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Eukaryotic Cell, April 2006, p. 672-682, Vol. 5, No. 4
1535-9778/06/$08.00+0     doi:10.1128/EC.5.4.672-682.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

High-Mobility-Group Box Nuclear Factors of Plasmodium falciparum{dagger}

Sylvie Briquet,1* Charlotte Boschet,1 Mathieu Gissot,1,{ddagger} Emilie Tissandié,1 Elisa Sevilla,1 Jean-François Franetich,1 Isabelle Thiery,2 Zuhal Hamid,1,§ Catherine Bourgouin,2 and Catherine Vaquero1*

INSERM, U511, Université Pierre et Marie Curie, Paris VI, Centre Hospitalo-Universitaire de la Pitié-Salpêtrière, Paris, France,1 Biologie et Génétique du Paludisme, CEPIA (Centre de Production et d'Infection des Anophèles), Institut Pasteur, Paris, France2

Received 30 November 2005/ Accepted 31 January 2006


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ABSTRACT
 
In eukaryotes, the high-mobility-group (HMG) nuclear factors are highly conserved throughout evolution and are divided into three families, including HGMB, characterized by an HMG box domain. Some HMGB factors are DNA structure specific and preferentially interact with distorted DNA sequences, trigger DNA bending, and hence facilitate the binding of nucleoprotein complexes that in turn activate or repress transcription. In Plasmodium falciparum, two HMGB factors were predicted: PfHMGB1 and PfHMGB2. They are small proteins, under 100 amino acids long, encompassing a characteristic HMG box domain closely related to box B of metazoan factors, which comprises two HMG box domains, A and B, in tandem. Computational analyses supported the conclusion that the Plasmodium proteins were genuine architectural HMGB factors, and in vitro analyses performed with both recombinant proteins established that they were able to interact with distorted DNA structures and bend linear DNA with different affinities. These proteins were detected in both asexual- and gametocyte-stage cells in Western blotting experiments and mainly in the parasite nuclei. PfHMGB1 is preferentially expressed in asexual erythrocytic stages and PfHMGB2 in gametocytes, in good correlation with transcript levels of expression. Finally, immunofluorescence studies revealed differential subcellular localizations: both factors were observed in the nucleus of asexual- and sexual-stage cells, and PfHMGB2 was also detected in the cytoplasm of gametocytes. In conclusion, in light of differences in their levels of expression, subcellular localizations, and capacities for binding and bending DNA, these factors are likely to play nonredundant roles in transcriptional regulation of Plasmodium development in erythrocytes.


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INTRODUCTION
 
Malaria is the most important parasitic disease in the world, and of the 300 to 500 million cases each year, approximately 2 million people die. Among the four species of malaria parasites infecting humans, Plasmodium falciparum causes the highest morbidity and mortality. Global efforts to eradicate malaria have failed, and there is presently no effective vaccine available. Therefore, a greater understanding of parasite biology throughout development is urgently needed in order for novel therapeutic strategies to control malaria to be proposed.

During the erythrocytic life cycle, intense multiplication of parasites takes place, as well as gametocyte differentiation associated with cell cycle arrest. These different developmental pathways require the coordinated and modulated expression of diverse sets of genes, involving transcriptional, epigenetic, and posttranscriptional regulation. Currently, it is commonly accepted that general mechanisms involved in gene regulation in eukaryotes also operate in P. falciparum (25, 32, 33). Nevertheless, elucidation of the molecular mechanisms involved in transcriptional regulation in Plasmodium is still challenging. Even if very little is known about the cis- and trans-regulatory elements of the parasite, Plasmodium genes exhibit the bipartite structure of eukaryotic promoters, i.e., a basal promoter regulated by upstream regulatory elements (25) that present some homology with the binding sites of eukaryotic transcription factors (TF). The recent completion of the genome sequence of P. falciparum revealed a high proportion of orphan proteins (60% of the open reading frames [ORFs] have no match with any of the annotated sequences listed in the data banks [18]). These data might contribute to the low numbers of recognizable, orthologous TF (11). However, it is reasonable to assume that in Plasmodium the interplay between regulatory elements and TF, whose availability (49) presumably modulated throughout parasite development, governs also the level of RNA synthesis.

In eukaryotes, in addition to general TF also annotated in Plasmodium (10, 23, 37, 38, 43, 44), the factors involved in transcriptional regulation can be divided into factors interacting either with specific DNA sequences (42) or with DNA structures. The latter include the nonhistone proteins of the high-mobility-group (HMG) superfamily (7, 58, 62), which is divided into three families of proteins in line with their characteristic functional motifs (8): HMGA, which interacts with the AT hook; HMGN, which interacts with the nucleosomes; and HMGB, which encompasses one or several copies of the HMG box DNA binding domain (for a review, see reference 7). HMG proteins are present in all metazoan phyla, plants, and yeast and have also been reported in unicellular parasites, including trypanosomes (15, 45), schistosomes (21), and Plasmodium (29). They are quite abundant proteins, one molecule for 10 to 15 nucleosomes in vertebrates. It is assumed that the wrapping of DNA by histones and nonhistone proteins, including the HMG proteins, controls the access of the TF to their target sites on nucleosomes (31).

HMGB factors are highly conserved throughout evolution, and their HMG box domain is composed of around 80 amino acids (aa) folded in three {alpha}-helices arranged in an L shape (3, 66). In vertebrates, the HMGB proteins generally present two boxes, A and B, and also basic N- and C-terminal extensions and a rather long C-terminal acidic tail (58). Despite their low sequence homology, both boxes (A and B) present a well-conserved L-shaped structure, even though their DNA binding and bending capacities may display some differences (28, 69). In lower eukaryotes, either the basic extension (Drosophila melanogaster) or the negatively charged tail (Saccharomyces cerevisiae) is missing, in contrast to plant HMGs (60), which possess both extensions, albeit of different lengths. The basic domains appear to play a role in the stabilization of HMGB-mediated DNA bending. In contrast, the role of the acidic tail remains elusive and may be shaped to interact with the positive charges of histones (for a review, see reference 62). Two subfamilies of HMGB, with either DNA sequence specificity (SOX, SRY, TCF, MATA) or structure specificity (HMGB per se), have been identified. The latter preferentially interacts with distorted DNA sequences and triggers DNA bending, hence altering the positioning of nucleosomes on the DNA fiber, thereby controlling the level of transcription (31). Finally, a linker histone H1 (27), via its interaction with the DNA linker between two nucleosomes, increases the compactness of the chromatin (53), impairing interactions between DNA and TF and therefore repressing gene transcription (24). In contrast, the HMGB proteins appear to be associated with active chromatin (48), increasing nucleosome sliding and target site accessibility and thereby enhancing transcription.

Lately, these proteins, historically known as nuclear proteins, have been reported to be released from mammalian cells and to act as mediators of the immune response and as potent macrophage-activating factors (for reviews, see references 14, 41, and 46).

In Plasmodium, very few TF have been annotated (PlasmoDB and our group [19]) and characterized (6, 20). In Plasmodium falciparum, four potential HMG factors have been annotated, including one previously reported for the FCQ27 (29) and FCC1/HN parasite clones. Two of these factors, PfHMGB1 and PfHMGB2, were investigated during the erythrocytic cycle to evaluate their molecular implications in transcription regulation.


