From the Departments of Microbiology,
§ Internal Medicine, and ¶ Pathology, University of
Virginia, Charlottesville, Virginia 22908
Received for publication, July 31, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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To study transcriptional regulation in the lower
branching eukaryote Entamoeba histolytica, we have
identified two sequence-specific DNA-binding proteins that recognize
the upstream regulatory element URE4, an enhancer that regulates
expression of the Gal/GalNAc lectin heavy subunit gene
hgl5. A chromatographic purification of E. histolytica nuclear extracts by gel filtration, cation exchange, and sequence-specific DNA affinity chromatography led to a 700-fold increase in URE4 binding activity and the appearance of two dominant protein species with molecular masses of 28 and 18 kDa. These proteins,
termed E. histolytica enhancer-binding proteins 1 and 2 (EhEBP1 and EhEBP2), were sequenced by tandem mass spectroscopy and
their corresponding cDNA clones identified. Recombinant EhEBP1 and
EhEBP2 were able to bind double-stranded oligonucleotides bearing the
URE4 motif in a sequence-specific manner, and antibodies raised against
EhEBP1 were able to interfere with the formation of URE4-protein
complexes in crude nuclear extracts. Overexpression of EhEBP1 in
E. histolytica trophozoites resulted in a 7-fold drop in
promoter activity in transiently transfected reporter gene constructs
when the URE4 motif was present, confirming its ability to specifically
recognize the URE4 motif and suggesting that additional cofactors may
be required for transcriptional activation by URE4. Further
characterization and identification of binding partners for EhEBP1 and
EhEBP2, the first proteins with demonstrated sequence-specific DNA
binding activity to be identified in E. histolytica, should
provide new insights into transcriptional regulation in this protozoan parasite.
Although control of gene expression in plants, animals, and fungi
has been extensively studied, little is known about transcriptional regulation in the early branching eukaryotes. Recent advances in
molecular biology techniques have led to the discovery of varying models of promoter architecture in this diverse group of organisms, which include Entamoeba histolytica, Acanthamoeba
castellanii, Trichomonas vaginalis, and Giardia
lamblia. A. castellanii is one of the later diverging organisms
among the protozoa. Its TATA and Inr elements are similar to metazoan
consensus sequences, and an A. castellanii nuclear extract
can initiate transcription correctly from an adenovirus promoter (1). A
DNA-binding protein that regulates TATA binding protein expression has
been identified in this organism, which recognizes DNA in a manner
similar to the metazoan tetrameric transcription factor p53 (2).
T. vaginalis is an earlier branching eukaryote than E. histolytica. Its core promoter contains an Inr element similar to
the metazoan Inr motif, but a TATA element has not been identified (3).
The T. vaginalis RNA polymerase that transcribes
protein-coding genes is E. histolytica is another protozoan parasite whose
mechanisms of transcriptional regulation are beginning to be
investigated. Causing amebic colitis and amebic liver abscesses,
E. histolytica is estimated to be responsible for an
estimated 50 million cases of invasive disease and 70,000 deaths per
year (6). It possesses a divergent core promoter, with nonconsensus
TATA and Inr elements, and a third core promoter element "GAAC"
unique to E. histolytica (7-9). Like T. vaginalis, transcription of protein-coding genes in E. histolytica is The 5'-flanking regions of a handful of E. histolytica genes
have been studied via truncation and mutational analysis (8, 12-14).
In the hgl2 promoter, a CCAAT-like motif was identified that
decreased reporter gene expression when mutated, but the existence of
proteins recognizing this element has not been demonstrated (3). In the
EhPgp1 and EhPgp2 genes, several metazoan-like upstream regulatory elements were identified by sequence homology (9,
21). Some of these elements were able to compete for DNA-binding in
electrophoretic mobility assay, but a mutational analysis was not
performed. The promoter that has been best characterized, however, is
the Gal/GalNAc lectin hgl5 promoter, which was subjected to
a linker scanner mutagenesis that revealed the presence of several
upstream regulatory elements (15). One of these upstream regulatory
elements, URE4,1 was shown to
function as a transcriptional enhancer, activating reporter gene
expression in either direction in front of a minimal promoter (16).
URE4 is composed of two direct nine base pair repeats of the sequence
AAAAATGAA, and the presence of sequence-specific URE4-binding proteins
in E. histolytica nuclear extracts was demonstrated by
electrophoretic mobility shift assay and UV cross-linking (16). We
report here the purification and cloning of two sequence-specific URE4-binding proteins, EhEBP1 and EhEBP2.
Cultivation and Transfection of Amebae--
E.
histolytica trophozoites of strain HM-1:IMSS were grown at
37 °C in TYI medium containing penicillin (100 units/ml) and streptomycin (100 µg/ml) (17). Amebae in logarithmic phase growth (~6 × 104 trophozoites/ml) were used for
transfection experiments and nuclear extract preparation. Stable or
transient transfections were performed as described previously except
that, for transient transfections, cells were lysed after 6 instead of
10 h (18, 19).
