Possible Role of the CCAAT/Enhancer Binding Protein in the Expression Regulation of the EhPgp1 Multidrug Resistance Gene in Entamoeba histolytica

Abstract

Several potential binding sites for transcription factors participating in gene expression regulation have been identified in the 5 9 -flanking region of the EhPgp1 gene involved in the multidrug resistance (MDR) phenotype of the emetine-resistant mutant clone C2. To begin the fine characterization of the cis -regulatory sequences of the EhPgp1 gene core promoter, we focused our attention on two highly conserved CCAAT/enhancer binding protein (C/EBP) motifs located at 2 54 and 2 196 bp positions at the EhPgp1 gene promoter. According to the results obtained from electrophoretic mobility shift assays (EMSA), these sites seem to be involved in the expression of this gene (1). The C/EBP family members are important in regulating many eukaryotic genes during differentiation processes. These nuclear proteins have between 150 and 360 amino acids. They present a bipartite DNA-binding domain composed of a positively charged region that contacts the DNA, an amphipathic helix at the conserved carboxy-terminal end that mediates dimerization through the formation of a leucine zipper, and a less well-conserved amino-terminal region containing regulatory and transactivation domains. All C/ EBPs share homology in their basic domain and, as a result, recognize the same DNA motif (T T / G TGG T / A T / A A / T ), forming homoor heterodimers with each other (2). In this report, we present the location and alignment of consensus C/EBP sequences in mdr gene promoters of mammals and protozoan parasites. By EMSA, we detected a nuclear factor able to bind the C/EBP sites in E. histolytica and propose it as a possible trans -regulatory element in MDR phenotype regulation, as described in mammals. Materials and Methods