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MATERIALS AND METHODS
 
Plasmodium falciparum culture. The 3D7 clone of P. falciparum was provided by D. Walliker and was grown in human erythrocytes, as described by Trager and Jensen (61), except that the culture medium contained 0.5% Albumax instead of human serum. Gametocytes were produced from the gametocyte-producing isolate NF54 (provided by W. Eling) as aforementioned, with 10% AB human serum, using the automated culturing system developed by Ponnudurai et al. (52). Parasitemia was monitored on Giemsa-stained blood smears. Thirteen to 15 days after induction of gametocytogenesis, gametocytes were monitored for maturity by observation under a microscope (1 x 100 immersion) of microgamete exflagellation, with the addition of 5 µl of gametocyte culture in a 10-µl drop of human serum.

Antibodies. Primary antibodies used to characterize the PfHMGB1 and PfHMGB2 proteins were obtained after immunization of BALB/c mice with either recombinant protein (50 µg, twice at 2-week intervals). Sera with high levels of anti-PfHMGB1 and anti-PfHMGB2 activity were collected after the first booster inoculation (day 30) and selected for immunochemical analyses.

Annotation. Before the sequence of the P. falciparum genome was completed in 2002, PfHMGB1 was annotated within chromosome 12 by homology to a consensus of the HMG box domains of around 50 disparate eukaryotic sequences. Then, the HMG box domain of PfHMGB1, used as a query against the Plasmodium database, allowed the annotation of PfHMGB2 and PfHMGB3 within chromosomes 8 and 12, respectively, while that of PfHMGB2 allowed the annotation of PfHMGB4 within chromosome 13. The presence of relevant HMG box domains in these proteins was checked with MotifScan (16).

Molecular cloning. The Pfhmgb1 and Pfhmgb2 ORF sequences were amplified from genomic DNA of the P. falciparum 3D7 clone, with forward (5'-GGTGGATCCATGAAGAATACAGGAAAAGAAG-3' and 5'-GGTCCCGGGGCCAATTTAAGCTTTCATTTTC-3') and backward (5'-ATTGGATCCATGGCTTCAAAATCTCAAAA-3' and 5'-ATTGGTACCTTATTCTTGATTTTTCTTTC-3') primers, respectively, using PCR conditions with an elongation temperature of 60°C, as described previously (57). Fragments of 294 bp and 300 bp, corresponding to the complete Pfhmgb1 and Pfhmgb2 ORF sequences, were cloned directly into the pGEM-T Easy vector (Promega) and pCR II-Topo (Invitrogen), respectively, and then sequenced with the ABI Prism kit (Perkin Elmer).

Northern blot analysis. Total RNA was purified from isolated parasites with TRIzol (Invitrogen), and the integrity of the RNA preparation was monitored by ethidium bromide staining on an agarose gel and analysis with an Agilent bioanalyzer. The Pfhmgb1 and Pfhmgb2 transcripts were characterized from 20 µg of 3D7 total RNA by Northern blotting, according to the Ultrahyb protocol of Ambion, with a 65°C hybridization temperature. Antisense {alpha}-32P riboprobes were prepared as previously described (50) from the pGEMT-pfhmgb1 and pCRII Topo-pfhmgb2 vectors by using T7 and SP6 RNA polymerase (Promega), respectively.

Expression and purification of recombinant proteins. The pQE30 vector (QIAGEN) was used to express the recombinant proteins (rePfHMGB1 and rePfHMGB2) in Escherichia coli as His6-tagged proteins with BamHI-XmaI- and BamHI-KpnI-digested inserts from the above-mentioned vectors, respectively. The bacterial strain SG 13009, harboring the Pfhmgb1 or Pfhmgb2 expression construct, was grown at 37°C in 200 ml of 2YT medium containing 100 µg/ml ampicillin as well as 50 µg/ml kanamycin. Expression was induced with 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 3 h at 37°C, and collected cells were solubilized in sonication buffer S (25 mM Tris-HCl [pH 8], 300 mM NaCl, 10 mM imidazole, 10 mM ß-mercaptoethanol, 0.5% Triton X-100), 1 ml per mg of dry pellet, in the presence of lysozyme and a 1/25 final dilution of a protease inhibitor cocktail tablet (Roche). Purification of His-PfHMGB proteins was performed essentially as previously described with Ni-nitrilotriacetic acid agarose beads (QIAGEN) (34). After three washes with buffer S supplemented with 20 mM imidazole, bound proteins were eluted with either 250 mM or 50 mM imidazole in 20 mM Tris-HCl (pH 8)-300 mM NaCl for rePfHMGB1 or rePfHMGB2, respectively.

EMSA with synthetic four-way DNA junctions. The partially complementary oligonucleotides 1 to 4, previously described (5, 67), were used to create four-way DNA junctions (4H), as well as 3H and 2H. In addition, the radiolabeled leg 1 containing cruciform 4H was fractionated and eluted from a 5% polyacrylamide gel in 0.5x Tris-borate-EDTA (TBE) buffer. Electrophoretic mobility shift assays (EMSA) were performed by incubating labeled 4H with increasing amounts (0 to 25 µM) of rePfHMGB1 or rePfHMGB2 in a 10-µl final volume for 20 min at room temperature. This dose-response experiment was carried out to determine the amounts of protein necessary to create a major 4H-PfHMGB complex. In the competition assay, 100- and 500-fold molar excesses of cold complete 4H or incomplete 3H and 2H were added to the reaction for an additional 20 min. Samples were run on a 6.5% polyacrylamide gel in 0.5x TBE buffer at 120 V. The vacuum-dried gels were autoradiographed with intensifying screens at –80°C overnight.

Ligase-mediated circularization assay. The circularization assay was based on existing protocols (9, 67). Linear DNA fragments of 123 bp were {gamma}-32P 5' end labeled and preincubated with increasing concentrations (0.25 to 100 µM) of rePfHMGB1 or rePfHMGB2 for 20 min at room temperature in 1x DNA ligase buffer (New England Biolabs [NEB]) in a final volume of 10 µl. DNA circularization was generated with T4 DNA ligase (NEB), and linear DNA was subsequently digested by exonuclease III (NEB). Samples were treated with proteinase K (Invitrogen) and run on a 6.5% polyacrylamide gel in 0.5x TBE buffer at 120 V. The gels were vacuum dried and autoradiographed.

Preparation of parasite NE, CE, and total extracts and Western blot analysis. Nuclear extracts (NE), cytoplasmic extracts (CE), and total lysates were prepared from 50 ml of red blood cells at 10 to 12% parasitemia infected with 3D7 asexual-stage cells, as described by Osta et al. (49). Proteins (12 µg) were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blotting experiments (see Fig. 4b) after transfer onto polyvinylidene difluoride membranes (Bio-Rad). For sexual-stage analysis, 2 ml of NF54 culture was harvested on day 13 after induction of gametocytogenesis, with 13.8% parasitemia including 55% mature gametocytes (stage V). After erythrocytic lysis in phosphate-buffered saline-0.15% saponin, gametocytes were collected and resuspended in 40 µl of Laemmli buffer. Half of this sample was fractionated for detection of either protein, PfHMGB1 or PfHMGB2. A similar amount of asexual-stage culture was run in parallel (see Fig. 4c). The blots were probed with a 1:2,000 dilution of anti-PfHMGB1 or anti-PfHMGB2 serum, followed by incubation with a peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibody (Sigma), and revealed by chemiluminescence (Perkin Elmer) or the Supersignal West Femto kit (Pierce). Negative controls were performed with preimmune sera and positive controls with the recombinant proteins. Finally, an anti-HSP70 serum (55) was used as a positive control for CE (see Fig. 4b) and for normalization of protein loading between asexual/sexual stages (QuantiScan Biosoft 2.1).


