We have shown that EhHMGB1 is a homolog of mammalian HMGB protein. EhHMGB1 was able to bend DNA, which is a characteristic feature of all HMGB proteins. Mutation of two key conserved residues in EhHMGB1, which are required for DNA bending in other HMGB proteins, inhibited its ability to bend DNA, whereas deletion of the C-terminal acidic tail did not. EhHMGB1 enhanced binding of human p53 to its target DNA and, as expected, was located in the parasite nucleus. Overexpression of EhHMGB1 resulted in modulation of 33 transcripts involved in a variety of cellular functions, including 20 altered in amebae invading the mouse intestine. Most notable was the modulation of parasite genes encoding two of the light subunits of the Gal/GalNAc inhibitable lectin.
HMG proteins are one of the most evolutionarily conserved proteins in eukaryotes. They are abundantly expressed nonhistone proteins which play a role in nucleosome remodeling (39
). All members of the HMG superfamily share a common structural motif called the HMG box. The HMGB proteins typically interact with specific DNA structures rather than sequence. They have been divided into three families: HMGA proteins, which interact with an AT hook; HMGN proteins, which interact with the nucleosomes; and HMGB (15
). Structurally the HMGB domain consists of approximately 80 amino acids that fold into three alpha helices forming an L-shaped structure (7
). The vertebrate HMGB proteins have two consecutive L-shaped domains due to the presence of two HMG boxes. They also possess basic N termini and long acidic C-terminal tails. The two P. falciparum
proteins which have been studied (PfHMGB1 and PfHMGB2) contain only one HMGB domain (13
). In contrast, the six HMGB proteins that have been identified in S. mansoni
and Schistosoma japonicum
) contain two HMGBs similar to mammalian HMGB proteins. The domain organization and amino acid sequence of EhHMGB1, which carries a single HMGB domain, is most similar to the Plasmodium
HMGBs. Although both single and double HMGB domain-containing proteins have the ability to bend and stabilize DNA, it has been suggested that proteins with two HMGBs have a greater affinity for DNA, but the significance of an increased affinity is not known (45
In metazoan eukaryotes the amino acids serine at position 10 and a hydrophobic residue at position 32 (domain numbering based on HMG-D) were predicted to be crucial for HMGB function by mutational and bioinformatics analysis (13
). In EhHMGB1 the equivalent residues are conserved (threonine, a conserved substitution, and phenylalanine, a hydrophobic residue). Point mutations of both these residues resulted in the loss of DNA bending ability, a proof of involvement of one or both of these residues in HMGB function (Fig. ). This allowed us to assign EhHMGB1 as a classical HMGB family transcription factor.
PfHMGB1 and PfHMGB2 lack the characteristic C-terminal acidic tail of HMGB proteins in metazoan eukaryotes. The EhHMGB1 C-terminal acidic tail is comprised of nine acidic residues interrupted by a glycine and a serine residue. The exact role of the acidic tail in HMGB proteins is unknown, although it has been suggested that it interacts with the positive charges of histones (63
). The much longer acidic tail of human HMGB1 has been shown to be repressive for DNA bending (31
). However, the deletion of the short C-terminal acid tail of SmHMGB1 did not affect the ability of this protein to support the ligation of a 123-bp DNA fragment (21
). Similarly, the deletion of the EhHMGB1's acidic C-terminal residues did not change the function of the protein in ligase-mediated circularization assays. This mutant appeared in fact to enhance the formation of circular DNA, as seen from the densitometry analysis.
HMGB proteins are known to facilitate the binding of a variety of transcription factors to their cognate DNA binding sites (1
). Proteins shown to physically interact with HMGBs include the HOX, p73, Rel, nuclear hormone receptor, and p53 transcription factors (2
). HMGB proteins operate as chaperones and augment the binding of p53 to linear DNA by presenting prebent DNA for p53 binding (47
). It has also been suggested that HMGB remains associated with DNA only transiently and is released when the cognate transcription factor has bound (44
). The wild-type EhHMGB1 possessed the capability to enhance the binding of human p53 to its target sequence in a manner similar to that of human HMGB1 (Fig. ). This suggests that EhHMGB1 could provide an active architectural role by interacting with many potential E. histolytica
transcription factors including the homolog of human p53, Ehp53 (17
The acid tail of EhHMGB1 may have an important role in this process because the Δ-Acid mutant, although not defective in DNA bending, was defective in the enhancement of p53 transcription factor binding. Therefore, this suggests that the absence of an acid tail traps EhHMGB1 into a stable DNA complex and thus prevents the binding of other transcription factors (1
). Bonaldi et al. have previously shown that the mutant HMGB1 lacking the acidic tail had 100-fold-increased DNA binding affinity but completely froze ACF (A
TP utilizing c
hromatin assembly and remodeling f
actor)-mediated nucleosome sliding (11
). Thus, the presence of the acidic tail seems to be important for transient interaction of HMGB proteins with DNA. As expected, the double mutant with mutations in the conserved amino acids Thr34Ala and Phe56Ala failed to bend DNA or to enhance the binding of p53. Future studies will be required to determine the biological role of EhHMGB1 in the assembly of E. histolytica
Microarray analysis of cells overexpressing EhHMGB1 showed a substantial overlap with the transcriptome profile of trophozoites isolated from the mouse model of amebiasis (Fig. ). However, these transcripts were modulated in opposite directions (Fig. ). One explanation for this unexpected result is the 100-fold overexpression of EhHMGB1.
Overexpression of EhHMGB1 alone without cooperating transcription factors could lead to a dominant-negative effect or another type of dysregulated gene expression. Nevertheless, these data can still provide an important insight into the EhHMGB1 regulon (see Table S2 in the supplemental material). These changes suggest that EhHMGB1 could be part of the trophozoite's response to sudden changes in the environment when it encounters the host. One could anticipate the need for the parasite to quickly adjust to changes in carbon source and the presence of existing microbial flora in the host gut as well as to host defense mechanisms. The EhHMGB1-modulated genes involved in cellular metabolism belong to various pathways including carbon metabolism. In addition, genes implicated in virulence were altered. Of particular interest were four members of the E. histolytica
-induced gene) gene family, which are similar to plant AIG genes involved in bacterial resistance. The function of AIG genes in E. histolytica
still needs to be defined (56
). The most striking observation was upregulation of the transcripts encoding light subunits of the Gal/GalNAc inhibitable lectin that mediates contact-dependent cytolysis of host cells, an important hallmark of amebiasis. The Gal/GalNAc lectin is composed of a transmembrane heavy subunit (170 kDa) linked by disulfide bonds to the light subunit (31 to 35 kDa) and associated with an intermediate subunit (150 kDa). EhHMGB1 overexpression resulted in a significant increase in the levels of two of the five homologous genes that encode the light subunits Lgl3 and Lgl4 (4
To our knowledge, this is the first report for a unicellular parasite of a role for an HMGB protein in the regulation of parasite genes involved in pathogenicity. This suggests a role of EhHMGB1 in parasite adaptation to, and destruction of, the host intestine.