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INTRODUCTION |
The MDR1 phenotype in
Entamoeba histolytica seems to be a consequence of an
increased expression of the P-glycoprotein, although the protein has
not been detected (1). However, E. histolytica drug-resistant mutants overexpress mdr-like genes
(EhPgp) (2, 3) and common characteristics are shared among
the MDR phenotype of this parasite, Plasmodium falciparum,
Leishmania tarentolae, and mammalian cells (Refs. 4-6 and
reviewed in Ref. 7). As in some mammalian transformed cells, where only
certain mdr genes are expressed, three out of the four
EhPgp genes are transcribed in the drug-resistant
trophozoites (clone C2) (3, 4). While the EhPgp1 and
EhPgp6 genes are transcribed in clone C2 grown in the
absence of the drug, the amount of EhPgp5 transcript
increases according to the emetine concentration (3, 4).
The overproduction of the P-glycoprotein has been proposed to be
mediated mainly by transcription, gene amplification, or both (8).
However, the amplification alone may not be sufficient to activate the
expression of a gene that is normally turned off or transcribed at very
low levels (9). Increased mdr gene transcription precedes
gene amplification in several mouse cell lines (10), and in human
breast cancer and neuroblastoma cell lines (11, 12), supporting that
the regulation of the mdr genes is principally controlled at
the transcriptional level.
As occurs in the EhPgp5 gene, the expression of the mouse
mdr1b gene is induced by the presence of the drug in the
medium. The functional analysis of the mdr1b promoter
demonstrated that three nuclear protein binding sequences from
82 to
59, from
123 to
101, and from
272 to
249, participate in the
mdr1b gene regulation (13). In addition, DNase I
footprinting experiments showed that the site from
272 to
249 is
recognized by the human AP-1 transcription factor, suggesting that this
factor plays a role in the transcriptional activation of the
mdr1b gene. On the other hand, deletion and mutation studies
of the pgp1 promoter in ovarian hamster cells showed that
the AP-1 binding sequence, located near the transcription initiation
site, is required for full promoter activity (14). The nuclear proteins
and sequences involved in transcription regulation of E. histolytica genes have not been identified as yet. However, due to
the high conservation of these proteins through evolution, it is
possible to find similarities between E. histolytica and
mammalian transcription factors. In fact, the TBP, the only
transcription factor cloned in this parasite, has 52% homology to the
DNA-binding domain of the mammalian TBP (15). In this work, to identify
the DNA regions and putative nuclear factors involved in the induced
expression of the EhPgp5 gene, we initiated the functional
and structural characterization of its promoter. By transfection
experiments, we show here that the EhPgp5 promoter isolated
from the drug-resistant clone C2 is active in this mutant, and its
activity increases in trophozoites grown in the presence of emetine,
while it is turned off in the drug-sensitive clone A. Gel shift assays
exhibited interesting differences in DNA-protein interactions of the
EhPgp5 promoter with nuclear proteins from clone C2 grown in
the presence and absence of the drug. Our results suggest that
different transcriptional factors may be participating as positive or
negative regulators of the EhPgp5 gene expression in
clone C2.
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EXPERIMENTAL PROCEDURES |
E. histolytica Cultures--
Trophozoites from clones A and C2
(strain HM1:IMSS) (16) were axenically cultured in TYI-S-33 medium
(17). Clone C2 was drug step selected to 40 (C2(40)) and 225 µM (C2(225) emetine.
Cloning and Sequencing of the EhPgp5 Promoter--
The
EhPgp5 promoter of clone C2 was obtained from the p12
recombinant pBluescript plasmid (pBS) (Stratagene, CA) containing the
first 3300 bp of the EhPgp5 ORF and 1087 bp upstream the ATG start codon (see Fig. 1A). This plasmid was obtained from a
genomic DNA library constructed in
Zap II vector with DNA from the
clone C2 (2). The EhPgp5 promoter of clone A was isolated by
PCR of total DNA using the primers EhPgp5-S30 and EhPgp5-AS36 described below, PCR product was cloned in pBS. As a negative control we used the
EhPgp5-AS36 primer and the reverse primer from pBS. Sequence was
performed with overlapping primers by the dideoxynucleotide chain
termination method (18) using Sequenase version 2.0 DNA polymerase
(U. S. Biochemical Corp.). Comparison of sequence data was carried
out by using the Fasta algorithm (19) in the EMBL and GeneBank
data bases. Identification of putative consensus sequences for
eukaryotic transcription factors were done using the software package
of the University of Wisconsin Genetics Computer Group (GCG)
(20).