Figure 4
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  		FIG. 4. Characterization of Pfhmgb1 and Pfhmgb2 transcripts by Northern blotting (a) and of their corresponding proteins, His-PfHMGB1 and His-PfHMGB2, by Western blotting (b). By SDS-PAGE, the apparent molecular mass of PfHMGB factors was approximately 12 kDa, whereas that of HSP70 was approximately 70 kDa. (c) Expression of PfHMGB1 and PfHMGB2 in total lysates prepared from asexual- versus gametocyte-enriched cultures. HSP70 protein expression was used for normalization of sample loading. AS, asexual stages; G, gametocytes.

Immunofluorescence assay. For localization of PfHMGB1 and PfHMGB2 in P. falciparum, asexual and sexual stages obtained as described previously were washed twice in RPMI, fixed in 2% paraformaldehyde for 20 min at room temperature, and laid on poly-L-lysine-coated multiwell glass slides. Endogenous fluorescence was quenched with 75 mM NH4Cl for 10 min. The parasites were permeabilized with 0.5% Triton X-100 for 3 min, blocked with 0.5% bovine serum albumin in phosphate-buffered saline for 1 h, and then incubated with a 1:200 dilution of primary antibodies (anti-PfHMGB1, anti-PfHMGB2, anti-HSP70) for 1 h at room temperature, followed by incubation with a fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (Sigma) and DAPI (4',6'-diamidino-2-phenylindole) for 1 h before examination by fluorescence microscopy using a Leica DM RD fluorescent microscope equipped with UV and fluorescein filters. The time of exposure was selected in relation to the intensity of the immunofluorescence obtained with each antibody. Images were acquired with Lucia 4.7 and merged with Adobe Photoshop.

Nucleotide sequence accession numbers. The nucleotide sequences of the genes encoding the following proteins have been assigned the corresponding PlasmoDB accession numbers: PfHMGB1, PFL0145c; PfHMGB2, MAL8P1.72; PfHMGB3, PFL0290w; PfHMGB4, MAL13P1.290.


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RESULTS
 
Several predicted factors in Plasmodium belong to the HMGB family. Four putative HMG proteins in P. falciparum were predicted by sequence homology, as described in Materials and Methods. The proteins appeared to belong to the HMGB family and were named PfHMGB1 to PfHMGB4. The first two, PfHMGB1 and PfHMGB2, are small proteins, under 100 aa in length, while PfHMGB4 has been predicted to encode a 160-aa-long protein. All three encompass only one HMG box domain. In contrast, PfHMGB3 is a larger protein (2,284 aa), with two HMG box domains and several additional putative functional motifs, including one Myb domain (2). The two small proteins, PfHMGB1 and PfHMGB2, are presented herein. It is worthy of note that in contrast to most eukaryotic HMGBs, displaying N- and C-terminal extensions of diverse lengths (58) and reported to bear functional roles, the two Plasmodium factors have only a short basic extension upstream of the HMG box domain and no acidic C-terminal tail.

Soullier et al. (56) revealed by phylogenetic analysis that the HMGB factors can be separated into two clearly defined subgroups: (i) the SOX/SRY/MATA/TCF family, whose members are able to bind specific linear DNA sequences, and (ii) the HMGB/UBF family, whose members interact with high affinity to distorted DNA structures. After the addition of Plasmodium and Babesia bovis sequences, the same analysis was performed several times with different random number seeds, and we were able to assign PfHMGB1 and PfHMGB2 to the subgroup of HMGB proteins characterized by DNA structure specificity (see supplemental material S1) (17, 59). This finding was strengthened by the alignment shown in Fig. 1, performed with several eukaryotic HMG box domains and two sets of complete HMGB1 and HMGB2 sequences issued from diverse Plasmodium species. The HMGB of Plasmodium possessed two of the three determinants reported to determine the structural DNA specificity (47), that is, the presence in positions 10 and 32 (according to the residue numbering of Drosophila HMG-D) of a serine and a hydrophobic residue, respectively. Therefore, we assigned the Plasmodium factors to the architectural HMGB family. Finally, a phylogenetic tree was issued from the HMG box domains of PfHMGB1 and PfHMGB2 and those of various metazoan proteins containing two boxes, A and B, in tandem (see supplemental material S2). This analysis, performed several times with different random number seeds, revealed that the HMG box sequences of both Plasmodium proteins are more similar to box B than to box A of proteins with two HMG box domains.


Figure 1
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  		FIG. 1. Multiple alignment of HMG box domains of several eukaryotic HMG proteins and complete sequences of Saccharomyces NHP6A and HMGB1 and HMGB2 of diverse Plasmodium species. Dashes and dots represent gaps and missing sequences, respectively. Uppercase letters represent HMG box domains identified by the program MotifScan (16). Identities shared by all HMGB sequences presented herein are indicated in bold blue print, by all Plasmodium sequences in green, by all Plasmodium HMGB1 sequences in red, and by all Plasmodium HMGB2 sequences in pink. The boxed amino acids are two crucial determinants that differ between the sequence-specific and the structure-specific HMG box domains: here, a serine and a hydrophobic residue, as found in all non-sequence-specific HMG proteins, whereas all sequence-specific HMG proteins present an asparagine and a hydrophilic residue at these positions. I, II, and III represent the three {alpha}-helices of the D. melanogaster HMG-D structure (PDB file 1qrv [47]), and asterisks indicate the two residues of D. melanogaster HMG-D that intercalate in the DNA minor groove. Underlined residues in PfHMGB1 and PfHMGB2 represent the four {alpha}-helices modeled by homology with box B of the Chinese hamster HMG1 protein, used as a template (PDB file 1hsn [54]). Abbreviations are as follows: Bb, Babesia bovis; Dm, Drosophila melanogaster; Gm, Glycine max; Os, Oryza sativa; Pb, Plasmodium berghei; Pf, Plasmodium falciparum; Pk, Plasmodium knowlesi; Pv, Plasmodium vivax; Py, Plasmodium yoelii; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Zm, Zea mays.

The three-dimensional structure of the two Plasmodium factors was modeled by homology using as template the structure of box B of the Chinese hamster HMG1 protein (PDB file 1hsn [54]). Four {alpha}-helices, called 1, 1', 2, and 3 (underlined residues in Fig. 1 corresponding to the three {alpha}-helices, I, II, and III, stated at the top of Fig. 1 for D. melanogaster HMG-D), were predicted to fold in an L shape (see supplemental material S3) (12, 30) in the PfHMGB1 sequence from His 19 to Tyr 90 and in the PfHMGB2 sequence from Ala 23 to Gln 98.

Therefore, all of these computational analyses agreed in suggesting that the two PfHMGB proteins were genuine factors of the HMGB family and may therefore behave as potential architectural factors.

