From the Departments of Internal Medicine,
§ Microbiology, and ¶ Pathology, University of
Virginia, Charlottesville, Virginia 22908
Received for publication, November 4, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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The Entamoeba histolytica
upstream regulatory element 3-binding protein (URE3-BP) binds to the
URE3 sequence of the Gal/GalNAc-inhibitable lectin
hgl5 and ferredoxin 1 (fdx) gene
promoters. This binding can be inhibited in vitro by
addition of calcium. Two EF-hand motifs, which are associated with the
ability to bind calcium, are present in the amino acid sequence of
URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in
the loss of calcium inhibition of DNA binding as monitored by
electrophoretic mobility shift assay. Chromatin immunoprecipitation
assays revealed that URE3-BP was physically bound to the
hgl5 and fdx promoters in vivo.
Parasite intracellular calcium concentrations were altered by changes
in extracellular calcium. Promoter occupancy was lost when
intracellular calcium levels were increased by coordinate increases in
extracellular calcium. Increased intracellular calcium also resulted in
decreased levels of URE3-BP mRNA. Together these results
demonstrate that changes in extracellular calcium result in changes in
URE3-BP mRNA and in the ability of URE3-BP to bind to
URE3-containing promoters. Modulation of URE3-BP by calcium may
represent an important mechanism of control of gene expression in
E. histolytica.
The early branching eukaryote Entamoeba histolytica is
a human parasite that is the etiologic agent of amebic dysentery and liver abscess. Only one of every 10 infections leads to disease (1),
and the parasite and host factors that control the outcome of infection
are not well understood. Alteration in transcriptional control of
certain crucial genes may contribute to the expression of a virulence
phenotype. Padilla-Vaca et al. (1) demonstrated that
co-cultivation of E. histolytica with Escherichia
coli O55 resulted in increased virulence and a decrease in the
expression of the Gal/GalNAc-inhibitable lectin
light subunit. Ramakrishnan et al. (2) have shown
alterations in the hgl genes transcribed in trophozoites
derived from liver abscesses compared with those transcribed in
established cell cultures. Bruchhaus et al. (3) have shown,
in similar work, changes in the expression of over 55 other E. histolytica transcripts.
There has been considerable divergence in the mechanisms of
transcription of the early branching E. histolytica from
later branching eukaryotes such as Homo sapiens and
Saccharomyces cerevisiae. For instance the E. histolytica core promoter for protein encoding genes
consists of a novel GAAC element, in addition to a TATA and
INR (4-6) and contains short regulatory 5' and 3' sequences (7). The RNA polymerase II is also unusual for a eukaryote in that it
is resistant to A yeast-one-hybrid screen of an E. histolytica cDNA
library using the URE3 element as "bait" identified the
URE3-binding protein (URE3-BP) (14). Analysis of the URE3-BP amino acid
sequence did not identify any canonical DNA binding motifs but did
reveal the presence of two EF-hand motifs, which are correlated with the ability of a protein to bind to calcium (15). This suggested that
the URE3-BP protein might function as both a calcium sensor and a
transcription factor. The only other reported sequence-specific DNA-binding protein known to contain EF-hand motifs is DREAM. DREAM is
a human neuronal protein (also known as Calsenilin and KChIP3) that
functions both as a transcription factor (16, 17) and as a calcium
sensor (16-20). DREAM contains four EF-hand sequence motifs.
Typically, the binding of Ca2+ to EF-hands induces
structural changes that alter the function of the protein. In the case
of DREAM, binding of Ca2+ to its EF-hands leads to a
reduced affinity for sequence-specific binding to its target downstream
regulatory element sequence (16).
Previous analysis of the recombinant URE3-BP protein sequence-specific
DNA binding by electrophoretic mobility shift assay (EMSA) showed that
recognition of a URE3-containing oligonucleotide was blocked in
vitro by calcium (14). In this report, we tested the in
vivo role of the EF-hand motifs of URE3-BP in protein-promoter interactions.
Cultivation of E. histolytica--
E.
histolytica strain HM1:IMSS trophozoites were grown at
37 °C in TYI-S-33 medium containing penicillin (100 units/ml)
(Invitrogen) and streptomycin (100 µg/ml) (Invitrogen) (21).
In experiments examining the effect of calcium on transcription,
E. histolytica was seeded at a concentration of 2-4 × 104/ml 18 h prior to the addition of medium
containing 5 mM MgCl2 and 5 mM EDTA
to sequester the serum calcium (1.4-2 mM Ca2+)
calculated using the dissociation constants of the chelating ligands
and the CaLBuf Program (ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip (22). The resulting calculated free calcium was in the order of 3-5
µM. The addition of 5.4 mM CaCl2
raised Ca2+ to 1 mM. Cells were harvested at
the time points indicated under "Results."
