkaline phosphatase gene (32).
The resulting synthetic promoter was cloned into pGL3 Basic. The
PYP-GATA-6 expression plasmid contains the GATA-6 coding region from
the sequence PVYVP (amino acid 42) to the stop codon in the expression
vector pR18 (33). Expression constructs containing the coding region of
the two chicken GATA-5 isoforms (GATA-5S and GATA-5L) inserted into
pcDNA3 (Invitrogen) have been reported previously (20). The
PYP-GATA-4 expression construct contains the largest cDNA isolated
for GATA-4 inserted into pR18 and begins at the conserved PVYVP found
in GATA-4-6 (18). The HA-GATA-5 and HA-GATA-6 contain the HA epitope (34) fused in-frame to the initiating methionine of the entire GATA-5(long isoform) and GATA-6 inserted into pcDNA3. The
MYC-GATA-5 and MYC-GATA-6 constructs contain five copies of the myc
epitope (35) from pCST+MT (kindly donated by D. Turner, R. Rupp, and H. Weintraub) fused in-frame to the initiator methionine of GATA-5(long) and GATA-6, respectively, inserted into pcDNA3. Expression
constructs for Xenopus GATA-4, -5, and -6 (16) contained the
entire coding region inserted into pcDNA3 (Invitrogen).
Western Blot Analysis-- Ten microliters of the whole cell extracts used for EMSA were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose using standard conditions (36). Duplicate blots were probed with either anti-HA (5 µg/ml final) or anti-myc (100 ng/ml final) monoclonal Ab. After incubation with horseradish peroxidase-conjugated secondary Ab, the blots were developed with the Renaissance chemiluminescent reagent (NEN Life Science Products) and exposed to Kodak X-Omat x-ray film.
Materials--
[-32P]ATP was purchased from
NEN. All restriction enzymes and T4 polynucleotide kinase were
purchased from New England Biolabs. Pfu polymerase was
purchased from Stratagene. The anti-HA monoclonal Ab (12CA5) was
purchased from Boehringer Mannheim, and the anti-c-myc monoclonal Ab
(9E10) was purchased from Santa Cruz Biotechnology Inc. Horseradish
peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit
antibodies were purchased from Amersham Pharmacia Biotech.
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RESULTS |
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Cotransfection of Chick GATA-6 Can Transactivate the m2 Promoter
via an Element Located within a 789-bp Region--
We utilized a
heterologous system consisting of the human choriocarcinoma cell line
JEG-3, which express very low levels of mAChR, to determine whether
chick GATA factors were capable of transactivating m2 reporter gene
constructs and what promoter region may be responsible for activation.
Preliminary experiments2 demonstrated that GATA-6
transactivated the m2 promoter at levels higher than GATA-4 or -5. Cotransfection of equimolar amounts of vector alone (pGL3) or either
the 789 bp, 2 kbp, 3.3 kbp, or 8 kbp m2 promoter reporter gene
constructs along with PYP-GATA-6 and an RSV--galactosidase plasmid,
to correct for minor differences in transfection efficiency, resulted
in an increase in m2 promoter-driven luciferase expression (45-63-fold
above pGL3 alone) compared with basal levels (2-7-fold above pGL3
alone) (Fig. 1). A positive control
plasmid pAP/GATA containing a minimal promoter region from the human
alkaline phosphatase gene downstream of a synthetic consensus GATA
binding site (see "Experimental Procedures") showed an increase
from 3- to 26-fold above pGL3 alone when PYP-GATA-6 was cotransfected.
Although the various m2 luciferase constructs displayed different basal
levels of expression (37), cotransfection of PYP-GATA-6 resulted in
similar m2-driven luciferase expression, indicating that the
GATA-responsive element is located within the 789-bp promoter
fragment.
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The m2 Promoter Contains Three Consensus GATA Factor Binding Sites
Located within 789 bp from Intron One That Are Sufficient for
Transactivation by GATA-4, -5, and -6 in a Heterologous
System--
Sequence analysis identified three consensus GATA factor
binding sites (37, 38) located at positions 150,
500, and
580 bp
in the m2 promoter (Fig. 2A).