Primer Extension--
Assays were carried out using a reverse
transcriptase sequencing kit (Promega, Madison, WI) (21) and an
oligonucleotide (5'-CACTAACCTTTCCTTCTG-3') complementary to nucleotides
+62 to +80. Ten µg of total RNA from clones C2 and C2(225) were
hybridized to the
-end-labeled oligonucleotide (5 × 105 cpm). Annealing was carried out at 47 °C for 25 min
and extension at 42 °C for 30 min with 15 units of avian
myeloblastosis virus reverse transcriptase. The products were separated
on denaturing 8% urea-polyacrylamide gels together with the sequence
reaction using the same primer.
Plasmid Constructions--
To assay the EhPgp5
promoter activity, two constructs were generated using the
promoter-less pBSCAT-ACT reporter vector (46). The 1108- and 259-bp
fragments containing 1084 and 235 bp of the EhPgp5 promoter,
respectively, and 24 bp downstream from the transcription initiation
site, were PCR amplified using the p12 plasmid as template and the
EhPgp5-S30 (5'-AAAACTGCAGTCATCGATTTAATTGAATGA-3') and EhPgp5-AS36
(5'-CCCAAGCTTGTATGCTGGTTCACTTGTCATGCGAAT-3'); and the EhPgp5-S18
(5'-TATAATAACCATTTTGGG-3') and EhPgp5-AS36 oligonucleotides, respectively. The 1108-bp fragment was cloned in PstI and
HindIII sites (p1108Pgp5) and the 259-bp fragment in the
SmaI and HindIII sites (p259Pgp5) into the
pBSCAT-ACT vector (see Fig. 3). As a positive control we used the
pA5'A3'CAT vector containing the actin promoter (22). The orientation
and sequence of each construct were confirmed by DNA sequence analysis
(18).
Transfection and CAT Assays--
Transfection assays were
carried out by the electroporation method as described previously (22).
Electroporated trophozoites were transferred into plastic flasks (Nunc)
containing 30 ml of TYI-S-33 medium and then, incubated at 37 °C for
48 h. CAT activity was determined by the 2-phase diffusion CAT
assay as described (23) after 16 h incubation of trophozoite
extracts (5 µg) with 200 µl of chloramphenicol (1.25 mM) and [14C]butyryl-CoA (NEN Life Science
Products Inc.). Protein concentration was determined by the Bradford
method (24). CAT activities were representative of three independent
experiments performed in duplicate and expressed as counts/min of the
butyrylated chloramphenicol derivatives. The background given by the
trophozoites transfected with the promoter-less pBSCAT-ACT vector was
substracted from results obtained with the plasmids containing the
promoters.
