Журнал бактериологии и паразитологии

Абстрактный

Transcriptional activation by a URE4-like sequence in the EhPgp1 ​​gene core promoter

M. E. Ramirez, D. G. Perez, E. Nader, and C. Gomez

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 are involved in the transcriptional activation of this gene. However, there is another corresponding region that also governs the regulation of EhPgp1 ​​expression in clone C2. In this report, we provide evidence that EhPgp1 ​​gene transcription is at least partially regulated by cis-acting repeat sequences R9 and the EhEBP1 protein. Structural analysis of the region from -234 to -197 bp reveals the presence of two 9-bp repeat sequences [R9(1) and R9(2)] located in the range from -226 to -203 bp. Analysis of deletions and mutations of R9 motifs significantly reduced promoter activity in trophozoites from clone C2. EMSA experiments revealed specific binding of E. histolytica core proteins to the R9 sequence, while competition assays showed that more than one R9 sequence is required for strong DNA-protein interactions. Moreover, Western blot experiments with partially purified R9 motif-interacting proteins and anti-EhEBP1 antibodies recognized a 28 kDa protein. Interestingly, this antibody prevented the formation of DNA-protein interactions between R9 sequences and amoeba core proteins in supershift assays, indicating that one of the proteins interacting with the R9 element is EhEBP1-like. In conclusion, we demonstrate that R9 motifs are recognized by EhEBP1 and activate EhPgp1 ​​gene expression.