RNA Isolation--
Approximately ~2.4-5 × 105 E. histolytica were lysed by the addition of
0.8 ml of TRIzol reagent (Invitrogen), and total RNA was purified
according to the manufacturer's directions. RNA of greater than 200 nucleotides in length was isolated from total RNA by the RNeasy
protocol (Qiagen) after first treating the total RNA preparation with
DNase I (Roche Molecular Biochemicals) to remove contaminating genomic DNA.
Cloning and Expression of His6-(wt)URE3-BP and
His6-EF(2)mutURE3-BP--
The wild-type URE3-BP
His6-tagged fusion protein expression vector was
constructed as described previously (14). Mutagenesis of the EF-hand
motif (2) of URE3-BP was done by a two-stage PCR procedure. The 3'
sequences of URE3-BP were amplified and mutated using the
oligonucleotides GTTATGAACGCTAGAGCTAGAAGTG and AAATGTCGACTTATTCCAAGAGGGAAGTAACAACG that replaced the conserved first
and third amino acids of the EF-hand motif with two alanine residues.
The resulting PCR product was denatured and used along with an
oligonucleotide AAAAGCTCTTCAAACATCA-ACCACCTGTAGCTAATTTC to amplify the
mutated copy of URE3-BP. This DNA was then subcloned into the
pCRT7/NT-TOPO expression vector (Invitrogen). The mutated gene was
sequenced to confirm the presence of the desired mutation. The
expression of the His6-tagged fusion protein was induced in E. coli BL21(DE3) (F Sequence and Sequence Analysis Software--
The upstream and
downstream non-coding sequences of URE3-BP were analyzed using the
Wisconsin Package Version 10.0, Genetics Computer Group (GCG) software
and the Basic Local Alignment Search Tool (BLAST) at NCBI (23, 24).
Sequence data pertaining to the URE3-BP genomic context were produced
by the TIGR Entamoeba histolytica Genome Project
(www.tigr.org/tdb/e2k1/eha1/) and the Pathogen Sequencing
Unit at the Sanger Institute
(www.sanger.ac.uk/Projects/E_histolytica/blast_server.shtml), which
are both part of the International Entamoeba Genome
Sequencing Project. These sequences were obtained from
ftp.sanger.ac.uk/pub/pathogens/E_histolytica/and the GSS division
of GenBankTM. The University of Virginia Biomolecular
Research Facility performed all other sequencing.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assays with oligonucleotides hgl5-URE3 and
hgl5-MUT were performed as described previously (10).
To create the radiolabeled hgl5-URE3 and
hgl5-MUT probes complementary oligonucleotides were annealed
and then labeled with the large DNA polymerase I subunit (Klenow) and
[ Immunodetection--
Mice were immunized as described previously
(14), hybridoma cell lines producing anti-URE3-BP mAb were prepared,
and monoclonal antibodies against URE3-BP (4D6 and 3E6) were produced.
Protein samples of interest were electrophoresed on a 12%
SDS-polyacrylamide gel, transferred to a polyvinylidene
difluoride membrane (Millipore) and incubated for 1 h at
room temperature in 5% nonfat dry milk in blot wash buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 0.1% Tween 20).
The blot was then incubated overnight in 2% nonfat dry milk in blot
wash buffer containing 10 µg/ml of mAb 4D6 and 5 µg/ml of mAb 3E6.
After three 5-min washes in 2% nonfat dry milk in blot wash buffer,
the membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) antibody (Sigma) at a dilution of 1:1500. The secondary antibody was detected using the ECL Western blotting detection system according to the manufacturer's directions (Amersham Biosciences) and was visualized by
exposure of the blot to BioMax MR-1 film (Eastman Kodak Co.).
Calcium Calibration--
Amebae were prepared and labeled with
the acetoxy-methyl ester of the Ca2+-sensitive fluorescent
dye indo-1 (indo/AM; Molecular Probes) as described by Carbajal
et al. (25) with the following modifications. Labeling was
performed in a buffer of 10 mM Hepes-HCl, pH 7.2, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 10 mM glucose, 0.1% bovine serum albumin, and 0.2% pluronic
F-127 (Molecular Probes) buffer containing 26.7 µM
indo-1/AM. The acetoxymethyl ester of indo-1 passively diffuses
across cell membranes, and once inside the cell this ester is cleaved
by intracellular esterases to release indo-1. This dye is a
cell-impermeable and UV light-excitable fluorescent Ca2+
indicator. Trophozoites were incubated in the loading buffer at a
concentration of 1 × 106 amebae per ml for 30 min at
37 °C followed by 30 min at room temperature. After incubation
amebae were washed as described (25) and then suspended at a
concentration of 2 × 105 amebae per ml in a modified
buffer, Buffer B (20 mM Hepes-HCl, pH 7.2, 140 mM NaCl, 5 mM EDTA, 5 mM
MgCl2) containing MgCl2 and EDTA to generate a
store of sequestrated calcium. Before measuring [Ca2+]i cells were warmed to
37 °C. Changes in [Ca2+]i after
the addition of CaCl2 were monitored in an SLM 8100C
spectrofluorometer (SLM/Aminco) using methods published previously
(26-28). CaCl2 was added as calculated by the CalBuf Program of Droogmans (22) to give external free calcium concentrations of 7 µM free Ca2+ (1.2 mM
CaCl2), 608 µM Ca2+ (5 mM CaCl2), and 1.1 mM
Ca2+ (5.7 mM CaCl2). After analysis
amebae were pelleted, and the fluorescence of the supernatant was
measured to control for contributions to the fluorescence measurements
from cell debris.