Gel Shift Assays--
Three different fragments of approximately
100 bp each, corresponding to the first 235 bp upstream from the ATG
codon were amplified and labeled by PCR using
[
-32P]dATP, 2 mM cold nucleotides, 50 ng
of the p12 plasmid, and 0.5 unit of Deep Vent DNA polymerase (New
England Biolabs, Beverly, MA), during 28 cycles (94 °C, 30 s;
42 °C, 30 s; and 72 °C, 35 s) in a Perkin Elmer 9600 Thermal Cycler. The oligonucleotides used as primers for each one of
the fragments were: Is (5'-CTCAAACTTTCTAAATTC-3') and Ias
(5'-TGCTGGTTCACTTGTCAT-3') for fragment I, IIs (5'-ATAGTAAATATAAATA-3') and IIas (5'-TACGGAGTTTGAAAG-3') for fragment II, and IIIs
(5'-GAATGAAAGAGAAA-3') and IIIas (5'-AGTATTATCATTTAT-3') for fragment
III. Nuclear extracts (NE) from clones C2(225), C2, and A and gel shift
assays were performed as described (46). Competition assays were done
using a 150-fold excess of the same unlabeled fragments or unlabeled double stranded oligonucleotides containing putative consensus sequences for the following transcription factors: AP-1
(5'-GGGCGTGAGTCATGGGCG-3'), HOX (5'-GTAAGAGTTATTATTGAT-3'),
OCT
(5'-AGCTAATTGCATACTTGGCTTGTAC-3'),
C/EBP
(5'-ATTCAATTGGGCAATCA-3'), CF-1
(5'-AAGAGAAAATGGTCGGGC-3') and TATA box (5'-AATTCTCTATTTAAAGAG-3') or
1.5 µg of poly[d(I-C)] as nonspecific competitor (350-fold molar
excess).
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RESULTS |
Sequence Analysis of the EhPgp5 Promoter--
To analyze the
upstream region of the EhPgp5 gene isolated from clones C2
and A, we selected the p12 plasmid which includes the first 3300 bp of
the ORF and 1087 bp upstream from the ATG start codon and the pBS
plasmid containing an amplified fragment obtained from total DNA of
clone A, respectively (Fig.
1A). Sequence data showed that
the EhPgp5 promoters isolated from both clones were 99.6%
identical, showing only five changes at
3,
250,
354,
500, and
1073 bases (Fig. 1B). The PCR reaction made with the pBS
reverse primer did not amplified any fragment from clone A. The 1087 bp
upstream the ATG start codon are 78% A/T-rich, with different sized
repeats and palindromic regions (underlined in Fig.
1B). The region from
472 to
700 bp has 61% identity to the
325 to
33 region of the E. histolytica EhPgp1
promoter (46) and 59% identity to the
119 to
265 region of the
dynein gene promoter from Dictyostelium
discoideum (25). Comparison of the 5'-flanking sequence with other
E. histolytica genes revealed an 8-bp TATA box-like motif
(TATTTAAA) (26) at
31 nucleotides upstream the transcription
initiation site, which was determined later in this paper (Fig.
1B, boxed sequence).

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Fig. 1.
Nucleotide sequence of the EhPgp5
promoter of E. histolytica clones C2 and A. A, schematic representation of the 4387-bp insert from the p12
plasmid containing 3300 bp of the EhPgp5 ORF and 1087 bp
upstream of the ATG. ATP-bs, ATP-binding sites.
Arrow, marks the transcription initiation site (+1).
B, nucleotide sequence of 1087 bp upstream the ATG. The
entire sequence for the resistant clone C2 promoter is shown. For the
sensitive clone A promoter, the identical nucleotides are marked with a
dash ( ). The nucleotide changes in the sequence are shown
by italic bold letters. A single gap in the clone A promoter
is represented by a slash (/). The translation initiation
codon is in bold. The TATA box (TATTTAAA) consensus sequence
is boxed and the palindromic and repeated sequences are
underlined. The sequence is numbered relative to the
transcription initiation site at position +1 (arrow).
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Transcription Initiation Site of the EhPgp5 Gene--
To find the
5' end of the EhPgp5 mRNA of clone C2, we determined the
transcription initiation site by primer extension assays in
trophozoites grown in the presence and absence of the drug. A single
product mapping at the ATTCG sequence, three bases upstream from ATG,
was detected in mRNA from clone C2(225) (Fig.
2, lane 1). Whereas this
product was not detected in mRNA from trophozoites grown without
the drug, instead, we found a minor primer extension product at 16 bases downstream the ATG, which has no ORF (Fig. 2, lane 2).
These results suggest that EhPgp5 gene expression could be
associated with the accurate selection of the transcription initiation
site.

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Fig. 2.