Nuclear Extract Preparation and Protein
Purification--
Nuclear extracts were prepared as described
previously (20). Approximately 5 × 108 amebae were
used as starting material, resulting in 22.8 mg of protein in 6 ml. The
extract was transferred to dialysis tubing, and the volume was reduced
to 2 ml by covering the tubing with polyethylene glycol 8000 for
several hours. Concentrated nuclear extract was loaded onto a HiPrep
16/60 Sephacryl S-200 column (Amersham Pharmacia Biotech). After the
void volume was discarded, outflow from the gel filtration column was
passed onto a 1-ml HiTrap SP cation exchange column (Amersham Pharmacia
Biotech). After fractions containing proteins with native molecular
masses of ~100 to 25 kDa (as calculated by column calibration using
standards of known molecular mass) were allowed to flow into the cation exchange column, the gel filtration column was disconnected. The cation
exchange column was then washed with several column volumes of
DNA-binding buffer (10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 1 mM EDTA, 5% glycerol). Bound
protein was eluted with an NaCl gradient of 0.2-0.6 M NaCl
over 20 ml. Fractions (0.5 ml) were collected, aliquoted, and stored at
Fractions enriched for gel shifting ability were purified further by
sequence-specific DNA affinity chromatography. The sequence specific
affinity resin was prepared as follows. A DNA construct containing four
head-to-tail copies of the URE4 motif served as template for PCR using
a primer with a 5'-biotin modification (Life Technologies, Inc.). The
PCR product was incubated for 16 h at 4 °C with a slight excess
of magnetic, streptavidin-coated beads in DNA binding buffer plus 2 M NaCl (Promega). Binding to beads was monitored by agarose
gel electrophoresis. The beads were then washed in DNA-binding buffer
and stored at 4 °C.
Affinity chromatography was performed by incubating a single fraction
of nuclear extract purified by gel filtration and cation exchange
chromatography (containing 48 µg of protein) with the URE4-complexed
beads in DNA-binding buffer. After 1 h at 4 °C, beads were
immobilized on a magnetic stand and flow-through material collected.
Beads were then washed twice with DNA-binding buffer before elution
with this buffer plus 1 M NaCl.
Electrophoretic Mobility Shift Assay--
This assay was
performed as described previously (20). Briefly, radiolabeled probe was
created by annealing complementary oligonucleotides to form
double-stranded DNA followed by "filling in" with Klenow and
[ Southwestern Blot--
Protein samples were separated by
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride (PVDF) membrane (Millipore). Bound proteins
were denatured briefly by exposure to 6 M guanidine
hydrochloride and renatured by incubation for 16 h in wash buffer
(10 mM Hepes (pH 7.9), 50 mM KCl, 1 mM EDTA, 6.4 mM MgCl2). A
radiolabeled oligonucleotide bearing the URE4 sequence prepared in the
same manner as for electrophoretic mobility shift assay was allowed to
bind to the immobilized protein in the presence of 0.01% Tween 20. After 3 h of incubation at 25 °C, the membrane was washed for
3 h in wash buffer. Radioactivity was detected by PhosphorImager
analysis (Molecular Dynamics).
Cloning of EhEBP1 and EhEBP2--
Peptides released from
SDS-PAGE gels by trypsinization were microsequenced by tandem mass
spectrometry by the W. M. Keck Biomedical Mass Spectrometry
Laboratory at the University of Virginia. Five to six amino acids from
tryptic peptide sequences were used to design degenerate PCR primers.
The forward primer used for amplification of EhEBP1 was
TA(T/C)GA(T/C)GTIGTIGA(A/G)GC(T/A/C)AC, based on the peptide sequence
YDVVEAT(I/L)VK). The forward primer used for amplification of EhEBP2
was CCAACIGAATT(C/T)TC(A/T)GA(C/T)GA, based on the peptide sequence
PTEFSDD(I/L)NFVK. The reverse primer annealed to downstream vector
sequences flanking the 3' end of cDNA inserts, which were cloned
into the vector pAD-GAL4 (Stratagene). PCR products were amplified
using HotStarTaq (Qiagen) and the E. histolytica cDNA
library was used as template. Authenticity of PCR products was checked
by examining the amplified sequence for the next few amino acids
predicted by the peptide sequence but not incorporated into the PCR
primer. Amino-terminal sequence was obtained by designing a new reverse
primer based on amplified sequences, which was
CTACCTTCTACTTCAAAGTTATCCATTTC for EhEBP1 and CCCTTTGATCTTACATTTCCTCTATG
for EhEBP2. The forward primer was complementary to vector flanking
sequences upstream from the cDNA inserts.