Immunoprecipitation and Chromatin Immunoprecipitation--
The
mouse anti-URE3-BP mAb 4D6 (described above) or a control mAb of the
same isotype (anti-lectin mAb 7F4) was bound to protein G Dynabeads
(Dynal). They were then used to immunoprecipitate chromatin from amebic
nuclear extracts prepared by a modification of methods described
previously (10). Briefly, nuclei were harvested from amebae that had
been cross-linked in 1% formaldehyde in either phosphate-buffered
saline or Buffer B + 1 mM Ca2+ (5.7 mM CaCl2). Nuclear extracts were prepared from
1 × 108 amebae preincubated in Buffer A (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 2 mM
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane, and 2 mM 4-(2-aminoethyl) benzenesulfonylfluoride,
HCl) for 20 min prior to sedimentation (900 × g
for 5 min) and resuspension in Buffer A containing 6% Nonidet P-40.
They were then spun through a Qiashredder (Qiagen) for 1 min and then
left in Buffer A containing 6% Nonidet P-40 for 5 min at room
temperature before four volumes of Buffer A were added. The nuclei were
collected as described previously (14) and suspended in an equal volume
of 20 mM Hepes-HCl, pH 7.9, 0.42 M NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane, and 2 mM 4-(2-aminoethyl) benzenesulfonylfluoride HCl. The
nuclei were sonicated for 10 s and then centrifuged (10,000 × g, 15 min). The supernatant was passed three times
through a 301/2-gauge needle to sheer DNA into
~100-800-bp fragments. Two volumes of Buffer A were added before the
sample was split into two equal volumes for the anti-URE3-BP and
control-mAb protein G Dynabead immunoprecipitation. After
immunoprecipitation and DNA purification, the samples were resuspended
in 100 µl of 10 mM Tris-HCl, pH 8.5, and 5 µl was analyzed by real time PCR.
Real Time Quantitative PCR--
Real time quantitative PCR
analysis of the hgl5 and fdx promoters and
srehp coding region was preformed in a Bio-Rad iCycler. The
fluorescent dye SYBR Green I (Molecular Probes) was used to detect double strand DNA. Continuous SYBR Green I monitoring during amplification was done according to the manufacturer's
recommendations. The 5 µl of the immunoprecipitated DNA was subjected
to 40 amplification cycles with Qiagen's HotStar Taq. To
amplify
Reverse transcription followed by real time PCR was implemented to
quantitate the calcium-dependant expression of fdx and URE3-BP genes and normalized to the level of the control
transcript, L10. The reverse transcription was primed using
random sequence hexamers and performed with the Superscript II enzyme
(Invitrogen). Real time amplification was preformed as described above.
To amplify the region between +3 to +151 of the URE3-BP
cDNA the oligonucleotides AAAAGATCTATGGGATAGTGTTAAGTAATGGGAACC and
AAAgcggaagcgatatccaccaatgcaaccacc were used. The ferredoxin
1 cDNA was amplified using the oligonucleotides TGATGACTGTGTCGCTTGC and ACTTATTCAACTTTAAGAACTCC, which amplify the DNA
between the nucleotides +45 to +200 of the coding sequences (31). The
region of the L10 cDNA +6 to +72 amino acids was
amplified via primers CTACTGGGATCCAGGAAGATGTTATAGACTTG and
CTACTGGAATTCTTAATTGAATACGTGCTGC. In all experiments utilizing real time
PCR the cycle threshold values (CT; the cycle number at
which fluorescence exceeds the threshold value) were linked to the
quantity of initial DNA after calibration of the amplification
efficiency of the primer pair utilized (32).
Mutation of the EF-hand Motif in URE3-BP Abrogates Calcium
Inhibition of in Vitro DNA Binding--
Recognition of URE3 by URE3-BP
is blocked in the presence of calcium, as measured by in
vitro EMSA. URE3-BP contains two EF-hand motifs. We mutated the
second EF motif to test its role in both DNA binding and calcium
sensitivity. The Prosite consensus pattern of the EF-hand domain
(PDOC00018) consists of 36 amino acids, with a twelve-residue loop,
Dx-[DNS]-[ILVFYW]-[DENSTG]-[DNQGHRK]-[GP]-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW], flanked on both sides by twelve-residue
The resulting mutant protein was tested in an EMSA assay (Fig.
1). The mutated URE3-BP protein was able
to form a protein-DNA complex with a radiolabeled double stranded
hgl5-URE3 oligonucleotide and was competed by the addition
of unlabeled double stranded oligonucleotide hgl5-URE3 (Fig.