PfHMGB1 and PfHMGB2 interact with 4H. Several sets of in vitro assays were used to validate the computational identification. After the genes were cloned, expression of rePfHMGB1 and rePfHMGB2 was carried out in Escherichia coli and His proteins were purified as described in Materials and Methods.

First, increasing amounts of both recombinant rePfHMGB1 and rePfHMGB2 (up to 25 µM) were incubated with radiolabeled complete 4H to analyze the formation of 4H-rePfHMGB1 and 4H-rePfHMGB2 complexes by EMSA. When 3 µM rePfHMGB1 (Fig. 2a) or 0.6 µM rePfHMGB2 (Fig. 2b) was added to the reaction, the 4H labeled cruciform became incorporated into a major 4H-PfHMGB1 or 4H-PfHMGB2 retarded band, respectively. These amounts of recombinant factors were used for the subsequent EMSA experiments.


Figure 2
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  		FIG. 2. EMSA interaction between cruciform DNA and either rePfHMGB1 (a) or rePfHMGB2 (b). Increasing concentrations (0 to 25 µM) of rePfHMGB proteins were incubated with radiolabeled 4H. Competition EMSA experiments were performed between the rePfHMGB1-4H (c) or rePfHMGB2-4H (d) complex and various DNA competitors. After incubation of the rePfHMGB proteins with labeled 4H, either cold incomplete junctions (3H and 2H) or complete 4H were added to the reaction at 100- and 500-fold molar excesses, as indicated above each lane. Figure 2, cruciform DNA. The first lanes correspond to cruciform DNA migration without protein interaction (a to d), and the second lanes correspond to a cruciform DNA-rePfHMGB complex retarded band without competition (c and d).

Second, the binding specificities of the 4H-PfHMGB1 and 4H-PfHMGB2 complexes were analyzed by competition experiments, that is, by adding a 100- or 500-fold molar excess of either complete (4H) or incomplete (3H and 2H) cold DNA junctions, after DNA-protein complex formation. A 500-fold excess of the integral 4H structure abolished the interaction of either protein, whereas a 100-fold molar excess shifted the 4H-PfHMGB2 interaction completely but the 4H-PfHMGB1 interaction only weakly (Fig. 2c and d). In addition, the competition with cold 3H and 2H was ineffective upon analysis of PfHMGB1, in contrast to the substantial competition observed in the presence of PfHMGB2. Thus, binding of PfHMGB1 to 4H required an intact crossover-containing structure. Altogether, these results indicated that the two recombinant proteins were able to bind distorted DNA in vitro, even though the nature of their interaction with 4H was quite different, being more efficient and specific for PfHMGB1 than for PfHMGB2.

PfHMGB1 and PfHMGB2 induce DNA bending. We compared the efficiencies of increasing concentrations of rePfHMGB1 and rePfHMGB2 to bend and in turn promote T4 DNA ligase-mediated circularization of a labeled synthetic linear DNA fragment. Indeed, when ligase was added to the labeled fragment of around 125 bp, several bands appeared, including a circular DNA form resistant to exonuclease III (Fig. 3a and b). In the presence of exonuclease III alone, which digests only linear DNA molecules, a marked decrease in all labeled bands was observed, showing that in the absence of PfHMGB proteins, only small amounts of minicircles, if any, were produced. In contrast, in the presence of ligase and increasing amounts of PfHMGB proteins, the quantity of minicircles was quite increased, suggesting that both proteins were capable of enhancing DNA flexibility and hence DNA circularization. The capacity for DNA bending is thus an intrinsic property of PfHMGB1 and PfHMGB2. Nevertheless, rePfHMGB1 once again showed greater efficacy (Fig. 3a), since it started to promote circularization at 0.25 µM, at a concentration 10-fold lower than that of rePfHMGB2 (3 µM), and the maximum signal was reached with 1 µM compared with 50 µM rePfHMGB2. Moreover, the signal observed with 50 to 100 µM PfHMGB2 (Fig. 3b) was far weaker than that observed with 2 µM PfHMGB1 (Fig. 3a).


Figure 3
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  		FIG. 3. DNA bending and ligase-mediated circularization assay with either rePfHMGB1 (a) or rePfHMGB2 (b). The {gamma}-32P 5' end-labeled 123-bp DNA fragment was preincubated with increasing amounts of rePfHMGB1 (0 to 2 µM) or rePfHMGB2 (0 to 100 µM), followed by ligation with T4 DNA ligase. The ligation products were subjected to electrophoresis after exonuclease III treatment. T4 DNA ligase was added to all samples except that loaded in the first lane. All samples were treated with exonuclease III except samples of the first two lanes. The migration positions of 123-bp linear and 123-bp minicircular DNAs are indicated at left.

Pfhmgb1 and Pfhmgb2 transcripts are expressed during the P. falciparum erythrocytic cycle. In order to determine the presence of Pfhmgb1 and Pfhmgb2 RNA and to characterize them molecularly, we performed a Northern blotting analysis of total RNA prepared from infected red blood cells (Fig. 4a). The lengths of Pfhmgb1 and Pfhmgb2 mRNA were estimated at 1.3 and 1.1 kb, respectively. The integrity and quality of total RNA extracts of 3D7 were verified after ethidium bromide staining of the gel. As already described for many other Plasmodium messengers, the 5' and 3' untranslated regions of both transcripts are quite long, as the coding regions comprise fewer than 300 nucleotides.

PfHMGB1 and PfHMGB2 proteins are present in the P. falciparum nucleus. Localization of PfHMGB1 and PfHMGB2 was analyzed by Western blotting analysis using CE and NE of P. falciparum asexual stages and specific antisera raised against each recombinant protein. The NE and CE prepared from 3D7 parasites (12 µg), as well as the recombinant proteins rePfHMGB1 and rePfHMGB2 (50 ng), were fractionated by SDS-PAGE, and after transfer, the membranes were developed with the specific antisera (Fig. 4b). The specificities of the two antibodies were verified. No cross-reaction was observed, as His-PfHMGB1 and His-PfHMGB2 were recognized only by their respective antisera (Fig. 4b, lanes 4 to 5 and lanes 8 to 9 for His-PfHMGB1 and His-PfHMGB2, respectively). The control experiments performed with the two preimmune sera gave no signal (data not shown). Both PfHMGB1 and PfHMGB2 were clearly detected in the NE (lanes 3 and 7), whereas same protein loading of CE does not give any detectable signal (lanes 2 and 6). The quality of the CE preparations was controlled by detection of the HSP protein (70 kDa) (lane l). The apparent molecular mass of both PfHMGB proteins in the nuclear extracts, around 12 kDa, was in good agreement with the theoretical molecular masses of 11.3 kDa and 11.5 kDa for PfHMGB1 and PfHMGB2, respectively (lanes 3 and 7), slightly smaller than the recombinant His proteins (12.7 kDa and 12.9 kDa, respectively).

Localization of the factors within the nucleus was confirmed by immunofluorescence analysis of unsynchronized parasite cultures containing all asexual stages of P. falciparum. Control experiments carried out in the presence of either preimmune sera or the secondary antibody alone showed no signal (data not shown), in contrast to the labeling obtained with both anti-PfHMGB sera coupled with nucleus-specific DAPI staining (Fig. 5). The two proteins appeared to be present mainly in the parasite nuclei, as shown by the merge of DAPI (lanes a and b), and, in contrast to the HSP70 signal, also readily detectable in the cytoplasm, as indicated by the red fluorescence observed in the superposition insert (lane c).


