Cis-Elements Upregulate the Activity of the Entamoeba histolytica EhPgp1 Gene Promoter

Abstract

EhPgp1 , one of the genes responsible for the MDR phenotype in Entamoeba histolytica , is differentially transcribed in trophozoites of the drug-sensitive clone A and in the drug-resistant clone C2. Our previous results suggested that the expression of this gene is mainly regulated at transcriptional level (1). EhPgp gene promoters have transcription factor binding sequences similar to those described in other organisms, mainly in mammals (1,2). In the EhPgp1 promoter, we found C/EBP, HOX, GATA-1, OCT, and Inr DNA binding sites. These sequences seem to be relevant for the expression of the EhPgp1 gene. Gel shift assays showed complexes formation between DNA promoter fragments and nuclear extracts. Competition assays using the consensus sequences for these transcription factors inhibited the formation of the DNA–protein complexes (1). The identification of the transcriptional regulation factors in E. histolytica genes is fundamental to understand the mechanisms controlling switching of parasite genes. Transcriptional selectivity of the eukaryotic genes is mediated by complex control regions composed of different combinations of elements to allow multiple distinct regulatory factors that function coordinately to potentiate RNA synthesis. However, unique promoter specificity and regulation can be conferred by the particular composition and spatial organization of the multiple elements constituting the complete set used by a given gene. The analysis of binding sequences found in the EhPgp1 promoter showed certain differences with the reported consensus sequences in other organisms. Thus, their functional role needs to be proven by transfection assays. Additionally, their precise positioning on the promoter is also important for the formation of stable complexes that permit the expression of this gene. To investigate the mechanisms regulating differential expression of the EhPgp1 gene, we generated several constructs deleting different sequences in this promoter. The DNA fragments were cloned in front of the CAT reporter gene. Then, the constructs were transfected in sensitive and resistant trophozoites and their ability to drive CAT expression was analyzed.