The site present at
150 bp, designated the 3' site, is located 208 bp
downstream of the most 3' start site of transcription. The two other
sites, located at 142 and 222 bp upstream of the most 3' start site of
transcription, were designated the MID and 5' sites, respectively. We
wanted to determine if the chick GATA-4, -5, and -6 were capable of
transactivating the m2 promoter in our heterologous system. Increasing
amounts of the expression constructs PYP-GATA-4, GATA-5L, GATA-5S, and PYP-GATA-6 (see "Experimental Procedures") were cotransfected with
the 789-bp m2 reporter gene construct into JEG-3 cells. An RSV-
-galactosidase plasmid was included to correct for minor differences in transfection efficiencies. The PYP-GATA-4, GATA-5L, and
PYP-GATA-6 were capable of transactivating the m2 789-bp promoter, albeit at different levels, whereas the GATA-5S did not transactivate the m2 789-bp promoter (Fig. 2B). The levels of
transactivation for PYP-GATA-6 and PYP-GATA-4 peak at 75 ng of
expression plasmid/well and decrease with higher amounts of expression
plasmid, whereas the GATA-5L shows the highest transactivation when 150 ng/well expression plasmid is cotransfected. In this system, PYP-GATA-6 transactivates the m2 promoter the greatest (43 ± 4-fold
increase) with PYP-GATA-4 and GATA-5L transactivating less (18 ± 2 and 6 ± 2). Neither the Xenopus GATA-4, -5, nor -6 were capable of transactivating the m2 promoter in this
system.2
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The 3' Consensus GATA Factor Binding Site Is Required for Basal Level Expression of m2 in Chick Heart Primary Cultures-- Site-directed mutagenesis was used to change the three consensus GATA sites to GGTA in the 789-bp m2 reporter gene construct. The wild type and mutant constructs were transfected into 9-day embryonic primary chick heart cultures grown in either defined media (Fig. 3A) or M199 supplemented with 5% FBS (Fig. 3B). Cultures were transfected using Transfectam reagent (Fig. 3A) or by the calcium phosphate method (Fig. 3B). The two culture and transfection methods were used to ensure that our results were not biased by selective transfection of a subpopulation of the multiple cell types found in chick heart primary cultures. Changing a single adenine to guanine nucleotide in the 3'-GATA site of the 789-bp promoter region reduced the basal level expression to 25 ± 6 and 22 ± 1% that of the wild type construct for cultures grown in defined media and M199, 5% fetal bovine serum, respectively (Fig. 3, A and B). Mutating the other consensus GATA sites resulted in little or no change in the basal level expression for cultures grown in defined media. However, mutating the MID-GATA site resulted in decreasing the basal activity to 67 ± 2% that of wild type for cultures supplemented with fetal bovine serum. Any combination of the 3'-GGTA with either the 5'-GGTA or MID-GGTA resulted in a similar decrease found with constructs containing only the 3'-GGTA. Mutating all three GATA sites resulted in slightly greater decreases in basal activity (20 ± 4 and 17 ± 3% that of wild type) for cultures grown in defined media and M199, fetal bovine serum, respectively. Similar results were seen when the mutant constructs were cotransfected with PYP-cGATA6 into JEG-3 cells.2
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A Nuclear Factor Isolated from 9-Day Primary Chick Heart Cultures Grown in Defined Media Shows Specific Binding to the 3'-GATA Site in Vitro-- Electrophoretic mobility shift assays were performed with nuclear extracts isolated from chick primary heart cultures grown in defined media to determine if the 3'-GATA site shown to be functionally important was a binding site for a nuclear protein. A 32P end-labeled double-stranded oligonucleotide for the 3'-GATA site was incubated with nuclear extracts isolated from 9-day embryonic chick heart primary cultures grown in defined media. The products of the binding reaction were separated on a native polyacrylamide gel and subjected to autoradiography. Two different protein-DNA complexes were detected (Fig. 4, A-C). Competition experiments demonstrated that the binding of the slower mobility complex could be competed off, whereas the binding of the faster mobility complex was decreased but not eliminated by 100-fold molar excess of unlabeled 3'-GATA probe (Fig. 4A). These results suggest that at least one specific protein binds the 3'-GATA site and that the faster mobility band may be composed of either a protein that has higher affinity for binding or two proteins, one of which shows specific binding to this site. Unlabeled 5' and MID-GATA probe showed much less competition for binding by both proteins to the 3'-GATA site but had some effect when a 100-fold excess was used (Fig. 4, A and B). The mutant 3'-GGTA did not compete for binding of either proteins even in a 100-fold molar excess (Fig. 4C). We were unable to reproducibly detect any protein-DNA complexes when the MID and 5'-GATA sites were used as probes.2 Although it is reasonable to postulate that one (or more) of the chicken GATA factors is the nuclear protein that binds to the 3' site, the lack of antisera against chick GATA-4, -5, or -6 prevents determination of whether the 3'-GATA protein-DNA complex contains either chick GATA-4, -5, or -6.