Transcription initiation site of the
EhPgp5 gene. Primer extension products were analyzed
by electrophoresis alongside sequencing ladder extended with the same
18-bp primer (see "Experimental Procedures"). The two
lanes at the left show the products from the
drug-resistant clones, C2(225) (lane 1) and C2 (lane
2). The transcription start site at the consensus sequence (ATTCG)
is indicated by a solid double arrow, a minor primer
extension product is indicated by a dashed single arrow. Met
indicates the ATG start codon.
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Transient Expression Analysis of the EhPgp5 Promoter--
Two
different plasmids (p1108Pgp5 and p259Pgp5) carrying 1084 and 235 bp of
the EhPgp5 promoter, respectively, and 24 bp downstream from
the transcription initiation site, were constructed to analyze their
functional activity in clones C2(40), C2, and A (Fig.
3). We used clone C2(40) for these
experiments because emetine-resistant trophozoites grown at drug
concentrations higher than 40 µM are very fragile and
most of them died after electroporation. The CAT activity was measured
after 16 h incubation of the trophozoites extracts with the
substrate, because when CAT assays were done after a 2-h incubation,
butyrylated chloramphenicol forms were poorly detected. In contrast,
trophozoites transfected with the p964Pgp1 and p268Pgp1 plasmids,
containing the EhPgp1 promoter of clone C2, expressed enough
CAT enzyme to be detected in the first 2-h incubation (46). The
p1108Pgp5 and p259Pgp5 plasmids were able to drive the expression of
the CAT reporter gene in C2 and C2(40) trophozoites, while no activity
was found when these plasmids were transfected into drug-sensitive
clone A (Fig. 3). Both plasmids showed a higher CAT activity in C2(40)
than in C2 (from 2084 to 3215 and from 2045 to 3854 cpm, respectively)
(Fig. 3). In all experiments, the p259Pgp5 plasmid presented similar activity to the p1108Pgp5 construction, strongly suggesting that the
full promoter activity is found in the first
235 bp. The positive
control carrying the actin promoter (22) showed similar CAT
activity in the three clones, independently if it was measured at
16 h (Fig. 3) or 2 h (data not shown) incubation. Thus, we identified a functional and inducible promoter within the p235Pgp5 plasmid which may control the expression of the EhPgp5 gene
in a drug dependent fashion.

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Fig. 3.
Transient transfection of the EhPgp5
promoter from clone C2. Left, schematic representation
of the relevant features in p1108Pgp5, p259Pgp5, and pA5'A3'CAT
plasmids. The p1108Pgp5 and p259Pgp5 contain +24 bp downstream and
1084 and 235 bp upstream the transcription initiation site of the
EhPgp5 gene, respectively. The pA5'A3'CAT contains 480 bp
of the actin promoter. All plasmids carry the CAT reporter
gene and the 3'-flanking actin region (3'ACT). Restriction sites are: B, BamHI; E,
EcoRI; K, KpnI; H,
HindIII; P, PstI; S,
SmaI; X, XhoI. Right, bars
show CAT activities (cpm) obtained by the two-phase diffusion assays,
after 16 h incubation of the CAT substrate and extracts from
trophozoites transfected with p1108Pgp5, p259Pgp5, or pA5'A3'CAT
(positive control) plasmids. Each bar corresponds to the
average of CAT activities ± S.D., representative of three
independent experiments performed in duplicate. The background given by
the trophozoites transfected with the pBSCAT-ACT was substracted in all
experiments.
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DNA-Protein Interactions on the Proximal
235 bp of the EhPgp5
Promoter--
As the first
235 bp of the promoter seem to contain
the core promoter, we focussed our studies on the structural analysis of this region (Fig. 4). Overlapping DNA
fragments of approximately 100 bp each (fragments I, II, and III) were
PCR amplified using specific oligonucleotides (Fig. 4A) and
DNA-protein interactions were studied by gel shift assays using NE from
clones C2(225), C2, and A. Four main complexes (Ia, Ib, Ic, and Id)
appeared on fragment I incubated with NE from clone C2(225) (Fig.
4B). Complexes Ia, Ic, and Id were also formed with NE from
clones C2 and A, although intensity of complexes varied among them.
