From the Departments of Patología
Experimental and § Genética y Biología
Molecular, Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional, CINVESTAV Instituto Politecnico
Nacional, AP 14-740, Mexico 07300, Mexico and ¶ Centro de
Investigación en Ciencia Aplicada y Tecnología Avanzada,
CICATA Ap 11-500, Mexico
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ABSTRACT |
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We present here the cloning and characterization of the EhPgp1 multidrug resistance gene promoter isolated from the Entamoeba histolytica drug-resistant mutant clone C2. The EhPgp1 promoter lacks the typical TATA box and the transcriptional initiation sequences described for other E. histolytica promoters. The major transcription initiation site of the EhPgp1 gene was located at the ATG start codon. The EhPgp1 core promoter located within the first 244 base pairs showed a higher chloramphenicol acetyltransferase expression in the transfected trophozoites of clone C2 than in those of the sensitive clone A. Gel shift assays revealed three specific DNA-protein complexes (Ia, IIa, and IIIc) using nuclear extracts from clone C2, whereas three main complexes (If, IIf, and IIg) were limited to clone A. Competition assays suggested the presence of C/EBP-like and OCT-like proteins in complexes Ia and IIa, respectively, probably involved in the expression of the EhPgp1 gene, whereas complex IIIc was competed by GATA-1, C/EBP, OCT, and HOX oligonucleotides. Thus, differential DNA-protein complexes may be formed by transcriptional factors involved in the regulation of the EhPgp1 gene expression.
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INTRODUCTION |
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Entamoeba histolytica is the protozoan responsible for human amoebiasis. Like other parasitic infections, amoebiasis is primarily controlled by drug treatment of symptomatic individuals using drugs such as metronidazole or emetine (1, 2). Differences in drug susceptibility have been found in several E. histolytica strains (3, 4) and clones (5). Case reports of failed drug treatments (6, 7) suggest that drug resistance can occur in this parasite. The multidrug resistance (MDR)1 phenotype first described in mammalian cells (reviewed in Ref. 8) has also been described for parasites, including Plasmodium falciparum, Leishmania tarentolae, and E. histolytica (9-11). E. histolytica emetine-resistant mutants (clone C2) (5) (i) present cross-resistance to several drugs, (ii) present increased efflux and decreased accumulation of radiolabeled emetine, (iii) present resistance reversion by calcium channel blockers, and (iv) overexpress a 4.0-kilobase mRNA transcript (11, 12). The transfection with the EhPgp1 gene, cloned in front of the actin promoter, also confers emetine resistance to sensitive trophozoites (13).
A membrane glycoprotein (Pgp), encoded by mdr genes, functions as an energy-dependent drug efflux pump, maintaining the intracellular drug concentration below cytotoxic levels (14). mdr genes are amplified or overexpressed in resistant cell lines exposed to drugs, hormones, or metals (15). In cell lines and cancers, the increased expression of the Pgp may be caused by transcriptional regulation alterations, gene amplification, promoter mutations, DNA rearrangements, or mRNA stability (16). The multigenic mdr families consist of three mdr genes in rodents and two in humans (17), whereas there are two mdr genes in P. falciparum (9) and three in L. tarentolae (10). In E. histolytica, four mdr genes (EhPgp1, EhPgp2, EhPgp5, and EhPgp6) have been cloned and sequenced (18, 19). They have between 61 and 67% homology among them and 41% homology with the human MDR1 gene (18). In the drug-resistant clone C2, the EhPgp1 gene is constitutively transcribed independently of drug concentration; the EhPgp2 transcript has not been detected but the EhPgp5 gene is transcribed at high emetine concentrations (19, 20), suggesting that their expression is regulated at the transcriptional level. Like the EhPgp1 gene, the human MDR1 gene is constitutively expressed. MDR1 mRNA has been detected in several normal tissues, suggesting a role for its encoded protein in toxin or steroid transport (21). Several tumors, including leukemia and lymphoma, express high levels of MDR1. Thus, tissue-specific factors appear to be important in the regulation of the Pgp expression in normal and transformed tissues (22).