Figure 5
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  		FIG. 5. Immunofluorescence localization of PfHMGB1 and PfHMGB2 in asexual (a, b, and c) and sexual (d, e, and f) stages of Plasmodium erythrocytic development. Paraformaldehyde-fixed parasites were labeled with mouse anti-PfHMGB1 and anti-PfHMGB2 antibodies (1:200) and FITC-conjugated anti-mouse IgG (1:100); DNA was stained with DAPI (1:100). Merged fluorescent signals are shown in the "superposition" column. Cells were visualized by phase-contrast (a and c) or transmission (b, d, e, and f) microscopy. Panels: a, trophozoites; b, trophozoite and schizont; c, trophozoites; d to f, gametocytes. Anti-PfHMGB and anti-HSP70 fluorescence is red for panels a to c and green for panels d to f.

PfHMGB1 and PfHMGB2 factors are expressed differentially in the asexual and gametocyte stages. We compared the expression levels of both factors within total lysates from mixed asexual versus gametocyte cultures by means of Western blotting experiments (Fig. 4c). Each factor was evaluated as detailed in Materials and Methods via HSP70 expression. The level of PfHMGB1 expression was clearly lower in gametocytes than in mixed asexual stages, in contrast to PfHMGB2, whose expression was higher in gametocytes. Indeed, after densitometric quantification of this representative experiment via HSP70 protein normalization (QuantiScan Biosoft 2.1), the normalized values of protein expression were 71%/29% for PfHMGB1 and 27%/73% for PfHMGB2, respectively, when asexual and gametocyte cultures were compared.

We also compared the localizations of both factors in asexual (red immunofluorescence) and gametocyte (green immunofluorescence) stages. As already mentioned, the two PfHMGB factors (Fig. 5, lanes a and b) appeared to be located mainly in the nucleus of the asexual stages (rings, trophozoites, and schizonts), whereas the HSP70 protein (lane c) was also found in the parasite cytoplasm. Surprisingly, in addition to its nuclear localization, PfHMGB2 could also be readily detected within the cytoplasm of different stages (IV and V) of gametocytes (lanes e), as also observed for the HSP70 protein (lane f), whereas PfHMGB1 was associated mainly with the nucleus of gametocytes, as in asexual parasites (Fig. 5, lanes d and a).


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DISCUSSION
 
The two Plasmodium HMG proteins belong to the HMGB subfamily and comprise only one HMG box domain, like proteins of plants and several proteins of yeast and Drosophila. The parasite HMG box domains are quite similar to those of all eukaryotic organisms and show characteristic residues that are important for DNA binding and bending, as shown for the Drosophila HMG-D protein by Murphy and colleagues (47). The presence of Ser-10 and Val-32 (boxed residues in Fig. 1, according to the numbering of HMG-D) allowed us to assign the Plasmodium factors to the architectural HMGB family, as also shown by the phylogenetic analysis performed with 159 HMG box domains (see supplemental material S1). Ser-10 forms water-mediated hydrogen bonds with DNA. The hydrophobic residues Val-32 and Met-13 (Fig. 1, residues with asterisks) partially intercalate between two base pairs, introducing two successive kinks into the bound DNA that enhance the more uniform bend associated with the widening of the minor groove. In addition to the HMG box domain, the parasite proteins exhibit a basic extension N terminal to the HMG box domain and apparently no acidic C-terminal tail, as also observed for the yeast NHP6A protein. The HMG box domains bind the DNA minor groove, and in the NHP6A and HMG-D proteins, the basic extension binds in the compressed major groove on the face of the helix opposite the widened minor groove (1, 13), so as to stabilize the HMG box domain-induced bending (39) and consequently facilitate circularization (22, 68).

Since the two Plasmodium HMGB factors exhibited only one HMG box domain, we asked whether the Plasmodium box was more similar to box A or box B of the metazoan HMGB that encompasses two HMG box domains in tandem. All analyses converge to the same conclusion: the Plasmodium HMG box domain more closely resembles box B. When the phylogenetic analysis (see supplemental material S2) was performed with the HMG box domains of PfHMGB1 and PfHMGB2 and of various proteins containing box A and box B, the Plasmodium factors clearly clustered with all B boxes. For the human HMG1, it was reported that structure-specific binding to the four-way DNA junction was mediated by the A domain (65) and that box B, flanked by the basic region, displayed a marked DNA recognition activity (69). Hence, the short N-terminal basic domain of the two Plasmodium nuclear factors might govern their interaction with distorted DNA and subsequent DNA bending.

In addition, box B of the HMGB of vertebrates was reported to behave as a potent proinflammatory cytokine (64). The tumor necrosis factor (TNF)-stimulating activity was mapped to the KDPNAPKRPPSAFFLFCSEY sequence, corresponding to the first 20 aa of box B in human HMG1 factor, according to the numbering of Li et al. (36). In Plasmodium, a domain sharing 75% and 70% identical or strongly similar residues with the TNF-stimulating domain of the human factor was found at the N-terminal position of the HMG box domains of PfHMGB1 and PfHMGB2, respectively. Presently, experiments are under way to analyze whether the two Plasmodium factors exhibit TNF-stimulating function.

An automatic three-dimensional structural prediction was performed, and for both Plasmodium factors, four {alpha}-helices, called 1, 1', 2, and 3, were predicted (underlined residues of PfHMGB1 and PfHMGB2 of Fig. 1), folding in an L shape in the HMG box domain. HMG box domains have actually exhibited only three {alpha}-helices (66), but even if four {alpha}-helices were predicted in the two parasite proteins, their positions would be in good agreement with those of Drosophila melanogaster HMG-D (PDB file 1qrv [47]), which are indicated (I, II, and III) at the top of Fig. 1. In addition, the sample of the reference template used for nuclear magnetic resonance contained a molecule of ß-mercaptoethanol attached to the single cysteine of the protein. The molecule of ß-mercaptoethanol, which reduces the affinity of the HMG box domain for the four-way DNA junction, also disrupted the usual first helix (see supplemental material S3). For that reason, helices 1 and 1' could be regarded as a single, kinked helix.

These computational analyses were concordant in suggesting that the Plasmodium proteins were genuine architectural HMGB factors close to box B in being able to bind and bend DNA. In vitro analyses performed with both recombinant proteins established that they were indeed able to interact with distorted DNA structures (Fig. 2) and bend linear DNA (Fig. 3), leading to the validation of the computational data (Fig. 1 and supplemental materials S1, S2, and S3). In contrast, EMSA performed with labeled linear DNA binding sites reported to interact specifically with members of the HMGB subfamily comprising the usual TF SOX and SRY (63) gave no detectable retarded complexes (data not shown).