Interestingly, complex Ib was strongly detected in clone C2(225), it
was fainter in clone C2 and not detected in clone A, whereas complex Ie
was strongly detected in clone A, diminished in clone C2, and very faint in clone C2(225). Fragment II formed four complexes with NE from
clones C2(225), C2, and A (IIa, IIb, IIc, and IId). NE from clones C2
and A produced complex IIe which varied in intensity from experiment to
experiment and was very faint or absent in clone C2(225) (Fig.
4C). There were no qualitative differences in the complex
formed with NE from clones C2(225), C2, and A on fragment III, although
complexes IIIb and IIIc were reproducibly detected in lower amounts in
clone A (Fig. 4D). All complexes were specifically inhibited
or diminished by a 150-fold molar excess of the same unlabeled
fragments, but were not affected by a 350-fold molar excess of
unspecific DNA. In summary, complex Ib is a candidate to be involved in
the EhPgp5 promoter activation, while complex Ie may be
participating in its repression. The functional role of these complexes
is currently under study by mutation and transfection experiments.

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Fig. 4.
Structure of the EhPgp5 promoter
and nuclear protein binding to fragments I, II, and III. A,
schematic representation of the first 235 bp of the EhPgp5
promoter and the putative consensus binding sequences
(boxes). Arrow indicates the transcription
initiation site. Inverted short arrows indicate sense and
antisense primers used for the PCR of fragments I, II, and III.
B-D, gel shift assays performed with 15 µg of NE from
clones A, C2, and C2(225) and 1 ng of -32P-labeled
fragments I, II, and III as described under "Experimental Procedures." B, fragment I; C, fragment II; and
D, fragment III. Lane 1, free probe; lane
2, no competitor; lane 3, specific competitor (Sc)
(150-fold excess of the homologous cold fragments); lane 4, unspecific competitor (Uc) (350-fold excess of
poly[d(I-C)]). The DNA-protein complexes are indicated by
lowercase letters.
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Competitive Binding Analysis of the Complexes Formed on the EhPgp5
Promoter--
Several sequences described in higher eukaryotes as
consensus for transcription factors were identified in the
EhPgp5 gene promoter isolated from clone C2, although their
function is still unknown (Fig. 4A and Table
I). Experiments were done to ascertain if
the putative binding sequences identified in the first 235 bp of the
EhPgp5 promoter could be associated with the complexes formed with NE from clones C2(225), C2, and A. Double stranded oligonucleotides containing reported consensus sequences for specific transcription factors were used as competitors (Figs.
5-7). The competition experiments of
fragment I were done with an AP-1 oligonucleotide (Fig. 5, C
and D), which competed the complex Ib in clone C2, but not
in clone C2(225). In contrast, the HOX sequence inhibited complexes Ia
and Ib in clones C2 and C2(225). We also observed that in clone A,
complex Ia diminished when it was competed by HOX and AP-1
oligonucleotides (Fig. 5A). Fragment I has two putative HOX
sequences, one is close to the AP-1 site, while the other overlaps with
the TATA box at
31 bp (Fig. 5, C and D), whose sequence differs only in two bases from the consensus HOX
oligonucleotides used for these experiments. To investigate which of
the complexes were formed by an HOX-like protein and which by other
factors interacting with the TATTTAAA box sequence, as could be the
TBP, we carried out competition experiments with 150- and 250-fold molar excess of the TATTTAAA oligonucleotide using NE from clone C2
(Fig. 5B). The oligonucleotide competed the complexes Ia and Ie, but not the complex Ib, which was competed by the HOX sequence, suggesting that complex Ib is formed by an HOX-like protein, while complex Ia and Ie may be formed by a protein related to TBP. The results from competition experiments showed that complex Ib could be
formed by an associated HOX and AP1-like factors in clone C2, reflecting the fact that both sequences are very close in fragment I
(Table I, Fig. 5C). Whereas in clone C2(225) qualitative or quantitative differences in the factors forming complex Ib could exist.

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Fig. 5.