Transcriptional regulation in E. histolytica and the
underlying mechanisms for the EhPgp genes activation are
poorly understood. This study presents the functional and structural
analysis of the EhPgp1 promoter isolated from clone C2 as a
step toward elucidating the mechanisms involved in the control of its
constitutive expression. Nucleotides 244 to +24 of the
EhPgp1 promoter efficiently directed the expression of the
chloramphenicol acetyltransferase (CAT) reporter gene in clone C2, but
reduced activity was detected in the drug-sensitive clone A. Gel shift
analysis showed interesting differences between nuclear factors from
clones C2 and A bound to the first
244 bp of the EhPgp1
promoter. This suggests that specific transcriptional regulators may be
involved in the constitutive expression of the EhPgp1 gene
in clone C2.
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EXPERIMENTAL PROCEDURES |
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E. histolytica Cultures-- Trophozoites of clones A and C2 (strain HM1:IMSS) (5) were axenically cultured in TY1-S-33 medium (23).
Cloning and Sequencing of the EhPgp1 Promoter-- The EhPgp1 promoter of clone C2 was obtained from a recombinant pBluescript (pBS) (Stratagene, CA) plasmid (p7) containing 2230 bp of the EhPgp1 coding region and 1770 bp upstream from the ATG start codon. This plasmid was previously isolated from a genomic DNA library constructed in Lambda Zap II vector with DNA from clone C2 (18). The EhPgp1 promoter of clone A was isolated by PCR of total DNA using the primers EhPgp1-S28 and EhPgp1-AS33 described below, and the PCR product was cloned in pBS. As a negative control, we used the EhPgp1-AS33 primer and the reverse primer from pBS. Sequence was done with overlapping oligonucleotides by the dideoxynucleotide chain-termination method (24) using Sequenase, version 2.0, DNA polymerase (U. S. Biochemical Corp.). Sequence data analysis and sequence alignments were done with Fasta algorithm (25) in the EMBL and GenBank data bases. The localization of consensus sequences was performed with the software package of the University of Wisconsin Genetics Computer Group (26).
Primer Extension--
Assays were done using a reverse
transcriptase sequencing kit (Promega, Madison, WI) (27). Ten µg of
total RNA from clones A and C2 were hybridized to a -end-labeled
18-bp primer (5'TACTCCTGCATACTGAAA3') (5 × 105 cpm)
complementary to nucleotides +110 to +128 of the EhPgp1 gene. Annealing was carried out at 45 °C for 25 min, and the
extension reaction was performed at 42 °C for 30 min with 15 units
of avian myeloblastosis virus reverse transcriptase (Promega). Nucleic acids were phenol-chloroform extracted, ethanol precipitated, and
separated by electrophoresis on 8% urea-polyacrylamide gels. The
product length was determined by comparison with the corresponding DNA
sequence obtained with the same primer.
Plasmid Constructions-- For transfection experiments several plasmids were constructed using PCR-amplified DNA fragments, inserted into the multiple cloning site of the pBS plasmid. The promoterless pBSCAT-ACT plasmid was constructed after PCR amplification of the bacterial CAT reporter gene (659 bp) and the 3'-flanking sequence of the actin gene (600 bp) from the pA5'A3'CAT vector (28). The CAT-ACT fragment was amplified using the sense CAT-S26 (5'-CCCAAGCTTATGGAGAAAAAAATCAC-3') and the antisense oligonucleotides Eh-Ac3'-AS29 (5'-CCGCTCGAGTTCTCTCTCCTGTGTACACC-3') (28), cloned into the HindIII and XhoI sites of the pBS vector. The 964-bp fragment was PCR-amplified using the p7 plasmid as template and the sense EhPgp1-S28 (5'-AAAACTGCAGTGAAGTGTCAGCACTTAA-3') and antisense EhPgp1-AS33 (5'-CCCAAGCTTAAACTCACTTTCAGTTATATCCAT-3') oligonucleotides. For the 268-bp fragment, the sense IIIs (5'-TAAATGAACTAAAAAATA-3') and the antisense EhPgp1-AS33 oligonucleotides were used. These fragments contained 940 and 244 bp of the EhPgp1 promoter, respectively, and 24 bp of its coding region. The 964-bp fragment was cloned into the PstI and HindIII sites (p964Pgp1), and the 268-bp fragment was cloned into the SmaI and HindIII sites (p268Pgp1) in front of the CAT gene, into the pBSCAT-ACT vector (see Fig. 3A). The orientation and sequence of constructions were confirmed by DNA sequencing (24).