Northern Blot Analysis--
Total RNA from either lab-passaged
or gerbil-passaged amebae was isolated using RNeasy columns (Qiagen).
Ten micrograms of RNA was separated on a formaldehyde-containing
agarose gel and transferred to a Zeta-probe GT Genomic blotting
membrane (Bio-Rad). Pre-hybridization, hybridization, and washes were
performed according to the manufacturer's instructions. DNA probes
consisted of the coding regions of either EhEBP1 or EhEBP2, (amplified
by PCR and purified over Qiaquick columns (Qiagen)) or the coding
region of the hexokinase gene (recovered from plasmid pSFL3 by
digestion with BamHI and XhoI) (21). Probes were
labeled using random primers, [ Expression in E. coli of Recombinant EhEBP1 and EhEBP2--
PCR
was used to introduce the coding regions of EhEBP1 and EhEBP2 into a
modified version of the vector pGEX-3X (Amersham Pharmacia Biotech)
that contained a tobacco etch virus (TEV) protease recognition site
separating the glutathione S-transferase (GST) tag from the
introduced protein. Recombinant protein was induced and purified from
bacterial cultures by glutathione-conjugated agarose affinity
chromatography. Homogeneity of the final product was ascertained by
Coomassie Blue-stained SDS-PAGE. For mouse immunization, EhEBP1 was
released from the GST fusion partner by overnight incubation with 100 units of polyhistidine-tagged TEV protease (Life Technologies, Inc.).
Protease was removed by a 1-h incubation at room temperature with 50 µl of nickel-coated magnetic beads (Qiagen).
Immunization, Immunoblot, and Immunoprecipitation--
Male
BALB/c and A/J mice were immunized intraperitoneally with 100 µg of
TEV protease-cleaved EhEBP1 emulsified in complete Freund's adjuvant.
Two and 4 weeks later, the mice were boosted with 100 µg of
TEV-cleaved EhEBP1 in incomplete Freund's adjuvant. One week after the
last boost, about 0.8 ml of immune sera was obtained by retro-orbital
puncture. Nonimmune sera was obtained from mice that had not been immunized.
Immunoblots were performed by electrophoresing 2 µg of recombinant
protein or amebic lysates from 80,000 cells through a 12% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane (Millipore), incubated overnight at 4 °C in 5% nonfat dry milk in
blot wash buffer (50 mM Tris (pH 7.4), 200 mM
NaCl, 0.1% Tween 20), and probed with mouse antisera diluted 1:1000 in
the same buffer for 1 h at room temperature. After three 5-min
washes, a 1-h incubation with Fc-specific, anti-mouse antibody
conjugated to horseradish peroxidase (Sigma) was performed (diluted
1:1500), and signal was detected by ECL (Amersham Pharmacia Biotech).
Immunoprecipitation of the Flag-EhEBP1 was performed by incubating a
lysate of 2.0 × 106 trophozoites with 1 µg of
anti-Flag antibody (Sigma) and 30 µl of Protein G beads (Sigma) for
1.5 h at 4 °C. Beads were washed and boiled in sample buffer,
and released protein complexes were analyzed by polyacrylamide gel
electrophoresis and immunoblot as described above.
Expression of EhEBP1 in E. histolytica--
An amino-terminal
Flag-EhEBP1 fusion protein was engineered by PCR. This fusion was
inserted into the BglII and SalI sites of the
pGIR209 vector, placing Flag-EhEBP1 expression under
tetracycline-inducible control (22, 23). Amebae bearing the two
expression constructs, Flag-EhEBP1 and tetracycline repressor, were
cultured by selection using G418 and hygromycin. Flag-EhEBP1 expression
was induced by the addition of tetracycline (5 µg/ml) 16 h prior
to transient transfection with reporter gene constructs. Reporter
activity was measured 8 h after transfection of the reporter (a
total of 24 h after induction of Flag-EhEBP1 expression).
Column Chromatography Was Used to Purify Two Sequence-specific
URE4-binding Proteins from E. histolytica Nuclear Extracts--
In a
pilot experiment, nuclear extracts were prepared from E. histolytica trophozoites and fractionated by gel filtration. URE4
binding activity was detected by electrophoretic mobility shift assay
in fractions with estimated molecular masses ranging from 25 to 100 kDa
(data not shown). For the final purification, nuclear extracts were
separated by gel filtration and fractions known to be enriched for URE4
binding activity were allowed to pass onto a cation exchange column.
Material bound to this column was eluted with a salt gradient.
Electrophoretic mobility shift assay analysis of crude nuclear extract
typically resulted in a pattern of three protein-DNA complexes.