Gel shift competition assays of the
DNA-protein complexes in fragment I. A, gel shift assays
were performed as described in the legend to Fig. 4 in the presence of
different unlabeled competitors: AP-1 and HOX oligonucleotides
(150-fold excess); Uc, unspecific competitor (350-fold
excess). Complexes formed are marked with lowercase letters.
Arrowheads show the complexes competed. B, competition
experiments performed with 15 µg of NE from clone C2 using 150 (+)
and 250 (++)-fold excess of the unlabeled oligonucleotide containing
the TATA binding sequence (TATTTAAA) and 150-fold excess of the Uc.
Arrowheads show the complexes competed. C,
schematic representation of fragment I with the putative consensus binding sequences HOX, AP-1, and TATTTAAA box. Numbers
indicate the base pairs at the 5' and 3' ends of the fragment.
D, HOX, AP-1, and TATA. EhPgp5, represents the
corresponding sequences found in the fragment; below them are the
oligonucleotides used as competitors. Consensus means the
reported consensus sequence for other organisms. Bold
letters in the sequences indicate the identical bases shared by
the oligonucleotides.
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In fragment II, we detected two putative binding sites for a C/EBP
factor, a sequence represented several times in the E. histolytica promoters (Fig. 6,
B and C). However, oligonucleotides containing
the C/EBP
consensus sequences did not eliminate any
complex (Fig. 6A). Fragment III has putative consensus
sequences for C/EBP, HOX, OCT, and CF-1 transcription factors (Fig.
7, B and C), but
oligonucleotides containing C/EBP
, OCT
,
and CF-1 sequences failed to eliminate any of the complexes (Fig.
7A), although in clone C2 a lower amount of complex IIIc was
detected when we used these oligonucleotides. The HOX oligonucleotide competed the complex IIId in experiments carried out with NE from clones A and C2, whereas the competence of this sequence with NE from
clone C2(225) was lower (Fig. 7A), suggesting that there are
qualitative or quantitative differences in the factors forming some of
the complex detected here, especially in complex IIId formed with NE
from clone C2(225).

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Fig. 6.
Gel shift competition assays of the
DNA-protein complexes in fragment II. A, gel shift assays
were performed as described in the legend to Fig. 4 in the presence of
unlabeled competitors: C/EBP , oligonucleotide
sequence represented several times in E. histolytica promoters (150-fold excess); and Uc, unspecific competitor
(350-fold excess). Complexes are marked with lowercase letters.
B, schematic representation of fragment II with the putative
consensus binding sequences. Numbers indicate the base pairs
at the 5' and 3' ends of the fragment. C, C/EBP:
EhPgp5, represents the sequence found in the fragment; below
it is the oligonucleotide used as competitor. Consensus
means the reported C/EBP consensus sequence for other organisms.
Bold letters in the sequence indicate the identical bases
shared by the oligonucleotide; n, represents any
nucleotide.
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Fig. 7.
Gel shift competition assays of the
DNA-protein complexes in fragment III. A, gel shift assays
were performed as described in the legend to Fig. 4 in the presence of
different unlabeled competitors: C/EBP , CF-1, HOX, and
OCT oligonucleotides (150-fold excess); and
Uc, unspecific competitor (350-fold excess).
Arrowheads show the complexes competed. The complexes formed
are indicated with lowercase letters. B, schematic representation of fragment III with the putative consensus binding sequences. Numbers indicate the base pairs at the 5' and 3'
ends of the fragment. C, C/EBP, CF-1, OCT, and HOX:
EhPgp5, represents the corresponding sequences found in the
fragment; below them are the oligonucleotides used as competitors.
Consensus means the reported consensus sequence for other
organisms. Bold letters in the sequence indicate the
identical bases shared by the oligonucleotides; n,
represents any nucleotide.
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DISCUSSION |
Factors involved in the EhPgp5 gene expression are
important for understanding MDR in E. histolytica.