Transfection and CAT Assays-- Transfection was carried out by electroporation as described previously (28). Briefly, 106 trophozoites were transfected with 100 µg of the p964Pgp1, p268Pgp1, pA5'A3'CAT, or pBSCAT-ACT plasmids. Electroporated trophozoites were transferred into plastic flasks (Nalgene, Rochester, NY) containing 30 ml of TYI-S-33 medium and incubated for 48 h at 37 °C. CAT activity was analyzed by thin layer chromatography (29) using 150 µg of trophozoite extracts, 0.5 mM acetyl coenzyme A, and 1 µCi (37 kBq) of [14C]chloramphenicol (50-60 mCi/mmol) incubated for 16 h at 37 °C. In other experiments, CAT activity was determinated by the two-phase diffusion assay (30) using 5 µg of trophozoite extracts and 200 µl of chloramphenicol (1.25 mM), which were incubated with [14C]butyryl-CoA (NEN Life Science Products) for 2 h. Protein concentration was determined by the Bradford method (31). CAT activities were expressed as cpm of the butyrylated derivatives. The background given by the pBSCAT-ACT plasmid transfected into the trophozoites was substracted from the positive results obtained with the other plasmids. CAT activity was determined in the linear range of the assay.
Nuclear Extracts--
NEs were prepared from trophozoites of
clones A and C2 by a modified Schreiber's protocol (32). Briefly,
107 trophozoites were harvested, washed twice with cold
phosphate-buffered saline, pH 6.8, resuspended in 4 volumes of Buffer A
(10 mM Hepes, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and incubated 20 min at
4 °C. The trophozoites were centrifuged at 8600 rpm in a JA-20
Beckman rotor and resuspended in 5 volumes of Buffer A supplemented
with a protease inhibitor mixture (0.5 mM
phenylmethylsulfonyl fluoride; 2 mM benzamidine; 5 µg/ml
of each aprotinin, pepstatin A, leupeptin, and E-64). The trophozoites
were homogenized with 25 strokes in an all-glass Dounce homogenizer
using a pestle. Integrity of the nuclei was monitored by phase-contrast
microscopy. The nuclei were lysed by incubation for 40 min at 4 °C
in 100 µl of Buffer C (20 mM Hepes, pH 7.9, 0.42 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol) in the presence of the protease
inhibitor mixture. After incubation, NEs were microcentrifuged at
14,000 rpm for 20 min at 4 °C. The supernatant was aliquoted and
stored at 70 °C. Protein concentration was determined by the
Bradford method (31). Reagents were purchased from Sigma.