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Chick GATA Factors Expressed in COS-7 Cells Bind Specifically to the 3'-GATA Site-- Since there are no antibodies available for chick GATA -4, -5, or -6, we epitope-tagged the chick GATA-5 and -6 at the N terminus with either one copy of the HA epitope (39) or five copies of the myc epitope (35). The epitope-tagged GATA-5 and -6 transactivated the 789-bp m2 reporter construct in JEG-3 cells with similar abilities relative to the non-tagged versions (GATA-5L, 6.6 ± 0.9-fold increase; HA-GATA-5, 10.3 ± 1.0; myc-GATA-5, 40.4 ± 4.6; PYP-GATA-6, 41.2 ± 3.6; HA-GATA-6, 75.4 ± 11.9; myc-GATA-6, 135.7 ± 23.3; mean ± S.E., n = 3). We were unable to express functionally active N-terminal myc or HA-tagged PYP-GATA-4.2 To determine if GATA-4, -5, and -6 were capable of binding to the 3'-GATA site in vitro, whole cell extracts isolated from COS-7 cells transiently transfected with expression constructs for chick PYP-GATA-4 and epitope-tagged GATA-5L and 6 were used for EMSA with the 3'-GATA site probe (Fig. 5). Experiments performed with extracts from cells transfected with PYP-GATA-4, myc-tagged GATA-5 or GATA-6 (myc-GATA-5, myc-GATA-6), and HA-tagged GATA-5 or -6 (HA-GATA-5, HA-GATA-6) show a single protein-DNA complex that is not present in binding reactions performed with extracts from cells transfected with empty expression vector or untransfected cells. The formation of this complex can be eliminated by including a 100-fold molar excess of unlabeled competitor in the binding reaction (Fig. 5). The addition of monoclonal antibodies to the HA or Myc epitope to the binding reactions produced a "supershift" of the protein-DNA complexes that was proportional to the number of epitopes present in the tagged GATA factors (1 epitope for HA versus 5 for myc). Competition experiments demonstrated that the specificity of chick GATA -4, -5, and -6 for the 3'-GATA site (Fig. 6) was similar to results observed with chick heart nuclear extracts (Fig. 4). Western blot analysis demonstrated that the myc-tagged GATA-6 was expressed at higher levels than the myc-tagged GATA-5, and the extent of transactivation of the m2 promoter by the myc-tagged constructs was proportional to the relative levels of protein expression (Fig. 7). The HA-tagged GATA-5 and GATA-6 are expressed at comparable levels and, in the experiment shown, HA-GATA-5 is expressed at slightly higher levels (Fig. 7). The ability of the HA-tagged GATA constructs to transactivate the m2 promoter do not appear to be dependent on the relative protein levels detected by Western blot analysis (Fig. 7).
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DISCUSSION |
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We have demonstrated that the chick GATA-4, -5, and -6 transcription factors are capable of transactivating the chick m2 promoter in a heterologous system. In this system, truncated versions of the chick GATA-4 and -6 were capable of transactivating the m2 promoter, in agreement with a recent report demonstrating that mouse GATA-4 containing an N-terminal truncation at the PVYVP motif conserved between mouse, human, chick, and Xenopus shows the same activity as the full-length protein when cotransfected into NIH-3T3 cells (40). In contrast to the GATA-5 long isoform, the GATA-5 short isoform was incapable of transactivating the m2 promoter. Similar results have been reported using a different promoter in COS-7 cells (20). Cotransfection of GATA-5L into chick heart primary cultures with the 789-bp m2 reporter plasmid resulted in an increase in m2-driven luciferase expression, whereas cotransfection of the GATA-5S had no effect.2 Therefore, it is unlikely that the short GATA-5S isoform acts as a negative regulator of m2 transcription in heart.