1B). The DNA-protein complex was also inhibited to a lesser
extent by the addition of hgl5-MUT, a double stranded DNA
oligonucleotide, in which the URE3 DNA sequence was mutated to
TTAGAATTC from the wild-type sequence of TTATCTTAT. The partial
inhibition of the protein-DNA complex with hgl5-MUT suggested some loss of specificity for DNA binding of the mutated URE3-BP. Strikingly the addition of 86 µM free calcium
had no effect on the ability of the mutated URE3-BP to form a
DNA-protein complex (Fig. 1B). These experiments
demonstrated that EF(2)mutURE3-BP was no longer sensitive to
Ca2+ inhibition.
Detection of in Vivo Promoter Occupation of
URE3-BP--
ChIP assays allow study of the interaction between
nuclear proteins and DNA sequences in the context of the chromatin
template (33). This technique was used to test whether nuclear URE3-BP was physically associated with the hgl5 gene promoter in the
chromosome. Trophozoites were treated with formaldehyde to cross-link
nuclear proteins with chromosomal DNA. DNA-protein complexes were then purified by immunoprecipitation. The cross-links were reversed, and
associated DNA was purified. Detection and measurement of the
precipitated DNA was achieved by use of real time PCR (9). Figs.
2 and 3
show representative ChIP experiments in which DNA purified by
immunoprecipitation of cross-linked nuclear DNA-protein complexes with
anti-URE3-BP mAb was amplified by real time PCR for the hgl5
and fdx promoters. The graphs in Fig. 2A and Fig. 3A show the calibration of the CT value of the
primer pairs used to amplify the hgl5 and fdx E. histolytica promoters against DNA concentration. Each experiment
was controlled with a ChIP using an irrelevant mAb. An additional
control was PCR amplification of an irrelevant gene segment (the
srehp coding sequence). There was no significant difference
observed between the CT for the srehp DNA
amplified from the control or anti-URE3-BP mAb ChIPs (data not shown).
Anti-URE3-BP mAb specifically immunoprecipitated DNA containing the
hgl5 and fdx E. histolytica promoters (see Figs.
2 and 3). This was consistent with URE3-BP being physically associated
with the DNA-chromatin complex at those promoters (see Figs. 2 and
3).
Manipulation of Intracellular Calcium in E. histolytica
Trophozoites--
Because of the importance of calcium in the
sequence-specific recognition of URE3 by URE3-BP, we wished to test the
effect of intracellular calcium on promoter binding by URE3-BP. We
estimated the level of intracellular calcium using the fluorescent dye
indo-1. Trophozoites were suspended in Buffer B (25), which contained 5 mM MgCl2 and EDTA to sequestrate calcium. The
baseline internal Ca2+ was measured by the fluorescence
ratio (398/480 nm) after excitation at 360 nm. The effects of altering
the external [Ca2+]o
concentration on internal Ca2+ was followed after the
addition of 1.2 mM CaCl2 (7 µM
[Ca2+]o), 5 mM
CaCl2 (608 µM
[Ca2+]o), and 5.7 mM
CaCl2 (1.1 mM
[Ca2+]o) (27). A rapid and
consistent increase in internal Ca2+ was observed (as
measured by the ratio of indo-1 fluorescence emission at 398/480 nm)
when the external Ca2+ concentration was increased to 608 µM or 1.1 mM (Fig.
4). The change in indo-1 fluorescence
induced by increases in extracellular calcium was not because of
leakage of the dye from the trophozoites, as indo-1 fluorescence was
not detected in the cell-free supernatant (data not shown). This
indicates that in these conditions, changes in extracellular calcium
resulted in alterations of intracellular calcium in the amebic
trophozoites.
Changes in URE3-BP Occupancy of the hgl5 and fdx Promoters in Vivo
upon Alteration of Calcium Levels--
The in vitro EMSA
experiments (Fig. 1A) demonstrated that the
sequence-specific interaction of URE3-BP with URE3 was blocked by
calcium. To test the importance of calcium for URE3-BP binding to the
promoter in vivo we performed ChIP after modulating
intracellular calcium. Trophozoites were suspended in
Buffer B + 5.7 mM CaCl2 (1.1 mM [Ca2+]o) before
formaldehyde treatment, ChIP, and real
time PCR analyses. As demonstrated in Figs. 5 and
6 the increase in intracellular calcium
(caused by the addition of 5.7 mM extracellular calcium to
the trophozoites) resulted in inhibition of URE3-BP binding to both the
hgl5 and fdx promoters as measured by ChIP and
real time PCR. The failure to immunoprecipitate the hgl5 and fdx promoters with the anti-URE3-BP mAb was not because of
calcium-mediated interference in immunoprecipitation of the URE3-BP, as
similar amounts of the protein were immunoprecipitated under both
conditions (Fig. 6A). We therefore concluded that in the
presence of elevated intracellular calcium URE3-BP was unable
to occupy promoters that contained the URE3 DNA motif.