Fractionation by cation exchange chromatography resulted in a nearly
complete loss of complex 3, the largest protein complex, and a
separation of complex 1- and complex 2-forming activities. Both complex
1 and complex 2 were produced by URE4-specific DNA binding, as
confirmed by competition by excess URE4 but not unrelated
double-stranded oligonucleotides (data not shown).
URE4-binding proteins purified by gel filtration and cation exchange
chromatography were further purified by sequence-specific DNA affinity
chromatography. Individual fractions from the cation exchange step were
incubated with four copies of the URE4 sequence immobilized on magnetic
beads under conditions similar to the electrophoretic mobility shift
assay. After two washes, DNA-binding proteins were eluted with 1 M NaCl and tested for URE4 binding activity by
electrophoretic mobility shift assay. When fraction 28, a sample
enriched for complex 2-forming activity, was used as input material,
enrichment of sequence-specific URE4 binding activity was detected in
the elution fraction by electrophoretic mobility shift assay (Fig.
1). This final step resulted in an ~800-fold increase in specific activity as compared with the starting material, crude nuclear extract (Table
I). When fractions enriched for complex
1-forming activity, such as fractions 22 and 23 (Fig. 1), were used in
the same affinity purification procedure, however, URE4 binding
activity was detected in the input and flow-through fractions but not
in the elution fraction (data not shown).
SDS-PAGE analysis of the affinity chromatography fractions shown in
Fig. 1 revealed the presence of two dominant bands with molecular
masses of 28 and 18 kDa in the salt-eluted fraction (E, Fig.
2A). A parallel purification
using fraction 27 as input material resulted in a similar increase in
sequence-specific URE4 binding activity as measured by electrophoretic
mobility shift assay in the elution faction (data not shown), which
also contained two dominant proteins with molecular masses of 28 and 18 kDa (E', Fig. 2A). The URE4-binding ability of
these proteins was tested by Southwestern blot; proteins were
transferred to a PVDF membrane and allowed to interact with
radioactively labeled double-stranded URE4 oligonucleotide (Fig.
2B). This assay confirmed the URE4-binding ability of the
28- and 18-kDa proteins and also identified a third protein with a
molecular mass of 45 kDa. The 45-kDa protein was not present in
sufficient quantities for sequencing and was not further analyzed.
Cloning of EhEBP1 and EhEBP2--
The 28- and 18-kDa proteins
derived from sequence-specific DNA affinity chromatography, named
E. histolytica enhancer-binding protein 1 and 2 (EhEBP1 and
EhEBP2), were trypsinized and the resulting peptides sequenced by
tandem mass spectroscopy. Degenerate oligonucleotides were designed
based on the first five or six amino acids of several tryptic peptides
and used as PCR primers to amplify two cDNA clones from an E. histolytica cDNA library (Fig.
3). The authenticity of amplified PCR
products was determined by identification of amino acids predicted by
the peptide sequence but not incorporated into the PCR primer.
Furthermore, for both EhEBP1 and EhEBP2, the sequence of every tryptic
peptide identified by mass spectroscopy was found in the predicted
protein sequence, confirming that the cloned genes corresponded with
the sequenced URE4-binding proteins. For EhEBP1, which had an apparent
molecular mass of 28 kDa as determined by SDS-PAGE (Fig.
2A), an open reading frame was predicted that corresponded
to a protein with an approximate molecular mass of 28 kDa. For EhEBP2,
which had an apparent molecular mass of 18 kDa as determined by
SDS-PAGE (Fig. 2A), an open reading frame was predicted that
corresponded to a protein with an approximate molecular mass of 22 kDa.
This discrepancy may reflect an aberrant SDS-PAGE mobility for EhEBP2,
or it may result from a post-translational modification.
EhEBP1 and EhEBP2 Contain Regions Homologous to the RNA Recognition
Motif--
Examination of the amino acid sequences of EhEBP1 and
EhEBP2 by searching computer data bases did not detect any motifs
commonly associated with sequence-specific DNA-binding proteins.
Surprisingly, both EhEBP1 and EhEBP2 contained regions of homology to
the RNA recognition motif (RRM), a nucleotide binding motif found in a large group of RNA-binding proteins (24, 25). The RRM is also present
in several sequence-specific DNA-binding proteins, such as
stage-specific activator protein (SSAP) (26). EhEBP1 contained two full
copies of the RRM as well as a 54-amino acid region with many acidic
and basic residues (Fig. 4A).
EhEBP2 contained one and a half copies of the RRM as well as a 58-amino
acid region with many acidic and basic residues. Alignment with
representative RNA- and DNA-binding, RRM-containing proteins revealed
the presence of many well conserved amino acids within the RRMs of
EhEBP1 and EhEBP2 (Fig. 4B). Sequence homology was also seen
between the RRMs of EhEBP1 and EhEBP2 in regions not well conserved
between different RRM-containing proteins, such as loop 3.