Mutation of the 3'-GATA site in the m2 promoter drastically decreased the basal level activity in chick heart primary cultures, suggesting that the results from our heterologous system are relevant to cardiac regulation of m2 in vivo. Several reports implicate GATA-4, -5, and -6 involvement in early cardiac development (15-17, 19, 41-44). Targeted gene disruption of GATA-4 in mouse results in an embryonic lethal phenotype with cardiac morphogenic defects (45, 46). In chick, the negative chronotropic effect of muscarinic agonists can be detected starting at embryonic day 5 (47). Expression of m2 mRNA did not vary greatly from embryonic day 4 through day 20 but was expressed at levels 10-fold higher than either m3 and m4 (6). Transcripts for chick GATA-4, -5, and -6 were detected in embryonic day 10 through post-hatched day 6 heart; however, expression at earlier stages has not been investigated (18). In mouse and Xenopus GATA-4, -5, -6, expression was present in mesoderm during gastrulation (15-17, 19, 44). Mutating the GATA sites in the m2 promoter does not completely eliminate basal level expression in chick heart primary cultures. Taken together, these results suggest that although GATA-4, -5, and -6, in combination with other transcription factors, may determine mAChR subtype-specific expression during cardiac differentiation, they are most likely required for but do not regulate chick m2 expression levels during development past embryonic day 4. Chick GATA-4, -5, -6 are incapable of transactivating a mouse m1 subtype mAChR promoter construct in JEG-3 cells,2 consistent with a specific role for GATA-4, -5, and -6 in the regulation of cardiac mAChR expression.
The identity of the GATA factor(s) that are required for maximal m2 expression in heart remains to be elucidated. Our data suggest that all three members of the GATA-4, -5, and -6 can family bind to and transactivate the m2 promoter. The abilities of the myc-tagged GATA constructs to transactivate m2 transcription in our heterologous system are proportional to the relative levels of protein demonstrated by Western blot analysis and by the intensity of the protein-DNA complexes seen by EMSA. In contrast, the relative abilities of the HA-tagged GATA constructs to transactivate m2 did not appear to be dependent on the amount of protein expressed as detected by Western blot analysis. These results suggest that GATA-6 may be better at transactivating transcription of m2 than GATA-5 when the two proteins are expressed at similar levels. This may reflect different binding affinities of GATA-5 or GATA-6 for the 3'-GATA element or differential interactions of GATA-5 and GATA-6 with other proteins required for transcription from the m2 promoter. We observed at least two protein complexes that can bind the 3'-GATA site in the m2 promoter when chick heart nuclear extracts were used for EMSA but only saw a single complex when GATA-4, -5, and -6 were overexpressed in COS-7 cells. The two complexes may contain different members of the GATA-4, -5, and -6 family or, alternatively, may contain a GATA factor and a cofactor. Recently, a novel factor, dubbed FOG, was found to associate with GATA-1 and synergistically activate transcription of a hematopoietic regulatory region (48). Therefore, the two protein-DNA complexes may contain different combinations of GATA factors and/or cofactors. Preparation of subtype-specific antisera for the chick GATA-4, -5, and -6 will be necessary to clarify which GATA factor(s) are involved with regulation of m2 receptor expression in heart.
The results presented here are the first identification of a transcription factor required for maximum basal level expression of a muscarinic acetylcholine receptor gene. Although we and others have identified promoter regions involved with the determination of neuronal expression of mAChR, the mechanisms determining cardiac-specific expression of mAChRs were previously unknown. Our results provide a framework for further studies to better understand the transcriptional regulation of the m2 gene in both heart and in neural tissue.
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ACKNOWLEDGEMENTS |
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We thank Dr. John B. Burch for his donation of the chick GATA-4, -5, and -6 expression plasmids and helpful discussions. We thank Dr. Todd Evans for donation of the Xenopus GATA-4, -5, -6 expression plasmids. We thank Dr. Monica Torres, Michael Schlador, and Dr. William P. Scheimann for critical reading of the manuscript and for many useful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL30639 (to N. M. N.) and NS7332 (to M. L. R.) and an American Heart Association of Washington postdoctoral fellowship (to M. L. R.)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 and reprint requests should be addressed:
Dept. of Pharmacology, University of Washington, Box 357750, Seattle,
WA 98195-7750. Tel.: 206-543-9452; Fax: 206-616-4230.
1 The abbreviations used are: mAChR, muscarinic acetylcholine receptor; G-proteins, GTP-binding regulatory proteins; EMSA, electrophoretic mobility shift assays; HA, hemagglutinin epitope YPYDVPDYA; MYC, myc epitope MEQKLISEEDLNE; Ab, antibody; RSV, Rous sarcoma virus; bp, base pair(s); kbp, kilobase pair(s); MID, middle.
2 M. Rosoff and N. Nathanson, unpublished observation.
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REFERENCES |
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