Identification of Two Entamoeba histolytica Sequence-specific URE4 Enhancer-binding Proteins with Homology to the RNA-binding Motif RRM*

Joanna M. SchaenmanDagger , Carol A. Gilchrist§, Barbara J. MannDagger §, and William A. Petri Jr.Dagger §||

From the Departments of Dagger  Microbiology, § Internal Medicine, and  Pathology, University of Virginia, Charlottesville, Virginia 22908

Received for publication, July 31, 2000, and in revised form, October 3, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

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 alpha -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).

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -70 °C for further analysis.

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 [alpha -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).

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, [alpha -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).

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).



View larger version (82K):
[in this window]
[in a new window]
 
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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Purification of URE4-binding proteins

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.



View larger version (51K):
[in this window]
[in a new window]
 
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.

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.



View larger version (67K):
[in this window]
[in a new window]
 
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.

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. 



View larger version (31K):
[in this window]
[in a new window]
 
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.

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.



View larger version (73K):
[in this window]
[in a new window]
 
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.

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.



View larger version (89K):
[in this window]
[in a new window]
 
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.

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.



View larger version (75K):
[in this window]
[in a new window]
 
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.

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).



View larger version (10K):
[in this window]
[in a new window]
 
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.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  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.

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.


    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.


    FOOTNOTES

* 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Liu, F., and Bateman, E. (1992) Gene 120, 143-149[CrossRef][Medline] [Order article via Infotrieve]
2. Huang, W., and Batman, E. (1997) J. Biol. Chem. 272, 3852-3859[Abstract/Free Full Text]
3. Liston, D. R., and Johnson, P. J. (1999) Mol. Cell. Biol. 19, 2380-2388[Abstract/Free Full Text]
4. Quon, D. V. K., Delgadillo, M. G., and Johnson, P. J. (1996) J. Mol. Evol. 43, 253-256[Medline] [Order article via Infotrieve]
5. Holberton, D. V., and Marshall, J. (1995) Nucleic Acids Res. 23, 2945-2953[Abstract]
6. World. (1995) Geneva
7. Bruchhaus, I., Leippe, M., Lioutas, C., and Tannich, E. (1993) DNA Cell Biol. 12, 925-933[Medline] [Order article via Infotrieve]
8. Buss, H., Lioutas, C., Dobinsky, S., Nickel, R., and Tannich, E. (1995) Mol. Biochem. Parasitol. 72, 1-10[CrossRef][Medline] [Order article via Infotrieve]
9. Singh, U., Rogers, J. B., Mann, B. J., and Petri, W. A., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8812-8817[Abstract/Free Full Text]
10. Lioutas, C. a. T., E. (1995) Mol. Biochem. Parasitol. 73, 259-261[CrossRef][Medline] [Order article via Infotrieve]
11. Luna-Arias, J. P., Hernandez-Rivas, R., de Dios-Bravo, G., Garcia, J., Mendoza, L., and Orozco, E. (1999) Microbiology 145, 33-40[Abstract]
12. Gilchrist, C. A., Streets, H. L., Ackers, J. P., and Hall, R. (1995) Mol. Biochem. Parasitol. 74, 1-10[CrossRef][Medline] [Order article via Infotrieve]
13. Gomez, C., Perez, D. G., Lopez-Bayghen, E., and Orozco, E. (1998) J. Biol. Chem. 273, 7277-7284[Abstract/Free Full Text]
14. Perez, D. G., Gomez, C., Lopez-Bayghen, E., Tannich, E., and Orozco, E. (1998) J. Biol. Chem. 273, 7285-7292[Abstract/Free Full Text]
15. Purdy, J. E., Pho, L. T., Mann, B. J., and Petri, W. A. J. (1996) Mol. Biochem. Parasitol. 78, 91-103[CrossRef][Medline] [Order article via Infotrieve]
16. Schaenman, J. M., Driscoll, P. C., Hockensmith, J. W., Mann, B. J., and Petri, W. A., Jr. (1998) Mol. Biochem. Parasitol. 94, 309-313[CrossRef][Medline] [Order article via Infotrieve]
17. Diamond, L. S., Harlow, D. R., and Cunnick, C. (1978) Trans. R. Soc. Trop. Med. Hyg. 72, 431-432[Medline] [Order article via Infotrieve]
18. Purdy, J. E., Mann, B. J., Pho, L. T., and Petri, W. A., Jr. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7099-7103[Abstract]
19. Vines, R. R., Purdy, J. E., Ragland, B. D., Samuelson, J., Mann, B. J., and Petri, W. A., Jr. (1995) Mol. Biochem. Parasitol. 265-267
20. Gilchrist, C. A., Mann, B. J., and Petri, W. A., Jr. (1998) Infect. Immun. 66, 2383-2386[Abstract/Free Full Text]
21. Ortner, S., Plaimauer, B., Binder, M., Scheiner, O., Widermann, G., and Duchene, M. (1995) Mol. Biochem. Parasitol. 73, 189-198[CrossRef][Medline] [Order article via Infotrieve]
22. Ramakrishnan, G., Vines, R. R., Mann, B. J., and Petri, W. A., Jr. (1997) Molecular and Biochemical Parasitology 84, 93-100[CrossRef][Medline] [Order article via Infotrieve]
23. Vines, R. R., Ramakrishnan, G., Rogers, J. B., Lockhart, L. A., Mann, B. J., and Petri, W. A., Jr. (1998) Molecular Biology of the Cell 9, 2069-2079[Abstract/Free Full Text]
24. Nagai, K., Oubridge, C., Ito, N., Avis, J., and Evans, P. (1995) Trends Biochem. Sci. 20, 235-241[CrossRef][Medline] [Order article via Infotrieve]
25. Birney, E., Kuman, S., and Krainer, A. R. (1993) Nucleic Acids Res. 21, 5803-5816[Abstract]
26. DeAngelo, D. J., DeFalco, J., Rybacki, L., and Childs, G. (1995) Mol. Cell. Biol. 15, 1254-1264[Abstract]
27. Kenan, D. J., Query, C. C., and Keene, J. D. (1991) Trends Biochem. Sci. 16, 214-220[CrossRef][Medline] [Order article via Infotrieve]
28. Maxson, J. M., and Woodson, W. R. (1996) Plant Mol. Biol. 31, 751-759[Medline] [Order article via Infotrieve]
29. Ding, J., Hayashi, M. K., Zhang, Y., Manche, L., Krainer, A. R., and Xu, R.-M. (1999) Genes Dev. 13, 1102-1115[Abstract/Free Full Text]
30. Scherly, D., Boclens, W., Dathan, N. A., and van Venrooij, I. W. (1990) Nature 345, 502-506[CrossRef][Medline] [Order article via Infotrieve]
31. Nagai, K., Oubridge, C., Jessen, T. H., Li, J., and Evans, P. R. (1990) Nature 348, 515-520[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.