Identification of EhEBP1 and EhEBP2 Message in E. histolytica
Trophozoites--
Northern analysis of RNA derived from E. histolytica trophozoites demonstrated the existence of single
transcripts for both EhEBP1 and EhEBP2 (Fig.
5A). Interestingly, both
EhEBP1 and EhEBP2 were approximately 2-fold more abundant in
animal-passaged versus laboratory-passaged trophozoites
after loading correction based on hexokinase message levels (Fig.
5B). Animal-passaged trophozoites have been shown to be more
virulent than laboratory-passaged trophozoites, but whether expression
of EhEBP1 and EhEBP2 is involved is a topic for further
investigation.
Recombinantly Expressed EhEBP1 and EhEBP2 Bind URE4 in a
Sequence-specific Manner--
The sequence-specific URE4 binding
activity of recombinant EhEBP1 and EhEBP2 was tested using the
electrophoretic mobility shift assay. Although purified GST protein
alone was unable to bind to the URE4 sequence, a GST-EhEBP1 fusion
protein was able to bind URE4 double-stranded oligonucleotide in a
sequence-specific manner (Fig.
6A). Binding specificity was
demonstrated by competition with excess of URE4 but not unrelated
oligonucleotides. EhEBP2 was also able to specifically bind
double-stranded URE4 when expressed as a GST fusion protein (Fig.
6B). Binding specificity for this protein was demonstrated
by competition with excess of URE4 but not by mutant oligonucleotides.
The combination of EhEBP1 and EhEBP2 GST fusion proteins did not result
in the appearance of any higher order complexes in an electrophoretic
mobility shift assay (data not shown). This failure to associate may
indicate that EhEBP1 and EhEBP2 do not form heterodimers.
Alternatively, it may result from steric hindrance by the GST epitope
tag or from a lack of posttranslational modifications required for
dimerization.
Anti-EhEBP1 Antibodies Recognize a 28- and 18-kDa E. histolytica
Proteins and Inhibit URE4-native Protein Complex Formation in Crude
Nuclear Extracts--
The GST epitope tag was removed from the
GST-EhEBP1 fusion protein by site-specific protease digestion, and the
freed EhEBP1 used for mouse immunization. Sera derived from this mouse
recognized the GST-EhEBP1 fusion protein but not GST protein alone in
an immunoblot assay. Additionally, this sera recognized several
proteins in E. histolytica lysates; the molecular masses of
the two predominant proteins recognized were 28 and 18 kDa (data not shown).
The involvement of proteins recognized by the anti-EhEBP1 antibodies in
forming complexes with URE4 was tested using E. histolytica nuclear extracts. Preincubation of nuclear extracts with sera from a
mouse immunized against EhEBP1 but nonimmune sera from a mouse of the
same strain resulted in an inhibition of URE4-protein complex formation
(Fig. 7) This suggests that EhEBP1 is a
critical part of the URE4-protein complex formed by native E. histolytica proteins.
Overexpression of EhEBP1 Leads to Repression at the hgl5
Promoter--
The function of EhEBP1 in E. histolytica
trophozoites was tested by inducible expression of a Flag-EhEBP1 fusion
protein. After 24 h of tetracycline induction, the induced protein
was detected by anti-Flag immunoprecipitation followed by immunoblot with anti-EhEBP1 antisera (Fig.
8A). The Flag-EhEBP1 migrated in SDS-page at its expected molecular mass of 29.9 kDa and was only
present in the induced amebae; the heavy chain of the anti-Flag antibody was visible in both lanes (Fig. 8A).
Transient transfection of a luciferase reporter gene construct under
transcriptional control of the hgl5 promoter was performed with induced and uninduced cells to test the effect of Flag-EhEBP1 overexpression on hgl5 promoter activity. Induction of
Flag-EhEBP1 expression for 24 h resulted in an approximately
7-fold drop in reporter gene expression (p < 0.0001)
(Fig. 8B). Inducible expression of an unrelated protein had
no effect on reporter gene expression. The effect of Flag-EhEBP1
expression was also tested with an hgl5 promoter-luciferase
construct that contained a mutated version of URE4. Although mutation
of URE4 resulted in a dramatic decrease in reporter gene expression
luciferase activity in the uninduced cells compared with the wild type
promoter, induction of Flag-EhEBP1 did not result in a statistically
significant further reduction of expression controlled by the mutant
promoter (Fig. 8B).