URE3-BP Genomic Sequence Context--
The URE3-BP cDNA
sequence was used to identify the equivalent genomic sequence in the
International Entamoeba Genome Sequencing Project data bases
(Fig. 7). A URE3 DNA consensus sequence
was present in the 3' sequence 1.3 kb distal to the URE3-BP gene stop codon. We have previously found functional regulatory sequences for
other Entamoeba genes more than 1 kb from the open reading frame.3 There were no
differences in genomic and cDNA sequences, which was not
surprising, because most (about 80-90%) E. histolytica genes lack introns. A sequence (ATTCG) that strongly resembled the INR
consensus ATTCA was located immediately before the cDNA start site
(7). The sequence GAACT, which is identical to the GAAC consensus
(GAACT), was located Calcium Effect on fdx and URE3-BP mRNA--
To investigate the
role of the URE3 in the transcriptosome's response to calcium in cell
culture the levels of fdx, URE3-BP and L10
ribosomal protein mRNA were followed by quantitative reverse transcriptase PCR (Fig. 8). The promoters
of fdx and 3' sequences of URE3-BP genes contain
the URE3 motif whereas the putative promoter sequences of L10 do not.
The L10 mRNA was utilized as a control transcript. The ratio of
fdx and the L10 mRNA transcripts isolated from trophozoites in 1 mM Ca2+ or
Ca2+-depleted medium showed no consistent
statistically significant variation. The URE3-BP transcript,
however, markedly decreased in 1 mM Ca2+. The
URE3-BP mRNA calcium:calcium-depleted ratio was
statistically significantly different (p This work demonstrates that URE3-BP is a calcium-binding protein
that can function as both a transcription factor and calcium sensor in
E. histolytica. Only one other calcium binding eukaryotic transcription factor, DREAM, has been described. DREAM (also known as
Calsenilin or KChIP3) also contains EF-hand motifs and is similar to
URE3-BP in that it exhibits sequence-specific binding to DNA. An
increase in calcium levels in vitro blocks DNA binding by
DREAM and URE3-BP. The mutation of EF(2) of URE3-BP had little effect on DNA binding but impeded the ability of Ca2+ to block
in vitro DNA binding to URE3. This showed that the second EF-hand motif of URE3-BP was essential for the calcium modulation of
URE3-BP DNA binding.
Promoter occupancy of URE3-BP in vivo on the native
chromatin of URE3-BP of the fdx and hgl5
promoters was tested in this work. To determine whether URE3-BP
interacted with URE3-containing promoters in the nucleus we utilized
the ChIP assay in conjunction with real time PCR. This allowed
measurement of URE3-BP bound to URE3-containing promoters in intact
trophozoites. Our results indicated that URE3-BP was located on both
the fdx and hgl5 promoters in the nuclear environment.
Internal changes in [Ca2+]i have
been observed in response to various stimuli (25, 34, 35), and
several important calcium-binding proteins have been discovered in
E. histolytica including a protein similar to calmodulin,
EhCaBP (36, 37). In addition, the multidrug resistance
phenotype mediated by the up-regulation of the EhPgp1 and
EhPgp5 genes of E. histolytica has been shown to
be reversed by calcium-channel blocker verapamil (38-41). To
investigate the role of URE3-BP as a calcium sensor protein we
investigated the impact of altering
[Ca2+]i on URE3-BP promoter
occupancy. A significant decrease in URE3-BP location at both the
hgl5 and fdx promoters was observed upon an
increase in [Ca2+]i. Therefore,
calcium not only prevented in vitro binding of URE3-BP to
URE3 but also blocked the occupancy of URE3-containing promoters by
URE3-BP in vivo.
Because calcium levels influenced the binding of URE3-BP to the
URE3-containing promoters the effect of calcium on the steady state
levels of fdx and URE3-BP mRNA was examined.
The hgl5 mRNA was not analyzed, because it was not
possible to design PCR primers that would amplify hgl5
mRNA without cross-hybridizing to other hgl genes that
lack URE3-containing promoters (2). In the case of the fdx
gene, its transcript was not significantly changed by increases in
intracellular calcium. This perhaps was not surprising as changes in
intracellular calcium would be predicted to have pluripotent effects on
the trophozoite that could have disparate effects on fdx
mRNA abundance.
In contrast to the situation for fdx mRNA, the addition
of calcium sharply decreased the level of the URE3-BP mRNA. This
decrease could be mediated by an autofeedback mechanism where URE3-BP
is displaced by calcium from the URE3 motif located at the 3' sequence of URE3-BP. Mutational analysis of the URE3 motif in the 3'
noncoding region of URE-3BP will be required to test directly whether
the gene is directly autoregulated, although one might expect, as is
the case for the fdx gene, that URE-3BP will fall under
complex regulatory responses to changes in intracellular calcium. In
either case these results indicate that increases in intracellular
calcium not only decrease the ability of URE-3BP to bind DNA but also decrease its mRNA abundance. This suggests that URE3-BP is both an
effector and a responder in a cascade of calcium-regulated gene
expression in the parasite.
