Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression

Here, we show the relevance of promoter regions (-74 to +24, -167 to -75 and -259 to -168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites (-54 to -43 bp and -198 to -186 bp) are cis-activating elements of gene expression in the drug-resistant (clone C2) and -sensitive (clone A) trophozoites. Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti-human C/EBPβ antibodies. UV cross-linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively. The nuclear factors that bind to C/EBP sites were semipurified by affinity chromatography. They were immunodetected by anti-human C/EBPβ antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at -54 bp stabilizes the transcription pre-initiation complex, whereas the other site at -198bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.