Transcriptional Activation by an URE4-like Sequence in the EhPgp1 Gene Core Promoter

Abstract

EhPgp1 is one of the multidrug resistance genes expressed in drug resistant trophozoites from Entamoeba histolytica. Previous studies in our laboratory have demonstrated that two C/EBP sites participate in the transcriptional activation of this gene. However there is other relevant region that also governs the regulation of EhPgp1 expression in clone C2. In this report we provide evidence that transcription of the EhPgp1 gene is at least partly regulated by the cis-acting R9 repeated sequences and EhEBP1 protein. Structural analysis of the region from -234 to -197 bp shows the presence of two repeated sequences of 9 bp [R9(1) and R9(2)] located at -226 to -203 bp. Deletions and mutations analysis of the R9 motifs significantly reduced promoter activity in trophozoites from clone C2. EMSA experiments revealed specific binding of nuclear proteins from E. histolytica to the R9 sequence. While competition assays showed that the presence of more than one R9 sequence is necessary for a strong DNA-protein interaction. Moreover, Western blot experiments with partially-purified proteins interacting with the R9 motif and antibodies against EhEBP1, recognized a 28 kDa protein. Interestingly, this antibody in supershift assays prevented the DNA-protein interactions formation, of the R9 sequences and nuclear proteins from amoeba, indicating that one of the proteins that interact with the R9 element is an EhEBP1-like one. In conclusion, we demonstrate that R9 motifs are recognized by an EhEBP1 protein and activate the EhPgp1 gene expression.