Gel Shift Assays--
Gel shift assays were performed as
described previously (33) with some modifications. Briefly, three
different overlapping ~100-bp fragments, corresponding to the first
244 bp upstream from the ATG start codon, were amplified and labeled by
PCR. The PCR mixture contained [-32P]dATP, 2 mM cold nucleotides, 50 ng of template DNA and 0.5 units of
Deep Vent DNA polymerase (New England Biolabs). The reaction was
carried out during 28 cycles (94 °C for 30 s, 42 °C for
30 s, and 72 °C for 35 s) in a Perkin-Elmer 9600 Thermal
Cycler. The oligonucleotides used as primers for each fragment
were as follows: Is (5'-TTTTAGATTTAATGTGTT-3') and Ias
(5'-CACTTTCAGTTATATCCA-3') for fragment I, IIs
(5'-TAACAAAGGAGAGAAAAT-3') and IIas (5'-ACCAAACACTAACACATT-3') for
fragment II, and IIIs and IIIas (5'-CTTATTATTTTCTCTCCT-3') for fragment
III. The labeled fragments were separated on 12% nondenaturing
polyacrylamide gels and purified after elution. DNA fragments (0.5-1
ng) were incubated with 15 µg of NEs from clones A or C2, 1 µg of
poly[d(I·C)] (Pharmacia Biotech Inc.) and 10% glycerol in
DNA-protein binding buffer (12 mM Hepes, pH 7.9, 60 mM KCl, 1 mM dithiothreitol, 1 mM
EDTA, 4 mM Tris-HCl, pH 7.9, 1 mM spermidine, 1 mM MgCl2) for 10 min at 4 °C. The bound and
unbound complexes were separated on 6% nondenaturing polyacrylamide gels in 0.5× TBE (44.5 mM Tris-HCl, pH 7.9, 44.5 mM boric acid, 1 mM EDTA) at 25 °C and 100 V
for 4 h and visualized by autoradiography. Competition assays were
performed using a 150-fold excess of the same unlabeled fragments or
unlabeled double-stranded oligonucleotides containing consensus
sequence for the following transcription factors: C/EBP
from rat albumin (5'-GGTATGATTTTGTAATGGGGTAGG-3') (34), a putative C/EBP
-like sequence represented several times in
different E. histolytica promoters
(5'-ATTCAATTGGGCAATCA-3'), GATA-1 (5'-GTTGCAGATAAACATT-3'), HOX
(5'-GTAAGAGTTATTATTGAT-3'), OCT
(5'-ACATAGTTTATGCAACCGAAA-3') and OCT
(5'-AGCTAATTGCATACTTGGCTTGTAC-3') oligonucleotides or 1.5 µg of
poly[d(I·C)] as a nonspecific competitor (350-fold excess).
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RESULTS |
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Sequence Analysis of the EhPgp1 Promoter--
The 942 bp upstream
from the ATG start codon from EhPgp1 gene were sequenced using as
template the p7 plasmid (18) or the pBluescript plasmid containing a
PCR-amplified fragment obtained from total DNA of clone A (Fig.
1). The EhPgp1 promoters
isolated from both clones were 99.7% identical, except for three
changes at the position 501,
928, and
930. The PCR made with the
pBS reverse primer did not amplify any fragment from clone A. This region was 75% A/T rich with different-sized repeated and palindromic sequences (Fig. 1B, underlined). The
325 to
33-bp region
presented 61% identity with the
472 to
700-bp region of the
EhPgp5 promoter (58). Homology of 53-66% with promoter
regions of discoidin (
448 to
1046 bp), D19
(
820 to
1164 bp), dynein (
360 to
886 bp) and
ecm A (
1530 to
1783 bp) genes from Dictyostelium
discoideum (35-38) was found. The TATA box-like motif (TATTTAAA)
described for other E. histolytica promoters (39) was not
detected, but two putative initiator (Inr) elements at positions
18
and
67 (Fig. 1B, boxes) were found. The Inr element found
at
18 bp (GAACTAA) contains the conserved sequence CE2 (GAAC)
recently reported for several 5'-flanking regions in E. histolytica genes (40). Interestingly, the 5'-flanking region of
the APorC gene of E. histolytica (40) also has
the GAACTAA sequence localized at
18 bp, whereas the putative Inr
element (TTAGATT) is identical to that described in mammalian cells
(41). In other gene promoters, mainly in the TATA-less promoters, Inr
elements interact with transcriptional factors to influence accurate
transcription (42).