Therefore, it can reasonably be assumed that these architectural HMGB factors might play a role in the remodeling of chromatin. In eukaryotes, one proposed mechanism is that the HMGB nuclear factors might change the nucleosome structure and relax the wrapped DNA so as to enhance the accessibility of the remodeling complexes to chromatin and facilitate interaction of TF with their binding sites (for a review, see reference 62). It has also been observed that the interplay between these factors and the linker histone H1 modulates the balance between alternative conformations of the chromatin, histone H1 enhancing chromatin compaction, in contrast to HMGB. In Plasmodium, even though the gene for the H1 linker histone has not yet been annotated, along with 60% of the 5,300 predicted genes, a putative histone H1-like protein might be present and counteract the function of Plasmodium HMGB. Indeed, the H1 histones are evolutionarily conserved in metazoans but substantially divergent in protists (51). Some protists appeared to have only a lysine-rich basic protein, whose composition is similar to some of the histone H1-like proteins from eubacteria, animals, and plants (26).

Le Roch et al. reported differential expression of both transcripts. Pfhmgb1 is preferentially expressed during the erythrocytic asexual stages, in contrast to Pfhmgb2, in which preferential expression occurs in gametocytes (35). Figure 4c shows that expression of the two corresponding proteins is closely related to the level of transcripts and is differentially expressed in mixed asexual and gametocyte stages. The Western blot of cytoplasmic and nuclear extracts prepared from asexual stages (Fig. 4b) and immunofluorescence of asexual and gametocytes stages (Fig. 5a, b, d, and e) revealed that the two factors are localized mainly in the nucleus. Furthermore, PfHMGB2 was clearly detected in the cytoplasm of gametocytes (Fig. 5d and e).

In addition to the differences in the levels of expression and localization within asexual and sexual parasites, these two factors exhibited different affinities when interacting and bending DNA (Fig. 3), PfHMGB2 being less efficient, at least when examined in vitro. All of these results argue in favor of little if any redundancy between the two proteins and in favor of a role in gametocytogenesis.

In summary, a combination of computational and molecular analyses is needed to increase our knowledge of transcriptional regulation of Plasmodium genes involved in crucial steps of asexual and sexual erythrocytic development. This report describes the characterization of two Plasmodium HMGB factors that appear to exhibit substantial similarity to architectural factors as regards their biological functions, at least when analyzed in vitro for the capacity to interact with distorted DNA and to bend DNA, even though their capacities to do so appeared to be quite different. As in eukaryotes, HMGB factors in Plasmodium, since they are highly conserved through evolution, are probably involved in chromatin remodeling. However, even though these proteins were observed in asexual and gametocyte stages, their levels of expression were clearly different, with PfHMGB1 likely implicated in proliferation and PfHMGB2 implicated in differentiation of Plasmodium. Much more work will be needed to understand the functions of these two proteins, both as nuclear factors and as cytokines. Invalidation of either the factors or the interaction between the PfHMGB and DNA via gene silencing strategies (20) and antagonists, etc., will increase our knowledge of transcriptional regulation as well as our control of the erythrocytic development of the parasite (proliferation and differentiation). It might provide exciting new therapeutic possibilities. In this regard, it is worthy of note that this type of approach, via disruption of DNA/factor interaction, is currently being evaluated for human cancer therapy with a special focus on HMG proteins (4, 40).


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ACKNOWLEDGMENTS
 
We thank Stephan Soullier for kind help in the phylogenetic studies, Olivier Silvie for the gift of anti-HSP70 serum, and Justine Masson (Inserm U677, Paris) for help with the immunofluorescence facilities. We are grateful to Jennifer Richardson for critical reading of the manuscript.

M.G. and C.B. were financially supported by the Ministère de l'Education Nationale, France. The project was supported by Inserm and UPMC funds to C.V.


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FOOTNOTES
 
* Corresponding author. Mailing address: INSERM, U511, Université Pierre et Marie Curie, Paris VI, Centre Hospitalo-Universitaire de la Pitié-Salpêtrière, 91 boulevard de l'Hôpital, 75013 Paris, France. Phone: 33 (0) 1 40 77 81 14. Fax: 33 (0) 1 45 83 88 58. E-mail for Sylvie Briquet: briquet{at}ext.jussieu.fr. E-mail for Catherine Vaquero: vaquero{at}ext.jussieu.fr. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back

{ddagger} Present address: Albert Einstein College of Medicine, New York, N.Y. Back