One of the mysteries of amebiasis is why only a minority of infected
individuals develop invasive disease. E. histolytica encounters differing levels of extracellular calcium in the human intestine where it lives. We have shown here that intracellular calcium
levels in the parasite are exquisitely sensitive to changes in
extracellular calcium. The extracellular calcium concentration in the
intestinal lumen is influenced by the amount of calcium ingested,
absorbed, and excreted. In the small intestine where the parasite
excysts, luminal calcium concentrations are well above 1 mM (42). E. histolytica trophozoites colonizing
the large intestine are exposed to extracellular calcium levels that vary depending upon the diet (13, 42). In hosts with a high calcium
diet trophozoites would be exposed to >1.25 mM calcium. We
would predict from our data that these calcium levels would be
sufficient to block URE3-BP binding and decrease URE3-BP synthesis. In
a low calcium diet, luminal calcium concentration may drop to ~1.25
mM. In this circumstance calcium absorption in the large intestine is considerably enhanced, and free Ca2+ will be
further decreased (42). URE3-BP-mediated gene regulation would then
play a significant role in the regulation of transcription. It is
possible that calcium and calcium sensors including URE3-BP influence
the expression of the virulence phenotype of E. histolytica in the large intestine and impact the development of disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amanitin (8). To increase our understanding of
transcriptional regulation in this organism we investigated the
mechanisms of transcriptional control of a well characterized virulence
protein, the galactose- and
N-acetyl-D-galactosamine-inhibitable lectin
(Gal/GalNAc-inhibitable lectin), which is essential for parasite
adherence and contact-mediated cytolysis. The promoter for one of the
genes encoding the lectin heavy subunit (hgl5) contains five
major regulatory regions (upstream regulatory elements 1-5
(URE1-5))1 upstream of the
core promoter (4) and a GAAC sequence motif that predominantly
influences the rate and site of transcription initiation (4-6). The
hgl5 UREs differ in whether they regulate transcription via
the TATA or the GAAC elements, with the URE3 exerting its effect on
mRNA transcription via GAAC (9). Interestingly, mutation of the
URE3 motif in the hgl5 promoter leads to an increase in
transcription and in the fdx promoter decreased promoter
strength (10). This indicates that, as is the case for the mouse
context-dependant Pax-5 transcription factor, the URE3 sequence can
mediate both positive and negative control in different milieu (11).
Two different genes encode the ferredoxin proteins of E. histolytica. The fdx URE3-containing promoter regulates
the gene that encodes the ferredoxin 1 protein. This transcript is
down-regulated in metronidazole-resistant amebae, in contrast with the
level of the ferredoxin 2 transcript (fd2), which
is unaltered (12). Bruchhaus et al. (3) discovered that
fd2 mRNA is one of the transcripts up-regulated in ameba
cultured from liver abscesses. These two papers suggest that
ferredoxins may be tightly regulated in E. histolytica.
Investigation of the sequence surrounding the fd2 gene
showed that a URE3 motif was located 1.2 kb 5' of the initiation codon
(International Entamoeba Genome Sequencing
Project)2, but it is
unclear whether this motif would play any role in fd2 regulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ompT
gal [dcm] [lon] hsdSB,
(rB-mB-)) cells grown in Luria-Bertani medium
with 100 µM isopropyl-thio-
-galactopyranose. The
recombinant protein was affinity-purified by use of nickel-chelate
resin according to manufacturer's directions (Qiagen) and then
dialyzed against DNA binding buffer (10 mM Tris-HCl, pH
7.9, 50 mM NaCl, 1 mM EDTA, 20% glycerol).
-32P]dATP. The URE3 DNA sequence was mutated to
TTAGAATTC from the wild-type sequence of TTATCTTAT in the
hgl5-MUT probe. EMSA reactions contained 3 fmol of
radiolabeled probe, 0.01 µg/µl poly(dI·dC), and 0.05 µg/µl of
recombinant URE3-BP protein purified from bacterial lysates (Qiagen) in
EMSA buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 0.5 mg/ml nonfat milk powder (Carnation), 3% glycerol, 0.05 mg/ml
bromphenol blue). When indicated 1 mM CaCl2 was
added that was calculated by the CalBuf Program of Droogmans (22) to
bring the Ca2+ concentration to 86 µM. The
reaction was incubated at 4 °C for 1 h prior to electrophoresis
on a non-denaturing polyacrylamide gel for 2-3 h. The gel was then
fixed, dried, and quantitated by PhosphorImager analysis.