Transcriptional analysis of the EhPgp5 promoter of clone C2
was undertaken in this paper to study elements that may play a role in
the differential expression of this gene. The EhPgp5
promoter was 99.6% identical in the sensitive clone A and in the
drug-resistant clone C2, and although one of the changes (A-G) was at
the first base of the consensus sequence of the transcription
initiation site, it could be not relevant in the promoter activity,
because the presence of a G has been reported in the same position for
the transcription initiation site motif in other E. histolytica genes (35). In contrast to the constitutively
expressed EhPgp1 gene, which does not contain a TATA box and
has several transcription initiation sites (46), the EhPgp5
gene expression is induced by the presence of emetine in the medium,
and it has the E. histolytica reported TATA-box like motif
at
31 bp (TATTTAAA) which may confer tighter regulation on the
selection of the transcription initiation at the ATTCG motif. Primer
extension showed that although no transcripts were detected in clone C2
by Northern blot assays (3, 4), a minor product which has no ORF was
evident, suggesting that the EhPgp5 gene could be
transcribed in an unfunctional product at a low amount in the absence
of drug. The presence of functional transcripts with a unique
initiation site mapping at sequence ATTCG correlates with
EhPgp5 gene overexpression in trophozoites grown in 225 µM emetine.
The p1108Pgp5 and p259Pgp5 plasmids, containing two different fragments
of the EhPgp5 promoter, drove the CAT gene expression in
trophozoites of clones C2 and C2(40), while no activity was detected in
clone A. Full promoter activity was kept in the p259Pgp5 plasmid that
was higher in the presence of emetine in the culture medium, strongly
suggesting that the minimal promoter and important drug responsive
elements could be located in this region. Similar CAT activity in the
sensitive- and drug-resistant clones was observed in extracts from
trophozoites transfected with the plasmid containing the
actin promoter, showing that the drug response was specific for the EhPgp5 promoter. Interestingly, at a low drug
concentrations, the EhPgp1 promoter activity was stronger
(46) than that presented by the EhPgp5 promoter, evidencing
the differences between the constitutively expressed actin
and EhPgp1 genes with the inducible expressed
EhPgp5 gene. Our previous results indicated that the amount
of the EhPgp5 transcript is much lower in trophozoites grown
in 40 µM emetine than in those grown in 225 µM (3, 4). Thus, we could expect a much stronger activity
when we will be able to efficiently transfect the trophozoites of clone
C2(225). The high identity between the EhPgp5 promoters of
clones C2 and A together with the transfection and gel shift
experiments strongly suggest that the expression of the
EhPgp5 gene in clone C2(225) is regulated by transcriptional
factors induced by the presence of emetine. This assumption is
supported by the fact that trophozoites of clone C2(225) revert to the
phenotype of clone C2 when they are cultured for a short period in the
absence of emetine, conditions in which the EhPgp5 gene is
poorly expressed, and its high resistance is recovered when
trophozoites are step cultured in increasing concentrations of the
drug. Additionally, the EhPgp5 promoter isolated
from clone C2 was unable to drive CAT activity in the sensitive clone
A.
Transcriptional regulation of mdr gene promoters in
mammalian cells is mediated by the interaction between specific nuclear factors and regulatory sequences present in the promoter region (36-40). In vitro studies with the human MDR1
promoter revealed that sequences located a few hundred base pairs
relative to the transcription initiation site influence mdr
gene transcription (41-43) and that the AP-1 factor plays an important
role in the positive regulation of the hamster pgp1 gene
(14). In the DNA-protein interactions on the first
49 bp, complex Ib
was competed by AP-1 and HOX oligonucleotides in clone C2, suggesting
that an associated protein complex constituted by HOX and AP-1-like
factors could be interacting with the DNA. In contrast, it is possible
that in clone C2(225), the AP-1 like factor could be synthesized in a
higher amount, and therefore it could not be competed by the concentration of the AP-1 oligonucleotide used in our experiments. It
is also possible that another unidentified factor could be forming
complex Ib in clone C2(225). Additional assays are in progress to
precisely identify and characterize factors forming complex Ib in
clones C2 and C2(225). We postulate that AP-1-like factor is present in
the NE of E. histolytica, because heterologous mammalian
antibodies against c-Jun and c-Fos recognized two 39- and 55-kDa bands
in Western blot experiments of E. histolytica proteins (data
not shown).