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Transcription Initiation Sites of the EhPgp1 Gene-- Primer extension experiments with total RNA were used to identify the 5' end of the EhPgp1 transcript. The major extension product of the EhPgp1 gene from clone C2 initiated at the base A of the ATG start codon, although other, fainter bands were visible at positions +16, +19, and +55 (Fig. 2, lane C2), but no open reading frame was found. In clone A, the major extension product also mapped at the ATG start codon; however, the amount of the main product was less than that in clone C2 (Fig. 2, lane A, right), indicating that the EhPgp1 gene is also transcribed in clone A, even though transcripts were not detected by Northern blot assays (19). These results suggest that the differential amount of the EhPgp1 transcript in sensitive and resistant clones may be the regulating mechanism of the MDR phenotype in E. histolytica. Results were highly reproducible, supporting the specific binding of the primer.
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Transient Expression Analysis of the EhPgp1
Promoter--
Transfection assays were done to examine whether the
region upstream from the EhPgp1 gene had a functional
promoter. The p964Pgp1 and p268Pgp1 plasmids, which contain 24 bp of
the open reading frame and 940 and
244 bp of the EhPgp1
promoter, respectively (Fig.
3A), were transfected into
clones A and C2. Both plasmids drove the CAT expression in the
resistant and sensitive clones. However, after 2 h of incubation
of the trophozoite extracts with the substrate, CAT activities were
significantly higher in trophozoites of clone C2 compared with clone A
(Fig. 3A), suggesting the presence of positive
transcriptional regulators in clone C2, which may be absent,
diminished, or modified in clone A. To define a shorter region with
promoter activity, the p268Pgp1 plasmid was transfected into
trophozoites of clones C2 and A. Interestingly, no differences in CAT
expression were detected in trophozoites of clone A transfected with
p964Pgp1 or p268Pgp1 plasmids, whereas when clone C2 was transfected
with p268Pgp1, it had more CAT activity than when it was transfected
with the p964Pgp1 plasmid (Fig. 3A). The latter proved that
the EhPgp1 core promoter is located within the first 244 bp.
In contrast to the strong EhPgp1 promoter activity, the EhPgp5 promoter presented little activity in clone C2 grown
without drug (Fig. 3B and Ref. 58). CAT activity of the
plasmid carrying the actin promoter was similar for clones A
and C2 but less than that obtained with the p964Pgp1 and p268Pgp1
plasmids when activity was measured after 2 (Fig. 3A) or 16 (Fig. 3B) h of incubation. In both experiments the negative
control, the promoterless pBSCAT-ACT plasmid and the cpm obtained from
the trophozoites transfected with this plasmid (basal activity) were
substracted from the activity obtained with the p964Pgp1, p268Pgp1, and
pA5'A3'CAT plasmids (Fig. 3).
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DNA-Protein Interactions on the Proximal 244 bp of the EhPgp1
Promoter--
The structural analysis of the EhPgp1
promoter was done using the 244 bp upstream from the ATG start codon of
the EhPgp1 gene because transfection experiments indicated
that the core promoter was located in this region. Overlapping DNA
fragments of approximately 100 bp each (fragments I, II, and III),
covering bp
244 to +19 were PCR-amplified using specific
oligonucleotides (Fig. 4A,
bottom) and their interactions with NEs from clones C2 and A were
studied by gel shift assays. NEs from clone C2 incubated with fragment I (
74 to +19 bp) formed five main complexes (Ia, Ib, Ic, Id, and Ie).
Interestingly, complex Ia was not detected in experiments carried out
with NEs from clone A (Fig. 4B). Additionally, NEs from
clone A formed complex If, which was not detected in clone C2 (Fig.
4B). Fragment II (
167 to
47 bp) formed five complexes with NEs from clone C2 (IIa, IIb, IIc, IId, and IIe) (Fig.