270 to
3 bp of the hgl5 promoter (4) the primers
CTACTGAAGCTTAGTAAAGAATAGTATTGA and
CTACTGGGATCCTTGAATTTCTAGTTCATTGTCT were used. To amplify the region
513 to +14 of the fdx promoter (29) oligonucleotides CTACTGAAGCTTTAAAAATACAAACAACTACC and
CTACTGTCTAGACATTAGATTTGAATGAATAA were used. To amplify the coding
sequences +120 to + 663 of the srehp gene (30) the
oligonucleotides GTCCTGAAAAGCTTGAAGAAGC and GGACTTGATGCAGCATCAAGGT
were used. All real time amplification reactions were
performed in duplicate on each ChIP experiment, and the resulting
fluorescent values were averaged.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical domains (15). In an EF-hand loop the calcium ion is bound by the six residues in
positions 1, 3, 5, 7, 9, and 12 or X, Y, Z, -Y, -X, and -Z (15). The
amino acid sequence of the second EF-hand loop in URE3-BP was
DRNRSGTLEPHEI
(conserved residues in bold). We replaced the residues at positions 1 and 3 in the second EF-hand to alter the sequence to
ARARSGTLEPHEI (changed residues underlined).
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Fig. 1.
Electrophoretic mobility shift
assay of the recombinant (wt)URE3-BP and EF(2)mut URE3-BP.
A, (wt)URE3-BP; B, EF(2)mut URE3-BP in
which two key residues in EF-hand 2 have been altered. In both
gels the first lane is an EMSA of the recombinant protein.
In the second lane EMSA was performed in the presence of
6-fold excess unlabeled hgl5-URE3(wt), and in the
third lane with 6-fold unlabeled hgl5-MUT
(mut) oligonucleotide competitors. EMSA in buffer containing
6.7 µM ((wt)URE3-BP) or 86 µM free
Ca2+ (EF(2)mutURE3-BP) is shown in the
fourth lane.
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Fig. 2.
ChIP analysis of the binding of the URE3-BP
transcription factor to the hgl5 promoter in
vivo. Trophozoites were treated with formaldehyde to
cross-link DNA protein complexes. These complexes were purified by
immunoprecipitation with either an anti-URE3-BP mAb ( ) or a control
antibody (
) of the same isotype. The hgl5 promoter
concentration was determined by real time PCR. A,
calibration of hgl5 primer efficiency. The y axis
represents quantity of input DNA, and the x axis represents
the cycle at which the fluorescent value exceeded the threshold value
(threshold value was set at 5-fold standard deviation of base line).
The equation derived from the data is displayed on the
graph. B, representative ChIP/quantitative real
time PCR amplification plots of the hgl5 promoter. The
y axis (Sybergreen fluorescence at 530 nm) represents the
change in the emission intensity of the SYBR Green I reporter dye after
subtraction of the base line (i.e. early cycles of PCR prior
to detectable levels of template). The x axis represents the
PCR cycle number. C, amount of the hgl5 promoter
immunoprecipitated utilizing the anti-URE3-BP mAb (URE) or
the control mAb (ctr) (mean ± S.E., n = 12, p = 0.0014).
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Fig. 3.
ChIP analysis of the binding of the URE3-BP
transcription factor to the fdx promoter in
vivo. Trophozoites were treated with formaldehyde to
cross-link DNA-protein complexes. These complexes were purified with
either an anti-URE3-BP mAb ( ) or a control antibody (
) of the
same isotype. The fdx promoter concentration was determined
by real time PCR. A, calibration of fdx primer
efficiency. The y axis represents quantity of input DNA, and
the x axis the cycle at which the fluorescent value exceeded
the threshold value (threshold value was set at 5-fold standard
deviation of base line). The equation derived from the data is
displayed on the graph. B, representative
ChIP/quantitative real time PCR amplification plots of the
fdx promoter. The y axis (Sybergreen fluorescence
at 530 nm) represents the change in the emission intensity of the SYBR
Green I reporter dye after subtraction of the base line
(i.e. early cycles of PCR prior to detectable levels of
template). The x axis represents the PCR cycle number.
C, amount of the fdx promoter immunoprecipitated
utilizing the anti-URE3-BP mAb (URE) or the control mAb
(ctr). The y axis indicates the DNA concentration
(ng), and the x axis indicates the mAb used in the
immunoprecipitation (mean ± S.E., n = 7, p = 0.04).
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Fig. 4.
Effect of changes in extracellular
Ca2+ on cytosolic Ca2+ in E. histolytica trophozoites. Amebae were loaded with the
INDO-1 dye, and the ratio of fluorescence at 398 and 480 nm was
determined as a measure of internal free calcium
[Ca2+]i in the ameba.
Arrow indicates time of addition of extracellular
calcium.
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Fig. 5.
Effect of intracellular calcium on URE3-BP
promoter occupancy. ChIP was performed before and after modulation
of trophozoite intracellular calcium. A and B
show data for the hgl5 promoter, and C and
D show data for the fdx promoter. Intracellular
calcium was raised in the trophozoites prior to ChIP in B
and D by placing the amebae in Buffer B containing 1.1 mM Ca2+. The y axis (Sybergreen
fluorescence at 530 nm) represents the change in the emission intensity
of the SYBR Green I reporter dye after subtraction of the base line
(i.e. early cycles of PCR prior to detectable levels of
template). The x axis represents the PCR cycle number.