To further our understanding of transcriptional regulation in the
lower branching eukaryote E histolytica, we have begun to characterize upstream regulatory elements and their cognate binding proteins. Using standard chromatography techniques, we have enriched for sequence-specific binding to the hgl5 enhancer URE4,
leading to the purification and cloning of two URE4-binding proteins, EhEBP1 and EhEBP2. These two proteins represent the first DNA-binding proteins with demonstrated sequence-specific binding activity identified in E histolytica. Both recombinant EhEBP1 and
EhEBP2 have sequence-specific URE4 binding ability as assayed by
electrophoretic mobility shift assay. Furthermore, antibodies raised
against EhEBP1 were able to inhibit the formation of the URE4-protein
complex in E histolytica crude nuclear extracts. The ability
of EhEBP1 to repress transcription by the hgl5 promoter in
an URE4-dependent manner further demonstrated the ability
of EhEBP1 to specifically recognize the URE4 sequence.
EhEBP1 and EhEBP2 are part of a growing class of sequence-specific
DNA-binding proteins that share homology with RNA-binding proteins
largely involved in splicing and export of pre-mRNA (25, 27). The
RNA-binding motif used by these proteins, the RRM, is found in a
diverse range of organisms, from animals to bacteria, and has the
flexibility to bind a diverse group of RNA templates, from hairpin
loops to stretches of poly(A). Two sequence-specific DNA-binding
proteins that contain RRMs are SSAP and carnation ethylene-responsive
element-binding protein (4, (28). The DNA recognition sites for both of
these proteins are AT-rich, and both proteins show sequence-specific
binding to both single-stranded and double-stranded DNA. It has been
suggested that these AT-rich sites exhibit some single-stranded
character in the cell, allowing for interaction between the conserved
aromatic residues within the Another interesting aspect of URE4 sequence is that it is composed of
two direct nine-base pair repeats. Crystallization of the
single-stranded DNA-binding protein hnRNP A1, which is involved in
regulation of telomere length, revealed that an hnRNP A1 homodimer could recognize two copies of its DNA recognition site simultaneously via its two RRM domains (29). Perhaps another determinant of URE4-binding specificity is interaction between EhEBP1 and EhEBP2 homo-
or heterodimers and both nine-base pair sequences.
In the RRMs of EhEBP1 and EhEBP2, loop 3 contains many regions of
sequence identity not seen in other RRM-containing proteins. This
conservation may point to residues important for sequence-specific DNA
binding. Swapping of amino acid sequences between U1A and U2B
snRNP-associated proteins identified loop 3 as important for specific
binding site recognition (30). Additionally, loop 3, which is not well
conserved among RNA-binding proteins, varying in length and amino acid
composition, was found for one RNA-binding protein to have a disordered
structure until complexed with RNA, suggesting an induced-fit mechanism
for nucleotide-protein interaction (31). Future studies of EhEBP1 and
EhEBP2 will include truncation and mutational analysis to map the
domains involved in sequence-specific recognition of URE4.
When overexpressed as a Flag fusion protein, EhEBP1 resulted in a
repression of transcription at the hgl5 promoter. There are
several possible explanations for this effect. EhEBP1 may have an
ability to activate transcription that is disrupted by fusion of the
Flag epitope to its amino terminus. Alternatively, excess amounts of
EhEBP1 could titrate out a cofactor, present in limiting amounts, which
is required for transcriptional activation by URE4, either by blocking
cofactor access to the DNA or by binding and sequestering the cofactor
away from the DNA. If this model were true, coexpression of the missing
cofactor would result in transcriptional activation at the
hgl5 promoter. Candidates for this coactivating protein
include EhEBP2 as well as the 45-kDa protein identified by Southwestern
blot of purified nuclear extracts (Fig. 2B).
Coimmunoprecipitation with anti-EhEBP1 antibodies could determine
whether EhEBP1 exists in a protein complex either with EhEBP2 or other proteins.
Another possibility that would explain the repressing effect of EhEBP1
overexpression is that EhEBP1 is a bona fide
inhibitor of transcriptional activation. The ability of URE4 to
function as a positive regulatory element may be due to the presence of other URE4-binding proteins that function as transcriptional activators and whose activity dominates over that of EhEBP1 in laboratory-cultured trophozoites. This possibility could be tested by assaying the transcriptional activity of nuclear extracts immunodepleted with anti-EhEBP1 antibodies in an in vitro transcription assay, a
technique that has not yet been adapted for E. histolytica.
The fact that EhEBP1 message is more abundant in the more virulent
animal-passaged trophozoites, however, seems to suggest that EhEBP1 has
an activating rather than a repressing function.
The identification of two sequence-specific enhancer binding proteins
in E. histolytica has opened many new avenues of
investigation into the transcriptional regulation of this nonmetazoan
eukaryote. Future studies will focus on further characterization of
EhEBP1 and EhEBP2 and identification of putative binding partners.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amanitin resistant, and cloning of the
polymerase gene demonstrated a lack of conserved amino acids in the
region thought to bind this drug (4). G. lamblia is another
early branching eukaryote about which little is known. Three conserved
promoter elements have been identified by comparing 5'-flanking
sequences, none of which share homology with metazoan core promoter
motifs (5). No sequence-specific DNA-binding proteins have been
identified in either of these two protists.