4C). Except for complex IId, all complexes were more
abundant in clone C2 than in clone A; this was particularly clear for
complex IIa, which was very strong in clone C2 and very faint in clone
A. In contrast, the doublet IIf and IIg, present in clone A, was not detected in clone C2 (Fig. 4C). Fragment III (
244 to
144
bp) (Fig. 4D) formed four complexes (IIIa, IIIb, IIIc, and
IIId) with NEs from clone C2, one of which (IIIc) was not detected in
clone A. All of the complexes were specifically competed by the same cold fragments and were maintained in the presence of poly[d(I·C)], used as nonspecific competitor. In summary, from gel shift assays, complexes Ia, IIa, and IIIc, detected mainly with NEs from clone C2,
were identified. These complexes may be involved in the constitutive EhPgp1 gene expression. On the other hand, complexes If,
IIf, and IIg, which were reproducibly detected only with NEs from clone A, may be acting as negative regulators and may be responsible for
suppressing the EhPgp1 expression. Their specific function is currently under study by mutation and transfection experiments.
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Competitive Binding Analysis of the Complexes Formed on Fragments
I, II, and III of the EhPgp1 Promoter--
Promoters share common
structural features, reflecting similar interaction with RNA
polymerase. This fact has allowed the identification of DNA consensus
sequences for different transcriptional factors. The EhPgp1
promoter was scanned for potential consensus sequences candidates that
might participate in the binding of transcriptional regulatory factors
(Table I and Fig. 4A). To investigate the identity of some of the sequences and complexes formed
by fragments I, II, and III and the NEs from clones A and C2, we
carried out competition experiments using double-stranded oligonucleotides containing consensus sequences for transcription factors present in these fragments (Table I and Fig. 4A).
Two different putative C/EBP binding sequences were used to compete the
complexes formed on fragment I: (i) the C/EBP binding
sequence from the rat albumin gene (34), and (ii) a C/EBP
-like, putative sequence represented several times
in some E. histolytica sequences upstream from the ATG start
codon. The C/EBP
oligonucleotide competed complex Ia
formed exclusively with NEs from clone C2, whereas the
C/EBP
oligonucleotide did not compete any complex (Fig.
5A). The C/EBP
sequence shares 9 of 12 bases of the putative C/EBP sequence found
between
43 and
54 bp in the EhPgp1 promoter (Fig. 5,
B and C), whereas the C/EBP
-like
sequence shares only 5 bases (Fig. 5C). These results
suggest the presence of a C/EBP-like transcription factor interacting with the EhPgp1 promoter that could be involved in the
expression of the EhPgp1 gene in clone C2 but not in clone
A.
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DISCUSSION |
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The structural and functional characterization of the EhPgp1 promoter isolated from clone C2 was analyzed. EhPgp1, one of the genes responsible for the MDR phenotype in E. histolytica, is differentially transcribed in the drug-sensitive clone A and in the drug-resistant clone C2. This work and the accompanying paper (58) represent one of the first attempts to study the concerted interplay of protein transcription factors and promoters on the regulatory regions of E. histolytica genes. The results suggest that the expression of the EhPgp1 gene involved in the MDR phenotype of clone C2 is finely regulated at transcriptional level.
The EhPgp1 promoters from the drug-sensitive and
drug-resistant clones were 99.7% identical in the 942 bp sequenced.
Sequence analysis revealed that the EhPgp1 promoter lacks a
TATA box motif found in other E. histolytica genes (39).
Instead, it has two putative Inr sequences that have been described as
being involved in the same functions as TATA boxes (41, 42). In fact,
the putative Inr sequence found at 18 bp is necessary for the
transcription of the hgl5 gene of E. histolytica
(40). The human MDR1 gene, which is constitutively expressed
in several tissues, also lacks a TATA box and has an Inr element (22).