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Fig. 6.
Calcium modulation of URE3-BP promoter
binding. A, Western blot of URE3-BP immunoprecipitated after
trophozoites were formaldehyde cross-linked in Buffer B ± calcium. B and C, compilation of ChIP/real time
PCR data (mean ± S.E.) expressed from 10 independent experiments
for hgl5 promoter (p = 0.02) (B)
and for the fdx promoter (p = 0.05)
(C).
40 bp from the ATG start sequence (
28 bp from
the previously published cDNA sequence). The sequence (TGATATAAAG)
with a very low similarity to the TATA consensus (GTATTTAAA(GyC) was
located
46 bp from the ATG (6). A pentanucleotide sequence (TAATT)
that was identical to the 3'-terminal consensus sequence TA(A/T)TT was
located 42 bp after the translation stop codon (7). The 3' non-coding
sequence contained an open reading frame in the opposite orientation
that terminated at +73 bp after the URE3-BP stop codon and initiated at + 720 bp on the opposite strand and thus intervened between the
termination of the URE3-BP open reading frame and the 3' URE3 sequence
motif. The intervening open reading frame could encode a highly basic 25-kDa protein but did not have an easily recognizable core promoter.
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Fig. 7.
Sequence context of the URE3-BP gene.
Sequences were obtained from the Entamoeba histolytica
Genome Sequencing Project at the Institute for Genomic Research web
site at www.tigr.org and the Wellcome Trust Sanger Institute at
www.sanger.ac.uk. The 5' sequence is Sanger Data base Ent774f10.q1c.
Sequences 3' included sequences derived from both the Institute for
Genomic Research (ENTLU68TR, ENTLU68TF) and the Sanger Institute
(Ent1246d08.p1k, Ent1123h08.q1c, Ent683f12.p1c). Numbering is as the
previously published cDNA sequence of URE3-BP, accession
number AF291721. The proposed TATA, GAAC, INR, terminal pentanucleotide
sequences, and 3' URE3 motifs are outlined and
underlined.
0.02) from
that of L10 mRNA at 4, 6, and 8 h post-calcium
modulation (Fig. 8). We concluded that the presence of a URE3 motif in
the URE3-BP non-coding region was consistent with the gene being under
autoregulation.
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Fig. 8.
Sequence expression of URE3-BP mRNA ± calcium. E. histolytica were seeded at a
concentration of 2-4 × 104/ml 18 h prior to the
addition of 5 mM MgCl2 and EDTA to sequestrate
free calcium (calculated to reduce TYI-S-33 medium
Ca2+ to 3-5 µM), and the addition of 5.4 mM CaCl2 was calculated to raise the free
calcium concentration to 1 mM and was added to calcium
treated trophozoites. RNA was purified from the trophozoites at the
times indicated, and the level of URE3-BP and L10 transcripts was
quantified by real time reverse transcriptase PCR. URE3-BP mRNA
values were then normalized to the level of L10 mRNA. The
graph shows the ratio of URE3-BP mRNA isolated from
trophozoites in 1 mM Ca2+ versus
calcium-depleted medium at the times indicated. A value of one
indicates that no change was observed. These results were obtained from
three independent experiments. The asterisks (*) indicate that a
statistical significant difference was observed from the L10 value at
this time point (mean ± S.E., n = 3, p 0.02).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Keller, D. M. Haverstick, and L. S. Gray for advice and helpful discussions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI 37941. Sequence data pertaining to the URE3-BP genomic context was obtained from the Entamoeba histolytica Genome Data base. The sequences were made freely available by the Pathogen Sequencing Unit at the Sanger Institute (ftp.sanger.ac.uk/pub/pathogens/E_histolytica) and the TIGR Entamoeba histolytica Genome Project (www.tigr.org/tdb/e2k1/eha1/). Preliminary sequence data for E. histolytica are deposited regularly into the GSS division of GenBankTM. The sequencing effort is part of the International Entamoeba Genome Sequencing Project and is supported by an award from NIAID, National Institutes of Health.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.
Burroughs Wellcome Fund Scholar in Molecular Parasitology. To
whom correspondence should be addressed: University of Virginia Health
System, MR4 Bldg., Rm. 2115, P. O. Box 801340, Charlottesville, VA
22908-1340. Tel.: 434-824-5621; Fax: 434-924-0075; E-mail: wap3g@virginia.edu.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M211271200
2 Z. Wang, J. Samuelson, C. G. Clark, E. Tannich, B. J. Mann, N. Hall, and B. Loftus, unpublished data.
3 C. A. Gilchrist, N. D. Missaghi, and W. A. Petri, Jr., unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: URE, upstream regulatory element; BP, binding protein; EMSA, electrophoretic mobility shift assay; MUT, mutant; mAb, monoclonal antibody; ChIP, Chromatin immunoprecipitation; wt, wild-type; DREAM, downstream regulatory element antagonist modulator.
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REFERENCES |
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