Based on the functional assays and DNA-protein interactions, we propose
a working model to explain a possible mechanism for the
EhPgp5 induced expression (Fig.
8): (i) complex Ib, which seems to be
formed by HOX and AP-1-like factors in clone C2 and by HOX and an
unidentified factor in clone C2(225), may be involved in the
up-regulation of the promoter activity. An AP-1 motif located near the
transcription initiation site in the human MDR1, mouse mdr1a, and hamster pgp1 gene promoters, is
involved in up-regulation of these genes (36). On the other hand, an
HOX-like protein, which recognized the ATATTAA motif has been
implicated in the developmental-specific activation of the
-globin
gene by promoting specific protein-protein interactions between factors
bound to the promoter region (44). (ii) Complexes IIIb and IIIc are
augmented in the trophozoites of clone C2(225) and could also be
proposed as candidates for the positive regulation of EhPgp5
gene expression. These complexes were always detected in a lower amount
in clones A and C2 grown without the drug. It is possible that drug
pressure provokes the overproduction of certain transcription factors
in clone C2(225) or the modification of pre-existing proteins, or both,
facilitating their DNA binding. It has been postulated in the
transcriptional regulation of the mouse mdr1b gene that
certain factors present in the drug-sensitive cells are activated or
modified by the drug pressure (13). (iii) An unidentified factor
forming complex Ie in clones A and C2 could be acting as a negative
regulator of the EhPgp5 gene transcription. Complex Ie was
competed (together with the complex Ia) by the TATA box
oligonucleotide, thus, it is possible that factors forming this complex
may interfere directly or indirectly with the TBP interaction at the
TATTTAAA sequence, provoking loss of promoter activity in clone A and
the wrong selection of the initiation site in clone C2, in comparison
with clone C2(225) in which complex Ie was poorly detected. This is
supported by the null or very low CAT activities showed by transfected
trophozoites of clones A, C2, and also by the primer extension
experiments, which detected an EhPgp5 transcript with no ORF
in the clone C2. However, more experiments are necessary to elucidate
the biological significance of these interactions and factors involved
in the complex formation. The expression of mdr genes in rat
is negatively controlled by an unidentified transcriptional repressor,
present in the sensitive cells (45). We do not know yet the identity of
factors forming complexes Ie, IIIb, and IIIc and our model does not
discard the participation of other unidentified factors in the
regulation of EhPgp5 gene expression. However, the findings presented in this paper provide the first basis for the identification and characterization of trans-acting factors and cis-acting elements mediating the EhPgp5 gene regulation. Further functional and
biochemical studies are currently in progress to define the role of
these regions and their corresponding factors.

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Fig. 8.
Working model proposing putative
transcription factors involved in the EhPgp5 gene
expression regulation. Complex Ib, formed by a putative HOX-like
and an unidentified factor in clone C2(225), as well as complex IIIb
and IIIc may be in abundance and be recruited to the EhPgp5
promoter in clone C2(225). These proteins could recognize the promoter
or basal factors of the transcription preinitiation complex
(TPC) and mediate the transcriptional activation of the
gene. In clone C2, complexes Ib, IIIb, and IIIc are in lower amount,
while complex Ie, a putative negative regulator may interact directly
or indirectly with the TATTTAAA sequence. Complex Ie could provoke
decrease of transcriptional activation and the wrong selection of the
initiation site. In sensitive clone A, the putative repressor (complex
Ie) was strongly detected, while complexes Ib, IIIb, and IIIc (putative
activators) were poorly found. Arrows represent the
transcription initiation sites. Undulated arrows represent
the EhPgp5 transcripts in clones C2(225), C2, and A.
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We thank Dr. García Carranca for
providing the oligonucleotides containing the consensus sequences for
the transcription factors. We also thank Francisco Paz for technical
assistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF010401.