By analogy, a similar regulatory mechanism may exist for the
EhPgp1 and MDR1 promoters. The function of these
putative Inr sequences is being investigated. The EhPgp1
promoter has several palindromic and repeated sequences that may
be also involved in transcriptional regulation, as has been reported
for Dictyostelium, Drosophila, and yeast genes
(35, 51, 52).
In contrast to other E. histolytica genes, which start their transcription at the ATTCA or ATCA motifs, located near the ATG start codon (39, 40), the major EhPgp1 mRNA initiates just at the ATG start codon, whereas other minor products initiate downstream from the ATG. In other systems, differences in the 5' end of mRNA influence the translation efficiency through the creation or elimination of binding sites for trans-acting factors or through the formation of stable secondary structures that can modulate the overall translation efficiency (53). Additionally, variations in the 5' upstream sequences may influence steady state mRNA levels (54). However, any of these mechanisms appear to act on the EhPgp1 promoter because the minor products lack open reading frames. In the EhPgp1 gene activation, the correct selection of the transcription initiation site and the amount of the transcript may function as regulatory mechanisms.
We demonstrated the functionality of the EhPgp1 promoter
isolated from clone C2 by transfection assays. The p964Pgp1 and
p268Pgp1 plasmids carrying 940 and 244 bp upstream and +24 bp
downstream from the ATG, respectively, were able to drive the
transcription of the CAT gene in clones C2 and A. However, CAT activity
was higher in the resistant clone C2, suggesting that different factors may be interacting with regulatory sequences in this promoter to
modulate the transcriptional repression or activation of the EhPgp1 gene. Because the sequences of the core promoters in
the sensitive clone A and the resistant clone C2 were identical, we suggest that clone A, in contrast to clone C2, does not have the elements to efficiently enhance the EhPgp1 gene expression.
Transfection experiments using the promoter from the resistant clone C2
and gel shift assays support this assumption. The p268Pgp1 plasmid contains all the necessary elements to efficiently drive the CAT expression in clone C2, even better than the p964Pgp1 construct, suggesting the presence of sequences acting as silencer regulatory elements between nucleotides 244 and
940. Both plasmids showed a
higher activity than the pA5'A3'CAT plasmid, which contains the
actin promoter, probably due to the presence of the +24 bp downstream from the ATG in the EhPgp1 promoter plasmids. It
has been suggested that a small sequence downstream from the ATG
enhances the transcription activity of other E. histolytica
promoters (55).
By gel shift assays, three complexes (Ia, IIa, and IIIc) were found on
fragments containing the first 244 bp of the EhPgp1 promoter
with NEs from clone C2. Complex Ia was competed by the C/EBP oligonucleotide, suggesting the presence of a
putative C/EBP-like protein in E. histolytica that could be
binding to the sequence found at
43 to
54 bp. We consider that a
C/EBP-like nuclear protein may be a good candidate with a positive role
in the EhPgp1 gene expression in clone C2 because (i)
complex Ia, competed by C/EBP
, was poorly detected in
clone A; (ii) a putative C/EBP binding sequence is close to the
transcription initiation site; (iii) in Western blot assays, antibodies
against the human C/EBP protein recognized a ~20-kDa band in NEs of
E. histolytica (data not shown), supporting the presence of
a C/EBP-like factor in this parasite; (iv) the C/EBP protein has been
described as an activator factor in mammalian genes; and (v) C/EBP
binding sequences have been found also in the human MDR3 and
in the mouse mdr1b promoters, probably acting as
cis-elements regulating their transcriptional activity (43, 56).
Complex IIa, also specific for clone C2, was competed by an
OCT oligonucleotide, even though no OCT binding
sequences in fragment II were detected. However, POU and Pit-1 binding
sequences are found, and POU and Pit-1 proteins share the DNA binding
domains with the OCT family proteins (45). OCT factors participate in the regulation of the expression of housekeeping genes. The
EhPgp1 promoter shares some characteristics with
housekeeping promoters, such as the presence of putative Inr elements
and its constitutive expression in clone C2.