-amanitin resistant (10). The only protein related to transcriptional regulation that has been identified in this
organism is TATA-binding protein. However, the ability of this protein,
which was cloned by sequence identity, to bind DNA or function in
assembly of the basal transcriptional apparatus remains to be
demonstrated (11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C for further analysis.
-32P]dATP. A typical reaction contained 0.01 pmol of
radiolabeled probe, 0.2 µg of poly(dI·dC), and 0.001-5 µg of
protein. Sense strands of oligonucleotides used were
TGAATTGTTATAAAAATGAATGGAAAAATGAAATGAATTA (URE4),
TGAATTGTTATAATCTAGAATGGAAAAATGAAATGAATTA (mutant), and TGTTCCAAAAAGATATATTCTATTGAAAATAAAAGAA (unrelated). For supershift analysis, antisera was preincubated with protein samples for 20 min on
ice before addition of other reaction components. Radioactivity was
detected by PhosphorImager analysis (Molecular Dynamics).
-32P]dATP, and the
Klenow fragment of DNA polymerase I (Life Technologies, Inc.). Message
levels were determined by PhosphorImager exposure and the ImageQuant
program (Molecular Dynamics).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electrophoretic mobility shift of
affinity-purified specific URE4-binding proteins. Proteins
enriched for URE4 binding activity by gel filtration and cation
exchange chromatography (fraction 28) was diluted to an NaCl
concentration of 150 mM and incubated with double-stranded
DNA containing four head-to-tail URE4 motifs immobilized on magnetic
beads. After washing in DNA-binding buffer (10 mM Tris-Cl
(pH 7.9), 1 mM EDTA, 50 mM NaCl, 3% glycerol),
URE4-binding proteins were eluted with DNA-binding buffer plus 1 M NaCl. Electrophoretic mobility shift assay was used to
analyze the URE4 binding activity of input (I), flow-through
(FT), wash (W1 and W2), and eluted
(E) material. The electrophoretic mobility shift was
performed with 0.2 µg of input material and 0.007 µg of eluted
material. Adding self (URE4) or unrelated oligonucleotides at 50- or
100-fold excess as indicated tested specificity of the eluted
fraction.
Purification of URE4-binding proteins
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Fig. 2.
Analysis of affinity-purified URE4-binding
proteins. A, Coomassie Blue-stained SDS-PAGE of the
input (I), flow-through (FT), wash (W1
and W2), and elution (E) fractions from affinity
chromatography. The first five lanes
represent protein fractions derived from a single affinity
purification; lane E' contains material obtained
in an independent purification. The two major bands seen in
lanes E and E' had estimated molecular
masses of 18 and 28 kDa. B, Southwestern blot of
affinity-purified material. Two µg of flow-through and 0.1 µg of
wash 1 and eluted protein were separated by SDS-PAGE prior to transfer
to PVDF. The membrane was probed with a Klenow-labeled double-stranded
oligonucleotide containing the URE4 sequence. Proteins detected had
molecular masses of ~45, 28, and 17 kDa.
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Fig. 3.
cDNA clones for the two purified
proteins. A, DNA sequence and protein translation of
the 28-kDa protein, EhEBP1. The longest open reading frame is
translated. The estimated molecular mass of the protein if the entire
ORF were translated is 28 kDa. Amino acids identified from sequencing
of the affinity-purified protein are shown in bold.
B, DNA sequence and protein translation of the 18-kDa
protein, EhEBP2. The longest open reading frame is translated. The
estimated molecular mass of the protein if the entire ORF were
translated is 22 kDa. Amino acids identified from sequencing of
affinity-purified protein are shown in bold.
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Fig. 4.
Sequence analysis of EhEBP1 and EhEBP2.
A, schematic of the primary amino acid sequences of EhEBP1
and EhEBP2. EhEBP1 contains a 54-amino acid amino-terminal domain
containing many acidic and basic residues, two complete RRMs separated
by a 19-amino acid linker, and a 10-amino acid COOH-terminal tail.
EhEBP2 contains an 8-amino acid amino-terminal domain, one half of an
RRM, a 58-amino acid linker section containing many acidic and basic
residues, a second RRM, and a 12-amino acid COOH-terminal tail.
B, sequence alignment of the RRMs of EhEBP1, EhEBP2, sea
urchin transcription factor SSAP, and human U1A snRNP-associated
protein. Both the amino-terminal and the carboxyl-terminal RRMs are
shown for each protein (1 and 2).
Shading highlights conserved residues. Black
shading indicates typically invariant residues, and
gray shading represents residues typically
occupied by a conservative grouping of amino acids. The secondary
structure is modeled after the crystallized RNA-binding protein U1A
snRNP-associated protein and is indicated above the
sequence. The starting amino acid for each RRM repeat is indicated to
the left of the amino acid sequence.