§ Present address: INMO, University of Gezira, Sudan. Back


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REFERENCES
 
    1
  1. Allain, F. H., Y. M. Yen, J. E. Masse, P. Schultze, T. Dieckmann, R. C. Johnson, and J. Feigon. 1999. Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding. EMBO J. 18:2563-2579.[CrossRef][Medline]
    2. 2
  2. Aravind, L., L. M. Iyer, T. E. Wellems, and L. H. Miller. 2003. Plasmodium biology: genomic gleanings. Cell 115:771-785.[CrossRef][Medline]
    3. 3
  3. Baxevanis, A. D., and D. Landsman. 1995. The HMG-1 box protein family: classification and functional relationships. Nucleic Acids Res. 23:1604-1613.[Abstract/Free Full Text]
    4. 4
  4. Beckerbauer, L., J. J. Tepe, J. Cullison, R. Reeves, and R. M. Williams. 2000. FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo. Chem. Biol. 7:805-812.[CrossRef][Medline]
    5. 5
  5. Bianchi, M. E., M. Beltrame, and G. Paonessa. 1989. Specific recognition of cruciform DNA by nuclear protein HMG1. Science 243:1056-1059.[Abstract/Free Full Text]
    6. 6
  6. Boschet, C., M. Gissot, S. Briquet, Z. Hamid, C. Claudel-Renard, and C. Vaquero. 2004. Characterization of PfMyb1 transcription factor during erythrocytic development of 3D7 and F12 Plasmodium falciparum clones. Mol. Biochem. Parasitol. 138:159-163.[CrossRef][Medline]
    7. 7
  7. Bustin, M. 1999. Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins. Mol. Cell. Biol. 19:5237-5246.[Free Full Text]
    8. 8
  8. Bustin, M. 2001. Revised nomenclature for high mobility group (HMG) chromosomal proteins. Trends Biochem. Sci. 26:152-153.[Medline]
    9. 9
  9. Bustin, M., D. A. Lehn, and D. Landsman. 1990. Structural features of the HMG chromosomal proteins and their genes. Biochim. Biophys. Acta 1049:231-243.[Medline]
    10. 10
  10. Callebaut, I., K. Prat, E. Meurice, J. P. Mornon, and S. Tomavo. 2005. Prediction of the general transcription factors associated with RNA polymerase II in Plasmodium falciparum: conserved features and differences relative to other eukaryotes. BMC Genomics 6:100.[CrossRef][Medline]
    11. 11
  11. Coulson, R. M., N. Hall, and C. A. Ouzounis. 2004. Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome Res. 14:1548-1554.[Abstract/Free Full Text]
    12. 12
  12. Douguet, D., and G. Labesse. 2001. Easier threading through web-based comparisons and cross-validations. Bioinformatics 17:752-753.[Abstract/Free Full Text]
    13. 13
  13. Dow, L. K., D. N. Jones, S. A. Wolfe, G. L. Verdine, and M. E. Churchill. 2000. Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA. Biochemistry 39:9725-9736.[CrossRef][Medline]
    14. 14
  14. Dumitriu, I. E., P. Baruah, A. A. Manfredi, M. E. Bianchi, and P. Rovere-Querini. 2005. HMGB1: guiding immunity from within. Trends Immunol. 26:381-387.[CrossRef][Medline]
    15. 15
  15. Erondu, N. E., and J. E. Donelson. 1992. Differential expression of two mRNAs from a single gene encoding an HMG1-like DNA binding protein of African trypanosomes. Mol. Biochem. Parasitol. 51:111-118.[CrossRef][Medline]
    16. 16
  16. Falquet, L., M. Pagni, P. Bucher, N. Hulo, C. J. Sigrist, K. Hofmann, and A. Bairoch. 2002. The PROSITE database, its status in 2002. Nucleic Acids Res. 30:235-238.[Abstract/Free Full Text]
    17. 17
  17. Felsenstein, J. 1989. PHYLIP—Phylogeny Inference Package (version 3.63). Cladistics 5:164-166.
    18. 18
  18. Gardner, M. J., N. Hall, E. Fung, O. White, M. Berriman, R. W. Hyman, J. M. Carlton, A. Pain, K. E. Nelson, S. Bowman, I. T. Paulsen, K. James, J. A. Eisen, K. Rutherford, S. L. Salzberg, A. Craig, S. Kyes, M. S. Chan, V. Nene, S. J. Shallom, B. Suh, J. Peterson, S. Angiuoli, M. Pertea, J. Allen, J. Selengut, D. Haft, M. W. Mather, A. B. Vaidya, D. M. Martin, A. H. Fairlamb, M. J. Fraunholz, D. S. Roos, S. A. Ralph, G. I. McFadden, L. M. Cummings, G. M. Subramanian, C. Mungall, J. C. Venter, D. J. Carucci, S. L. Hoffman, C. Newbold, R. W. Davis, C. M. Fraser, and B. Barrell. 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498-511.[CrossRef][Medline]
    19. 19
  19. Gissot, M. 2005. Etude de la régulation transcriptionnelle des gènes lors du cycle érythrocytaire de Plasmodium falciparum. Ph.D. thesis. Université Pierre et Marie Curie—Paris VI, Paris, France.
    20. 20
  20. Gissot, M., S. Briquet, P. Refour, C. Boschet, and C. Vaquero. 2005. PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation. J. Mol. Biol. 346:29-42.[CrossRef][Medline]
    21. 21
  21. Gnanasekar, M., R. Velusamy, Y. X. He, and K. Ramaswamy. 2006. Cloning and characterization of a high mobility group box 1 (HMGB1) homologue protein from Schistosoma mansoni. Mol. Biochem. Parasitol. 145:137-146.[CrossRef][Medline]
    22. 22
  22. Grasser, K. D., S. H. Teo, K. B. Lee, R. W. Broadhurst, C. Rees, C. H. Hardman, and J. O. Thomas. 1998. DNA-binding properties of the tandem HMG boxes of high-mobility-group protein 1 (HMG1). Eur. J. Biochem. 253:787-795.[Medline]
    23. 23
  23. Hirtzlin, J., P. M. Farber, and R. M. Franklin. 1994. Isolation of a novel Plasmodium falciparum gene encoding a protein homologous to the Tat-binding protein family. Eur. J. Biochem. 226:673-680.[Medline]
    24. 24
  24. Horn, P. J., and C. L. Peterson. 2002. Molecular biology. Chromatin higher order folding—wrapping up transcription. Science 297:1824-1827.[Abstract/Free Full Text]
    25. 25
  25. Horrocks, P., K. Dechering, and M. Lanzer. 1998. Control of gene expression in Plasmodium falciparum. Mol. Biochem. Parasitol. 95:171-181.[CrossRef][Medline]
    26. 26
  26. Kasinsky, H. E., J. D. Lewis, J. B. Dacks, and J. Ausio. 2001. Origin of H1 linker histones. FASEB J. 15:34-42.[Abstract/Free Full Text]
    27. 27
  27. Khochbin, S. 2001. Histone H1 diversity: bridging regulatory signals to linker histone function. Gene 271:1-12.[CrossRef][Medline]
    28. 28
  28. Knapp, S., S. Muller, G. Digilio, T. Bonaldi, M. E. Bianchi, and G. Musco. 2004. The long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes. Biochemistry 43:11992-11997.[CrossRef][Medline]
    29. 29
  29. Kun, J. F., and R. F. Anders. 1995. A Plasmodium falciparum gene encoding a high mobility group protein box. Mol. Biochem. Parasitol. 71:249-253.[CrossRef][Medline]
    30. 30
  30. Labesse, G., and J. Mornon. 1998. Incremental threading optimization (TITO) to help alignment and modelling of remote homologues. Bioinformatics 14:206-211.[Abstract/Free Full Text]
    31. 31
  31. Langst, G., and P. B. Becker. 2004. Nucleosome remodeling: one mechanism, many phenomena? Biochim. Biophys. Acta 1677:58-63.[Medline]
    32. 32
  32. Lanzer, M., D. de Bruin, and J. V. Ravetch. 1992. Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates. EMBO J. 11:1949-1955.[Medline]
    33. 33
  33. Lanzer, M., S. P. Wertheimer, D. de Bruin, and J. V. Ravetch. 1993. Plasmodium: control of gene expression in malaria parasites. Exp. Parasitol. 77:121-128.[CrossRef][Medline]
    34. 34
  34. Le Roch, K., C. Sestier, D. Dorin, N. Waters, B. Kappes, D. Chakrabarti, L. Meijer, and C. Doerig. 2000. Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue. J. Biol. Chem. 275:8952-8958.[Abstract/Free Full Text]
    35. 35
  35. Le Roch, K. G., Y. Zhou, P. L. Blair, M. Grainger, J. K. Moch, J. D. Haynes, P. De La Vega, A. A. Holder, S. Batalov, D. J. Carucci, and E. A. Winzeler. 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301:1503-1508.[Abstract/Free Full Text]
    36. 36