Complex IIIc, which is also specific for clone C2, was competed by
GATA-1, C/EBP, OCT
, and HOX binding
sequences but not by the OCT
oligonucleotide. GATA-1,
C/EBP
, Pit-1, and HOX sequences were located in fragment
III in a region of 37 bp, suggesting that some factors could be
interacting each other to form a multiprotein complex, which is
probably required for transcription regulation of the gene. This
assumption is supported by the fact that binding sequences for these
four putative transcription factors are very close. It has been
demonstrated that many transcription factors contain domains that
mediate the formation of homo- and heterodimers, forming multiprotein
complexes that could bind to the DNA and that may be involved in
transcriptional regulation (57). On the other hand, some of the
complexes found exclusively in clone A (If, IIf, and IIg) may be
candidates for negative regulation of EhPgp1 gene
transcription. Complexes IIf and IIg were competed by an
OCT
oligonucleotide using the NEs from sensitive clone
A, suggesting that the presence of a putative OCT transcription factor
could be a repressor when it binds to fragment II in clone A. When the OCT
oligonucleotide competes complex IIIe formed with
NEs from clone C2, the corresponding transcription factor could be
acting as an activator. Another possibility is that two different
members of the OCT family may be acting in different regions with
different sequences.
A combination of positive and negative control regulatory mechanisms is
frequently responsible for the inducible expression of certain genes.
Based on the results obtained, we propose a working model to explain
the regulation of the EhPgp1 gene expression in the
resistant clone C2, which may be mediated by the interaction of
transcription factors with regulatory elements present in the EhPgp1 promoter (Fig. 8). We
postulate the presence of (i) activators in clone C2, which may be
absent, modified or diminished in clone A. These factors may be related
to complexes Ia (C/EBP-like protein) and IIa (OCT-like protein). (ii)
The other possible activator could be a multiprotein complex (IIIc),
which was competed by GATA-1, C/EBP, OCT
,
and HOX sequences. (iii) Repressors in clone A may be absent, modified
or diminished in clone C2. These proteins could be related to complexes
If, IIf, and IIg, which are formed mainly with NEs from clone A. The
existence of other factors participating in the positive or
negative regulation of the EhPgp1 gene cannot be
discarded by the proposed working model. Additionally, detection of a
viable product in clone A by primer extension assays and the low
promoter activity in transfection assays suggest that basal
expression level of the EhPgp1 gene is mediated by
nuclear factors that may be present in different amount in
drug-sensitive and drug-resistant trophozoites. Mutations analysis of
the binding sequences found are currently carried out to define the
precise role of these DNA regions in the EhPgp1 promoter.
Furthermore, the identification of the nuclear proteins involved and
the knowledge of their expression pattern in sensitive and resistant
clones will allow a better understanding of the regulation of the
EhPgp genes.
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ACKNOWLEDGEMENTS |
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We thank Dr. García Carranca for providing the oligonucleotides containing the consensus sequences for the transcription factors. We thank Drs. Joseph DiPaolo, Rima Gharaibeh, and Patricio Gariglio for critical readings of the manuscript. We also thank Francisco Paz for technical assistance.
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FOOTNOTES |
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* This work was supported in part by Consejo Nacional de Ciencia y Tecnologia (CONACyT) (México).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF010402.
Present address: Laboratory of Biology, NCI, National
Institutes of Health, Bethesda, MD 20892.
** International Fellow of Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 52-5-747-7000, ext. 5650; Fax: 52-5-747-7108; E-mail: eorozco{at}gwpat.pat.cinvestav.mx.
1 The abbreviations used are: MDR, multidrug resistance; Pgp, P-glycoprotein; CAT, chloramphenicol acetyltransferase; pBS, pBluescript; bp, base pair(s); PCR, polymerase chain reaction; NE, nuclear extract; Inr, initiator.
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