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Fig. 5.
Northern blot analysis of EhEBP1- and
EhEBP2-encoding RNA. A, laboratory-passaged
(lanes 1 and 3) or animal-passaged
(lanes 2 and 4) trophozoites were used
to prepare total RNA, which was separated on an agarose gel,
transferred to Zeta-Probe (Bio-Rad) membrane, and hybridized with the
DNA coding for EhEBP1 (lanes 3 and 4)
or EhEBP2 protein (lanes 1 and 2).
Molecular masses from an RNA ladder are indicated on the
left in kilobases. B, the same membrane was
re-hybridized with DNA coding for the E. histolytica
hexokinase gene (1.4 kilobases) as a loading control.
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Fig. 6.
Analysis of sequence-specific DNA-binding
abilities of recombinantly expressed EhEBP1 and EhEBP2. Proteins
were expressed in bacteria as GST fusion proteins and purified by
glutathione-agarose affinity chromatography. A,
electrophoretic mobility shift assay was performed with radiolabeled
URE4 oligonucleotide and 3 µg of recombinant EhEBP1 protein, except
for the first two lanes, which
contained either no protein or 3 µg of purified GST protein as
indicated. Competitor oligonucleotides were added at 100- and 500-fold
excess as indicated. B, electrophoretic mobility shift assay
with radiolabeled URE4 oligonucleotide and 4 µg of recombinant EhEBP2
protein. Competitor oligonucleotides were added at 1000-, 2000-, and
3000-fold excess as indicated.
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Fig. 7.
Mouse antisera raised against recombinant
EhEBP1 inhibits formation of the URE4-protein complexes.
Electrophoretic mobility supershift assay was performed with
radiolabeled URE4 oligonucleotide and ~10 µg of crude nuclear
extract. Indicated reactions were preincubated with a 1:20 dilution of
either anti-EhEBP1 or nonimmune sera for 15 min on ice before addition
of the radiolabeled DNA.
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Fig. 8.
Induction of EhEBP1 expression results in
repression of transcription at the hgl5 promoter.
A, tetracycline-inducible expression in E. histolytica of the Flag-EhEBP1 fusion protein. A Flag-EhEBP1
fusion protein was placed under control of the tetracycline-inducible
promoter in E. histolytica. Trophozoite lysates from either
uninduced cells ( ) or cells induced with tetracycline for 24 h
(+) were immunoprecipitated with anti-Flag antibody (Sigma). Immunoblot
was performed with anti-EhEBP1 antisera. B, expression of
Flag-EhEBP1 decreases luciferase reporter gene activity in an
URE4-dependent manner. After 17 h of tetracycline
induction, induced (gray bars) and uninduced
cells (white bars) were transfected with either
the wild-type (WT) or mutant (mut) luciferase
reporter constructs. Six hours later, cells were lysed and luciferase
activity measured. As a control, expression of an unrelated protein was
induced from the same tetracycline-inducible promoter and its effect on
wild-type reporter gene activity measured.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sheets of the RRM and nucleotides in
the recognition site (26). Interestingly, the URE4 motif recognized by
EhEBP1 and EhEBP2 is also AT-rich, which might pose problems for
sequence-specific recognition in the context of the AT-rich E. histolytica genome. Perhaps the URE4 sequence assumes a partially
melted structure, facilitating specific recognition by its
RRM-containing binding proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael Kinter and Nicholas E. Sherman of the W. M. Keck Biomedical Mass Spectrometry Laboratory of the University of Virginia for the determination of the tryptic peptide sequences of p28 and p18. Drs Timothy Bender, Ann Beyer, Joel Hockensmith, and Robert Kadner gave valuable advice and discussion.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant AI 37941.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) AF224345 and AF224346 (for EhEBP1 and EhEBP2, respectively).
A Burroughs Wellcome Fund Scholar in Molecular Parasitology.
To whom correspondence should be addressed: University of Virginia HSC,
MR4 Bldg., Rm. 2115, 300 Park Pl., Charlottesville, VA 22908. Tel.:
804-924-0075; Fax: 804-924-0075; E-mail address: wap3g@virginia.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M006866200
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ABBREVIATIONS |
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The abbreviations used are: URE4, upstream regulatory element 4; Gal/GalNAc, galactose and N-acetyl-D-galactosamine; EhEBP1, E. histolytica enhancer-binding protein 1; EhEBP2, E. histolytica enhancer-binding protein 2; hgl, heavy subunit of the galactose and N-acetyl-D-galactosamine inhibitable lectin; TEV, tobacco etch virus; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; SSAP, stage-specific activator protein; RRM, RNA recognition motif; snRNP, small nuclear ribonucleoprotein.
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