  36. Li, J., R. Kokkola, S. Tabibzadeh, R. Yang, M. Ochani, X. Qiang, H. E. Harris, C. J. Czura, H. Wang, L. Ulloa, H. S. Warren, L. L. Moldawer, M. P. Fink, U. Andersson, K. J. Tracey, and H. Yang. 2003. Structural basis for the proinflammatory cytokine activity of high mobility group box 1. Mol. Med. 9:37-45.[Medline]
    37. 37
  37. Li, W. B., D. J. Bzik, H. M. Gu, M. Tanaka, B. A. Fox, and J. Inselburg. 1989. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains. Nucleic Acids Res. 17:9621-9636.[Abstract/Free Full Text]
    38. 38
  38. Li, W. B., D. J. Bzik, M. Tanaka, H. M. Gu, B. A. Fox, and J. Inselburg. 1991. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III. Mol. Biochem. Parasitol. 46:229-239.[CrossRef][Medline]
    39. 39
  39. Lnenicek-Allen, M., C. M. Read, and C. Crane-Robinson. 1996. The DNA bend angle and binding affinity of an HMG box increased by the presence of short terminal arms. Nucleic Acids Res. 24:1047-1051.[Abstract/Free Full Text]
    40. 40
  40. Lotze, M. T., and R. A. DeMarco. 2003. Dealing with death: HMGB1 as a novel target for cancer therapy. Curr. Opin. Investig. Drugs 4:1405-1409.[Medline]
    41. 41
  41. Lotze, M. T., and K. J. Tracey. 2005. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat. Rev. Immunol. 5:331-342.[CrossRef][Medline]
    42. 42
  42. Martinez, E. 2002. Multi-protein complexes in eukaryotic gene transcription. Plant Mol. Biol. 50:925-947.[CrossRef][Medline]
    43. 43
  43. McAndrew, M. B., M. Read, P. F. Sims, and J. E. Hyde. 1993. Characterisation of the gene encoding an unusually divergent TATA-binding protein (TBP) from the extremely A+T-rich human malaria parasite Plasmodium falciparum. Gene 124:165-171.[CrossRef][Medline]
    44. 44
  44. Meissner, M., and D. Soldati. 2005. The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites. Microbes Infect. 7:1376-1384.[CrossRef][Medline]
    45. 45
  45. Morales, M., E. Onate, M. Imschenetzky, and N. Galanti.1992 HMG-like chromosomal proteins in Trypanosoma cruzi. J. Cell. Biochem. 50:279-284.[CrossRef][Medline]
    46. 46
  46. Muller, S., P. Scaffidi, B. Degryse, T. Bonaldi, L. Ronfani, A. Agresti, M. Beltrame, and M. E. Bianchi. 2001. New EMBO members' review. The double life of HMGB1 chromatin protein: architectural factor and extracellular signal. EMBO J. 20:4337-4340.[CrossRef][Medline]
    47. 47
  47. Murphy, F. V. T., R. M. Sweet, and M. E. Churchill. 1999. The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition. EMBO J. 18:6610-6618.[CrossRef][Medline]
    48. 48
  48. Nemeth, A., and G. Langst. 2004. Chromatin higher order structure: opening up chromatin for transcription. Brief. Funct. Genomics Proteomics 2:334-343.[Abstract/Free Full Text]
    49. 49
  49. Osta, M., L. Gannoun-Zaki, S. Bonnefoy, C. Roy, and H. J. Vial. 2002. A 24 bp cis-acting element essential for the transcriptional activity of Plasmodium falciparum CDP-diacylglycerol synthase gene promoter. Mol. Biochem. Parasitol. 121:87-98.[CrossRef][Medline]
    50. 50
  50. Paillard, F., G. Sterkers, and C. Vaquero. 1990. Transcriptional and post-transcriptional regulation of TcR, CD4 and CD8 gene expression during activation of normal human T lymphocytes. EMBO J. 9:1867-1872.[Medline]
    51. 51
  51. Parseghian, M. H., and B. A. Hamkalo. 2001. A compendium of the histone H1 family of somatic subtypes: an elusive cast of characters and their characteristics. Biochem. Cell Biol. 79:289-304.[CrossRef][Medline]
    52. 52
  52. Ponnudurai, T., J. H. Meuwissen, A. D. Leeuwenberg, J. P. Verhave, and A. H. Lensen. 1982. The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 76:242-250.[CrossRef][Medline]
    53. 53
  53. Ragab, A., and A. Travers. 2003. HMG-D and histone H1 alter the local accessibility of nucleosomal DNA. Nucleic Acids Res. 31:7083-7089.[Abstract/Free Full Text]
    54. 54
  54. Read, C. M., P. D. Cary, C. Crane-Robinson, P. C. Driscoll, and D. G. Norman. 1993. Solution structure of a DNA-binding domain from HMG1. Nucleic Acids Res. 21:3427-3436.[Abstract/Free Full Text]
    55. 55
  55. Silvie, O., J. F. Franetich, S. Charrin, M. S. Mueller, A. Siau, M. Bodescot, E. Rubinstein, L. Hannoun, Y. Charoenvit, C. H. Kocken, A. W. Thomas, G. J. Van Gemert, R. W. Sauerwein, M. J. Blackman, R. F. Anders, G. Pluschke, and D. Mazier. 2004. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites. J. Biol. Chem. 279:9490-9496.[Abstract/Free Full Text]
    56. 56
  56. Soullier, S., P. Jay, F. Poulat, J. M. Vanacker, P. Berta, and V. Laudet. 1999. Diversification pattern of the HMG and SOX family members during evolution. J. Mol. Evol. 48:517-527.[CrossRef][Medline]
    57. 57
  57. Su, X. Z., Y. Wu, C. D. Sifri, and T. E. Wellems. 1996. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 24:1574-1575.[Free Full Text]
    58. 58
  58. Thomas, J. O., and A. A. Travers. 2001. HMG1 and 2, and related ‘architectural’ DNA-binding proteins. Trends Biochem. Sci. 26:167-174.[CrossRef][Medline]
    59. 59
  59. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    60. 60
  60. Thomsen, M. S., L. Franssen, D. Launholt, P. Fojan, and K. D. Grasser. 2004. Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein. Biochemistry 43:8029-8037.[CrossRef][Medline]
    61. 61
  61. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675.[Abstract/Free Full Text]
    62. 62
  62. Travers, A. A. 2003. Priming the nucleosome: a role for HMGB proteins? EMBO Rep. 4:131-136.[CrossRef][Medline]
    63. 63
  63. van de Wetering, M., M. Oosterwegel, K. van Norren, and H. Clevers. 1993. Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J. 12:3847-3854.[Medline]
    64. 64
  64. Wang, H., O. Bloom, M. Zhang, J. M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, K. R. Manogue, E. Faist, E. Abraham, J. Andersson, U. Andersson, P. E. Molina, N. N. Abumrad, A. Sama, and K. J. Tracey. 1999. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285:248-251.[Abstract/Free Full Text]
    65. 65
  65. Webb, M., and J. O. Thomas. 1999. Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain. J. Mol. Biol. 294:373-387.[CrossRef][Medline]
    66. 66
  66. Weir, H. M., P. J. Kraulis, C. S. Hill, A. R. Raine, E. D. Laue, and J. O. Thomas. 1993. Structure of the HMG box motif in the B-domain of HMG1. EMBO J. 12:1311-1319.[Medline]
    67. 67
  67. Wu, Q., W. Zhang, K. H. Pwee, and P. P. Kumar. 2003. Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently. Arch. Biochem. Biophys. 411:105-111.[CrossRef][Medline]
    68. 68
  68. Yen, Y. M., B. Wong, and R. C. Johnson. 1998. Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine. J. Biol. Chem. 273:4424-4435.[Abstract/Free Full Text]
    69. 69
  69. Yoshioka, K., K. Saito, T. Tanabe, A. Yamamoto, Y. Ando, Y. Nakamura, H. Shirakawa, and M. Yoshida. 1999. Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein. Biochemistry 38:589-595.[CrossRef][Medline]

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  • Abhyankar, M. M., Hochreiter, A. E., Hershey, J., Evans, C., Zhang, Y., Crasta, O., Sobral, B. W. S., Mann, B. J., Petri, W. A. Jr., Gilchrist, C. A. (2008). Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection. Eukaryot Cell 7: 1565-1572 [Abstract] [Full Text]  
  • Gissot, M., Ting, L.-M., Daly, T. M., Bergman, L. W., Sinnis, P., Kim, K. (2008). High Mobility Group Protein HMGB2 Is a Critical Regulator of Plasmodium Oocyst Development. J. Biol. Chem. 283: 17030-17038 [Abstract] [Full Text]  

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